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CYTOGENETIC RELATIONSHIP BETWEEN CULTIVATED RICE AND FOUR DIPLOID WILD RICE

RAJ KUMAR NIROULA

April 2003

CYTOGENETIC RELATIONSHIP BETWEEN CULTIVATED AND NEPALESE WILD SPECIES OF RICE

RAJ KUMAR NIROULA

THESIS SUBMITTED TO THE TRIVHUVAN UNIVERSITY INSTITUTE OF AGRICULTURE AND ANIMAL SCIENCE RAMPUR, CHITWAN, NEPAL

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE (PLANT BREEDING)

APRIL 2003

II

ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude and appreciation to his major advisor, Dr. Lakshmi Prasad Subedi, whose guidance was genuine and continuous from germplasm collection to end of the laboratory experiment. He is grateful to Prof. Dr. R.C. Sharma, Dr. Madhusudan Prasad Upadhyay (NARC) and Mr. Nav Raj Adhikari (members of the advisory committee) for their constant guidance, inspirations, timely suggestions, constructive criticisms, keen interest and continuous cooperation throughout the course of this study. The author is also greatly indebted to Prof. Dr. Ram Chandra Sharma, for making financial support, relevant journals, assistantship, and some experimental materials beside his role of committee member. Similarly, the effort made by Dr. M. P. Upadhyay to provide fund from IPGRI (International Plant Genetics Resource Institute, Rome, Italy) is highly appreciated. The author is greatly indebted to Dr. S.D. Brar (Scientist, Plant Breeding, Genetics and Biochemistry Division, IRRI) for his valuable suggestions, timely help, and providing a volume of literatures. The author appreciates his great patience to handcarry the some of the valuable and expensive chemicals from Philippines to Kathmandu (Nepal). The author also extends his special thanks to Mr. Madhav Prasad Pandey for his valuable suggestions, interest, sharing of his experience on the part of this study and his cooperation. He is very grateful to Dr. Sarah Johnson (Cornell University) for her kindly help and suggestions to improve the language of this manuscript. The partial grant (C11G/6800-C11G) support from IPGRI is highly appreciated. Similarly, the help and timely suggestions provided by Dr. Bhuwan Ratna Sthapit

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(Scientist, IPGRI), and Dr. Toby Hodgkin (Principal Scientist, IPGRI) are highly appreciated. Without their cooperation the research would not be possible. He would also like to express his gratitude to the following individuals for providing the constant inspiration, timely suggestions, and co-ordination: Dr. G.L. Shrestha (Director, GEM/Nepal), B.K. Joshi (Scientist, NARC), Dr. S.R. Shakya (Botany Division, TU). He would also like to thank the library of IAAS, NARC, Central library TU, and IRRI, Philippines. The author also grateful to Prof. Dr. Tej Bahadur K. C. (Dean) and Prof. Dr. D.D. Dhakal (Ex Dean), IAAS, Rampur, for providing the favorable environment that allowed success of this study. Likewise, the author is grateful to all the department members and his colleagues for their interest. He would like to express his sincere appreciation to the following individuals: Mr. Madan Adhikari, Mr. Bhanu Pokhrel (NARC), Anju. Baral, R. Upadhyay. S. Bhandari, Mr. Bharat Khanal, Ram and Shyam Basnet, Benup Aryal, Ram Kumar B.K, Deepak and Binod Ghimire, R. Burlakoti, S. Bhushal and Tulsi Photo Studio (Rampur) for their financial support and research assistance during study. At the last but not least the author extends his deep love to his parents Ram Bahadur and Muna Maya, brothers Shesh Kumar and Prem Kumar (Rajesh) and all sisters and brothers in law, without whose inspiration and moral support this piece of work would have been left undone. To them, he has no words to thank.

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DEDICATED TO

MY BELOVED PARENTS

RAM BAHADUR AND MUNA MAYA

TABLE OF CONTENTS Title Acknowledgements Table of contents List of tables List of figures and plates List of appendices Abbreviations Abstract 1. Introduction 1.1. Background 1.2. Objectives 2. Literature Review 2.1. Taxonomy and origin of rice 2.2. Rice biodiversity in Nepal 2.3. Description of Nepalese wild species of rices 2.3.1. Oryza granulata 2.3.2. O. officinalis 2.3.3. O. nivara and O. rufipogon 2.4. Genome and Genepool of rice 2.5. Importance of wild germplasm 2.6. Progress towards the utilization of wild relatives of rice 2.7. Wide hybridization 2.7.1. Barriers to wide hybridization 3-6 7 8 8-9 10 10 10-11 11 11 12 12-15 15-17 17-18 18 III-IV VI-IX XI XII XIII XIV 1-2

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2.7.1.1.Prefertilization barriers 2.7.1.2. Postfertilization barriers 2.8.1. Crossability 2.8.2. Causes of embryo abortion in wide crosses 2.8.3. Seed and embryo differentiation in hybrids 2.9. Embryo rescue in rice

18-19 19-20 21 21-22 22-23 23-24 24 25 25-26 26 26-27 27 27 27-28 28-29 29-30 30-31 31-32 33 33-34 34-35 35-36 36 37

2.10. Hardening of regenerated seedlings before field transfer 2.11. Hybrid embryo derived callus culture 2.12. In vitro fertilization 2.13. Media composition 2.14. Hybrid sterility 2.15. Chromosome pairing in haploid rice 2.16. Chromosome behavior between AA genome species hybrids 2.16.1. Meiosis in intervarietal crosses 2.16.2. Pachytene analysis 2.16.3. Diplotene, Diakinesis, and Metaphase I 2.16.4. Chiasma frequency 2.16.5. Anaphase bridge: cytological view 2.17. Meiotic behavior in intergenomic hybrids 2.18. Pachytene analysis in intergenomic crosses 2.19. Asynapsis and/or desynapsis 2.20. Auto and allosyndetic pairing 2.21. Nucleolus: number, type, and shape 2.22. Recent progress in rice chromosome research

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3. Materials and Methods 3.1. Preparation of parental materials 3.1.1. Germplasm collection 3.1.2. Germination and green house rearing 3.2. Hybridization 3.2.1. Cross combination 3.2.2. Forced anthesis 3.2.3. Emasculation 3.2.4. Artificial pollination 3.3. Embryo rescue 3.3.1. Aseptic excision of immature hybrid embryo 3.3.2. Aseptic preparation of mature hybrid embryo 3.3.3. Inoculation of embryo 3.3.4. Media preparation 3.3.5. Media employed 3.3.6. Media efficiency determination 3.3.7. Incubation of culture\ 3.3.8. Seedlings transfer 3.4. 3.5. 3.6. 3.7. Regeneration of plants from callus of hybrid embryos In vitro pollination Harvesting of F1 seeds Determination of crossability 3.7.1. Crossability between O. sativa and common wild rice species 38 38 38-41 41 41 41 42 42 42-43 43 43 44 44 44-45 45 45 45-46 46-47 47 47-48 48 48

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3.7.2. Crossability determination in intergenomics crosses 3.8. 3.9. Morphological characterization Chromosome preparation 3.9.1. Chemical preparation 3.9.1.1.Stain preparation 3.9.1.2.Fixative preparation 3.9.2. Standardization of suitable stages for meiotic study 3.9.3. Meiotic behavior study 3.9.3.1. Chromosome analysis 3.9.3.2. Pachytene analysis 3.9.3.3. Chiasma frequency determination 3.9.3.4. Detection of univalents, bivalents, trivalents, and quadrivalents 3.9.4. Pollen fertility and sterility determination 3.9.5. Spikelet fertility and sterility determination 4. Results and Discussions 4.1. Description of Nepalese wild rice speices 4.2. In vitro manipulation 4.2.1. Media efficiency determination 4.2.2. Embryo rescue 4.2.3. Hybrid embryo derived callus culture 4.2.4. In vitro fertilization 4.3. Crossability 4.3.1. Crossability in intergenomic cross combination

48-49 49 49 49 49-50 50 50-51 51 51-52 52 52 53

53-54 54-55

56-57 58 58 59-62 62-63 63-66 66 66

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4.3.2. Crossability between intragenomic species crosses 4.4. Morphology of the interspecific hybrids 4.5. Meiotic behavior in parents and their hybrids 4.5.1. Pachytene analysis in AA genome hybrids and their parents and their parents 4.5.2. Pachytene analysis in intergenomic (A and C genome) 4.5.3. Variation in nucleolus shape in parents and their hybrids 4.5.4. Diakinesis and Metaphase I. 4.5.4.1.Chiasma frequency in parents and their hybrids 4.5.4.2. Chromosome number and ploidy level 4.5.5. Meiotic behavior at Anaphase I and Telophase I in parents and their hybrids 4.6. Hybrid fertility and sterility: cytological dissection

66-68 68-69 69 69-75

77-79

79-81

81-91 91-97

97-98 98-102

105-109 110-112 113 114-143 144-156

Summery and conclusion Recommendation Literature cited Appendices

LIST OF TABLES

2.1. 2.2. 3.1. 3.2. 4.1. 4.1a. 4.1b. rice

Chromosome number, Genomic composition, geographical distribution and useful traits of Oryza species. Progress in gene introgression and transfer from wild Oryza species into elite lines of cultivated rice Description of germplasm collection, collection site, type of collection and their species name. Crossing scheme employed during experiment. Morphological characters of four Nepalese wild species of rice recorded during study period 2001/2002. Results of embryo rescue and crossability between O. sativa and two distant relatives; O. granulata and O. officinalis. Seed set, and crossability between O. sativa L. and two common wild species (O. nivara and O. rufipogon).

13-14 16 39 40 57 65 67 76 80 83-84 88-89 94-95 96

4.2.1. Chromosome behavior at pachynema in hybrids and their parents 4.2.2. Percent frequency of nucleolus shape variation in pachytene stage 4.2.3a. Meiotic configuration at diakinesis and metaphase I. from the parents used in the hybridization 4.2.3b. Meiotic configuration at diakinesis and metaphase I from the interspecific hybrids involving three different species. 4.2.3c. Percentage of normal and abnormal chromosome behavior at diakinesis and metaphase I in the parents and F1s 4.2.3d. Percentage of normal and abnormal chromosome behavior at diakinesis and metaphase I in the hybrid involving O. sativa L. and O. officinalis. Wall ex Watt. 4.2.4. Meiotic behavior at anaphase and telophase I in the parents and hybrids 4.2.5. Mean percentage of pollen and spikelet fertility in the parents and F 1 hybrids

103-104 108

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LIST OF PLATES AND FIGURES

Fig. 2.1. Evolutionary pathway of the two cultivated species of rice. Fig. 2.2. Schematic representation of stages in normal sexual reproduction and related barriers to interspecific hybridization. Plate1.Invitro manipulation: efforts made to regenerate intergenomic hybrids, figure a-l. Plate 2.Comparative morphology of parents and their hybrids. Plate3. Morphology of hybrid plants, figure 1-8, and wild species O. granulata. Plate4. Figure showing meiotic behavior at pachynema in hybrids, figure 1-3. Plate5. Figure showing meiotic behavior at pachynema in hybrids, figure 1-3. Plate6. Figure showing meiotic behavior at pachynema in hybrids, figure 1-3. Plate7.Figure showing meiotic behavior at pachynema in hybrids and parents, figure1-5. Plate8. Meiotic behaviour at diplotene and diakinesis in hybrids and parents, figure 1-11. Plate9. Meiotic configuration at metaphaseI in different hybrid combinations, figure 1-12. Plate10.Representative microphotograph of meiotic configuration at MI, TI in hybrid plant, figure 1-9. Plate11.Representative microphotograph of meiotic configuration at AI, and TI in hybrid plants, figure 1-11 and figure 12, showing hybrids. partial pollen sterility in AI, and rice

9 20 64 60 61 71 72 73 74 85 86 99 100

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LIST OF APPENDICES

3.1.

Nutritional components of different media employed for embryo culture during study period.

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3.2. 4.1. 4.2.

Nutrient constitution for the preparation of hardening solution. Comparative results of embryo culture on five different sterile media (preliminary report). Comparative results of different hardening techniques employed.

145 146-147 148 149-156

4.3a-4.3h. Phenotypic characters relationship between parents and their hybrids.

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ABBREVIATION AND SYMBOL

AFLP Ag. AI Dl Dp FISH GISH Hm I IAAS II III In IRRI ISH ISSR IV Lp M2 MI NARC NB RAPD RFLP TI Tl TU VI

Amplified Fragment Length Polymorphism Agriculture Anaphase I Deletion Duplication Fluorescence In situ hybridization Genomic In situ hybridization Heteromorphocity Univalent Institute of Agriculture and Animal Science Bivalent Trivalent Inversion International Rice Research Institute In situ Hybridization Inter Simple Sequence Repeat Quadrivalent Loose Pairing Second generation mutant progeny Metaphase I Nepal Agriculture Research Council Nucleolar Bodies Randomly Amplified Polymorphic DNA Restriction Fragment Length Polymorphism Telophase I Translocation Tribhuvan University Hexavalent

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CYTOGENETIC RELATIONSHIP BETWEEN CULTIVATED AND NEPALESE WILD SPECIES OF RICE ABSTRACT Name: Raj Kumar Niroula Semester and Year of enrollment: Ist semester, 2000 Major Subject: Plant Breeding Major advisor: Dr. Lakshmi Prasad Subedi ID. No: R-2000-PB-21-M Degree: M. Sc. (Ag.) Department: Plant Breeding

Four wild species (O. rufipogon, O. nivara, O. officinalis, and O. granulata) and nine cultivated varieties (IR 64, IR 72, Manshara, Jhinuwa, Pokhreli, Jethobudo, Kalanamak, Ghaiya, and Masuli) were used to study the crossability between species, morphology, pollen and spikelet fertility, and meiotic behavior of chromosomes in the parents and their hybrids, during 2001-2002 at IAAS, Rampur. Embryo rescue, callus culture, and in vitro fertilization were attempted to produce intergenomic hybrids and to overcome both pre and post fertilization barriers through manipulating different media. Among these methods, embryo rescue followed by new hardening technique was found to be capable of regenerating the intergenomic hybrids. However, embryo rescue technique was not able to overcome strong post fertilization barrier. Based on the embryo rescue results, the crossability between O. sativa and O. officinalis ranged from 0-2.44%. Strong crossability barrier was found between O. sativa and O. granulata, and hence no hybrids were obtained. The crossability between O. sativa and common wild rice varied from 7.5851.05%, and on an average landraces had closer cross affinity than advanced cultivars. Comparative study of morphology in parents and hybrids revealed that wild traits were found dominant to cultivated forms. Percent mean pollen and spikelet fertility in parents and hybrids varied from 3378.16 and 0-49.93, and 30-91.46 and 0-86.49, respectively. Although, Manshara/O. nivara had low pollen fertility, seed set was higher than its female parent. No significant associations were observed between mean percentage of stainable pollen and spikelet fertility. The meiotic behavior at different stages of meiosis in intragenomic hybrid was more or less normal, and the frequency of aberrations was comparable to their respective parents. Only six out of eleven hybrid combinations had cryptic structural hybridity in 4-22.58% cells at pachytene stage. In these hybrids existence of loose pairing, inversion, translocation, deletion, duplication, and heteromorphocity were 1

observed. Similarly, intergenomic hybrid (O. sativa/O. officinalis) showed high abnormality in later stages of meiosis, although pachytene was normal in more than 50% cells. Therefore, it is inferred that the abnormality in these hybrids was brought about by desynapsis rather than absence of complete homology between their genome (A and C). Most of the chromosomes in these hybrids appeared as univalents (24) at diakinesis and metaphase I, and laggards and bridges at anaphase I and telophase I. Based on chiasma frequency, O. granulata was found more primitive than other species. All the analyzed wild species of Nepal were diploid (2n = 24). At diakinesis and Metaphase I, no sharp reduction in chiasma frequency was observed except in O. sativa/O. officinalis derived hybrids. The mean frequency of ring bivalent was always high and varied from 10.42-11.25 and had >21-<24 chiasmata/cell in most of the hybrids. Other minor irregularities such as presence of univalents, quadrivalents, presence of nucleolar bodies, bridges and laggards, laggards, bridges and fragments, and unequal segregation at diakinesistelophase I were observed and were comparable to their parents. However, cells with bridges + fragments were highest in IR64/O. rufipogon (9.40%). On an average, no sharp structural differentiation was found between common wild rice and landraces of Nepal. Thus the partial sterility accounted in hybrids did not associate with chromosomal abnormalities except in IR64/O. nivara, IR 64/O.rufipogon, Jethobudo/O. nivara, and O. sativa/O. officinalis hybrids. Some of the landraces like Pokhreli, Jethobudo, and Kalanamak showed high crossability, F1 fertility and close meiotic affinity with O. rufipogon, and those of Manshara with O. nivara.

Dr. Lakshmi Prasad Subedi Major advisor

Raj Kumar Niroula Author

INTRODUCTION 1.1. Background Rice is the principal lifeline food of more than half of the worlds population (Mallik, 2002). Among cereals, rice is the major source of calories for most of the Asian people where 90% of rice is produced and consumed. At present demand for rice is rapidly increasing with the increase in population and will be increasing continuously, especially in less developed countries (Virmani et al., 1997). In spite of several yield limiting factors, it is expected, so much so, that by 2025, 800 million tones of rice will have to be produced annually to feed ever increasing population (Swaminathan, 1998). Similarly, Rice farming in Nepal is the foremost industry that contributes about 24% of the total gross agricultural products (Pokhrel, 1997), and the actual national economy still depends on what happens to rice cultivation. It is widely grown in different agro-ecological zones accounting for more than 50% of the total area and production (Upadhyay, 1996). Although, rice (Oryza sativa. L.) in Nepal has been cultivated prior to Vedic time (Upadhyay et al., 2002) and its cultivation ranges from the seasonal deep-water river basin to steep sloppy upland field of over 2700 m. altitude, the increment in average productivity during the past decays has been small. However, variations exist in its landraces and wild relatives, from the floating rice of tropical climate to chill tolerant upland rice varieties, indicating that Nepal has rich rice genetic resources (Shahi, 1999). In spite of its low productivity, at present, it is estimated that Nepal has over 2000 local landraces (Shrestha and Shrestha, 1999), and four species namely O. rufipogon, O. nivara, O. granulata, and O. officinalis out of 22 valid wild species of rice around the world (Upadhayay et al., 2002; Shrestha and Vaughan, 1989) and these constitute the all three basic gene pool of rice (Khush, 2000). Beside, Nepal also harbors weedy rice: Oryza

sativa f. spontanea, and Oryza related genera: Leersia hexandra, and Hygroryza aristata. These landraces and wild relatives of cultivated crop plants constitute the excellent insurance against the genetic vulnerability (Chang, 1976b). It is because of wild relatives have been used as gene/s source, against the biotic and abiotic stresses, to provide alternative cytoplasm and develop cytoplasmic sterility systems, to widen adaptation, to improve stature, increase crossability between, and increased yield. (Harlan, 1976). It is also note worthy that some crops could not maintain commercial status without genetic support of their wild species (Harlan, 1976). Beside these wild species of rice can be used in hybridization and varietal development and improvement programmes (Brar et al., 2002), and to isolate the apomictic gene block/s for sustaining and the commercialization of the hybrid rice production throughout the world through various biotechnological tools (Li and Yuan, 2000). Experiences at IRRI and in China and other countries have made remarkable contributions to recognize the worth of wide hybridization and wild relatives of rice in rice breeding and cultivar development. Considering the above mentioned facts, Nepal has still great opportunity to exploit these wild relatives, however, their proper utilization and exploration has not been carried out yet. Only very few landraces have been used in varietal improvement and development programme. On the other hand, its tremendous amount of genetic diversity is declining day by day (Lu, 1999a; Chang et al., 1982). Therefore, to cope with such burning problems for present and future necessitates several breeding strategies. Among them, varietal improvement and development through wide hybridization including modern varieties, land races and wild relatives is one of the viable options within country to extend the rice crop into new management regimes, new habitats, or regions of marginal climate, edaphic adaptation and bridging the yield gap in its production. However, their inclusion in the breeding program always necessitates the study 4

of crossability, genome relationship, and meiotic pairing between wild and cultivated species (Murthy and Reddy, 1993; Bennett, 1966). Understanding of the evolutionary change is essential to increase our efficiency as plant breeder engaged in promoting change to meet human needs (Hutchinson, 1970). Not only, but also genome and evolutionary relationship are crucial aspects for directing our effort to search for beneficial genes in wild species of rice (Chen, 2002). But several reproductive isolating mechanisms restrict the production of distant hybrids and the transfer of useful genes (Khush and Brar, 1992; Sitch, 1990). They occur in interspecific hybridization as a result of either sexual incompatibility or hybrid inviability (Willims et al., 1987). From the viewpoint of breeding, abortion of hybrid embryos at different stages of development is a more important and frequently encountered postfertilization barrier in interspecific hybrids than others (Khush and Brar, 1992). It is, however, possible to overcome this post fertilization barrier through rescue of immature embryo (Jena and Khush, 1984; Laibach, 1929), and other in vitro techniques (Raghavan, 1985). Once a distant hybrid was established, a carefully planned pre-breeding program and repeated backcrossing through cytogenetical manipulation is necessary to transfer useful genes from many wild species to an improved plant type (Singh et al., 1990). On the other hand, knowledge of the cytogenetic relationships between cultivated species and their allied wild relatives has proved to be essential in utilizing the desirable germplasm in the most effective manner (Bernham, 1966; Hey and Henderson, 1962). Although in the past, progress in crop production and productivity has been mainly due to conventional approaches and techniques, we cant expect to continue improving crop production indefinitely by only conventional techniques. Several innovative approaches in breeding crop plants have, therefore, been developed. One of these approaches in plant breeding 5

demands constant reference to the chromosomal status of the breeding materials. Such approaches involve interspecific hybridization, alteration in ploidy, aneuploidy, structural changes in chromosomes and regulation of recombination, and yield a wealth of information about the genetic architecture of crop plants, which may become the basis for future researches in plant breeding (Swaminanthan and Gupta, 1983). Likewise, meiotic study is inevitable to generalize the background information on evolutionary history, mode of speciation, cytological traits, species relationships etc. such informations have been greatly emphasized (McClintock, 1978; Hawkes, 1977; Morinaga, 1964; Li et al., 1961). That are essential for a conceptual approach to and design of a breeding program and to introduce the alien variation from allied wild and weedy species to the cultivated ones (Swaminanthan and Gupta, 1983). Similarly, chromosome pairing study is a preliminary aspect, because it determines the degree of variability, viability and stability of a variety and/or hybrid and progress of breeding program when develop through wide hybridization. Brar and Khush (1997) pointed out that there is urgent need to reinvestigate the chromosome pairing at earlier stage, particularly at pachynema. It is because of strong desynapsis mechanism existed, at later stages of meiosis, in the chromosome of cultivated and wild species having different genome. Therefore, karyomorhological study, banding pattern, which is chromosome specific (Griffiths et al., 2000), and meiotic behavior and its affinity studies at different stages are the fundamental and preliminary aspect in rice cytogenetic study to utilize these diverse germplasm in effective manner. This approach is one of the invaluable options to all the plant breeders. Hence, keeping these facts about the value of wide hybridization adopting various supplementary techniques of tissue culture and their cytogenetic study in plant breeding, the present study was undertaken with the following objectives:

1.2. i) ii)

Objectives To produce the intergenomic F1 hybrids through in vitro manipulation. To examine the crossability, pollen fertility and sterility, and morphological characters in the parents and F1 hybrids.

iii)

To study meiotic behavior, meiotic affinity, and chiasma frequency in parents and their hybrids.

iv)

To determine the chromosomal status of Nepalese wild rices.

2 LITERATURE REVIEW 2.1. Taxonomy and origin of rice Rice is classified under the family Poaceae (gramineae), sub-family Oryzodeae, tribe Oryzae. There are 12 genera under Oryzae tribe. Under the genus Oryza 24 spcecies with ten different genomes including four valied section and three gene pools have been reported so far (Khush, 2000). However, the number of species under genus Oryza delimit the taxonomist to determine the actual number of species around the world. As a consequences the number of species always vary with the taxonomist (Lu, 1999b) (Table 2.1). The present day cultivated rice is probably originated in the humid tropics of GONDWANALAND before that super continent began to fracture and drift apart about 135 million years ago to south and southeast Asia, Madagascar, Africa, Australia, South America and Antartica (Chang, 1976c). Based on the molecular analysis several authors suported this speculation (Joshi et al., 2000; Aggrawal et al., 1999). However, Cheng (1993) considered the Asia is the place where the origin of the O. sativa occurred. Shrestha and Upadhayay (1999), Shai (1999) also claimed that monsoonal swampy and tropical south-faced foothills of Nepal are the most probable original home land of the Asian rice. Khush (2000) also noted that most of the terai region of Nepal, and UP, Bihar of India are the pimary centres for Asian aromatic rice. Regarding the ancestral species of Asian cultivated rice, there are no single opinion: some of the taxonomists, such as Chatterjee (1951), Ramiah and Ghose (1951), and Shastry (1964) considered the annual taxa of common wild rice, O. nivara, must probably the immmediate ancestor of Asian cultivated speceis. On the other hand, authors like Morishima et al. (1961, 1963), Oka and Chang (1962), Oka and Morishima (1971), and

Nayar (1973) considered that the common perennial wild rice (O.rufipogon) is the direct ancestor of Asian rice, and differentiaton in the cultivated rice resulted in part from the geographical differentiation of the races of O. rufipogon (Second, 1982). However, Chang (1976c) and Chang and Vaughan (1991) later proposed evolutionary trends of cultivated rice, in which differentiation of perennial to annual first took palce and finally further differentiation resulted into present cultivated Asian rice. However in contrary, Shao et al. (1986 ) and Morishima (1986) pointed out that the intermediate of perennial-annual form are the immediate prototype of rice. Similar trends also took place in the Africa from other common wild rice (O. longistaminata), resulted in to african cultivated O. glaberrima. Their proposed path way is postulated in figure 2.1.
GONDWANALAND Common ancestor

South and South- East Asia


Wild perennial Wild Annual rufipogon (AA) Weedy annuals nivara (AA) spontanea stapfii sativa (AA) Cultivated annual
indica javanica japonica

Tropical Africa
longistaminata (A1A1)

barathii (AgAg)

glaberrima (AgAg)

Figure 2.1. Evolutionary pathway of the two cultivated species of rice. Taxa boxed by solid lines are wild perennials. Taxa boxed by broken lines are annuals. Arrow with solid line indicates direct descent. Arrow with broken line indicates indirect descent. Double arrows indicate introgression hybridization (adapted from Chang, 1976c). 9

2.1.

Rice biodiversity in Nepal Nepal represents the unique variability in geographic pattern, and a similar variation

have been reported in its crop plants, particularly, in the case of rice (Shahi, 1999). Its cultivation ranges from tropic to terperate regions and its allied related taxa pose accrodingly a similar pattern in genetic variability. It has been reported that, Nepal endowed with more than 2000 local rices and more than four wild related species namely O. rufipogon, O. nivara, O. officinalis, and O. granulata (Shresthat, 2002; Shrestha and Upadhayay, 1999; Shrestha and Vaughan, 1989). Within cultivated landraces differentation extend from indica to japonica (Nayar, 1973). Likewise intermediates form, O. sativa f.spontanea, have been reported throughout the terai region of Nepal (Lu, 1999a). Beside, two Oryza related genera are also abundantly found in wide range of altitude from1601640 meter altitude (Shrestha and Vaughan, 1989). Based on molecular analysis, Sharma et al. (1999) reported that the landraces of Nepal are highly diversed and most of the landraces posseessed one or more economic traits.

2.3. 2.3.1

Description of Nepalese wild rice species Oryza granulata Griff. O. granulata is one of the only two wild rice species in the genus Oryza that occurs

in the upland condition (Vaughan, 1994). In Nepal, this species is restricted to Chure hill of Chitwan and Jhapa district (Shrestha and Vaughan, 1989). It is a small bamboo-like miniature, perennial, and found under the dense vegetation. Morphology of O. granulata is unique from other rice species by its small and dark green plants, tightly compact panicles, and round-elliptical and awnless grains and flowers throughout the year (Vaughan, 1994).

10

The ploidy level of this species reported from other accessions (other countries) is diploid with GG genome (Aggrawal et al., 1997)

2.3.2. O. officinalis Wall ex Watt. This species was routinely observed in different parts of terai region of Nepal and identified as O. officinalis just a decay ago (Shrestha and Upadhayay, 1999). It is usually rhizomatous herb of variable height; basal panicle branches and is diploid with CC genome. However, the Nepalese O. officinalis either diploid or teraploid, it is still unknown. It is frequently grown in seasonally wet areas, ditches, swampy places, near small water holes, and along lakeside, streams, or rivers and found under full sun or partial shade (Vaughn, 1994).

2.3.3. O. nivara Shrama et Shastry, and O. rufipogon Griff. O. nivara and O. rufipogon are widely distributed in Southeast Asia and other rice growing countries (Vaughan, 1989). In the recent year, they are frequently known as common wild rice and are recognized as the immediate prototypes of cultivated rice (Khush, 2000; Chang, 1976a). Similarly, the large population of these two species has been frequently reported throughout the terai region of Nepal (Shrestha, 2002; Upadhyay and Gupta, 2000; Shrestha and Upadhyay, 1999). O. nivara is annual and it has short plant stature with many basal tillers, compact panicles, broad and well filled grains with long and tough awns and small anthers (Shrestha and Upadhyay, 1999). On the other hand, O. rufipogon is perennial and It has distinct nature of tallness and long awns along with black husk and long slender grain, larger anther and procumbent type of growth habit and both the species are diploid (Vaughan, 1994).

11

2.4.

Genome and genepool Various approaches including morphological differentiation, meiotic chromosome

pairing in F1 hybrids, and molecular divergence analysis, have been used in genome analysis, and in determining species relationships in Oryza (Khush and Brar, 2001). At present, ten different genomes have been identified and assigned under the genus Oryza based on meiotic pairing study and DNA based technology (Table 2.1). Based on ease of gene transfer from wild related species to cultivated rice, Khush (2000) divided Oryza species into primary, secondary and tertiary gene pool. According to him, Primary gene pool consists of species having AA genome, secondary includes the O. officinalis complex and tertiary gene pool comprises the O. meyeriana, O. ridleyi and O. schlechteri complex (Table 2.1). Hybrid production and subsequent gene transfer from this complex are rare or extremely difficult.

2.5. Importance of wild germplasm Frankel and Brown (1984) stated that wild germplasm are donors of genetic material rather than for direct use except some instances. They provide safe guard against unpredictable future genetic vulnerability (Alcazar, 1993; Harlan and Starks, 1980; Chang, 1976b; Harlan, 1975). The wild relatives of the cultivated cereals form an important reservoir of genetic variability for various economic characters such as disease and insect resistance, tolerance for abiotic stresses, increased biomass and grain yield, and improved quality characteristics (Harlan, 1976). The use of wild relatives is relatively demanding in recent year as in some instances, the genetic variability for some of these traits are limited or unavailable in the cultivated germplasm (Stalker, 1980). Not only, but also for example, in case of rice biotic stress resistance seems to follow an approximately 1:50 rule i.e. wild taxa offer 50 times more resistance genes than cultivated ones (Vaughan, 1989). 12

Table 2.1. Chromosome number, Genomic composition, geographical distribution and useful traits of Oryza species. Species O. sativa complex O. sativa L. O. nivara Sharma et Shastry O. rufipogon Griff. O. breviligulata A. Chev. et Roehr. O. glaberrima Steud. O. longistaminata A. Chev. et Roehr. O. meridionalis Ng O. glumaepetula Steud. O. officinalis complex O. punctata Kotschy ex. Steud O. minuta J.S. Presl ex C.B. Presl O. officinalis Wall ex Watt O. rhizomatis Vaughan O. eichingeri A. Peter 2n 24 24 24 24 24 24 24 24 24,48 48 24 24 24 Genome AA AA AA AA AA AA AA AA BB,BBB C BBCC CC CC CC Distribution Worldwide Tropical and subtropical Asia Tropical and subtropical Asia, tropical Australia Africa West Africa Africa Tropical Australia South and Central America Africa Philippines and Papua New Guinea Tropical and Subtropical Tropical Australia Sri Lanka South Asia and East Africa 13 Useful or potentially useful traitsa Cultigen Resistance to grassy stunt virus Blast , drought avoidance Elongation ability, source of CMS, resistance to BB Resistance to GLH, BB, drought avoidance Cultigen Resistance to BB, drought avoidance, source of perennial rice Elongation ability and drought avoidance Elongation ability and, source of CMS Resistance to BPH, zigzag leaf hopper Resistance to sheath blight, BB, BPH, GLH Resistance to thrips, BPH, Asia, GLH, WBPH Drought avoidance, rhizomatous Resistance to yellow mottle virus, BPH, WBPH, GLH

Table continues O. latifolia Desv. O. alata Swallen O. grandiglumis (Doell) Prod. O. australiansis Domin. O. meyeriana complex O. granulata Nees et Arn. ex Watt O. meyeriana (Zoll et Mor. ex Steud.) Baill O. ridleyi complex O. longiglumis Jansen O. ridleyi Hook. F. Unclassified O. brachynatha A. Chev. et Roehr. O. schlechletri Pilger
a

48 48 48 24

CCDD CCDD CCDD EE

South and central America South and central America South and central America Tropical Australia

Resistance to BPH, high biomas production Resistance to striped stemborer, high biomass production High biomass production Drought avoidance, resistance to BPH Shade tolerance, adaptation to aerobic soil Shade tolerance, adaptation to aerobic soil Resistance to blast, BB Resistance to stemborer, whorl maggot, blast, BB Resistance to yellow stemborer, leaf folder, whorl maggot, tolerance to laterite soil Stolonniferous

24 24

GG GG

South and South East Asia Southeast Asia

48 48 24 48

HHJJ HHJJ FF HHKK

Irian Java (Indonesia) and Popua New Guinea South Asia Africa Papua New Guinea

BPH = brown plant hopper, GLH = green leafhopper, WBPH = white-backed planthopper, BB = bacterial blight, CMS = cytoplasmic male sterility Source: Khush and Brar (2001) and Khush (2000) 14

From time to time, the roles of wild relatives of cultivated forms have been emphasized either theoretically or practically at greater extent in different crop species (Singh et al.; 1990; Moss, 1985; Frey et al., 1984; Swaminathan and Gupta, 1983; Stalker, 1980; Hawkes, 1977; Harlan, 1976; Knott and Dvorak, 1976) A few notable examples that raised the common concern in the utilization of wild relatives are rust resistance in wheat (Sears, 1984; Knott, 1971), grassy stunt resistance in rice (Khush, 1977) and transfer of CMS source for hybrid rice (Li and Yuan, 2000), mildew and crown rust resistance in oats (Aung and Thomas, 1976; Browning and Frey, 1969), increased biomass and grain yield in oats, perlmillet, and sorghum (Frey et al., 1984) and in rice (Xiao et al., 1998), and successful transfer of southern corn leaf blight resistance gene into maize from a wild related species (Mann, 1997; Chang, 1976b; Hooker, 1974). Recent advances in tissue culture and molecular marker technology and insitu hybridization technique have enabled to the utilization of wild species of crop plants to fuller extent (Brar et al., 2002).

2.6. Progress towards the utilization of wild relatives of rice Rice crop is grown world wide under a wide range of agroclimatic conditions therefore its productivity is affected by several biotic and abiotic stresses. The genetic variability for some of the traits, such as resistance to tungro, sheath blight, yellow stem borer, tolerance to acid sulfate condition and drought tolerance, is limited in the germplasm of cultivated rice. More over, continuous changes in insect biotypes and disease races are a continuing threat to increase rice production. Under such conditions, wild species of rice are a good source of useful variability (Brar et al., 1996). Thus there is urgent need to broaden the rice gene pool through genes introgression for such traits from diverse rice germplasm (Brar and Khush, 1995). However, in the past breeders did not utilize these 15

Table 2.2. Progress in gene introgression and transfer from wild Oryza species into elite lines of cultivated rice Trait transferred to O. sativa (AA) Grassy stunt resistance Bacterial blight resistance Name of gene/s donor Genome wild species symbol O. nivara O. longistaminata O. officinalis O. minuta O. latifolia O. australiensis O. brachyantha O. minuta O. officinalis O. minuta O. latifolia O.australiensis O. officinalis O. sativa.f. spontanea O. perennis O. glumaepatula O. nivara O. rufipogon O. rufipogon O. officinalis O. rufipogon O. rufipogon O.australiensis O. longistaminata 16 AA AA CC BBCC CCDD EE FF BBCC CC BBCC CCDD EE CC AA AA AA AA AA AA CC AA AA EE AA References Khush (1977) Brar et al. (2002); Brar et al. (1996) Brar et al. (2002) Brar et al. (2002); Amante et al. ( 1992) Brar and Khush (1997) Multani et al. (1994) Brar et al. (1996) Amante et al. (1992) Jena and Khush (1990) Brar and Khush (1997) Brar and Khush (1997) Multani et al. (1994); Ishii et al. (1994) Jena and Khush (1990) Lin and Yuan (1980) Dalmacio et al. (1995); Brar et al. (1998) Dalmacio et al. (1996) Brar et al. (1998) Brar et al. (1998) Khush et al. (2000); Khush et al. (1990) Kobayashi et al. (1992). Brar et al. (2002) Martinez et al. (2002); Xiao et al. (1998) Ishii et al. (1994) Soriano et al. (1999)

Blast resistance Brown plant hopper resistance

Whitbacked planthopper resistance Cytoplasmic male sterility

Tungro tolerance Tolerance to acid sulfate soil Yield improving trait Earliness Resistance to rootknot nematode

potential germplasm due to great difficulties to deal with (Khush, 1994). These difficulties mostly attributed to strong pre and post fertilization barrier and limited recombination between homoeologous chromosome of two different genomes is the most problematic one (Brar et al., 1996) However, successful transfer of grassy stunt virus resistance genes and CMS source from common wild rice are the two historical corner stone that rose the interest in the utilization of wild species. Now, utilization of wild taxa of rice is a component research at IRRI. At International Rice Research Institute (IRRI), a series of hybrids and monsomic alien addition lines (MAALs) have been produced following hybridization and embryo rescue technique. At present, utilization of wild species is highly advanced in rice breeding program and become a routine tool. To date more than dozens of genes have been transferred (Brar and Khush, 1997; Brar et al., 2002). Details about the gene transfer from wild taxa to cultivated forms of rice were shown in Table 2.2.

2.7.

Wide hybridization The meaning of wide hybridization and distant hybridization is quite different.

Chang and Vaughan (1991) defined wide hybridization as hybridization between species having the same genome and on the other hand hybridization between species having different genomes, or unknown genome in the same genus is called distant hybridization. Thomas Fairchild was the first who produced wide cross hybrids plants, between Carnation and the Sweetwilliam, in 1717 (Allard, 1960). Similarly in rice, the first hybrids plants was produced in 1898 by Takahashi in Japan (Morinaga, 1964). Since then several wide and distant hybrids in rice have been produced to introgress the segment of particular genes and/or chromosome in to cultivated ones following the simple tissue culture approach (Brar

17

et al., 2002). Now, wide hybridization in rice has become a significant plant breeding tools for the incorporation of genes of interest from wild to cultivated ones in current rice breeding (Jena and Khush, 1984). Many breeders apparently have concluded that the interspecific path is necessary for achieving their goals. A common justification for this choice is that wild relative and progenitors of our crops can be tapped for genes carrying special attributes not apparently in the cultivated forms (Bates and Deyoe, 1973). Briggs and Knowles (1967) have emphasized the interspecific path to transfer one or a few genes from one species to another, achieve new character expression not found in either parent, produce new alloploid species, and to determine the relationship of one species to another.

2.7.1. Barriers in species hybridization Although a few species combinations are highly interfertile, hybridization between the majority of related but distinct Oryza species is normally prevented in nature by prefertilization and/or postfertilization barriers (Sitch et al., 1989a.b; Sitch and Romero, 1990). Williams et al. (1987) diagrammatically presented these barriers in his review paper to illustrate the possible phases of incompatibility occurring during interspecific pollination for pasture legumes. However, these barriers are similar to those seen in many other grasses families (Harrison, 1982 in William et al., 1987). Therefore, this diagram is also presented to cover better understanding (Figure 2.2).

2.7.1.1.Prefertilization barriers in interspecific cross No so much research has been conducted regarding the prefertilization barrier in species cross of rice. However, Sitch et al. (1989 a, b); Sitch (1990) and Sitch and Romero (1990) made special treatment in this field and reported that prefertilization incompatibility 18

in interspecific and intergeneric crosses involving O. sativa. According to Sitch and Romero (1990), pollen germination was normal in crosses of O. sativa with O. brachyantha, O. eichingeri, O. officinalis, and O. ridleyi and slightly inhibited in crosses with Rhynchoryza subulata. Pollen tubes of O. eichingeri and O. officinalis, and O. ridleyi penetrated the stigma but growth was frequently inhibited in the style. Similar, stigmastyle incompatibility has been reported in intergeneric hybrids involving O. sativa and Proteresia coaractata (Sitch et al., 1989a). 2.7.1.2.Post fertilization barrier in interspecific cross Among post fertilization barriers hybrid embryo abortion at an early stage is the characteristics feature of wide crossing in rice (Brar and Khush, 1994). However, postfertilization barrier includes hybrid inviability, hybrid weakness and hybrid break down and some times fertility distortion (Brar and Khush, 1986) Hybridization among AA genome species generally gives rise to viable seeds and plants, but the rate of successful crossing is very variable and embryo rescue has sometimes been used (Nowick, 1986). In some cases, as reported by Chu and Oka (1970), when O. sativa was pollinated by O. longistaminata, the F1embryos and endosperm begin. to deteriorate about 6 days after fertilization, and when O. longistaminata is used as maternal parent, hybrid embryos fail after 3 days due to presence of a barrier controlled by a set of complementary dominant lethal genes. Interspecific hybrids involving AA group are generally highly sterile but their chromosomes nevertheless homologous (Bouharmont, 1991). Their fertility can be easily restored and introgression is possible through back crossing. On the other hand, distant hybridization among rice genomes necessitates the embryo culture (Bouharmont, 1991). For example, as stated by Jena and Khush (1986) production of interspecific hybrids between O. sativa and O. officinalis, embryo rescue is essential as embryo abortion started after 10-14 days of fertilization. 19

Normal Reproduction Pollen hydration and germination on stigma

Prefertilization Barriers Failure of pollen germination eg. by osmotic mismatch of pollen and stigmatic fluid

Pollen tube growth through stigma to reach style

Pollen tube growth through style to ovary

Pollen tube growth through ovary and into ovule

Interspecific pollen / pistil incompatibility or incongruity expressed at a number of possible levels in the pistil from stigma surface to penetrated embryo sac

Pollen tube penetration of embryo sac, and double fertilization

Postfertilization Barriers Embryo and endosperm development to seed maturation and germination

Seed abortion

Seedling growth

Seedling lethality

Vegetative growth

Poor vigor or abnormal growth Hybrid sterility/ hybrid break down*

Reproductive success

Figure 2.2. Schematic representation of stages in normal sexual reproduction (left) and related barriers to interspecific hybridization (right) Williams (1987) and Headly and Openshaw (1980).

20

2.8.1. Crossability between intra and inter genomic species cross Different investigators from time to time obtained differences in crossability between species crosses depending upon the genotypes of female parent and geographical races of wild species used. The crossability between O. sativa and O. rufipogon was greatly varied from 10-30% depending upon the geographical race of wild species and genotype of O. sativa used (Shastry, 1964; Henderson, 1964a.; Nezu et al., 1960; Ghose et al., 1960). Sitch et al. (1989c) obtained 23% seed set in cross involving IR64/O. rufipogon and 42.2% seed set in IR64/O. perennis. Not only, low crossability genes (Lcr) have been reported in certain geographical races of common wild rice, O. rufipogon resulted in low seed set (Sano, 1991). Like wise, Jena and Khush (1986, 1984) reported that crossability between three lines of O. sativa and O. officinalis varied from 1.0-2.3 %. However, Brar et al. (1991) obtained quite low crossability which was ranged from (0-1.1%). Similarly, other workers (Brar et al., 1991; Morinaga, 1964; Ghose et al., 1960) also obtained non or few hybrids in this set of cross.

2.8.2. Causes of embryo abortion in wide cross Successful development of an embryo depends upon the accompanying development of endosperm tissue capable of nourishing the embryo and a harmonious interaction of related embryo, endosperm, and maternal tissues (Singh et al., 1990). Based on the comparative study regarding the endosperm development and cytokinin biosynthesis in the normal and hybrid endosperm, Singh et al. (1990) suggested that the abortion of the embryo is attributed to embryo and endosperm incompatibility and reduction in the cytokinenis biosynthesis in hybrids endosperm. Similarly, Hadley and Openshaw (1980)

21

suggested that the differences in the dose effects of the genes as they act in the hybrid tissue of the resultant genomic combination could also lead endosperm disintegration. Hadley and Openshaw (1980) mentioned the three probable cause of hybrid weakness or inviability: disharmonies between genes of the parental species, disharmonies between genome of one species and the cytoplasm of the other, and disharmonies between the genotype of the F 1 zygote and the genotypes of endosperm or the maternal tissue with which the developing F1 embryo is associated or due to the physiological upset (Skrim, 1942). Kobayashi and Sano (1996) reported Lcr in wild rice O. rufipogon in chromosome 6 that cause unidirectional cross incompatibility when crossed with japonica strains. In such situation Lcr acts as postfertilization barrier at which hybrid embryo undergo to degenerate within 4-5 days after fertilization (Sano and Kobayashi, 1996). Copper and Brink (1940 in Hadley and Openshaw, 1980) purposed somatoplastic sterility in wide cross hybrid. In this case the excessive growth of maternal tissues impairs the capacity for endosperm development and leads to starvation and collapse of the embryo.

2.8.3. Seed and embryo differentiation in wide hybrids In rice and other grass families fertilization and growth of the embryo are rapid and provides the useful feature for the application of embryo culture and for the rescue of hybrid progenies from incompatible crosses (Bouharmont, 1991). In case of normal pathway, rice embryo is completely organized in less than two weeks. On the other hand when widely related species are crossed, fertilization takes place in some spikelets, the differentiation of the embryo can be normal for several days and the lower part of the ovary is swollen. In contrast, the rest of the ovary remains slender and collapses sooner or later due to some disturbances occurring during endosperm development (Ragahvan, 1985).

22

In some cases, an embryo can develop for several days in the absence of endosperm but the differentiation of the organs is disturbed and delayed as compared to normal (Zhang, 1978). In this situation malformation is prominent feature particularly in the scutelum (Bouharmont, 1991). Zhang (1978) reported that irregular cell division in O. sativa and O. officinalis cross and found endosperm degeneration started 5 th day after pollination and considerable deformity was found in 7-10th day of pollination. Similarly, Kobayashi and Sano (1996) and Sano and Kobayashi (1996) found three cross incompatible genes, which causes embryo abortion in unidirectional cross.

2.9. Embryo rescue in rice Embryo culture in plant breeding refers to the culture of excised immature embryos on artificial culture medium to obtain the normal viable plants with different objectives. The credit of embryo culture goes to the Laibach (1929) and Skirm (1942) for demonstrating the important application of the embryo culture technique to overcome interspecific incompatibility in Linum and Prunus, respectively. In vitro culture of hybrid embryo is useful in wide crosses where fertilization occurs and embryo begins to develop but aborts before reaching full maturity (Nandan, 1997). This technique has considerable application as a method of obtaining novel gene combination from interspecific or intergeneric hybridization. In the Genus Oryza, Niles (1951) has firstly been adopted the method of culture of intraspecific hybrids seeds on artificial medium. However, detail events of rice embryo culture have been noticed by Amemiya et al. (1956a.b). They studied the culture condition and first germinative stage of immature rice embryos. Nakajina and Morishima (1958) have obtained various interspecific hybrids by using the techniques designed by the

23

Amemiya et al. (1956a.b). Since then several workers have been obtained F 1 hybrids from otherwise unsuccessful interspecific cross in rice through embryo culture in a number of cross combinations (Bouharmont, 1991; Iyer and Govilla, 1964; Li et al., 1961). On the other hand authors like Katayama (1966a.b), Gopalkrishinan and Shastry (1966), Nowick (1986), and Mariam et al. (1996) have also been adopted the embryo rescue to obtain the interspecific rice hybrids for their cytogenenetic study by using different nutrients media. Likewise, Yie and Liaw (1975) studied the growth and development aspects of excised embryos of rice under in vitro and Ko et al. (1983) described a simplified fast and efficient method of rice embryo culture particularly excision of the embryo. Similarly Jena and Khush (1984), and de Guzman (1983) advanced the embryo culture techniques at IRRI. Following these technique wide arrays of intersectional hybrids have been produced at IRRI and in other countries (Abdullah, and Somantri, 1995; Brar and Khush, 1995; Brar et al., 1991; Sitch et al., 1989c).

2.10.Hardening of regenerated seedling before field transfer No much report have been found to raise the embryo rescue regenerated seedling of rice in field condition despite of its great importance to establish the plants in the field conditions. Only two reports paid special attention in the concerned subject (Iyer and Govilla, 1964, Bouharmont, 1961). Transferring cultural seedlings direct to potted soil can pose a serious survival problem. Bouharmont (1961) hardened seedlings by keeping their roots in petridishes containing distilled water and growing under shade. On the other hand, Iyer and Govilla (1964) improved survival rates by growing cultured seedlings in a nutrient solution as suggested by Karim and Vlamis (1962) before transferring them into soil.

24

2.11. Hybrid embryo derived callus culture This technique was increasingly utilized in a number of crosses involving distant wild relatives of different crop species and a number of successes have been reported other wise embryo culture technique failed to give hybrid plants (Brar and Khush, 1994). Embryo derived callus culture in rice was suggested by Bajaj and Bidani (1980) and reported wide range of genetic variability was recovered in the regenerants. Likewise, Nowick (1986) successfully employed this technique in O. sativa/O. latifolia hybrids and reported similar results (1980).

2.12. In vitro fertilization Any manipulation of excised maternal and paternal tissue to accomplish pollen tube penetration to the embryo sac for affecting fertilization is referred to as in vitro fertilization. It is an important technique for overcoming the barrier inhibiting the pollen tube growth and very early stage embryo abortion. To overcome these difficulties, in vitro fertilization followed by culturing of fertilized ovules to maturity is a promising approach and may be a viable alternative even to parasexual hybridization (Brar and Khush, 1986, 1994). Similarly, the method has been widely used in other crop species such as soybean (Tilton and Russel, 1982), and maize (Gengenbach, 1982). This is one of the viable options to attempt wide hybridization and alternation of cytoplasmically controlled traits. As stated by Zhang (1985) fertilization of ovules in test tube was first time reported by Kanta et al. (1962) in Papaver somniferum . Yeh et al. (1980) was also made in vitro fertilization in distantly related species using this technique in tobacco and wheat. However no much research have been made yet regarding the cereals. Dhaliwal and King (1978) obtained 5% seed set when Zea mays ovules fertilized by Z. mexicana pollen through this technique. Zhang (1985) suggested 25

that sterilization of parental flowers in rice for in vitro fertilization was difficult, despite of that he obtained 5.8% success in inter varietal cross.

2.13. Media composition A primary consideration for transplant survival is seedling vigor, which can be determined by the growth medium and other culture conditions (de Guzman, 1983). Several attempts have been made to standardize the media composition for in vitro rice embryo culture, however, most of the reports shown that any media can successfully be employed by slight modification (Ko et al., 1983; Yiew and law, 1975; Bouharmont, 1961; Nakajima and Morishima, 1958; Amemiya, 1956a.b).

2.14. Hybrid sterility Hybrid sterility in general normal phenomenon in wide crosses, and is attributed to either chromosomal differences or genetic, and genetic-cytoplasmic interaction (Allard, 1960, Stebbins, 1958). In wide cross hybrids most of the cause of sterility is mainly attributed to structural differences and limited pairing of the parental chromosome (Allard, 1960). In wide cross rice hybrids too sterility was brought about by similar phenomenon (Henderson, 1961; Shastry and Misra 1961a.b; Yao et al., 1958). However, under normal chromosome pairing conditions, sterility is attributed to genetic, and genetic and cytoplasmic factors of the parents (Sage, 1976; Meyer, 1969; Plamer and Hadley, 1968). But third one can be easily recognized through reciprocal crossing and no much important (Hadley and Openshaw, 1980). The sterility; the spikelet sterilities in the hybrids are mainly caused by the pollen sterility (Guiquen et al., 1994). The sterility genes determined by allelic interaction seem to be of wide occurrence (Sano, 1990) between distantly related taxa and serve as one of the 26

genetic mechanism for hybrid sterility (Sano, 1983). Three different genes causing F1 hybrids sterility were extracted and analyzed between the two species O. sativa and O. glaberrima (Sano, 1983). Of them, two genes acted as gamete eliminator and the other acted as pollen killer, which suggests that gametic abortion due to allelic interaction frequently occurred between them.

2.15. Chromosome pairing in Haploid rice Based on chromosome pairing study in haploid rice of O. sativa. L, Hu (1957) reported existence of intragenome pairing and this was due to true pairing. Again Hu (1960) analyzed the meiotic behavior in haploid rice variety belonging to O. glaberrima. Stued. and reported that about 34.2% of the cells at diakinesis, metaphase I and anaphase, I showed pairing association having 1II is common. However, he inferred later that the intragenome pairing in later was due to secondary association. Rao (1984) reported similar results in haploid indica rice variety T1242 and observed 90.6% cells showed chromosome association of 8I+2II, followed by 10I+1II in 9.4% of the cells studied by him. Based on his observation and previous observation of Hu (1957, 1960), he concluded that consistent synapsis in haploid rice indicates partial homology or residual homology between the chromosomes.

2.16. Chromosome behavior between AA genome species hybrids 2.16.1. Meiosis in inter-varietal crosses Hsieh and Oka (1958, in Nayar, 1973) found also no disturbance in chromosome pairing in several hybrids. Occasionally, univalents, stretched chromosomes, and anaphase bridges were found. The authors then attributed to precocious separation or reunion of sister chromatids after a break at the diplotene stage. Sampath and Mohanty (1954) found 27

low frequencies of bridges and fragments indicating the presence of paracentric inversions. Venkataswamy (1963) found quadrivalent in certain crosses, which showed the presence of reciprocal translocation. They observed univalents, quadrivalents, and anaphase bridges in low frequencies in some crosses. Meiotic analyses in the pachytene stage was done by Yao et al. (1958) for the first time they observed loops, which were interpreted as resulting from inversions, in less than 10% of the cells examined in 5 out of 7 crosses. Diakinesis and MI were normal. In further studies, Henderson et al. (1959) obtained bridges with fragments in 0.28% of AI cells in 9 out of 12 combinations. Anaphase bridges without fragments were noticed in both parents and hybrids. They concluded that a genetic basis for the cause of sterility was improbable, and that instead it could be attributed to cryptic structural differences caused by inversion of an included type (Henderson, 1964b). Shastry and Misra (1961a.b) repeated pachytene analyses and reported that meiosis was highly abnormal, showing the presence of inversions, translocations, deletions, and differential segments. In three semisterile hybrids, up to 31% of the chromatin length was unpaired. MI and AI stages were normal. They proposed that the main cause of sterility was cryptic structural differences of the chromosomes caused mostly by translocation (Shastry, 1964). In contrast to these findings, Wu et al. (1964) found pairing abnormalities only once during pachytene analyses of four hybrids. Likewise, there are number of investigator who reported considerable paring anomalies in hybrids involving indica and javanica varieties (Nayar, 1973).

2.16.2. Pachytene analysis Yao et al. (1958) was the first who initiated the pachytene analysis however, detailed analysis was made by Shastry and Misra (1961b) in indica-japonica rice hybrids. Based on the pachytene analysis Yao et al. (1958) observed inversion loops in the five inter 28

varietal crosses and reported that cells having loops did not exceed the 10 % of the cells examined. Similarly, Shastry and Misra (1961a.b) able to pin point the deletion, duplication, inversion and translocation in the pachytene chromosome in indica -japonica hybrids. They reported that about 31% of the chromatin length was unpaired in three semisterile hybrids. During meiotic division translocated chromosomes appeared as crossed shape quadruple at pachytene stage (Schulz, 1985) or small interstitial gap (Shastry and Misra, 1961a.b). Dolores et al. (1979) reported that normal chromosome pairing, in hybrids of O. sativa/O. nivara, on the basis of complete chromosome pairing at pachytene stage with low frequency of structural difference. Misra and Shastry (1969) also studied the chromosome association in reciprocal hybrids and showed that in one of the hybrid combination (O. glaberrima/O. sativa), normal pairing was restricted only to pachytene stage and showed univalents and trivalents at low frequency. Based on the results they reported that the both species are identical but differentiated by few chromosome structural changes. On the other hand its reciprocal combination showed normal pairing at all stage.

2.16.3. Diplotene, diakinesis, and Metaphase I Based on meiotic behavior study, Yeh and Henderson (1961) reported that meiosis in all hybrids, involving cultivated rice O. sativa and five diploid wild rice (O. sativa var. fatuwa, O. sativa.var. formosana, O. balunga, O. perennis. subspp. cubensis and O. perennis, was basically normal. They observed no more (0-11% and 0-4% irregular divisions at diakinesis and metaphase I (MI), and anaphase I (AI), respectively) irregular cells than observed in inter-varietal hybrids. Yao et al. (1958) reported higher frequency of rod shaped bivalents in five of 7 inter-varietal hybrids than homozygous controls and found 8% of the cells contained univalent at MI. The mean chiasmata frequency per bivalent was 29

2.7 with range 1.8-2.9. In 1954, Sampath and Mohanty made large scale cross between indica and japonica varieties in which sterility was found 25-99 %. Meiotic behavior in all hybrids was normal except stretched chromosome, and bridges with fragments at AI. Shastry and Misra (1961b) reported no reduction in chiasma frequency having 1 -3 chiasmata/bivalent at diakinesis and MI in four indica and japonica hybrids. Similarly, Shastry and Misra (1961b) also observed quadrivalents association in two of the hybrids and suggested that quadrivalent association is an indisputable indication of translocation heterozygosity. During these stages the shape of the structure depends on the frequency of crossover, size of the interchange fragments and location of chiasmata and most frequently appeared as 8, ring or rod shaped at diakinesis (Schulz, 1985). Brar et al. (1996) reported that meiotic behavior in O. sativa cv. IR64/O. rufipogon hybrids was normal. They observed 11.97 average bivalent per PMC at diakinesis and MI. Similarly, Dolores et al. (1979) studied the chromosome pairing in the F 1 hybrids between O. sativa including series of landraces and improved varieties, and O. nivara, and suggested that normal chromosome pairing with certain degree of abnormality. They reported only few quadrivalents and univalents at diakinesis and Metaphase I in some of the PMCs. Lu et al. (1998) also observed normal meiotic behavior, at Diakinesis and MI, in hybrids involving four AA genome species from different geographical origin. The mean bivalent observed by them was ranged from 11.51-12. 2.16.4. Chiasma frequency During 1910, Kuwada first studied meiosis in rice and reported that, the paired chromosomes were being ring or x shaped, later becoming square or dumbbell shaped (Nayar, 1973). These observations later lay that they had either two or one chiasmata/bivalent depending upon the shape of the chromosome. Soriano (1961) obtained

30

14-22 chiasmata/cell in MI of five indica varieties. Similarly, Jena and Misra (1984) studied chiasmata frequency in connection with evolutionary trends at diplotene and metaphase I of meiosis in eleven diploid species of Oryza and found that the evolutionary trend in that series was towards self-pollination and annual growth habit. In the recent studied, Lu et al. (1998) reported that both intra and interspecific hybrids between AA genome species from New world, Asia, and Australia showed > 23 average chiasmata/PMC except for one hybrid (O. glumaepatula/O. nivara) which had 22.26/PMC.

2.16.5. Anaphase bridges: cytological view Two types of inversions have been reported to date, paracentric and pericentric inversions. Among them paracentric inversions are more frequently occurred in plant kingdom (Schulz, 1985). It has been suggested that the occurrence of anaphase bridges and fragments during meiosis be brought about by inversion in species chromosome, mostly paracentric inversion while pericentric inversions lead karyotypic differences, if single chiasma occurs within inverted regions. However, formation of bridge and acentric fragments dont always indicate the occurrence of paracentric inversion as these can be brought about by spontaneous breakage and fusion of chromosomes during meiosis (Haga, 1953). Rees and Thompson (1955) reported that for about 27 generation, inbred rye showed bridges and fragments and concluded that such structural change can also be brought about by chromosome breakage and sister chromatid reunion. A heterozygous inversion may lead to bridges at anaphase I, however, if the chromosomes involved in the bridge fail to separate completely in the first meiotic division, the bridge may persist until the second division. Therefore, diagonal type bridge notice in second division may be the remnants of a bridge at anaphase I (Ahmad et al., 1977). On the other hand direct bridges at anaphase II could originate in two different ways: a) the aberrant bivalent may pass intact 31

to one pole in the first meiotic division, so that separation at anaphase II would give rise to the bridge; or b) if one chiasma occurred in the inversion lop and another in the region between the centromere and the inversion loop, then a monocentric loop chromatid appears at anaphase I which will give rise to a direct type anaphase II bridge (Ahmad et al., 1977). Likewise Schulz (1985) also suggested that formation of bridge at anaphase II can be due to four strand double crossover combined with two strand single cross over in the region between the centromere and the inversion loop, which results in bridges in each of the two AII. A bridge will appear during meiosis only if there was at least one crossover in the inverted segment. Such bridges and fragments were commonly observed in different type wide hybrids of crop plants. Henderson et al. (1959) reported that 0-0.8% cells having anaphase bridges with small fragments in inter-varietal crosses of rice. Anaphase bridges without fragments and fragments and laggards were also noticed in both parents and hybrids involving O. sativa and O. rufipogon hybrids and their hybrid swarm (Majumder et al., 1997). Similarly, Sampath and Mohanty (1954) found Anaphase bridges and fragments in 11 of the 85 intra-specific hybrids, and suggested that these were indication of inversions. Similar trend of results has been reported in inter-varietal rice hybrids by Demeterio et al. (1965). Likewise, Dolores et al. (1979) reported 0.5-7.4 % percent PMCs had bridges and fragments in F1 hybrids of O. sativa and O. nivara cross. Beside rice, anaphase bridges have been frequently reported in interspecific hybrids of soybean involving wild and cultivated forms (Palmer et al., 2000; Ahmad et al., 1977) and suggested that wild and cultivated forms were differentiated mainly due to structural differences caused by paracentric inversion.

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2.17. Meiotic behavior in intergenomic hybrids Several authors reported large amount of irregularities in the intergenomic hybrids between O. sativa and other wild species having genomes other than AA, and showed that limited or if at all no pairing based on late stage of meiotic analyses, particularly diakinesis and MI stage (Mahapatra et al., 2002; Brar et al., 1996; Jena and Khush; 1986; Ranganadhacharyulu and Yesoda Raj, 1974; Wuu et al., 1963). Jena and Khush (1986) reported that absence of complete chromosome pairing in O. sativa and O. officinalis hybrids. They observed only occasional bivalents with mean ranged from 0 -4 and frequently 20-24 univalents/cell. However, Shastry et al. (1961) based on pachytene analysis reported that complete chromosome pairing in satival-officinalis hybrids followed by desynapsis in later stage and also found completely 24 univalents at Metaphase I. Based on the meiotic observation (MI), Brar et al. (1996) also reported the similar results in O. sativa/O. brachynatha and O. sativa/O. granulata hybrids. They observed only 0.06, and 0.29 bivalent/PMC, respectively and suggested that limited chromosome pairing between these taxa. Likewise, limited chromosome pairing (0.05 bivalents/cell and 0.03 bivalents/cell) has been observed by Abbasi et al. (1998) and Wuu et al. (1963) respectively in O. sativa and O. brachyantha hybrids. Similarly, Shastry and Rao (1961) reported that timing imbalance existed between O. sativa and O. australiensis genomes.

2.18. Pachytene analysis in intergenomic hybrids At pachytene stage in the F1 hybrid of the cross, O. sativa/O. officinalis, Shastry et al. (1961) observed complete pairing between the chromosome complement of the O. sativa and O. officinalis and they concluded that the A genome was homologous to the C genome. However, Li et al. (1964) questioned the observation of Shastry et al. (1961) and reported that O. sativa/O. officinalis hybrids had 24 univalents at pachytene stage as well. 33

However, Katayama (1965) observed normal pairing in the F 1 hybrids of O. sativa/O. officinalis at pachytene and speculated that A genome was partially homologous to the C genome. Similarly he postulated the similar phenomenon in his series of paper (Katayama, 1966a.b.). He also observed that the chromosome behavior in later stages was very variable and showed that 3-12 II and 1-11 I in interspecific hybrid with the ABC, and ADC genome. Shastry (1966) again concluded that failure of pairing in intergenomic O. sativa/O. officinalis hybrids was due to desynapsis. However, he pointed out that it needs to be investigation whether this phenomenon common in all known stocks or varietal variation exists in the manifestation of this character. On the other hand, at pachytene stage Ranganadhacharyulu and Yesoda Raj (1974) observed irregular meiotic behavior and subsequently in later stage in hybrids involving O. Punctata and O. eichingeri.

2.19. Asynapsis and/or Desynapsis Plants having reduced amounts of chromosome pairing during meiosis have been reported in number of crop plants such as, in wheat (Li et al., 1945), in rice (Ramanujam and Parthasarathy (1935, cited by Nayar, 1973); Chao et al. (1960); Chao and Hu (1961), Misra and Shastry (1969), and Wang et al. (1965). Asynaptic refers to the condition during which pairing event falls apart particularly pachytene stage of meiosis. On the other hand desynaptic refers to the condition during which pairing event is normal but in the later stages chromosome fail to pair. Both conditions favor the production of non-functional gametes and hence caused higher sterility. Chao et al. (1960) obtained seven sterile plants in a M2 progeny of 34 plants after the treatment of neutron irradiation. They set less than 1% seeds while their sib plants set about 80% seeds. Their chromosome pairing was normal at pachytene, but they showed 10 I at diakinesis and 7 I at MI. Later, they concluded that the sterile plant arose as a mutation, 34

which was monogenetically controlled, for desynaptic behavior and its expression mainly depended on the certain temperature regime (Chao and Hu, 1961;Wang et al., 1965). Similar observation was found by Li et al. (1945) in desynaptic mutant wheat obtained through the varietal cross, and reported that some modifier genes act upon, and showed stable and unstable type. In wheat, Li et al. (1945) found that higher temperature favors the bivalent formation and low temperature acts opposite. Similar, phenomenon was made by (Misra and Shastry, 1969) in O. glaberrima/O. sativa hybrids, as well, and the fertility of desynaptic and asynaptic mutant had significantly lower. But the occurrence of asynaptic phenomenon is less frequent than desynaptic (Nayar, 1973). Similarly, Brar and Khush (1997) reported that chromosomes of cultivated and wild species of rice showed strong desynapsis at later stages of meiosis.

2.20. Autosyndetic and allosyndetic pairing According to Stebbins (1950 in Li et al., 1961) autosyndesis refers to the pairing of chromosomes derived from the same parental gametes of a particular plant, regardless of the similarity or difference from each other, while alllosyndesis refers similarity to pairing between chromosomes derived from different parental gametes. Li et al. (1961) explained this phenomenon in interspecific hybrids between O. praguaiensis/O. australiensis and O. australiensis/O. alata, and showed that frequency of alllosyndesis was predominant type of chromosome pairing. Similarly, Shastry and Rao (1961) observed 9.87% PMCs having autosyndetic pairing within sativa chromosomes in O. sativa/O.australiensis hybrids. Based on the chromosome size differences between O. australiensis and O. sativa Li (1964), and Li et al. (1961) also observed both auto and alllosyndesis phenomenon. It has been frequently

35

reported that O. australiensis and O. officinalis have larger chromosome than others (Morinaga, 1964; Bouharmont, 1962; Ghose et al., 1960). However, Kurata (1986) and Kurata and Omura (1982) did not observed any significant karyotype differences between O. sativa and O. officinalis. However, Abbasi et al. (1999) detect auto and allosyndetic pairing among A and E genomes through, molecular cytogenetical techniques, genomic in situ hybridization.

2.21. Nucleolus: number, type, and shape Kuwada (1910) found one nucleolus in the majority of PMCs. Selim (1930) reported two nucleoli in PMCs of indica varieties and one in japonica varieties. Since then different authors found differences in number, shape and size of nucleoli during the course of mitosis in several varieties of rice (Nayar, 1973). Misra and Shastry (1967) studied nucleolus variation in five indica varieties, two javanica varieties and two japonica varieties and found 1-2 big nucleolus and 2-5 smaller ones, even up to 18 some times and stain similarly as the nucleolus. Latter these were identified as supernumerary nucleoli (Misra and Shastry, 1967). They are either free or attached to pachytene chromosome. However, most of them lay free in cytoplasm. They compared them to the nuclear bodies observed by Walters (1963) in several Bromus species. About a third of these were attached to chromosomes and the rest lay free. They proposed that evolutionarily advanced species might show more of these bodies consequent to increase competition in nucleoar activity in them as a result of chromosomal structural changes undergone by them. Walters (1963) found usually only one nucleolar body in a cell, and they were not present in somatic cells.

36

2.22. Recent progress in rice chromosome study Rice karyotype analysis is the basis of cytogenetics (Wu and Chung, 1986). Unfortunately, smallness of the rice chromosome, it was found very difficult in the past to karyotype it. Shastry et al. (1960), and Shastry Misra (1961a.b.) initiated rice karyotyping based on the pachytene analysis in rice. Since, then improved techniques of rice chromosome preparation have been emerged (Hamoud et al., 1991; Faridi and Sitch, 1989; Quy and Phai, 1985; Wu and Chung, 1986; Kurata and Omura, 1982; Kurata et al., 1981a; Kurata et al., 1978; Khan, 1975; Hu, 1964; Sen, 1963). These technique have been employed to study the karyotypic variation in Oryza sativa and chromosome pairing at pachytene stage to analysis of interspecific hybrids (Dolores et al., 1979; Reddi and Reddi, 1977; Katayama, 1966a.b.; Ranganadhacharyulu and Raj, 1974) and to identified translocation and trisomics (Kurata, 1986;Kurata et al., 1981b; Sato et al., 1980). Similarly, these earlier developed techniques have been extended to construct the linkage maps in rice (Iwata et al., 1984; Sato et al., 1980). In mid 1980s, Fuki developed analytical method to characterize the all chromosomes of rice by image analyzing system and further utilized in developing somatic rice chromosome map (Fukui, 1996, Fukui and Ijijima, 1991; Fukui et al., 1988; Fukui, 1986). In the recent years, cytogenetic techniques in rice have been highly developed and still growing up. Now techniques to visualize the genes and nucleotide sequence within chromosomes at different stages have been reported (Fukui and Ohimido, 2000, de Jong et al., 1999; Singh et al., 1996; Gustafson and Dille, 1992). However, the principles of these techniques have already been exploited in rice such as GISH, FISH and ISH to characterize the parental genomes in interspecific hybrids including different taxa with O. sativa (Fukui and Ohimido, 2000; Asghar et al., 1998; Abbasi et al., 1999, 1998; Fukui et al., 1994).

37

3 MATERIAL AND METHODS The whole experiment was laboratory based and therefore, all the study was conducted at green house and at Central laboratory of Institute of Agriculture and Animal Science (IAAS), Rampur, during the academic year of 2001-2002. IAAS is located at 84 0 29 E and 270 37' N and, 244 m above sea level.

3.1.Preparation of parental materials 3.1.1. Germplasm collection Male parents used in the hybridization consisted of four wild species, namely O. rufipogon, O. nivara, O. granulata, and O. officinalis. Similarly, female parents consisted of six local land races belonging to O. sativa viz. Kalanamak, Jethobudo, Pokhreli, Manshara, Jhinuwa, and Ghaiya, two improved IRRI varieties viz. IR 64 and IR 72 and one commercial varieties; Masuli. Three of the four wild species of rice and five landraces were collected from different parts of Nepal. Seeds of one wild species, O. officinalis, was kindly provided by Dr. M.P. Upadhaya, (Botany division, NARC, Khumaltar, Kathmandu). The species name, site of collection, type of collection, and collection date were summarized in Table 3.1.

3.1.2. Germination and greenhouse rearing All the collected live male parent plants were planted in plastic buckets (buckets were provided by Prof. Dr. R.C. Sharma) filled with soil sterilized by formaldehyde. Each species was planted in ten buckets. On the other hand, collected seeds of O. officinalis and O. nivara were germinated by keeping dehulled treated seeds in incubator at 33 0 C for 7-10 days. Dehulled seeds were kept in sodium hypochlorite @ 1% (v/v) for 15 minutes

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Table 3.1. Description of germplasm collection during study period of 2001 Sn. Name of species Site of collection Ajighara swampy area of Rupendehi district (Plain) and Bulbule park of Surkhet (Canal) Valley 2 3 4 5 a c d O. sativa. cv. IR 64 and IR 72 Masuli Ghaiya* Pokhreli * Jethobudo* Manshara* and Jhinuwa* Kalanamak* Batule Chaour, Pokhara Municipality 16, Kaski (Valley) Lamjung (Hill) Siddhartha Adarsha VDC, Rupendehi (Plain) Seeds Seeds seeds
a a a

Type of collection

Season August 24-25

O. rufipogon O. nivara O. granulata O. officinalis

Plants

Canal, road side ditches , swampy area, and farmers field (Nepalgunj, Banke district), Latikoili VDC (2) of Surkhet district (Valley) and Khumber forest ponds (Bardiya district) River bank forest (Chure hill)of Piple-6, Chitwan district (Valley) NARC, Botany division, Khumaltar, Kathmandu IAAS, Rampur campus, Chitwan Nepal Plants and rhizome May 19-21 April 15-18 Seeds Seeds May 24-25 May 24-25 May 26-27 May 26-27 Plants and Seeds August 22-24

a ,*

improved varieties and Landraces of Nepal, respectively

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Table 3.2. Crossing scheme employed during the study period 2001-2002 Female parent O. sativa cv. Manshara Male parent Wild species Oryza nivara O. rufipogon O. officinalis O. granulata x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x Jhinuwa Jethobudo Pokhreli Kalanamak Masuli Ghaiya IR 64 IR 72

crossing did not make

40

following the procedure of Vaughan (1994). Then germinated seeds were planted into ten buckets filled with same material. Staggered planting of all female parents (including six landraces, commercial variety Masuli and two IRRIs varieties) was made continuously for two months at four days of interval, to synchronize the flowering time of parents. Each female seed was planted in two replication; one bucket/replication, and two seeds/bucket at a time. Usual agronomical practices were adopted as needed. All these mentioned activities were done in normal rice growing season of 2001 at IAAS green house.

3.2.

Hybridization

3.2.1. Cross combination Each female parent was crossed with all four wild species except Ghaiya and Masuli, which were crossed only with O. granulata in the second season of 2002. In general twenty-eight hybrid combinations were expected at the end of the crossing. However, due to non- synchronization of flowering, crossing of Manshara and IR 72 with O. rufipogon was not carried out. The one way cross schemes employed during the crossing were shown in the Table 3.2.

3.2.2. Forced anthesis Out of four wild species, two species O. nivara and O. rufipogon, often bloomed after 1.30 p.m. but the entire female parents bloomed after 9.30 a.m. and completely ceased by 1.30 p.m. at Rampur environment during October-November 2001. Therefore, forced anthesis was done by keeping these two male parents in well-illuminated and heated room (36-380C) by fluorescent light and electric heater for an hour. Water was also sprayed to facilitate the blooming and maintain constant temperature by hand sprayer.

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3.2.3. Emasculation Hand emasculation of female parents to be crossed was done without clipping the flag leaf prior to 1-2 days of crossing continuously for one and half months. The number of emasculated spikelets in each panicle ranged from 1093 depending upon the availability of pollen source. The emasculated panicles were bagged by Glassine bag and pollination was done in the next day.

3.2.4. Artificial pollination Both approaches and hand pollination methods were adopted for pollination. During hand pollination, fresh anthers ready to dehisce were collected and immediately crushed in the petridish and then pollens were shaded on the stigma with the aid of brush repeatedly, without disturbing stigmatic surfaces particularly, for low amount of pollen sources, for 2-3 days. In approach method, emasculated spikelets of female and male panicles were bagged together within a single glassine bag, and occasional shaking was provided for 2-3 days mostly at 9 -11 oclock. On the other hand, pollinated spikelets, of distant crosses involving O. officinalis and O. granulata, were sprayed by GA3 and NAA @ 75 ppm (1:1) once a day regularly up to five days of pollination.

3.3.

Embryo rescue At the beginning of the experiment, embryo rescue was partly practiced for all

hybrid caryopsis. However, it was later found that hybrid between intragenomic (AA type) cross, (crosses of O. sativa with O. rufipogon and O. nivara) set good amount of mature seeds without noticing abortion of embryo. Due to inviability of zygotic embryo, later rescue work was extensively practiced for intergenomic cross hybrids only. When hybrid

42

embryos came to age of 7-10 days, they were cultured in sterile nutrient medium. During embryo rescue work, following sequential activities were followed:

3.3.1

Aseptic excision of immature hybrid embryos Excision of the embryo was carried out by adopting the procedure suggested by Ko

et al. (1983) and Bouharmont (1991). The method consisted of removing the envelope (husk) from the cut made at the top of the spikelets at the time of emasculation with the aid sterile forceps. Then immature ovaries were excised without disturbing their structural integrity. Excised ovaries were then surface sterilized in freshly prepared solution of sodium hypochlorite (1%). The soft green ovaries were shaken up in this solution for 15 minutes. Then isolated ovaries were thoroughly washed thrice in sterile water and the lower part of the ovary was cut and pressed out with the help of sterile forceps without touching by hand. The excised embryos were washed thrice with sterile distilled water to free them from chlorine. After that, embryos were soaked in the same sterile water for two to five hours in an aseptic inoculation chamber i.e. under Laminar Bench.

3.3.2. Aseptic preparation of mature hybrid embryo Mature seeds of hybrid obtained from crosses involving O. rufipogon and O. nivara, as male parents with other female parents were also cultured. Similarly, the mature embryos of the wild species were also cultured in the nutrient medium. Such mature caryopsis were first surface sterilized after removing the husk in freshly prepared solution of sodium hypochlorite (1%) for 35 minutes. Then either a part or the whole endosperm with embryo was inoculated, after peeling off a portion of the pericarp opposite to the embryo.

43

3.3.3. Inoculation of embryo After providing the proper time for imbibition of excised embryos, embryos were then placed on the sterilized filter paper for 30-45 seconds to remove the excess surface water from embryos. Then isolated embryos were aseptically inoculated into the tube containing respective culture medium by the help of a needle with a loop.

3.3.4. Media preparation At the beginning of the experiment all the media were prepared without making stock solution. However, later, Bouharmont, (1991) and
th

MS media (Murashige and

Skoog, 1962) were prepared by making stock solution. Each ingredient was first divided into four groups and stock solution of them was prepared following the procedure mentioned by Razdan (2001). Stock solution, consisting of major salts (20X concentrated), minor salts (200X concentrated), organic nutrients (200X), and iron (200X concentrated), were made by weighing the respective nutrient composition as given in the protocol (appendix.3.1). Each stock solution was then stored in refrigerator at 5 0 C until used.

3.3.5. Media employed In the beginning of this study, five different Media namely Nistchs (1969), Whites (1953), MS (Murashige and Skoog, 1962), SR (Ko et al., 1980) and Bouharmont (1991) were employed to determine the media efficiency for hybrid embryo regeneration at IAAS tissue culture laboratory environment. Each B, W, N, MS and SR medium was supplemented with 10% coconut milk. All the media were gelled by 0.7% Agar Agar, after adjusting the appropriate pH (appendix 3.1). Finally prepared medium was then boiled in the micro-oven and dispensed into culture tubes, 20-25 ml. in each, stoppered with cap or

44

closed by aluminum foil and finally tubes were autoclaved at 15 psi. for 20 minutes. Then media were kept in laminar hood for two hours and allowed for setting.

3.3.6. Media efficiency determination Media efficiency was determined by repeating each experiment twice at IAAS, tissue culture laboratory. The preliminary results were recorded (appendix 4.2) and best media was choosen for further embryo rescue work. After findings the best media for complex hybrid embryo germination and subsequent development, the final embryo rescue study was conducted mostly on Bouharmont (1991) and partly on Murashige and Skoog (1962) media supplemented with 10% coconut milk as an organic supplement.

3.3.7. Incubation of culture The cultures were maintained in a temperature controlled chamber at 2510C under dark until germination and then continuous light (~110-foot candles) up to 2-4 weeks. Regenerated seedlings were removed out from the culture tubes after three-leaf stage. The roots of the seedlings were thoroughly washed to remove agar in the tap water and finally washed with sterilized distilled water.

3.3.8. Seedlings transfer Hardening of seedlings was done following the four methods: 1) direct transfer, 2) petridish method, 3) Iyer and Govill (1964) method, and 4) new method to enhance rooting and to make plants hardened in external environment. First one is the direct transfer of seedling obtained after embryo rescue. Second approach was hardening of seedlings in the moist filter paper over the petridish as suggested by Bouharmont (1961). Third method

45

consisted of hardening in the nutrient solution at temperature controlled chamber as reported by Iyer and Govilla (1964). The fourth and last method was the latest one that was developed during experimentation of this study. The newly developed hardening medium and procedures consist of sterilized 2:1 sand and soil (300 gm. in each 13 x 10cm 2 plastic bag) floated with a nutrient solution (Karim and Vlamis, 1962) up to 1.5 cm above the mixture surface in the bags. In which the molybdenum source (H 2 MoO4 ) was modified as sodium molybdate (Na2Mo4 .2H2O). Stock solution of each of the chemical was made and finally the complete nutrient solution was prepared (appendix 3.2) The whole small plastic bags with fragile seedlings were kept in plastic bucket, then covered by large white plastic bag with the aid of stakes to maintain humidity. The covered plastic bags were removed every day for 1-2 hours after 6-7 days of hardening up to three days and finally whole plastic bags were removed and seedlings left for two more days in the same medium. This hardening activity was undertaken in a well illuminated room and even on open field. Then finally hardened seedlings were planted to plastic buckets filled with well fertilized sterilized soil, and grown in the glass house of IAAS during winter season in 2001/2002.

3.4.

Regenerating plants from callus of hybrid embryos In most distant crosses, hybrid embryos (7-14 days old) are manipulated to produce

F1 plants directly. In some distant crosses, however, the hybrid embryos fail to differentiate in to plants and embryo abortion started earlier. To over come this barrier, and to maximize the chances of getting hybrids plants, the undifferentiated embryos were induced to proliferate as callus on a culture medium at an early stage and hybrid plant were regenerated from the callus following the technique of (Nowick, 1986). Immature embryo 46

to be cultured were aseptically excised and placed on the hybrid medium as proposed by Chen et al. (1991). The hybrid medium consisted of Murashige and Skoog (1962) organic salts and N6 mineral salts and supplied with 6% sucrose, 4mg/l NAA, and 2mg/l kinetin. Culture was maintained with 12 h light 12 h dark at 2510C. After 35-40 days of inoculation seedlings were removed following the same procedure as practiced for the embryo culture. Other further activities were also similar to the method as method described in embryo culture.

3.5.

In vitro fertilization In vitro fertilization was also carried out for O. granulata and O. sativa cross

combination following the procedure of Zhang (1985). The spikelet of the female parents O. sativa cv. Jhinuwa was emasculated by the hot water treatment method (45 0) for five minutes, and the spikelets ready to bloom were sterilized by wiping with a piece of ethanolmoistened gauge several times, soaked in 70 % ethanol for 5-10 seconds, and were washed with sterile water three times. A spikelet with a piece of peduncle was then placed upright on solid medium of type N6 containing 4% sucrose and pH 5.8. The spikelets of male parent O. granulata was surface sterilized by presoaking in 70% ethanol for 7 seconds, and finally soaking in 1% sodium hypochlorite for five minutes and thorough washing was provided. After this treatment, anthers were drawn out of the spikelet with a set of sterilized forceps and each was inserted in the maternal spikelet in a test tube. Cultures were maintained under darkness at 25 0 for 15days and then transferred to the Bouharmont (1991) media for regeneration following the embryo rescue technique. 3.6. Harvesting of F1 seeds

Mature and perfectly set hybrid seed was harvested separately at 24 - 28 days after

47

fertilization. Harvested seeds were immediately kept in dessicator at 30 0 for 3 days after proper tagging and labeling. Then these seeds were again germinated and sown as mentioned previously (3.1.2). 3.7. Determination crossability

3.7.1. Crossability between O. sativa and common wild rice Crossability percent of common wild species (O. nivara, and O. rufipogon) with O. sativa was determined by counting the total number of true F1 hybrids in proportion to the total number of florets pollinated during pollination (Wuu et al., 1963). However, in this study, all F1 plants were not reared at first due to lack of space and hence only 5 F 1 seeds were planted for cytological analysis and characterization purpose. All F 1 hybrids exhibited the purple basal leaf sheath color at an early stage of seedling. Since then, the morphological marker was used to identify the F1 hybrids for those crossed combinations involving O. sativa/O. nivara and O. sativa/O. rufipogon. On the next season, all the seeds harvested from these cross combinations were germinated and reared up to 20 days in the laboratory condition and looked for the presence of basal leaf sheath color as purple. It is because of all the cultivars belonging to O. sativa used here had green basal leaf sheath color. Therefore, hybrids were ascertained by observing the presence and absence of purple basal leaf sheath color at an early stage of seedlings. In this connection earlier finding was used to identify the hybrids, some of them again reared up to maturity and finally confirmed that all were hybrids. Non germinated seeds, and albino plants which were later died, were not considered in the calculation of crossability percent. 3.7.2 Crossability determination in intergenomic crosses The crossability between intergenomic species were also determined, based on the

48

hybrid plant establishment in the field condition after embryo rescue, as method reported by (Li, 1964). 3.8. Morphological characterization For the purpose of morphological characterization, each different female parent and their hybrid combinations were planted to five plastic buckets and wild species were planted in ten plastic buckets during winter season of year 2001/2002. A number of different observations regarding the morphology of included study materials were done from early vegetative stage to until harvest. About > 30 observable characters were characterized during study period. Each observation was taken from random sample of size five i.e. one sample from each bucket. The observation of each character was taken based on the standard characterization procedure (IBPGRI- IRRI, 1980; Sharma and Shastry, 1965).

3.9.

Chromosome preparation Chromosome, of the 15 F1 hybrids, 7 cultivars and four wild species of rice, was

prepared from immature anthers following the usual squashing technique as mentioned by (Wu and Chung, 1986; Khan, 1975). In some of the study enzymatic maceration was also employed as described by Kurata et al. (1982). Most of the observations were made from temporary slide.

3.9.1. Chemical preparation 3.9.1.1. Stain Most of the chromosomes were stained by 1% acetocarmine solution and this solution was prepared by following the procedure of Evans and Reed (1981). Their

49

procedure involves: One gram of acetocarmine powder weighted in electronic balance was dissolved in 100 ml glacial acetic acid solution having strength 45%. Before dissolving, 45% solution of glacial acetic acid was heated up to boil. For dissolving the powder continuous heating and stirring was provided for 5-10 minutes under hot Plate with magnetic stirrer. Prepared solution was allowed to cool and finally one drop of 45% GAA and trace amount of ferric chloride was supplied as mordant That prepared solution was then stored at 50C in refrigerator until used.

3.9.1.2. Fixative During study, two kinds of fixative were used: 1. Caryonys fluid, and 2. Aceticalcohol solution. Caryonys fixative was prepared by mixing Glacial acetic acid, Chloroform, and 95% Ethanol in the proportion of 1:3:6, respectively. Then finally prepared solution was provided with trace amount of ferric chloride. On the other hand, Acetic-alcohol fixative was prepared following the procedure of Khan (1975). This was prepared by mixing one part of Glacial acetic acid with 3 parts of 95% ethanol. This prepared solution was also supplied with trace amount of ferric chloride to ensure the better stain during staining. Every time these fixatives were freshly prepared, just before fixing the materials.

3.9.2. Standardization of suitable stages The suitable stage for studying the meiotic behavior were first standardized by harvesting the young panicles from early booting, varying in length below the junctura of the flag leaf (Khan, 1975). These harvested young panicles were fixed for 24 hours at 140250C in freshly prepared fixative and then tested under microscope by staining freshly prepared 1% acetocaramine solution. Since then different stages of meiotic behavior were 50

studied by harvesting the young panicles (the distance between boot and junctura of the flag leaf was 0-6.5 cm depending upon the species and their F1 derivatives) based on the results of standardization of suitable stage for meiotic study.

3.9.3. Meiotic behavior study Young spikelets at suitable stage were fixed in acetic alcohol (1:3 v/v) to which traces of ferric chloride was added to intensify the staining of the chromosomes. The materials were kept in the fixative for 24 hours at low temperature (14 0 -250C) and then transferred to 70 % ethanol until used for smearing. Fixed anthers were removed from the spikelets and smeared in one or two drops of 1 % acetocarmine. Slight warming and gentle tapping were provided to promote the excellent spreading and differentiation of the chromosomes. Data were recorded based on the analyses of 10 randomly selected immature spikelets of parents, including five F1 plants of each cross for each stages of studied. Data concerning the meiotic behavior included only from those PMCs that yield well spread and distinct configuration after screening a large number of dividing cells. Microscopic photographs, and drawings, if necessary, were made from temporary slides with the aid of microscopic camera and Camera Lucida at table level with the mirror at 45 0 inclination and using 100X oil immersion objective and 10X eye piece of Olympus Microscope, respectively.

3.9.3.1. Chromosomes analysis Meiotic chromosomes associations were mostly studied at, diakinesis, metaphase, anaphase, and telophase I. Similarly, meiotic associations at pachytene stage were also

51

studied in a selected number of the cells. Diplotene stage was also occasionally analyzed in some cells of the few hybrids.

3.9.3.2. Pachytene analysis Detailed karyotypic analysis (individual chromosome length, arm ratio etc.) of pachytene stage chromosomes was not carried out due to stickiness and poor resolution of the staining. The pattern of pairing in this stage was determined as normal and abnormal only from those cells having well spread and distinct pairing pattern. Percent normal cells and cells showing cryptic structural hybridity (Shastry and Misra, 1961a.b) were calculated based on the microscopic observation. Microscopic photograph and necessary drawings were made in limited PMCs after screening the large number.

3.9.3.3. Chiasma frequency determination Chiasma frequency in the parents including wild rice and hybrids was determined based on the meiotic association at the diakinesis and metaphase I following the procedure of Lu et al. (1998). During diakinesis and metaphase I chromosome were highly condensed and easily recognized as rod and ring shaped and proposed 1 and 2 chiasmata/bivalent, respectively. Similarly, two (Bothmer et al., 1988) and four chiasmata/bivalent were considered for each trivalent and quadrivalent association, respectively. The chiasma frequency/PMC and per bivalent were determined only from those cells that showed normal complete set of chromosome. Observed cells under microscope were numbered randomly as first observed cell numbered 1, second observed 2 and so on. Each cell was characterized by observing meiotic association at particular stages. Observed bivalents were further characterized as rod or ring.

52

3.9.3.4. Detection of univalents, bivalents, trivalents, and quadrivalents No rigid techniques was employed to detect the chromosome association at diakinesis and metaphase I. However, chromosome, simple rule as described in every photographs and drawings in the rice cytogenetics paper were thoroughly concerned and based on these observed photographs, all most all the univalents, bivalents, trivalents and quadrivalents were detected during microscopic observation. When chromosome number exceeds 12 and some of the chromosomes found comparatively smaller in size than remaining within the same PMC, were considered as univalents. Similarly, bivalents were uniform and comparatively larger than univalents and showed 12 in numbers. Bivalents were mostly either ring or rod shaped but univalents were almost ring shaped and small. On the other hand trivalents were found to be larger than bivalents and overall counts should meet 24 univalents (for example 9II + 2III). Similarly, quadrivalents were large enough to detect and doubled in size of bivalents.

3.9.4. Pollen fertility and sterility Pollen fertility and sterility of included materials were determined following the method described by Virmani et al. (1997). The stain used in this method was I-KI, which was prepared by dissolving one gm each of potassium iodide and iodine in 100 ml of deionized water. Randomly selected 15-20 spikelets were taken from the just emerged panicles of selected tillers of five plants in a vial containing 70% ethanol. All the anthers from at least 6 spikelets were taken and placed on the fresh slide and anthers were then crushed with 1% iodine potassium iodide (I-KI) stain. After removing the debris, a cover slip was placed and observed under the light microscope (10X). The numbers of pollens were counted in four random microscopic fields and observed pollen grains were classified

53

based on their shape, size, and extent of staining (Young et al., 1983; Chaudhary et al., 1981) as follows: Categories of pollen and their features

Categories of pollen Unstained withered sterile (UWS)

Shape and staining behavior Withered and undeveloped unstained

Classification Sterile

Unstained spherical sterile (USS)

Spherical and smaller unstained

Sterile

Stained round sterile (SRS)

Round and small, lightly or incompletely stained, rough surface

Sterile

Stained round fertile (SRF)

Round and Large, darkly stained, smooth surface

Fertile

Based on the classification, mean percent pollen fertility and sterility were calculated. Then plants were classified on the basis of pollen fertility as fertile (61-100% pollen stained), partially fertile (31-60%), partially sterile (1-30%) and sterile (<1%) (Virmani et al., 1997; Subedi, 1982).

3.9.5. Spikelet sterility Spikelet sterility in the respective parents and their hybrids were determined by selecting and counting the filled and unfilled grains in the main tillers of selected five plants of each. The spikelets of the selected plants of all the parents and their F 1 hybrids 54

were bagged before flowering up to maturity. At maturity, the bagged panicles were examined for seed set. The mean percent spikelet sterility was determined by counting the total number of seed set in proportion to the total number of spikelets/plant. Then the percentage sterility from each panicle was added and divided by five to calculate the mean sterility percent. Then parent and hybrids were classified as fertile (81-100% seed set), partially fertile (31-80%), partially sterile (1-30%) and sterile (<1%) (Virmani et al., 1997; Ikehashi and Araki, 1984).

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4 RESULT AND DISCUSSION 4.1. Description of Nepalese wild species of rice Out of four species, three species, namely O. rufipogon, O. officinalis, and O. granulata had perennial growth type and profuse tillering (Table 4.1). All the species exhibit specific morphological features depending on the species, however, most of them had spreading growth habit, high stigma exertion rate, high spikelet shattering, photoperiod sensitive to insensitive, fully awned to awnless; dark green leaves with basal leaf sheath coloration. The flowering period of these wild species was quite longer than cultivated species and two species, O. granulata and O. officinalis, mostly bloomed at early in the morning, and ceased by 8-9 oclock. Similarly, two common wild species, O. nivara and O. rufipogon bloomed at noon and often later than that cultivated forms. The plant height was varied depending upon the species and was shortest in O. granulata and the tallest in O. rufipogon. O. granulata and O. officinalis did better under partial shade, where as O. nivara and O. rufipogon showed well in open sun shine. O. granulata, in its natural state, was mostly found in upland field under the shade of dense vegetation. Other diagnostic features of these wild species are presented in Table 4.1. and illustrated in photograph (Plate 2 and Plate 3.). Similar observation were made by earlier researchers (Jackson et al., 2000; Vaughan, 1994; Sharma, 1986; Sharma and Sampath, 1985; Shrestha and Vaughan, 1989, Sharma and Shastry, 1965, Gopalkrishinan, 1962). In some of the species, marked ecotype specific morphological variation was observed for example, in O. rufipogon, collection from Surkhet had open panicle with creeping nature of culm, but from Kapilbastu, it had compact panicle with procumbent type of culm. Vaughan (1994) and Sharma and Shastry (1965) reported similar observation with minor geographic differentiation within this taxa.

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Table 4.1.Morphological characters, of four Nepalese wild species of rice, observed during study period 2001/002 Characters Apiculus color 200 grain wt Anther color Auricle color Awn color Awn density Awn length BLSC Blade pubescence Branching of culm Culm angle culm diameter Culm length Culm number Flag leaf angle Habit Heading days Height Internode length Internode color Leaf color Leaf angle Leaf length, Leaf width Ligule color Ligule length Ligule shape MNBLNP Panicle exertion Panicle length Panicle shattering Panicle type Pedicel length Rachilla SB Seed coat color SLC Stigma color TNPB Underground stem Photoperiod reaction O. nivara black 4.9 gm yellow pale green brown long & fully 5.21cm purple lines intermediate present(2-3) intermediate 0.34cm 46.1cm >25 descending annual 80 57cm 19cm purple lines DGLPT dropping 50.5cm 1.23cm white papery 1.15cm 2-cleft 2 just exerted 15.3cm high open 3.4mm minute heavy black brown dark purple 6 absent insensitive O. rufipogon black 3.89 gm deep yellow pale green brown long & fully 5.3cm purple lines pubescent present(2-3) procumbent 0.3cm 73.25cm >40 horizontal perennial 135 92.3cm 16.0cm purple lines DGLPT dropping 37.2cm 1.2cm white papery 2.15cm 2-cleft 1 well exerted 19.04cm high compact - open 3mm prominent absent black brown dark purple 7 absent sensitive O. officinalis brownish grey 1.35gm Pale-white light green brown short & partly 0.82cm brownish green pubescent present (3-4) intermediate 0.33cm 89.11cm >35 horizontal perennial 109 107.14cm 30.0cm DBPL dark green dropping 32.5cm 1.47cm white 0.35cm truncated 3 well exerted 27.16cm high open 3.8mm minute heavy brownish grey brownish green DPWT 8 present sensitive O. granulata brownish white 2.63 gm white light green Awnless brownish white pubescent present (2-3) open <600c 0.21cm 44.55cm >45 horizontal perennial 50-70 (continuous) 54.85 13.0cm dark green dark green horizontal 17.67 1.72cm whitish green small truncated 1 or (sessile) well exerted 10.3cm high compact minute minute absent brownish white blackish white white and large absent present insensitive

TNPB = total number of panicle branches, DPWT = dark purple with white tip

DGLPT = dark green with light purple tip, SB = secondary branching, TNPB = total number of panicle branches, SLC = sterile lemma color, MNBLNP = maximum number of branches at the lowest node of panicle 57

4.2.

In vitro manipulation

4.2.1. Media efficiency In the preliminary study, hybrid as well as two selected parental embryos subjected to different cultural media showed quite variation in germination and other observations depending on the composition of media employed (Appendix 3.1). Although, the parental embryos germinated within two days irrespective of the media used, significant variation was observed in intergenomic hybrids depending on the media composition (5-13 days). The effect of media composition was apparently seen in the case of intergenomic hybrid embryos, and some of the seedlings died even after germination (Appendix 4.1). The frequency of dead seedlings after germination was highest in Whites, Nistch and SR Medium. The observed variation in the germination and subsequent growth in the media employed, suggest that complex hybrid embryo needs complete media as the normal endosperm supplied (Singh et al., 1990; de Guzman, 1983; Hadley and Openshaw, 1980; Murashige, 1979). Therefore, it was found that successful culture of embryo needs complete medium and success will be depend on the choice of media (Brar and Khush, 1994; Yeung and Thorpe, 1981). In other words, the more complex the hybrid, complex media will be needed to foster the better germination and vigorous growth (de Guzman, 1983; Yiew and Law 1983). However, the cultural conditions may also affect the germination, and subsequent growth and development of the seedlings (Amemiya, 19656a.b; Ko et al., 1983), and it may also be varied from laboratory to laboratory and expertise in handling of the materials (Razdan, 2001).

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4.2.2. Embryo rescue. Embryo rescue was extensively practiced for crosses involving O. officinalis and O. granulata. Embryo degeneration after fertilization was varied with remoteness of the wild species used. It was quite earlier in O. sativa/O. granulata (5-10 days) than O. sativa/O. officinalis hybrid (9-17days) depending on the female genotype used. The germination of hybrid embryo was varied from 0-66.67% (Plate 1 Fig. e-h.). In some of the combinations the germination of hybrid embryos obtained from O. sativa/O. granulata, was also found good, however most of them were died after germination. Therefore, hybrid between O. sativa and O. granulata were not obtained. Most of the caryopsis were without embryo or only filled with watery endosperm (Table 4.1a) however, very few embryos were obtained in the year 2002. They were cultured, but most of the embryos did not respond. About 127 embryos were cultured and only 34 hybrids were germinated, however, all were died after germination. Only one hybrid seedling was obtained, but that was also died during hardening process indicating that there should be strong pre and post crossability barriers, or mostly handicapped by post germination barrier. Similar prefertilization barrier was found by Sitch et al. (1989a.b), Sitch (1990) in the genus Oryza and reported in other taxa like O. brachyantha, O. minuta. O. ridleyi, when crossed with O. sativa, they all showed strong prefertilization barriers. On the other hand hybrid involving O. sativa and O. officinalis gave 16 hybrids plants.however, field establishment of regenerated seedlings was found very difficult. At initial stage 150 interspecific hybrids died due to lack of appropriate hardening technique. However, later proposed method as described in materials and methods significantly improved the field establishment and gave nearly 100% success (Plate 1. fig. j-l) (appendix 4.2). Similar results were presented by several researchers (Brar et al., 1991, Sitch et al., 1989c; Jena and Khush, 1986, 1984; Iyer and Govilla, 1964) regarding the hybrid caryopsis 59

Plate 2. Figure 13. Morphology of parents and their hybrids. 1. Female parent (Manshara), F 1 (Manshara/O. officinalis), male parent (O. officinalis), from left to right respectively. 2. Jethobudo, F1, and O. rufipogon, respectively 3. Pokhreli, F1, and O. nivara, respectively.

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Plate 3. Figure 1 8. Morphology of interspecific hybrids. 1. IR 64/O. nivara., 2. Pokhreli/O. rufipogon., 3. Jhinuwa/O officinalis,, 4. Kalanamak/O. nivara., 5.Pokhreli/O.officinalis., 6. Kalanamak/O. officinalis. 7. Jethobuo/O. nivara., 8. IR 72/O. nivara, respectively., 9. Wild species O. granulata.

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formation and their in vitro germination, however, except Iyer and Govilla (1964) others did not mention any details about the hardening process. Jena and Khush (1986) obtained variation in germination and their range were 56.57-70.05% in 1/4th MS medium. The successful hybrids obtained in O. sativa and O. officinalis suggests that there might be less crossability barrier than that found in O. sativa/O. granulata. They mostly showed post fertilization barrier and can be overcome by embryo culture. Several investigators have successfully been overcome such barriers by adopting embryo culture in different species hybrids (Philips1992; Sitch et al., 1989c; Williams, et al., 1987; Jena and Khush 1986, 1984; de Guzman, 1983; Keim, 1953). However, Brar et al. (1991) obtain 3 hybrid plants from O. sativa/O. granulata, in one combination out of three following this usual technique. This suggested that degree of incompatibility is varying with genotypes used in the crossing program. During study, O. granulata was crossed with nine genotypes (Table 3.2) up to two seasons and several hormonal manipulations were also made. However, most died after germination at single leaf stage. Therefore, after several attempts, it is concluded that there should be strong crossability barrier between cultivars. of O. sativa studied and O. granulata. There is no proof towards such slight discrepancy obtained in this study and that results found by earlier investigators, except to say differences in strains used in crossing. This over all result suggest that production of hybrid plants from O. sativa/O. granulata cross is very difficult (Khush, 2000; Ghose et al. 1960).

4.2.3. Hybrid embryo derived callus culture Out of 35 hybrid embryos (Pokhreli/O. officinalis) cultured, 15 embryos immediately differentiated into callus. Most of the callus was whitish and friable (Plate 1. fig.a-b). Responsive embryos induced callus within 8-15 days of culture. However, 5 test tubes containing callus did not differentiate into plantlets. Only 5 tube gave plantlets and 62

remaining 5 test tube were heavily contaminated with fungal and bacterial origin. Within 25-30 days of culture, these went to rhizogenesis at first and then caulogenesis (Plate 1. fig. c and d). Bajaj and Bidani (1980), and Nowick (1986) obtained similar results in rice. Nowick (1986) was able to isolate hybrid plants from O. sativa/O. latifolia and grown up to maturity. However, he did not report detail result in his paper about the success rate. Slight discrepancy in the result obtained might be due to cultural condition, laboratory environment and genotypic factors, as they are crucial to regenerate plants (Misso et al., 1989; Ko et al., 1983). Bajaj and Bidani (1980) reported that most of the callus went rhizogenesis after 5-15 days, however, in this experiment most of the callus formed shoot, but rhizogenesis was slightly inhibited. These slight discrepancies in the mode of regeneration might be attributed to hormonal difference in medium and genotypic differences. Other than genotypic difference, age of embryo also plays crucial role from formation of callus to whole plant regeneration. Similar type of research in rice was also conducted by Bouharmont and Dekeyser (1985) in African rice and reported that only embryo derive calli gave successful plant regeneration Thus it was found potential tool to regenerate hybrid plants when embryo abortion started quite earlier.

4.2.4. In vitro fertilization Out of 65 spikelets pollinated only 20 spikelets showed swelling of ovules after 5 days of pollination (Plate 1.fig. i). At ten days after of pollination, 35 tubes were heavily infected by fungal and bacterial pathogens. Nine ovaries were excised after 12 days of pollination and found that only 3 had embryo and endosperm. Remaining ovaries were full of watery endosperm and some were empty. No seedlings were obtained after culturing these three embryos on Bouharmont (1991) medium. However, after 5 days of incubation,

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Plate 1. Figure ac. Efforts employed to regenerate intergenomic rice hybrids. a-d. very young hybrid embryo subjected to induce callusing and subsequent regeneration i.e. regeneration of hybrid plants through callus culture, Pokhreli/O. officinalis., (direct regeneration without subculture on medium proposed by Chen et al., 1991)., c. regeneration of the shoot, and d. figure showing profuse rooting in the callus derived seedlings some of the seedlings are albino in origin which were died after transplanting in the pot., e-h embryo rescue Manshara/O. officinalis, Kalanamak/O. Officinalis, and Jhinuwa/O. officinalis, respectively., g. In vitro pollination O. sativa/O. granulata, showing swelling of ovary after 10 days of pollination., j-l new hardening technique employed to reduce the mortality in field transplantation.

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Table 4.1a. Results of embryo rescue and crossability between O. sativa, and O. officinalis, and O. granulata No. of floret pollinated parental combination Ghaiya/O. granulata1 Jhinuwa/O. granulata Masuli/O. granulata IR 72/O. granulata IR 64/O. granulata Manshara/O. granulata Pokhreli/O. granulata Jethobudo/O. granulata Kalanamak/O. granulata Jhinuwa /O. officinalis IR 72 /O. officinalis IR 64/O. officinalis Manshara/O. officinalis Pokhreli/O. officinalis Jethobudo/O. officinalis Kalanamak/O. officinalis
1, 2 2

Seed set %

Seeds without embryo 91 116 143 73 72 21 26 60 15 23 21 8 24 13 2 42

Total no. of embryo culture 18 24 26 7 13 4 10 15 10 18 15 3 20 13 8 18

Germinati on %

Died aft germination 5 5 7 1 3 3 8 2 2 3 2 7 4 3 4

No. of seedling well grown

Hardening done 1 5 5 4 4 -

No of F1 Crossab plant ility % obtained 5 4 3 4 0 0 0 0 0 0 0 0 0 2.44 0 0 1.97 1.72 0 1.99

508 411 513 413 389 211 221 341 147 205 115 163 203 174 125 201

21.46 34.06 32.94 19.37 21.85 11.85 16.29 21.99 15.65 20.00 31.30 6.75 21.67 14.94 8.00 29.85

27.78 20.83 26.92 14.29 23.08 30.00 53.33 20.00 50.00 20.00 66.67 55.00 61.54 37.50 33.33

1 5 5 4 4

crossing were made only with O. granulata in the second year of 2002

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only one embryo was germinated and that was also finally collapsed at single leaf stage. This result still suggested that existence of a strong crossability barrier, between O. sativa and O. granulata.

4.3.Crossability 4.3.1. Crossability in intergenomic combinations Regardless of the other factors, based on the embryo rescue worked the crossability between O. sativa/O. officinalis was ranged from 0-2.44% and average pooled mean was 1.35 % among all crosses with cultivars (Table 4.1a.). Similarly, it was zero for cv. of O. sativa and O. granulata, although a large number of florets pollinated and a lot of in vitro efforts were carried out up to 2 years. However, the present result was comparable to report of Jena and Khush (1986, 1984). They found the crossability between three lines of O. sativa and O. officinalis varied from 1.0-2.3 % and pooled mean was 1.7% (Jena and Khush, 1984). However, Brar et al. (1991) obtained quite low crossability which was ranged from (0-1.1%) and the pooled mean was 0.15% in five varieties of O. sativa. They got only two hybrid plants from two varieties, pollinating 1301 spikelets of five cultivars. Similarly, other workers (Brar et al., 1991; Morinaga, 1964; Ghose et al., 1960) also obtained non or few hybrids in this set of cross. 4.3.2. Crossability between intragenomic cross The seed set and crossability percentage between O. sativa and two common wild species of rices were shown in Table 4.1b. Among combination seed set and crossability was varied from 7.58-53.15and 7.58-51.05 percent, respectively. Among O. sativa/O. rufipogon hybrids, highest crossability was scored for Jethobudo/O. rufipogon and was 66

Table.4.1b. Seed set, and crossability between O. sativa L. and two common wild rice species (O. nivara and O. rufipogon).

Cross combination Female/ Male

Total spikelets pollinated

Seed set %

No. of F1 plants obtained

Crossability %

Kalanamak/O. nivara Kalanamak/O. rufipogon Jhinuwa/O. nivara Jhinuwa/O. rufipogon Manshara/O. nivara Jethobudo/O .rufipogon Jethobudo/O. nivara Pokhreli/O. rufipogon Pokhreli/O. nivara IR 64/O.nivara IR 64/O. rufipogon IR 72/O.nivara
a

120 133 108 132 181 143 208 250 320 140 138 101

40 49.63 45.37 7.58 45.86 53.15 32.4 48.80 30.00 35.00 50.00 16.83

48 65 49 (2a) 10 81 (3a) 73 (3b) 67 (4a , 3b) 122 (5a, 2b) 96 (15a) 49 69 (6a, 2b ) 14 (3a)

40.00 48.87 43.51 7.58 44.75 51.05 28.84 46.00 25.31 35.00 44.20 13.86

and b for non germinated seeds, and non viable albino plant, respectively

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least in Jhinuwa/O. rufipogon. Similarly, among O. sativa and O. nivara hybrids, crossability was highest in Manshara/O. nivara (44.75%) and was least in IR 72/O.nivara (13.86%). Based on crossability result, it was clear that the landraces like Pokhreli, Jethobudo, and Kalanamak showed close cross affinity with O. rufipogon and Manshara with O. nivara. In most of the combinations landraces of Nepal showed good comparable crossability than the improved varieties (IR64 and IR72). However, degree of cross affinity varied with genetic makeup of the female genotypes (Table 4.2.1b.) suggest that different cultivars have different cross affinity with their ancestral wild species. Similar such variation in crossability have been reported in literatures (Brar et al., 1991; Sitch et al., 1989c; Chu et al., 1969; Shastry, 1964; Henderson, 1964a.; Nezu et al., 1960; Ghose et al., 1960). They all reported that the crossability between O. sativa and O. rufipogon was greatly varied from 10-30% depending upon the geographical race of wild species and genotype of O. sativa used. Therefore, this slight discrepancy obtained in this study in seed set might be attributed to differences in geographical race of wild species used as male parent.

4.4.

Morphology of the interspecific hybrids All the hybrid plants, obtained from the crosses involving O. sativa with O. nivara

and O. rufipogon, were intermediate between the two parents in some traits but showed a preponderance of the characters of wild species, such as basal leaf sheath coloration, spikelet related traits, apiculus colour, seed coat colour, awning character, leaf color, panicle exertion, panicle shattering, panicle type, culm number, internode colour branching of culm and spreading growth habit, stigma color, size of anther, etc. (Appendix 4.3a-3h, and Plate 2-3. fig. 1,2,3, and 1-8, respectively). On the other hand, F 1 plants from four 68

landraces of O. sativa and O. officinalis, obtained through embryo rescue showed intermediate traits in some cases, but wild traits of the species were highly dominant as mentioned above. They were perennial and had rhizomes in their roots. They were robust and highly vigorous than the either parent (Appendix 4.3a-3h, and Plate 2. fig.1-3). Many workers, in several intragenomic and intergenomic hybrids, (Jena and Khush, 1986; Li, 1964; Morinaga, 1964; Hey and Henderson, 1961; Morinaga and Kuriyama, 1957) reported similar dominance of most of the wild type traits.

4.5.

Meiotic behavior in parents and their hybrids

4.5.1. Pachytene analysis in AA genome species hybrids and their parents The meiotic behavior at pachytene stage is presented in Table 4.2.1. Except IR 64 all female parents including wild forms showed complete pairing (Plate.4, fig. 1) IR 64 showed lose pairing at two of the bivalents in one PMC out of 25 studied. Five hybrids showed complete pairing in all bivalents out of eleven studied in this series. Other remaining hybrids showed absence of complete homology, and the frequency was quite high in Jethobudo/O. nivara, Kalanamak/O. nivara, IR 72/O. nivara and IR 64/O.rufipogon. However, it was not too large as reported by Shastry and Misra (1961a.b) in interracial hybrid between Japonica/Indica. They reported that 31.04% of the hybrid did not show normal pairing and most of them had terminal deficiency, heteromorphocity, duplication (mostly reverse repeat), interstitial differential segment (small translocation), inversion, translocation and lose pairing, even though later stages were normal. In this study, the frequency of such structural hybridity was ranged from 4.0-22.58% of the cells observed (Plate.4-7. except figure 2-5 in Plate 7). In IR64/O. rufipogon hybrid seven PMCs showed structural hybridity in 2-6 bivalents mostly with terminal unpaired region

69

and small interstitial translocation, frequently appeared with unpaired gap (Plate 4. figure. 2) On the other hand, inversion was observed in one of the bivalent of IR72/O. nivara hybrid (Plate 4. fig.1), but the frequency was quite low and only two PMCs showed inverted region out of 24 studied. One of the PMCs had only heteromorphocity in more than one bivalents. Similarly, Pokhreli/O. nivara hybrid showed interstitial gap with heteromorphocity and duplication in three bivalents in one PMC out of 19 observed (Plate 4, fig.3). On the other hand, Kalanamak/O. nivara hybrid showed structural abnormality in 13.04% cells out of 23 studied and included abnormalities were interstitial small translocation and mostly heteromorphocity (Plate. 5,.fig.2). Similar irregularities were recorded for Jethobudo/O. nivara hybrid in 14.29% of the PMCs out of 14 studied. Among irregularities, the heteromorphocity, lose pairing and small interstitial translocation was modal class (Plate.5, fig. 3). But it was unable to resolve the causes of heteromorphocity, however, such differences might also be occurred due to deletion (deficiency) or tandem repeats in along the length (Shastry and Misra, 1961b) or sometimes condensation differences between species chromatin (Misra and Shastry, 1967). But what ever the causes of such chromatin differences, on an average pairing was essentially normal along chromomere to chromomere in hybrids between O. sativa and two common wild species of rice. Other hybrid combination in this series showed complete pairing and did not record any of the irregularities, indicates that very close relationship between their chromosomes, even the number of cells was increased. Such structural hybridity have been frequently observed in intragenomic hybrids involving O. sativa and O. nivara (Dolores et al., 1979). They observed 32.4% structural hybridity including loose pairing (23.5%), reciprocal

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Plate 4. Figure 1-3. Representative photomicrograph of chromosomes behaviour during meiosis at pachynema and Camera Lucida drawings (right side). 1. Showing inversion (i, appeared as ), IR 72/O. nivara 2. Showing heteromorphocity and terminally unpaired bivalent (single arrow), quadrivalent association (q), small translocation (double arrow), reverse repeat (d), and loose pairing (l), respectively. IR 64/O. rufipogon ., 3. Showing interstitial translocation (double arrow), reverse repeat (duplication) (d), terminally unpaired (single arrow) Pokhreli/O. nivara.

71

i d i i

i d

Plate 5. Figure 1-3. Representative photomicrograph (left) and drawings (right) of chromosome behaviour during meiosis at pachynema. 1. 12 II, showing small inversion (inverted segment mostly appeared as ) and reverse repeat (d), IR 64/O.nivara., 2. 12 II, showing small interstitial translocation and terminally unpaired segments in two bivalents (single and double arrow, respectively), Kalanamak/O. nivara., 3. Showing interstitial translocation (double arrow), terminally unpaired (single arrow) and loose pairing (l) along length in two of the bivalents, Jethobudo/O. nivara. .

72

Plate 6. Figure 1-3. Representative photomicrograph (left) and

drawings (right) of chromosome behaviour during meiosis at pachynema. 1. Showing loose pairing in one of the 12 bivalent in IR 64 parent., 2. Showing translocation (double arrow) in two of the 12 bivalents of Jhinuwa/O. officinalis, intergenomic hybrids., 3. 12 normal bivalents in Manshara/ O. officinalis intergenomic hybrids. In this series of hybrids most of the normal bivalents had complex networking.

73

1.014

Plate 7. Figure. 1-5. Showing heteromorphocity in 2 of the 12 bivalents (single arrow), Manshara/O. officinalis., 2. 12 II Manshara parent., 3. 12 II, O. rufipogon., 4. Showing well differentiated heterochromatic region of chromosome, O. granulata., 5. 12 II, O. nivara.

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translocation (3.9%), and trivalent association (2.9%) out of 34 cells studied in IR8/O. nivara. Many investigators studied the pachytene chromosomes in interracial and intervarietal rice hybrids, and reported the similar results (Shastry and Misra, 1961a.b; Yao et al., 1958). Yao et al. (1958) observed inversion loops, mostly pericentric in five of the intervarietal rice hybrids in < 10 % of the PMCs. Further more, Shastry and Misra (1961a.b) reported about 11.9-31.04% of the chromatin length had such structural hybridity and suggested that such structural hybridities are the sole cause of sterility rather than other factors. Therefore, summing up the present results the structural hybridity observed in this study was very low or comparable to interracial and intervarietal differention. Based on this stage it is concluded that Nepalese common wild rices are not cytologically so differentiated as much differentiation found in the Japonica/indica rice hybrids. Thus pairing was essentially normal.

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Table 4.2.1. Chromosome behavior at pachynema in the parents and hybrids


Number cells observed Total PMCs Normal Hybrid studied IR64/O. nivara IR64/O. rufipogon Pokhreli/O. rufipogon Pokhreli/O. nivara Kalanamak/O. nivara Kalanamak/O. rufipogon IR 72/O. nivara Jhinuwa/O. nivara Manshara/O. nivara Jethobudo/O. rufipogon Jethobudo/O. nivara Manshara/O. officinalis Jhinuwa/O. officinalis IR72 IR64 Kalanamak Jhinuwa Manshara Pokhreli Jethobudo O. nivara O. rufipogon O. officinalis observed 25 31 35 19 23 38 24 39 32 53 14 11 15 21 25 31 9 36 31 17 23 21 19 pairing 24 (96 %) 24 (77.42%) 35 (100%) 18 (94.74 %) 20 (86.96%) 38 (100%) 21 (87.50) 39 (100 %) 32 (100 %) 53 (100 %) 12 (85.71%) 6 (55%) 9 (60%) 21 (100%) 24 (96%) 31 (100%) 9 (100%) 36 (100%) 31 (100%) 17 (100 %) 23 (100 %) 21 (100 %) 19 (100 %) Cryptic structural hybridity in one or more than one chromosome ( a, b, c, d, e, f)* 1 (4%) e and d 7 (22.58%) a, b, c, and f 0 (0%) 1 (5.26 %) b, d, and f 3 (13.04%) a, b, and f 0(0%) 3 (12.50 %) a, and e 0 (0%) 0 (0%) 0 (0%) 2 (14.29%) a, b, and f 5 (45%) a, b, and f 6 (40%) b, and f 0 (0%) 1 (4%) a 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Loose pairing (a), deletion (c), duplication (d), inversions (e), translocation (f), and heteromorphocity (b).

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4.5.2. Pachytene analysis in intergenomic hybrids (A and C genomes) The pairing pattern of chromosome between O. sativa and O. officinalis is shown in Table 4.2.1. Out of four combinations, only two hybrids were analyzed at this stage. Both hybrid combination showed partial homology between A and C genomes. The complete normal pairing was found in 15 cells out of 26 observed. It was 54.54% in O. sativa. cv. Manshara/O. officinalis and 60.0% in Jhinuwa/O. officinalis. However, the degree of homology varied from cell to cell and most cells possessed 2-3 cryptic differential bivalents in 11 of the cells (Plate. 6, fig. 2 and Plate 7, fig. 1). Similar ranges of results have reported by several researchers (Shastry et al., 1961; Katayama, 1966a. b, 1964). Shastry et al. (1961) obtained complete pairing, however, Katayama (1966a. b., 1965) observed partial homology. On the other hand, Li et al. (1964) was only one who reported 24 univalents at this stage in O. sativa/O. officinalis hybrids. However, Shastry (1966) again claimed that the pairing was normal between these species hybrid. In this study too there was no evidence to report the abnormal pairing, instead that nearly normal pairing was found. Pairing was normal in more than 50 percent cells in both sativa-officinalis hybrids studied, however, in this study most of the PMCs showed complex networking of the chromosome and very few of the cells were observable. No any univalents were detected, although, 11 cells had large unpaired interstitial differential segment in 2 -3 (frequently 1-2) bivalents (Plate 6,.fig.2). From this observation it is concluded that certain segment of the bivalents are subjected to evolutionary change, most probably due to small translocation. Remaining hybrid PMCs showed complete 12 bivalent. Thus the normal pairing shown by more than 50 percent cells in sativa-officinalis hybrids clearly indicates that A and C genomes should be partially homologous. Neither 100 percent complete pairing nor presence of univalents found in this study suggests that definitely certain structural change between these taxa 77

might be operated with the trends of evolution. This result was closely associated with findings of Shin and Katayama (1979). They reported that two of the hybrids, which have an additional chromosome of O. officinalis, formed univalent, other six hybrids studied by them did not show univalent and frequently formed trivalents at diakinesis and metaphase I. Based on the occurrence of many univalents in the PMCs of two alien addition lines of O. officinalis, Shin and Katayama (1979) suggested that two chromosomes of O. officinalis (W0065) have genes which are responsible to desynapsis of the pairing between species homologous chromosomes. However, they did not observe the pachytene chromosome, therefore they could not deduced the genetic control of univalent formation in the two types of alien addition lines. Such asynaptic and desynaptic genes controlling chromosome pairing at MI of PMCs have been reported in Zea (Beadle, 1933), Triticum (Li et al., 1945; Riley and Law, 1965), and in rice (Misra and Shastry, 1969). Beside, desynaptic mutant in rice has been artificially induced (Yasui et al., 1993). The asynapsis or desynapsis was in most cases controlled by a single recessive gene (Shin and Katayama, 1979) and such genes are more active particularly at low temperature (Li, 1945). In this study, also two of the twelve bivalents mostly did not show complete pairing in O. sativa. cv. Manshara/O. officinalis hybrid. Five of the PMCs in this hybrid showed such cryptic behavior. This differential region observed in this study indicates that certain evolutionary change might have evolved and the desynapsis mutant further erecting the strong reproductive barrier between these two complex. Not only at pachytene, even at diplotene, 3-6 bivalents and trivalents and rarely quadrivalents were observed (Plate. 8,.fig. 1-3). Among these bivalents some were loosely paired with a single chiasmata (Plate.8,. fig. 2). This latter stage analysis also supported the results of pachytene analysis. Such frequent bivalent formation at this stage have been reported by Katayama (1966a., b). He

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also frequently observed bivalents in O. sativa (AA)/O. minuta (BBCC) and O. sativa (AA)/O. latifolia (CCDD) hybrids. Based on this analysis he concluded that such bivalent mostly due to partial homology between A and C genomes; not due to pairing between A and B or D genomes. Thus the data from the present study, also clearly shows that three is partial homology between A and C genomes and did not support the earlier generalization as either very limited pairing or pairing is completely failure made by Li (1964), Morinaga (1964), (Jena and Khush, 1986), Nezu et al. (1960). However, all of them drew their conclusion based on data from diakinesis and Metaphase I. except Li et al. (1964). On the other hand these events might not be always true, as pairing event is largely effected by plant growing environmental condition (Palmer et al., 2000; Ahmad et al., 1977; Henderson, 1964a.; Dowrick, 1957; Li, 1945) or geographical differentiation in the race and genotype of the female parent used (Shin and Katayama, 1979; Shastry, 1966). Therefore, The discrepancy found in the pairing of pachytene chromosomes among these workers might be due to their use of different strains of O. officinalis. However, whatever the findings in the past had, the included materials in the present study showed partial homology between A and C genomes. The result obtained herein also clearly indicates that the failure of bivalent formation at later stage in distant hybrids always not a proof of lack of homology. Such failure of synapsis can possibly be brought by many external and internal factors (Misra and Shastry, 1969). Thus the present study revealed that, A and C genomes, more or less completely pair but differentiated by few chromosome structural changes, although these species are morphologically highly differentiated. 4.5.3. Variation in nucleolus shape in parents and their hybrids Shapes of nucleolus at pachytene were studied in all the parental forms and their possible hybrid derivatives (Table 4.2.2). Most of the landraces had inconsistent shape and 79

Table 4.2.2. Percent frequency of nucleolus shape variation in parents and hybrids
Parents/ hybrids Kalanamak Manshara Jethobudo Pokhreli Jhinuwa IR64 IR72 Manshara / O. nivara Jethobudo/ O. rufipogon Jethobudo/O. nivara Pokhreli/O. nivara Pokhreli/ O. rufipogon Jhinuwa/ O. rufipogon Jhinuwa/ O. nivara Jhinuwa/ O. officinalis Pokhreli /O. officinalis Kalanamak/O. nivara Kalanamak/O. rufipogon Manshara/ O. officinalis IR64/O.nivara IR64/O. rufipogon IR72/ O. nivara O. nivara O. rufipogon O. granulata O. officinalis Total PMCs observed 205 214 242 214 190 148 142 132 246 261 206 96 171 225 180 170 181 211 202 234 273 269 280 183 339 201 Single large nucleolus 43.41 36.45 19.42 47.66 52.63 67.57 95.77 38.64 73.98 70.88 49.03 98.96 45.61 44.44 11.11 47.06 13.81 61.61 39.60 51.28 99.27 14.13 25.00 87.98 100.00 97.51 Semidoubled 10.24 44.86 19.83 11.68 7.89 2.03 1.41 1.52 0.81 1.92 9.71 14.62 17.78 38.89 38.24 4.97 1.42 35.64 10.68 9.29 0.00 7.10 1.49 Single with Single with two one Bud bud 45.37 18.69 60.74 40.65 39.47 28.38 2.82 59.09 24.39 27.20 41.26 1.04 38.01 37.78 44.44 8.82 81.22 36.97 19.80 38.03 0.37 76.21 74.64 4.92 1.00 0.98 0.76 0.81 1.75 5.56 5.88 3.96 Single with three bud 0.99 Two Nucleolus /cell 2.03 0.37 0.37 0.36 -

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mostly represent single, semi-doubled and single with small one bud. However, the single with large and single with one bud type were commonly observed in most of the parents except Manshara, which had semi-doubled type in 44.86% cells. On the other hand IR 72 only showed single large nucleolus in 95.77% cells. Among wild species: O. granulata and O. officinalis contained single large nucleolus, however, small frequency of cells with other type of shape in O. officinalis was also observed. It was found interesting that O. granulata had only one single large nucleolus than all the species studied, even after screening large number of cells. On the other hand high frequency of single nucleolus with one bud type was observed in O. nivara in 74.64% cells. Most of the hybrid showed similar variable structure with preponderance of single type except O. sativa/O. nivara hybrid, which showed quite dominance over O. sativa nucleolus. Only two of the hybrid combinations showed two nucleolus/cell. No sharp effect of hybridization stimuli was detected in this object. Misra and Shastry (1967) carried out similar study. They observed variation in pachytene nucleolus in the shape within sativa varieties. However causes of such structural changes during this stage was still mysterious. But these variations can be treated as marker to determine the diversity among wild and cultivated forms.

4.5.4. Diakinesis and Metaphase I The chromosome behaviour at these stages in parents and hybrids are shown in Table 4.2.3a, 3b, 3c, and 4.2.3d and illustrated in photomicrograph (Plate 8 and 9). Among wild species, O. officinalis scored highest percentage (96.64%) of normal bivalent (12II) and O. nivara showed least one (89.24%) (Table 4.2.3c). Similarly, among cultivated forms IR 72 had highest normal cells (97.65%) followed by Pokhreli was least (80.39%). On an average all the parental forms showed normal cells having 12II and were ranged in 80.3981

97.65% of the PMCs. On the other hand, highest univalents (15.03%) was scored in Pokhreli among parents and least in IR 64 (0.35%). Similarly, almost parents, except, O. rufipogon, O. officinalis, IR72, and Kalanamak had quadrivalent association, and these were scored in 0.63-5.72% cells. The highest (5.72%) quadrivalent association was observed in Jethobudo among parents, although pachytene stage was normal. Similarly, Jethobudo and Kalanamak showed VI association at low frequency in 0.44 -1.91% cells. On the other hand, it was found quite interesting that Jhinuwa parents scored highest 1112II with nucleolar bodies (7.88% cells). All other parents except IR 64 had such cells and its occurrence was varied in 0.65 -7.88% cells. Aneuploids were also observed at low frequency in Jethobudo and Pokhreli parent (0.44% and 0.65% cells, respectively) (Table 4.2.3b and 3c.).

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Table 4.2.3a. Meiotic configuration at Diakinesis and Metaphase I from the parents used in the hybridization

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10

11

Plate 8. Figure 1-11. Representative photomicrograph of meiotic behavior at diplotene and diakinesis in interspecific rice hybrids. 1-3 Diplotene. 1. 1 III (double arrows, fork shaped) + 4 II + 13 I, Kalanamak/O. officinalis., 2. 1 IV (shown by single arrow, cross shaped, indicator of typical case of translocation) + 2 III (double arrows, frying pan shaped) + 3 II + 8I, Pokhreli/O. officinalis., 3. 1 IV (single arrow) + 2 III (double arrows) + 2 II +10 I, Manshara/O. officinalis. 4-11 diakinesis. 4. 12 II (early diakinesis) appeared as (three chiasmata), or 8 (2 chiasmata) and V or (mono chiasmatic bivalent), and ring (dichiasmatic bivalent), O. granulata., 5. 12 II O. officinalis., 6. 12 II + nucleolar bodies, Pokhreli/O. nivara., 7. 11 II only, IR 64/ O. nivara., 8. 11 II + 2 I (single arrow), Jhinuwa/O. nivara., 9. 24 I, Kalanamak/O. officinalis., 10. 24 I, Manshara/O. officinalis., 11. 24 I, Jhinuwa/O. officinalis.

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10

11

12

Plate 9. Figure 1-12. Representative photomicrograph of chromosome behavior during meiosis at MI in interspecific rice hybrids. 1. Showing 12 ring II, Kalanamak/O. rufipogon.., 2. Showing 12 II, four of the IIs are rod shaped, Manshara/O. nivara., 3. 12 ring II, Jhinuwa/O. nivara., 4. 11 II + 2 I (single arrow), IR 72/O. nivara., 5. 11 II + 2 I (single arrow), precocious separation of one bivalents resulted into univalent like structure, Pokhreli/O. rufipogon., 6. 11 II + 2I (single arrow), IR 64/ O. rufipogon., 7. 11 II + 2 I (single arrow) Kalanamak/O. nivara., 8. 11 II + 2 I (single arrow), Kalanamak/O. rufipogon., 9. 24I, Pokhreli/O. officinalis, 10. 2 II (1 rod + 1 pseudo-bivalents) + 20 II, Manshara/O. officinalis., 11. 51 chromosomes, showing hybridization stimuli, Pokhreli/O. officinalis., 12. 25 I, Pokhreli/O. officinalis.

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Similar, such abnormalities in true parental forms were reported by Nandi (1936), Sasaki (1935). They frequently observed one quadrivalents and 2-4 univalents. However, in this study too such irregularities were more or less similar, the frequency of univalents observed in Pokhreli were quite high (0-8) (Table.4.2.3b). This event might be either attributed to heterogeneity of its population or to winter effect, because, the analysis of this material was carried out on the second week of December 2002. Such effect of environment particularly low temperature was greatly emphasized by Li (1945), Ahmad et al. (1977). Among hybrids, involving O. sativa and common wild rice (O. nivara and O. rufipogon), chromosome association was essentially normal except Pokhreli/O. nivara and IR72/O. nivara; they showed mark reduction in normal bivalent association. More than 17% of the cells were with aberration in these hybrids. In other hybrids, reduction in the normal bivalent formation was found in < 11% cells except IR 64/O. nivara. IR 64/O. nivara had 16.26% cells without normal bivalent. On the other hand, it was found interesting that normal bivalent formation was slightly increased by 0.57%, 6.71% and 2.34% in hybrid forms of Jethobudo, Pokhreli, and Kalanamak with O. rufipogon, respectively. As compared to female parent (IR 64), IR 64/O. rufipogon showed reduction in normal bivalent in 12.07% cells. Similarly, highest univalents were observed in Pokhreli/O. nivara, and 18.18% cells had univalents with ranged from 2-6 (Table 4.2.3c) and was least in Kalanamak/O. rufipogon (4.56%). Almost all hybrids showed quadrivalent association except Kalanamak/O. rufipogon and Manshara/O. nivara, and it was highest in Pokhreli/O. nivara in 6.82% cells. Other abnormalities includes were, formation of hexavalent except Jhinuwa/O. nivara, Pokhreli/O. nivara, Pokhreli/O. rufipogon, and Kalanamak/O. rufipogon, presence of nucleolar bodies (0.91-12.27%), (Plate. 8,. fig. 6), 87

Table 4.2.3b. Meiotic configuration at Diakinesis and Metaphase I from the interspecific hybrids involving three different species

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Table 4.2.3b. Meiotic configuration at Diakinesis and Metaphase I from the interspecific hybrids involving three different species

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and occurrence of aneuploids (0.40-7.17%) in all hybrid combinations (Plate. 8, fig. 7). However, in Pokhreli/O. rufipogon, the aneuploidy found in parental forms was slightly reduced. The abnormalities observed at diakinesis and MI were also partly illustrated in microphotograph (Plate. 8, fig.1-11, Plate.9,. fig. 1-12, and Plate.10,. fig. 1-2). Similar, observations were reported by Dolores et al. (1979) in cross involving O. sativa and O. nivara. They reported that a series of abnormality including univalents trivalents, quadrivalents, nucleolar bodies and aneuploidy, but their frequencies were comparable to their respective parents. Although, no trivalents were observed in this study. Not only these small one, high frequency of abnormalities such as frequent 0-3 quadrivalents formation, univalents and chromosomal elimination have been reported in O. sativa and O. rufipogon hybrids (Majumder et al., 1977; Majumder and Ram, 1992) and even in true lines of partially sterile rice variety (Ram and Majumder, 1996). The frequent occurrence of nuclear bodies in this study at diakinesis in both parents and hybrids, however, necessitates that the new speculation needs to be made to generalize the causes of such abnormalities. There are several proposition about the occurrence of such bodies, like structural changes (Walter, 1963) or due to fragmentation of chromosome by translocation (McClintock, 1934 in Parthasarathy, 1938). However, the nature of the data observed here showed that there might be other causes too. It is because of the frequency of such bodies is quite high even in parent like Jhinuwa (7.88%), although pachytene pairing was normal. Similarly, univalents, and quadrivalents were frequently observed not only in interspecific hybrids, but also commonly reported for interracial and intervarietal hybrids (Sampath and Mohanty, 1954; Yao et al., 1958). Similar reports have been put forward in interspecific hybrid involving intragenomic cross hybrids (Yeh and Henderson 1961, 1962, Morinaga, 1964, Misra and Shastry, 1969).

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Likewise hybrid involving O. sativa and O. officinalis showed pronounced irregularities at these stages. Nearly 100% cells had irregularities and the mean frequency of bivalent was ranged from 0.98-1.59 in most of the crosses (Table 4.2.3d). The irregularities were characterized as formation (14-24 I) univalent with modal class 24 I (Plate. 8., fig.9, 10,11, and Plate. 9,. fig. 9 and 10). Besides, at low frequency, some of the PMCs had haploid-polyploid chromosomes complement. There are voluminous literatures that deal about such abnormality at these stages, although pachytene was normal in O. sativa and O. officinalis hybrids (Jena and Khush, 1986, Nayar; 1973; Hu and Chang, 1966, Katayama, 1966a. b; Morinaga, 1964; Li 1964; Ghose et al., 1960, Nezu et al., 1960). Similar irregularities have been reported in other intersectional species hybrids (Brar et al., 1996; Morinaga, 1964; Li 1964; Shastry and Rao, 1961; Ghose et al., 1960; Nezu et al., 1960). Most of them observed 24 univalents and occasionally 1 -5 bivalents. On the other hand, hyperploidy and hypoplody was reported by Hu and Chang (1966), Dolores et al. (1979). Hu and Chang (1966) observed more than 48 chromosomes in two of the geographical races of diploid O. officinalis hybrids and Nayar (1973) concluded that such phenomenon is due to the hybridization stimuli (Plate. 9, .fig. 11 and 12 and Plate 10,. fig. 2). Therefore, the over all result and discussion regarding these two stages showed that except intergenomic hybrids, pairing events obtained in the hybrids was comparable to their parents in one or more respects thus pairing was essentially normal. 4.5.4.1. Chiasma frequency Chiasma frequencies among parents included in the hybridization are shown in Table 4.2.3a and representative illustrations were shown in Plate. 8., for diakinesis and 9. for MI. Among wild species O. granulata showed least number of chiasmata/cell (20.962.03, 22.161.95) and per bivalent (1.750.18,1.850.17) at diakinesis and

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metaphase I, respectively with pooled mean 21.56/cell and 1.8/bivalent. Similarly, O. rufipogon and O. officinalis scored second and third position interms of chiasmata, respectively. On the other hand, annual wild species O. nivara showed highest chiasmata/cell and per bivalent, which is comparable to cultivated Oryza sativa forms (>23 chiasmata/cell and >1.9/bivalent). However, in all forms, ring bivalent was predominantly found (8.94-11.17) and rod bivalent varied from 0-8, and was highest in O. granulata. Some bivalents were visibly larger than others, they had 2 or 3 chiasmata and their shape was or . Some others had only 1 or 2 chiasmata and structure formed like V, X, or ring shape (Plate. 8,. fig.4.). Similar trends in chiasma frequencies was reported by Jena and Misra (1984) and they suggested that perennial and cross pollinated species in general showed lower chiasma frequency than their related annual self pollinated species. This generalization was also found evident in this study as well, particularly, in the case of O. rufipogon, which is perennial and occurrence of certain degree of cross-pollination due to its high stigma exertion for long duration than O. nivara. It is frequently reported that the out crossing rate in this species was diverged and quite high (30-50%) (Rao et al., 1997; Morishima and Oka, 1995). O. granulata and O. officinalis are perennial, however, based on chiasma frequency, it was appeared that O. granulata and O. rufipogon were more primitive than O. officinalis. Similar proposition is made by Shastry and Sharma (1985) based on high amount of heterochromatic segment at pachytene stage. In this study also O. granulata had highest amount of heterochromatic regions than other species (Plate 7, fig.4). On the other hand annual species O. nivara scored higher chiasma frequency (>23), which was the indication of its highly evolutionary advanced. Thus it is concluded that evolutionary changes would be going on in the genus Oryza and general trends seemed to perennial to annual and cross pollinated to self

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pollinated through effective genetic recombination (higher chiasma frequency) (Jena and Misra, 1984). Similarly, all the cultivated forms of Oryza sativa showed more or less same level of chiasmata frequency (>23/cell), except Pokhreli landraces, which had (22.59 chiasmata/cell), indicate that the landraces are so advanced as much advanced found in improved ones. However, highest chiasma frequency was found in IR72 (23.36/cell). Among landraces Kalanamak was more advanced than others. However, the chiasma frequency/ bivalent was found in Pokhreli also showed same level of evolutionary advance and low chiasmata frequency/cell was mainly attributed to more univalent formation at these stages. The mean frequency of rod bivalents in these series varied from 0 -9 however, 0-3 was the mode (Table 4.2.3a.). Similar ranged of results were presented by several authors (Soriano, 1961; Chu et al., 1969). On the other hand, among hybrids, involving common wild rice (O. rufipogon and O. nivara) and O. sativa did not showed sharp reduction in chiasma frequencies as expected and were comparable to their either parent indicated that significant association between their genomes Table 4.2.3a. The highest pooled chiasmata frequency/cell and per bivalent was found in Jethobudo/O. nivara (22.965 and 1.945, respectively). Another interesting event was that, the chiasma frequency of hybrids (Pokhreli/O. rufipogon) slightly exceeds the frequency of female parent, this suggests that chromosome pairing can be improved by genes from wild species (Table 4.2.3b). The mean number of rod shaped bivalent in these series of hybrids varied from 0.53-1.80 Similar result of increased in meiotic pairing from diploid wild relatives of Triticum have been reported by Sears (1976). Not only in the wheat Nowick (1986) also suggested that some of the AA genome cultivated rices have pairing promoter genes. 93

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Similar results were reported by other several investigators in hybrid among AA genome species (Yao et al., 1958; Yeh and Henderson, 1962, 1961; Shastry and Misra, 1961a.b.). However, Chu et al. (1969) obtained slight reduction in the chiasmata/bivalent in AA genome hybrids. According to him the mean number of chiasmata in the sativa variety was 1.980.65 and was 1.910.61 in O., rufipogon but chiasmata among twenty hybrids was varied from 1.080.62 to 1.830.59. Although, marked reduction in chiasma frequency was observed in O. sativa and O. officinalis hybrids, this does not imply the lack of pairing it was due to desynapsis and was varied from1.33-2.57/cell and 0.72-1/bivalent, respectively. The average mean ring bivalent was varied from 0.53-0.89. Such sharp reduction in the chiasma frequency and there by frequently formation of rod shaped bivalents was reported by Sarkar et al. (1994) in Maize/Teosinte hybrids. This implies that very limited pairing between these genomes at diakinesis and Metaphase I stages due to desynapsis. Jena and Khush, (1986), Morinaga (1964) Lu (1964) observed similar pattern of pairing in these species hybrids. Similar, events were reported in number of distant crosses of rice (Mahapatra et al., 2002, Brar et al., 1996, Li, 1964; Morinaga, 1964), however, they did not calculate chiasma frequency.

4.5.4.2. Chromosome number and ploidy level From the cytological observation it was confirmed that all the wild species from Nepal discovered so far, had a consistent chromosome number of 2n = 2x = 24 in both meiotic PMCs and pollen mitosis (Table.4.2.3a.). Therefore, it is reported that all the wild species, O. nivara, O. rufipogon, O. granulata, and O. officinalis, from Nepal are diploid (Plate. 8, and 9.). This observation showed the similarity with the results of Bouharmont (1962), and Vaughan (1994). Similar result was presented by Lu et al. (1997) and reported 97

that O. nivara, O. rufipogon, and O. granulata had 2n=24. On the other hand, 2 Indian, out of 138 accession of O. officinalis showed tetraploidy (2n = 4x = 48), and known as O. malampuzhaensis (Vaughan, 1994). In this study too minor meiotic irregularities was observed and meiotic abnormalities includes were few univalent and PMCs with abnormal nuclear bodies in all the species, however, the frequency was too small. Similarly, Lu et al. (1998) suggested that four AA genome species, O. nivara, O. rufipogon, O. glumaepatula and O. meridionalis, from different geographical origin of a world also showed consisted number of chromosome, 2n = 24. 4.5.5. Meiotic behavior at Anaphase and telophase I The chromosome behaviors at these stages are shown in Table 4.2.4. Among parents the percentage of normal cells varied from 87.88-98.54, which was highest in IR 72 and lowest in Pokhreli. All parents showed a minimum degree of all arrays of abnormalities except bridges and fragments and bridges and laggards Observed abnormalities includes were unequal segregation, bridges, laggards, late disjunction and early division of chromosomes. Their existence was observed in 0.69-3.93, 0.72-3.03,0.692.96, 0.85-4.24 and 0.85-1.95 percent cells, respectively (Table 4.2.4, Plate. 10,. fig. 6-9 and Plate 11,. fig.1-11). All types of anomalies were not recorded within single parent however more than two abnormalities were commonly observed. Among hybrids between O. sativa and common wild rices, Jethobudo/O. rufipogon scored highest percentage normal cells and was lowest in IR64/O.nivara. In this series of crosses, percentage of normal cells varied from 70 -95%. Other abnormalities include were more or less comparable to their respective parents except bridges + fragments and bridges + laggards (Plate.11,.fig.8). Four (Kalanamak/O. nivara, Jethobudo/O. nivara, Pokhreli/O. nivara and IR64/O.rufipogon) of the 11 hybrids had bridges + fragments. The percentage cells with

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Plate 10. Figure 1-9. Representative photomicrographs of chromosome behavior during meiosis at metaphase I, and anaphase I in interspecific rice hybrids. 1. Prometaphase, 24 I, Kalanamak/O. officinalis., 2. Prometaphase, 33 I, 9 II + 15 I, 4 are pseudobivalents due to e-e and s-s contact, Jhinuwa/O. officinalis., 3. AI, showing normal disjunction, Jethobudo/O. rufipogon., 4. AI, showing normal disjunction, 12 chromosomes are ready to go one pole, Jethobudo/O. rufipogon., 5. AI, normal disjunction, IR 72/O. nivara., 6. AI, showing two lagging fragmented univalent and single univalent, Kalanamak/O. nivara., 7. Two pairs of chromosome have formed bridges, apparently the results of delayed terminalization of chiasmata (sticky bridges), Kalanamak/O. nivara., 8. AI, delayed disjunction, Pokhreli/O. rufipogon., 9. AI, unequal segregation, Manshara/O. officinalis.

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11

12

Plate 11. Figure 1-11. Representative photomicrographs of chromosome behavior during meiosis at anaphase I, and telophase I in interspecific rice hybrids.1. MI-AI, showing sticky bridges and laggards, Kalanamak/O. officinalis., 2. AI, showing laggards Manshara/O. officinalis., 3. MI-AI, showing laggards Jhinuwa/O. officinalis., 4. MI-AI, 24 I randomly distributed along cell plate, Kalanamak/O. officinalis., 5. AI, sticky bivalent showing possibility of unequal segregation, IR 64/O. rufipogon., 6. MI-AI, 18 I were appeared in metaphase plate, Manshara/O. officinalis., 7. AI, showing bridges + fragments and laggards, Pokhreli/O. officinalis., 8. Showing typical anaphase bridges + fragments, indication of paracentric inversion, IR 64/O. rufipogon., 9. TI, 2 lagging, and 1 laggards, Pokhreli/O. nivara., 10. TI, showing laggards, Kalanamak/O. officinalis., 11. AI, showing anaphase false bridge due to delayed terminalization of chiasmata, Manshara/O. nivara., 12. Showing partial pollen sterility, IR 64/O. rufipogon.

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bridges + fragments were ranged from 0.44-9.40, which was quite higher in IR64/O. rufipogon (9.40%). On the other hand, bridges + laggards frequently observed in all hybrids except Jethobudo/O. nivara, IR64/O.nivara, Jethobudo/O. rufipogon and Pokhreli/O. rufipogon and their occurrence varied in 0.71-2.76% PMCs. Highest percentage of cells with late disjunction was found in IR 64/O. nivara (9.09%). On the other hand, hybrid involving O. sativa/O. officinalis showed high abnormalities at these stages and most of them had high frequency of laggards and it was observed in 33 -49% PMCs studied. All hybrids in this series showed bridges with fragments and its existence varied in 6.70-9.23% cells (Plate.11, fig. 7). Cells with laggard +fragments were observed in 16.50-21.86 % cells (Plate.11,. fig.9-11). Similar results were reported in many investigators in intragenomic cross hybrids (Majumder et al., 1997; Majumder and Ram, 1992; Dolores et al., 1979 Misra and Shastry, 1969; Shastry, 1964; Hey and Henderson, 1961, 1962; Nezu et al., 1960; Ghose et al., 1960), and in intervarietal and interracial rice hybrids (Ram and Majumder, 1996; Demeterio et al., 1965; Henderson et al., 1959, Yao et al., 1958; Sampath and Mohanty, 1954). Dolores et al. (1979) observed bridges and fragments in five of the 11 crosses involving O. sativa and O. nivara. However, they observed at low frequency in 0.3-0.7% PMCs. According to them, the maximum abnormalities at these stages were bridges, late disjunction and early division of chromosome. On the other hand, Sampath and Mohanty (1954) found bridges and fragments in 11 of the interracial hybrids out of 33 studied by them. Such irregularities was most frequently observed in <10% cells, and they concluded that the occurrence of anaphase bridges and fragments mostly due to presence of inversion in some of the chromosomes. Henderson et al. (1959) also observed 0.28% cells with bridges and fragments in interracial rice hybrids. Other several investigators reported 101

occurrence of anaphase bridges in interspecific crosses without mentioning their frequencies (Hey and Henderson, 1961, 1962; Misra and Shastry, 1969). However, in this study a quite high bridges and fragments formation at these stage might be due to genotypic differences in the female parents used or probable explanation for such abnormalities would be the induced inversions. High frequency of bridges formation at anaphase mostly attributed to delay terminalization of chiasmata or sticky bivalent formation or breakage and randomly reunion of the chromatids at earlier stages regardless of homology as reported by Walter (1963) (Plate.10, fig. 7 and Plate.11, .fig.11). Likewise, the quite high frequency of laggard formation at these stages undoubtedly the results of univalents at diakinesis and metaphase I. Besides, these aberrations, unequal separation, early disjunction, and late disjunction observed in this study also confirmed the results of Dolores et al. (1979) (Plate. 10, fig. 8-9 and Plate. 11, fig.5). These commonly occurred anomalies might be precocious separation of bivalent at metaphase (Plate.9,. fig.5) or anaphase or due to timing imbalance (Shastry and Rao, 1961) to separate the bivalent (Plate.10,.fig.5) or might be due to effect of hybridity. Besides these anomalies some of the hybrids showed improvement in pairing in these stages than their parental forms indicating that genes from wild rice improved the pairing efficiency as reported by Sears (1976) in diploids wild wheat. On the other hand high abnormalities in later stages of meiosis in O. sativa and O. officinalis hybrids have been frequently reported by several researchers (Jena and Khush, 1986; Katayama, 1966a.b., 1965; Morinaga, 1964; Li, 1964). However, they did not report bridges and fragments, as their study only confined to diakinesis and metaphase I except Katayama (1966a.b). He only demonstrated laggards and bridges in his figure but not reported the fragments (acentric) and bridges.

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In this study also most of the cells showed laggards (Plate.11,.fig. 1-4) and bridges as a result of same phenomenon as described above (Plate.11, fig.7). Formation bridges and fragments at these stages was quite different from the previous study, indicates that inversion would be certainly occurred during evolution in one three chromosome or these might be due to effect of hybridity. Probably inversion should be true as even in the pachytene frequently 2 of the bivalents did not completely paired. Most of the hybrids showed univalents either at the equatorial Plate as laggards or distributed randomly throughout the nucleus (Plate.11,.fig.1, 2, 3, 4, and 6). Very few of the bivalents separated and go to opposite poles (fig.6). Thus in these stages was pairing was normal, except for O. sativa and O. officinalis hybrids, as the aberration found in this stages were similar to previous results of other investigators, and even low as reported in interracial hybrids having similar genome. Not only, but also the frequencies of irregularities were more or less comparable to their parents as well.

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4.6.

Hybrid fertility and sterility: cytological dissection Pollen and spikelet fertility in F1 hybrids and their parents are shown in Table 4.2.5.

The F1 hybrids, among O. sativa and O. nivara, Manshara/O. nivara had highest pollen and spikelet fertility (25 and 86.49%), respectively. Pokhreli had the lowest pollen fertility (45.22%) among female parents. O. officinalis showed lowest pollen fertility (33%), and it was the highest Kalanamak (71.30%) among male and female parents, respectively. IR 72/O.nivara, on the other hand, showed lowest pollen fertility among hybrids in this series. Similarly, the F1 Pokhreli/O. rufipogon had the highest mean percentage for both pollen and spikelet fertility (49.53 and 71.69%), and it was least for IR64/O.rufipogon (23.0 and 2%, Fig. 12, Plate. 11), respectively, in this series of hybrids. It was found quite interesting that two of the hybrid Kalanamak/O. rufipogon and Jethobudo/O. rufipogon did better interms of spikelet fertility, although, the pollen fertility had lower than their respective female parents. On the other hand, among O. sativa and O. officinalis hybrids, most of the hybrids had average pollen fertility <4.55% (0-4.55%). All the F 1 hybrids had 100% spikelet sterility however, partiality fertility was recorded for their female parents (Table 4.2.5). Similar range of F1 sterility have been reported in intragenomic crosses, involving O. sativa and different AA genome species hybrids (Dolores et al., 1979; Hey and Henderson, 1961,1962; Richharia, 1960; Morinaga and Kuriyama, 1957). Dolores et al. (1979) observed 35-60% fertility as lowest in two of the O. sativa/O. nivara hybrids. Other hybrid studied by them showed normal pollen fertility according their classification (>60%). Morinaga and Kuriyama (1957) observed 4.3% averages pollen fertility and found 100% sterility in winter grown O. sativa and O. glaberrima hybrids. On the other hand, Hey and Henderson (1961) obtained 8.2-99.99% fertile pollen and 0-94.8% spikelet fertility in hybrid involving O. sativa and five wild species of Oryza having AA genome. However in 105

this study, most of the hybrid involving O. sativa and common wild rice as well as parents showed comparatively less pollen fertility (partial fertility). Such discrepancy might solely due to large environmental effect, because pollen fertility and sterility was determined from the winter grown plants. Such sterility have been largely reported not only for interspecific crosses, but for intraspecific crosses as well (Shastry and Misra, 1961a.b; Henderson et al., 1959; Sampath and Mohanty, 1954; Yao et al., 1958). Henderson et al. (1959) observed 28.7-98.4% sterility in intervarietal crosses. In some of the hybrid pollen fertility was quite low as compared to spikelet fertility for example, Manshara/O. nivara. On the other hand, the observation made by IKI staining technique did not reflect any significant correlation between pollen and spikelet fertility and it was found dubious to generalize the results based on this technique. Such dubious results have been frequently reported in literatures (Song et al., 2001; Joshi, 2000) There was a large deal about causes of pollen and spikelet sterility in many crop species and many of them reported that chromosomal causes (cryptic structural hybridity) (Yao et al., 1958 Shastry and Misra, 1961a.b.; Allard, 1960; Stebbins, 1958), or genic (Sano, 1993; Ikehashi and Araki, 1986; Ikehashi, 1991, 1986; Oka, 1974) or cytolopalsmic (Dolores et al., 1979) and or environmental (Palmer et al., 2000; Dolores et al., 1979; Ahmad et al., 1977). Dolores et al. (1979) found significant differences in 3 of the 6 reciprocal crossed hybrids involving O. sativa and O. nivara, and demonstrated the apparent effect of cytoplasm on varying degree of sterility. But, in this study reciprocal crosses did not carry out and degree of sterility caused by cytoplasmic differentiation could not be assessed. However it was clear that the partial sterility obtained in O. sativa and common wild species did not attribute to chromosomal hybridity except IR64/O. rufipogon, Jethobudo/O. nivara, Kalanamak/O. nivara and IR 72/O. nivara. Although most of these 106

hybrids showed 12.50-22.58% cells with cryptic structural hybridity, the degree of abnormality was quit far below than reported for interracial rice hybrids. Therefore, comparative analysis of the present data and those from interracial hybrids, other causes of sterility should certainly be occurred as reported by other investigators. However, Stebbins (1958) speculated that small inversion involving 1-5 genes would be sufficient to cause sterility. Similarly, many investigators obtained 100% sterility in O. sativa/O. officinalis hybrids (Li, 1964; Morinaga, 1964; Jena and Khush, 1986) and reported that complete sterility was due to limited or complete failure of chromosome pairing. However they never analyzed the pachytene stage and their evidence mostly based on the diakinesis and metaphase I. But from this present study, the 100% sterility for both pollen and spikelet in

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O. sativa/O. officinalis hybrids is not due to failure of chromosome pairing. It was solely due to desynapsis of chromosome followed by timing imbalance (Shastry and Rao, 1961) at later stages and a few part by cryptic structural hybridity rather than lack of homology. Thus, the present study supported the results of Shin and Katayama (1979), Shastry et al. (1961) and suggests that earlier generalization should be re-examined. Now summing up the results in most of the intragenomic hybrids, pairing was essentially normal, although, no significant association between normal pairing at pachytene stage and latter stages of meiosis was found. Therefore, based on the present result, sterility could not be accounted for by structural differentiation among AA genome species. The partial sterility might be due to complex action of other causes such as genetic, cytoplasmic or genetic-cytoplasmic, and or environmental.

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SUMMARY AND CONCLUSION

In vitro manipulation for the regeneration of intergenomic hybrids, crossability between species, morphology, pollen and spikelet fertility, and meiotic behavior in three distant hybrids and parental species were studied during 2001-2002 at IAAS, Rampur. For this study four Nepalese wild species of rice (O. nivara, O. rufipogon, O. officinalis, and O. granulata) and nine cultivars of O. sativa. L were used. In vitro manipulations: embryo rescue, callusing, and in vitro fertilization was attempted to overcome both pre and post fertilization barrier through manipulating different media. Among them embryo rescue followed by latest hardening technique was found potential to regenerate the intergenomic rice hybrids. However, embryo rescue technique could not be able to overcome the strong post fertilization barrier. Based on the embryo rescue results, the crossability between O. sativa/O. officinalis was ranged from 02.44%. Strong crossability barrier was found between O. sativa/O. granulata, and hence no hybrids were obtained. On the basis of direct crosses, the crossability of O. sativa with O. nivara and O. rufipogon was varied from 7.58%-51.05%. However, on an average landraces were showed close cross affinity than advanced cultivars. Comparative study of morphology in parents and hybrids showed that wild traits were dominant to cultivated forms. Among parents and hybrids, percent mean pollen and spikelet fertility was varied from 3378.16 and 0-49.93, and 30-91.46 and0-86.49, respectively. However, Manshara/O. nivara hybrid set more seed than its female parents despite of its low pollen fertility. No significant association between percentage of stainable pollen and spikelet fertility was observed. The meiotic behavior at different stages of meiosis in intragenomic hybrid was more or less normal, and the frequency of aberrations were comparable to their respective 110

parents. Only four out of eleven hybrid combinations showed cryptic structural hybridity including loose pairing, inversion, translocation, deletion, duplication, and

heteromorphocity at pachytene and their occurrence varied from 0-22.58% of the cells observed. Similarly, intergenomic hybrid (O. sativa/O. officinalis) showed high abnormality in later stages of meiosis due to desynaptic gene/s present in the genome of O. officinalis, although pachytene was normal in more than 50% cells. Therefore it was inferred that the abnormality in these brought about by desynapsis rather than lack of complete homology between their genome (A and C). Most of the chromosome in these hybrids appeared as univalents (24) at diakinesis and metaphaseI, and laggards and bridges in anaphase I. Based on chiasma study in wild species, O. granulata was found to be more primitive than other species included in this study. It was also known that all the wild species of Nepalese wild rices were diploid (2n = 24). At diakinesis and Metaphase I, no sharp reduction in chiasma frequency was observed except O. sativa/O. officinalis derive hybrids. The mean frequency of ring bivalent was always high and varied from 10.42-11.25 and had >21-<24 chiasmata/cell in most of the hybrids. Other minor irregularities such as formation of univalents, quadrivalents, presence of nucleolar bodies, bridges and laggards, laggards, bridges and fragments and unequal segregation at diakinesistelophase I were comparable to their parents. However, cells with bridges + fragments was quite high in some of the intragenomic hybrids and the percentage of cells having such irregularity varied from 0.44 9.40 and proved the existence of inversion. It was highest in IR64/O. rufipogon (9.40). On an average, based on all the stages of observation, no sharp structural differentiation was found between common wild rice and land races of Nepal. Therefore, partial sterility accounted in hybrids did not associated with chromosomal abnormalities

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except IR64/O. nivara, IR 64/O.rufipogon, Jethobudo/O. nivara, and O. sativa/O. officinalis hybrids. Beside some of the landraces like Pokhreli, Jethobudo, Kalanamak showed high crossability, F1 fertility and meiotic affinity with O. rufipogon and Manshara with O. nivara. Not only pairing efficiency, certain stages of meiosis were also improved and indicated that pairing can be enhanced by genes from wild rice. Therefore, based on crossability, F1 fertility, and meiotic affinity, Pokhreli, Jethobudo, Kalanamak leads the two speculations: either these land races descended directly from this perennial taxa or large amount of genes introgression should be occurred between these landraces and O. rufipogon. Similar case should exist between O. sativa. cv. Manshara with O. nivara. Therefore it was found evident that both annual and perennial forms of common wild rice were the immediate prototype of these and they should originate at the swampy areas of Pokhara valley and Kapilbastu of Nepal. Not only these evidences, these landraces were still frequently co-exist with the population of these common wild rice in the farmers field. Thus, it is better to generalize that both annual and perennial forms of common wild rice, such as found in Nepal, are the prototype of cultivated species. However, this speculation was mainly generalized based on the three mentioned affinity, further study will be needed to generalize the results to full extent in the same materials through recent developed molecular technology.

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RECOMMENDATION

Thorough study should be conducted based on pachytene stage through traditional, as well as molecular techniques in more number of cells as far as possible. The original population of O. nivara and O. rufipogon found in Nepal, particularly population from the foothills, should be studied in both natural as well as artificial environment through better sampling strategies. Also include hybrid swarms between cultivated and these common wild forms as far as possible. Parasexual hybridization should be carried out to produce the hybrids between O. sativa and O. granulata.

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143

Appendix 3.1. Nutritional components of different media employed for embryo culture during study period 2001/002
Amount (mg l 1) Components MgSO4.7H2O KH2PO4 NaH2PO4.H2O NH4NO3 CaCl2.2H2O (NH4)2. SO4 Ca(NO3 ) 2. 4H2 O KNO3 KCl MgSO4 CaHPO4. 2H2 O Micro components FeSO4. 7H2 O H3BO3 MnSO4. 4H2O MnSO4. 5H2O MnSO4. H2O ZnSO4. 7H2O Na2MO4. 2H2O CuSO4. 5H2O CoCl2. 6H2O KI Na2 EDTA. 2H2O Na2 EDTA Sucrose Agar Vitamins Nicotinic acid Thiamine HCl Pyridoxine HCl Myo-inositol Folic acid Glycine Biotin Growth regulators NAA Kinetin Casein hydrolysate pH
a

Whitesa 750 19 80 1.5 5 3 0.01 0.75 2% 0.8% 0.05 0.01 0.01 3 5.8

Nistchsa 185 68 720 950 27.8 25 10 0.25 0.025 0.025 37.3 2% 0.8% 5 0.5 0.5 100 0.5 2 0.05 5.8

SRb 296 179.52 720 352.8 2730 27.8 3. 10 2. 0.25 0.025 0.025 0.75 37.3 6% 0.9% 1 10 1 100 0.2 0.2 250 5.8

MSb 370 170 1650 440 1900 27.8 6.2 22.2 8.6 0.25 0.025 0.025 0.83 37.3 3% 0.8% 0.5 0.5 0.5 100 2 5.8

Bouharmontb 3000 1000 800 500 120 25 5% 0.7% 1 10 1 100 0.2 0.2 5.9

[Whites (1953), Nistchs (1969) ] adopted from Razdan (2001) SR (Ko et al.,1983), Murashige and Skoog (1962), and Bouharmont (1991) media, respectively .

144

Appendix 3.2. Composition of the nutrient solution (Karim and Vlamis, 1962) employed to prepare hardening solution Nutrients (molar concentration)

KNO3 Ca (NO3)2 KH2 PO4 MgSO4 FeSO4

,, ,, ,,

,, ,, ,,

1.77 0.27 0.274 0.131 0.032

ppm ppm ppm ppm ppm

1 ml./ l. 1 ml./ l. 2 ml./ l. 4 ml./ l. 1 ml./ l. 1 ml./ l.

1.112 gm / l

Johnsons Micronutrient solution KCL H3BO3 MnSO4. H2O ZnSO4. 7 H 2O CuSO4. 5 H2 O H2MoO41

0.0096 ppm

H2MoO4 was modified by Na2 MO4. 2H2O

145

Appendix 4.1. Comparative results of embryo culture on five different sterile media

Hybrid combination

No. of embryo Culture

No. of embryos germinated

No. of embryos not germinated

No. of tube attacked by Microorganisms 1 1 1 2 1 2 1 2 1 2 2 -

No. of seedlings died after germination 1 1 3 2 3 2 1 1

No. of seedlings well grown

Bouharmont (1991) IR 64 / O. nivara Manshara / O.officinalis Kalanamak / O. officinalis Kalanamak / O. nivara Kalanamak / O. rufipogon IR 72 / O. granulata IR 72 O. granulata SR (Ko, et al., 1983) IR 64 / O. nivara Manshara / O.officinalis Kalanamak / O. officinalis Kalanamak / O. nivara Kalanamak / O. rufipogon IR 72 / O. granulata IR 72 O. granulata Whites (1953) IR 64 / O. nivara

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

7 6 5 6 5 4 7 7 5 3 4 4 6 2 6 5 5

1 1 3 2 3 3 1 5 1 2 2

5 6 5 6 4 5 6 3 2 3 6 4 4

2 3 Appendix continued..

146

Appendix continues.. Manshara / O.officinalis Kalanamak / O. officinalis Kalanamak / O. nivara Kalanamak / O. rufipogon IR 72 / O. granulata IR 72 O. granulata Nistchs (1969) IR 64 / O. nivara Manshara / O.officinalis Kalanamak / O. officinalis Kalanamak / O. nivara Kalanamak / O. rufipogon IR 72 / O. granulata IR 72 O. granulata MS (1962) IR 64 / O. nivara Manshara / O.officinalis Kalanamak / O. officinalis Kalanamak / O. nivara Kalanamak / O. rufipogon IR 72 / O. granulata IR 72 O. granulata 7 7 7 7 7 7 7 7 7 6 7 7 7 7 7 7 7 7 7 7 7 7 7 4 2 4 3 1 7 7 4 1 3 6 5 3 7 6 7 5 4 7 7 2 7 7 3 4 3 4 6 3 4 4 1 2 4 1 2 3 5 1 1 1 2 3 1 2 2 2 1 2 1 2 1 1 2 2 1 2 1 2 1 2 1 2 1 2 1 4 4 3 3 3 1 1 5 5 4 4 6 2 4 5 7 6 5

147

Appendix 4. 2. Comparative results of different attempts employed to establish hybrids plants in the field from embryo culture regenerated seedlings

Techniques employed

Cross combination and success rate (%)

Total seedlings hardened

Direct

Bouharmont

Iyer and Govilla Present method

transfer (1961) method (1964) method

IR 64/O. nivara Success rate Kalanamak/O. officinalis Success rate Kalanamak/O .nivara Success rate Kalanamak/O. rufipogon Success rate Manshara/O. officinalis Success rate Total Mean success rate

19 12 24 26 11 92

7 0.0 6 0.0 6 0.0 5 0.0 3 0.0 27 0.0

4 25.0 2 0.0 5 20.0 7 0.0 3 0.0 21 9.0

5 60.0 2 100.0 8 50.0 8 75.0 3 66.67 26 70.33

3 100.0 2 100.0 5 80.0 6 100.0 2 100.0 18 96.0

148

Appendix.4.3a. Phenotypic characters relationship between parents and their hybrids Characters Male parent Female parent O. nivara (8) Manshara (1) dark brown 4.9gm long and Fully 4.0cm present(2-3) 80 57cm purple lines dark green with light purple tip just exerted 15.3cm high open dark brown dark purple purple >30 Brown weak red 3.46gm present(1-2) 96 112.14cm pale green green moderately exerted 19cm intermediate dark reddish Yellow white green 25 Female parent Jhinuwa (2) black 4.25gm absent 102 127.43cm pale green green well exerted 23.6 intermediate dark brown white green 15 F1 (1/8) dark brown 4.5gm long and fully 3.78cm present(2-3) 74 76.43cm purple lines dark green with light purple tip Just exerted 15.65cm high open dark brown dark purple purple >30 F1(2/8) Black 4.9gm long and fully 3.38cm present(2-3) 99 119.57cm purple strips dark green with light purple tip just exerted 23.46 high open dark brown dark purple purple >35 Dominance of character of F1(1/8) F1(2/8) 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 I I 8 8 8 2 8 8 I 8 8 8

Apiculus color 200 grain wt. Awn density Awn length Branching culm Heading days Height Internode color Leaf color Panicle exertion Panicle length Panicle shattering Panicle type Seed coat color Stigma color BLSC Culm number

BLSC = basal leaf sheath coloration 149

Appendix. 4.3b. Phenotypic characters relationship between parents and their hybrids Characters Male parent O. nivara (8) Female parent R72(6) Female parent IR64(7) Dominance of character of F1(6 / 8) F1(7/ 8) 8 I 8 8 8 I 8 8 8 8 I 8 8 8 8 8 8 8 8 8 8 8 I I 8 8 2 I 8 2 8 8 8 8

F1 (6 / 8)

F1(7/8)

Apiculus color 200 grain wt. Awn density Awn length Branching culm flowering days Height Internode color Leaf color Panicle exertion Panicle length Panicle shattering Panicle type Seed coat color Stigma color BLSC Culm number

dark brown 4.9gm long and fully 6.01cm present(2-3) 80 57cm purple lines dark green with light purple tip Just exerted 15.3cm high open brown dark purple Purple >25

light yellow 4.36gm 103 64.42cm whitish green green just exerted 21.14cm compact light golden white green 21

light golden 3.91gm minute partly 0.5mm 104 112.28cm light green green

dark brown 4.5gm long and fully 3.19cm present (2-3) 99 74.42 purple lines dark green with purple tip well exerted Just exerted 23.84cm 17.36cm high intermediate open straw yellow dark brown white dark purple green purple 22 >25

dark brown 4.9gm long and fully 3.8cm present(2-3) 100 123cm purple line dark green with purple tip just exerted 16.95 high intermediate dark brown dark purple purple >25

BLSC = basal leaf sheath coloration 150

Appendix.4.3c. Phenotypic characters relationship between parents and their hybrids


Characters Male parent O. rufipogon (9) black long and Fully large deep yellow 4.02. cm present(2-3) 125 procumbent 92.8cm purple lines dark green with light purple tip well exerted 19.04cm compact brown dark purple purple >40 Female parent Pokhreli (5) dark purple small light yellow 112 erect 146.42cm light green green well exerted 22.8cm open GRB white green 16 Female parent Jethobudo (4) dark purple very short, partly small light yellow 0.48cm 109 erect 119cm pale green green well exerted 23.6 compact light golden white green 22 F1 (5/ 9) F1(4/9) black long and fully large deep yellow 3.65cm present (2-3) 118 procumbent 123cm purple lines dark green purple tip well exerted 22.61cm high compact compact dark brown dark purple purple .>30 Dominance of character of F1(1/8) F1(2/8) 9 9 9 9 9 I 9 I 9 9 9 I I 9 I 9 9 9 I 9 9 9 9 9 I 9 I 9 9 9 I I 9 I 9 9 9 I

Apiculus color Awn density Anther size Anther color Awn length Branching culm Heading date Culm angle Height Internode color Leaf color Panicle exertion Panicle length Panicle shattering high Panicle type Seed coat color Stigma color BLSC Culm number

black long and fully large deep yellow 4.07cm present (2-3) 120 procumbent 117.57cm purple lines dark green purple tip well exerted 18.78cm high intermediate dark brown dark purple purple >25

BSLC = basal leaf sheath coloration GRB = golden reddish brown, I = intermediate, H = heterosis

151

Appendix. 4.3d. Phenotypic characters relationship between parents and their hybrids
Characters Male parent O. nivara (8) dark brown long and fully small yellow 6.01cm present (2-3) 80 intermediate 57cm purple lines dark green with light purple tip purple >25 just exerted 15.3cm high open dark brown dark purple Male parent O .rufipogon (9) black long and fully large deep yellow 4.02cm present (2-3) 125 procumbent 110cm purple lines dark green with light purple tip purple >40 well exerted 19.04cm high compact-open brown dark purple Female parent Kalanamak (3) F1 (3/8) black small yellow 109 intermediate 132.72cm pale green with green dot green green 24 well exerted 22.73cm compact black white F1 (3/9) Dominance of character of F1(3/8) F1(3/9) 8 8 I I 8 8 I I I 8 8 8 8 8 I 8 8 8 8 I 9 9 9 9 9 I 9 I 9 9 9 9 I I 9 9 9 9

Apiculus color Awn density Anther size Anther color Awn length Branching of culm Heading days Culm angle Height Internode color Leaf color BLSC Culm number Panicle exertion Panicle length Panicle shattering Panicle type Seed coat color Stigma color

dark brown Black long and long and fully fully small large yellow deep yellow 5.09cm 4.27cm present (2-3) present (2-3) 101 113 intermediate procumbent 82.6cm 122.57cm purple lines light green with purple lines dark green dark green with purple with purple tips tip purple purple >30 >40 just exerted well exerted 19.05cm 15.7cm high high open compact-open dark brown brown dark purple dark purple

BLSC = basal leaf sheath coloration 152

Appendix. 4.3e. Phenotypic characters relationship between parents and their hybrids Characters Male parent Female parent O. nivara (8) Pokhreli (5) Female parent Jethobudo (4) F1 (5/8) F1(4/8) Dominance of character of F1(5/8) F1(4/8) 8 8 8 8 8 I I 8 8 8 I 8 8 8 8 8 8 8 8 8 8 8 I I 8 8 8 I 8 8 8 8 8 8

Apiculus color 200 grain wt. Awn density Awn length Branching of culm Heading days Height Internode color Leaf color Panicle exertion Panicle length Panicle shattering Panicle type Seed coat color Stigma color BLSC Culm number

dark brown 4.9gm long and fully 6.01cm present(2-3) 80 57cm purple lines dark green with light purple tip just exerted 15.3cm high open dark brown dark purple purple >25

dark purple 3.45gm 112 146.42cm light green green well exerted 22.8cm compact golden reddish white green 16

dark purple 3.1gm very short and partly 0.4cm 109 119cm pale green green well exerted 23.6 intermediate light golden white green 22

dark brown 4.0gm long and fully 5.09cm present (2-3) 104 101cm purple lines dark green with purple tips just exerted 20.05cm high open dark brown dark purple purple >25

dark brown 3.98gm long and fully 5.38cm present(2-3) 96 98.57cm purple lines dark green just exerted 19.46 high open dark brown dark purple purple >25

BSLC = basal leaf sheath coloration 153

Appendix. 4.3f. Phenotypic characters relationship between parents and their hybrids Characters Male parent O. rufipogon (9) black long and fully large deep yellow 4.02. cm present(2-3) 125 procumbent 92cm purple lines dark green, purple tip well exerted 19.04cm high compact - open dark brown dark purple purple >40 Female parent IR64 (7) light golden very short and partly small light yellow 0.5mm 104 erect 112.28cm light green green well exerted 23.84cm intermediate straw yellow white green 22 Dominance of character of F1(7/9) 9 9 9 9 9 9 H 9 9 6 9 9 9 9 9 9

F1(7 / 9) black long and fully large deep yellow 3.65cm present (2-3) 115 procumbent 123cm purple lines dark green, purple tip well exerted 22.61cm high open dark brown dark purple purple >35

Apiculus color Awn density Anther Anther color Awn length Branching of culm Heading days Culm angle Height Internode color Leaf color Panicle exertion Panicle length Panicle shattering Panicle type Seed coat color Stigma color BLSC Culm number

BLSC = basal leaf sheath coloration 154

Appendix. 4.3g. Phenotypic characters relationship between parents and their hybrids Characters Male parent O. officinalis (10) black short and partly reddish grey 0.82. cm present(3-4) 110 107.14cm light green with dense dark green dots dark green 27.16cm high open brownish grey dark purple with white tip present brownish green >35 Female parent Kalanamak(3) black light yellow 109 132.72cm pale green with green dots green 22.73cm compact black white green 24 Female parent Manshara (1) brown weak red light yellow 96 112.14cm pale green green 19.00cm intermediate dark reddish yellow white green 25 F1 (3 / 10) black short and partly light yellow 0.38cm present (2-3) 107 130.2 cm dark green with purple lines dark green 19.67cm high open dark purple present BG >35 F1 (1 / 10) black short and partly light yellow 0.37cm present (2-3) 107 123cm dark green with purple lines dark green 22.61cm high open dark purple present BG >35 Dominance of character of F1(1/8) F1(2/8) 10 10 10 10 10 I H 10 I 10 10 10 10 10 10 10 10 10 10 10 10 10 10 I 10 10 I 10 10 10 10 10 10 10 10 10

Apiculus color Awn density Anther color Awn length Branching of culm Heading days Height Internode color Leaf color Panicle length Panicle shattering Panicle type See d coat color Stigma color Under ground stem BLSC Culm number BG = brownish green

155

Appendix. 4.3h. Phenotypic characters relationship between parents and their hybrids Characters Male parent O. officinalis (10) black short and partly light yellow 0.82. cm present(3-4) 110 107.14cm light green with dense dark green dots dark green >35 27.16cm high open dark purple with white tip present BG >35 Female parent Pokhreli (5) dark purple yellow 112 146.42cm light green green 16 22.8cm compact white green 16 Female parent Jhinuwa (2) black yellow 102 127.43cm pale green with dark green dots green 15 23.6cm intermediate white green 15 F1 (5 / 10) F1 (2 / 10) black short and partly light yellow 0.38cm present (2-4) 105 123cm light green with purple lines dark green >35 24.76cm high open dark purple present BG >35 Dominance of character of F1(1/8) F1(2/8) 10 10 10 10 10 I I 10 10 10 I 10 10 10 10 10 10 10 10 10 10 10 I 10 10 10 I 10 10 10 10 10 10

Apiculus color Awn density Anther color Awn length Branching of culm Heading days Height Internode color Leaf color Culm number Panicle length Panicle shattering Panicle type Stigma color Under ground stem BLSC Culm number

light red short and partly light yellow 0.32cm present (2-3) 108 130.2 cm light green with green dots dark green >40 22.98cm high open dark purple present BG >40

BLSC = basal leaf sheath color, BG = brownish green 156

BIOGRAPHICAL SKETCH

The author was born in 1974, August 1 at Wolane V.D.C, Panchthar, East Nepal. He got his primary education partly from Shree Wolane Primary School and partly from Shree Janajyoti Primary School, Damak- 7, nearby his village. He joined his secondary level of education at Shree Manohar Janta M.V., Madhumalla, Morang, but he completed S.L.C. with first division in 1990 from Shree Radhika M.V., Uralabari, Morang, Nepal. He joined IAAS at Rampur, Chitwan and graduated in first division with plant breeding as major subject in 1999. Immediately the author enrolled in 1999 at the IAAS, Rampur to pursue his Master of Science in Agriculture (Plant Breeding) as general student. During MSc he was awarded research assistantship for two years under the CDR/USAID Wheat breeding and Rockefeller Foundation funded Rice Breeding Projects, and he gained valuable knowledge in many aspects of genetics and plant breeding. In addition, he was also actively engaged in volunteer teaching for undergraduate and graduate student on his subject matter. He also presented some experimental findings in a scientific journal. Now he is very interested in molecular biotechnology and cytology, and his future research dream is on apomictic breeding in rice.

Raj Kumar Niroula

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