See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/282462738

APPLICATIONS OF CITRUS SHOOT-TIP GRAFTING IN VITRO

Article  in  Acta horticulturae · January 2015

DOI: 10.17660/ActaHortic.2015.1065.79

CITATIONS READS

8 1,583

3 authors:

José Juárez Pablo Aleza

National University of San Marcos Valencian Institute for Agricultural Research
100 PUBLICATIONS   2,838 CITATIONS    81 PUBLICATIONS   1,175 CITATIONS   

SEE PROFILE SEE PROFILE

Luis Navarro
Valencian Institute for Agricultural Research
273 PUBLICATIONS   6,866 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Development of semisolid systems of topical application, with extracts of antioxidant activity from native species of Peru: Solanum tuberosum and Zea mays L View
project

All content following this page was uploaded by Luis Navarro on 18 January 2017.

The user has requested enhancement of the downloaded file.

 

Applications of Citrus Shoot-Tip Grafting In Vitro

José Juárez, Pablo Aleza and Luis Navarro*
Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigación
Agrarias (IVIA), Moncada, Valencia, Spain
*Corresponding author: lnavarro@ivia.es

Abstract
The main application of shoot-tip grafting in vitro (STG) is to control graft-
transmissible pathogens that requires the use of healthy trees in the new plantings.
The routine application of STG using 0.1-0.2 mm shoot tips is very efficient for
elimination of all citrus graft-transmissible pathogens from local or imported
varieties. It has allowed the recovery of hundreds of healthy cultivars and the
planting of hundreds of millions of healthy certified trees worldwide. Only in Spain
about 140 million certified nursery plants propagated from micrografted plants
have been planted. STG is also a very useful technique for regeneration of elite
genotypes in several research areas. In vitro grafting for these purposes may be done
using larger shoots (up to 1 cm). STG is being routinely used for the following
purposes: (i) Regeneration of somatic hybrids from embryos difficult to germinate;
(ii) Regeneration of plants from irradiated shoots to produce seedless varieties; (iii)
Regeneration of plants from haploid embryos that are very difficult to germinate.
STG was used to regenerate the ‘Clemenules’ haploid plant that has been used by
the International Citrus Genome Consortium to sequence the whole citrus genome;
(iv) Production of stable tetraploid plants of non-apomictic genotypes, which are
very useful for triploid breeding; (v) Regeneration of transgenic plants from shoots
that are very difficult to root in vitro. STG has become a routine application in
citrus genetic transformation.

Keywords: micrografting, certification of nursery plants, regeneration, somatic hybrids,

irradiation, pathogen elimination, quarantine, haploid, tetraploid, genetic
transformation

INTRODUCTION
Citrus graft-transmissible diseases produced by viruses, viroids, some bacteria,
spiroplasmas, and phytoplasmas result in serious economic losses in most citrus growing
regions of the world. They cause decline, loss of vigour and low yields and poor fruit
quality. In addition, they restrict the use of some superior rootstocks susceptible to one or
several diseases. Thus, they may potentially become the main limiting factors of
production. Only preventive measures are efficient to control graft-transmissible
pathogens, such as the utilization of tolerant or resistant germplasm, exclusion of
potential diseases from the citrus area, and establishing new plantings using pathogen-free
high quality nursery trees.
Pathogen-free plants of many cultivars are often not available and it is necessary
to recover healthy plants from infected ones. In this situation, a method able to recover
citrus plant free of all graft-transmissible pathogens and without juvenile characters was
required to produce healthy trees for commercial propagation. Murashige et al. (1972)
were able to recover a few citrus plants by grafting shoot tips from diseased plants on
young rootstock seedlings growing in vitro. Some of these plants were free of the
exocortis viroid and did not have juvenile characters. This procedure was studied in detail
by Navarro et al. (1975), who named it shoot tip grafting in vitro (STG), and developed a
routine procedure that allowed a 30-50% incidence of successful grafts that were
transplanted to soil, with over 95% survival rate. The resulting plants did not have
juvenile characters, and most of them were free of graft-transmissible pathogens.

Proc. XIIth Intl. Citrus Congress 635

Eds.: B. Sabater-Muñoz et al.
Acta Hort. 1065, ISHS 2015
 

STG has allowed the recovery of hundreds of healthy cultivars and the planting of
hundreds of millions of healthy certified trees worldwide. Only in Spain about 140
million certified nursery plants propagated from micrografted plants have been planted.
STG is being routinely used for regeneration of plants from elite genotypes that
otherwise are extremely difficult to regenerate in vitro, as somatic hybrids, plants from
irradiated shoots, haploid and tetraploid plants, and regeneration of transgenic plants from
transgenic shoots difficult to root in vitro. In this paper, the different steps of the
technique of STG are described, and the elimination of pathogens and its research and
industry applications are reviewed.

TECHNIQUE OF SHOOT TIP GRAFTING IN VITRO

The STG technique includes the following steps: rootstock preparation, scion
preparation, grafting, culture in vitro of grafted plants, and transfer to soil (Fig. 1). The
standard procedure was described by Navarro et al. (1975) and Navarro and Juárez
(2007).

Rootstock Preparation
Seedlings produced by seed germination in vitro are used as rootstocks. Seeds are
peeled eliminating the outer and inner seed coats (Fig. 1a), surface sterilized, and cultured
in tubes containing 25 ml of the plant cell culture salt solution of Murashige and Skoog
(1962), with 1% Bacto Agar. Cultures are incubated at 27°C in darkness for two weeks
(Fig. 1b). Seedlings can be stored at 4°C for at least two weeks without any detrimental
effect on grafting success. This allows an easy selection of uniform seedlings for
experimental purposes.
‘Troyer’ or ‘Carrizo’ citranges (Citrus sinensis × Poncirus trifoliata) are the most
commonly used rootstocks for STG. It has the advantage that its trifoliate leaves serve as
a morphological marker to distinguish the shoot produced from the grafted shoot-tip from
the adventitious shoots produced by the rootstock. However, any rootstock which is graft-
compatible with the scion variety could be used for STG.
Under aseptic conditions the seedling is removed from the test tube and it is cut
back, leaving about 1.5 cm of the epicotyl.
The root is shortened to a length of 4-6 cm and the cotyledons and their axillary
buds eliminated (Fig. 1c). The most common method of grafting is to place the shoot tip
in an inverted-T incision, made by a 1-mm-long vertical incision, starting at the point of
decapitation, and a 1-2-mm-wide horizontal cut (Fig. 1i). The cuts are done through the
cortex reaching the cambial area and the flaps of the incision are slightly lifted to expose
the cortex.

Scion Preparation
Shoot tips can be excised from different sources: growing vegetative flushes (Fig.
1d) or dormant buds from field or greenhouse plants and from shoots produced by
budwoods (Fig. 1e) or nodal buds cultured in vitro. Growing vegetative flushes from
greenhouse plants or budwood cultured in vitro are the recommended sources since the
incidence of successful grafts and pathogen elimination was higher in comparison to other
sources (Navarro et al., 1975, 1976).
The most commonly used procedure in our laboratory is to culture budwoods in
vitro at constant 32°C and exposed 16 h daily to 80 μE m-2 s-1 illumination in a culture
medium containing the plant cell culture salt solution of Murashige and Skoog (1962)
solidified with 1.2% Bacto Agar. New flushes are produced in 8-16 days (Fig. 1e) and
used for isolation of shoot tips for STG. This source is routinely used in our laboratory for
plant introduction through the Citrus Quarantine Station (Navarro et al., 1984) and also
for local genotypes, since it is faster than propagation of plants in the greenhouse, and it
gives a better grafting success (Navarro et al., 2002).

636
 

Fig. 1. Standard shoot-tip grafting in vitro technique description. a-c. Rootstock

preparation. d-f. Scion preparation. g-m. Grafting and culture in vitro of grafted
plants. n. Plant recovered by STG.

637
 

Grafting
Flushes 2-3 cm long are used. Larger shoots should not be used to avoid the
abscission stage. Larger leaves are eliminated, cut to about 1 cm long (Fig. 1f), and
surface sterilized. Larger leaf primordia of shoots are removed with the aid of dissecting
tools and a microscope, and shoot tips composed of the apical meristem with three leaf
primordial, measuring 0.1-0.2 mm in length from the cut surface to the tip of the larger
leaf primordia, are excised with a razor blade sliver attached to a surgical handle (Figs. 1g
and h). These small shoot tips do not have vascular connection with the rest of the plant
(Fig. 1g), and this is one of the explanations why most pathogens are eliminated by shoot-
tip grafting. The shoot tip is placed inside the incision of the rootstock with its cut surface
in contact with the cortex exposed by the horizontal cut of the incision (Fig. 1i) made at
the top of decapitated epicotyl (Navarro et al., 1975). Following this procedure in our
laboratory we obtain about 50% successful grafts and more than 95% of the regenerated
plants are free of the pathogens that infected the original plant. The frequency of
successful grafts increases with the size of shoot tip, but the incidence of recovery of
healthy plants decreases (Navarro et al., 1976).

Culture In Vitro of Grafted Plants

Micrografted plants are cultured in a liquid nutrient medium composed of the
plant cell culture salt solution of Murashige and Skoog (1962), modified White's vitamins
and 75 g L-1 sucrose. The medium is distributed into 25×150-mm test tubes in 25-ml
aliquots. A folded paper platform, perforated in its centre for insertion of the root portion
of the rootstock, is placed in the nutrient solution (Fig. 1j). The cultures are kept at
constant 25°C and exposed 16 h daily to 40-50 μE m-2 s-1 illumination.
Histological studies showed that three days after grafting there was already some
callus development between the rootstock and the shoot tip, and five days after grafting
the callus completely developed at the graft union (Fig. 1k). Initiation of vascular
differentiation was observed seven days after grafting, and there was a complete vascular
connection between the shoot tip and the rootstock eleven days after grafting (Li, 1997).
Growing small leaves from the shoot tip coming out from the incision can be
observed about 3-4 weeks after grafting (Fig. 1l) and 4-6 weeks after grafting the
successful grafts already have two to four expanded leaves and they can be transplanted
to soil (Fig. 1m). In the Citrus Variety Improvement Program in Spain (CVIPS) carried
out in our laboratory this standard procedure has been done with more than 600 genotypes
of at least 44 Citrus species, and with species of the citrus-relative genera as Afraegle,
Glycosmis, Poncirus, Fortunella, Clausena, Severinia, Atalantia, Microcitrus, and
Swinglea. It is also possible to get successful grafts of citrus relative species as Murraya
paniculata (L.) Jack, Severinia disticha (Blanco) Swing. and Triphasia trifolia (Burm.
F.) P. Wils. grafted on rootstocks of the same species.

Transplanting to Soil
Scions of successful grafts should have at least two expanded leaves before being
transplanted to soil. This stage is usually reached four to six weeks after grafting (Fig.
1m). Micrografted plants are transferred to pots containing steam-sterilized artificial soil
mix suitable to grow citrus (Fig. 1n). Alternatively, the epicotyl of the micrografted plants
can be regrafted on vigorous seedlings growing in the greenhouse (De Lange 1978). With
both procedures, we usually obtain over 95% survival rate, but growth is faster with the
latter method that is now being used routinely in our laboratory.
Plants recovered by STG do not have juvenile characters if the shoot tips are
excised from adult plants. They usually flower and set fruits within two years after
grafting (Fig. 1n). Several thousand plants have been obtained by STG in different
laboratories, and all available data indicate that they are true to type.

638
 

APPLICATIONS OF SHOOT-TIP GRAFTING IN VITRO

Control of Graft-Transmissible Pathogens

To control diseases produced by graft-transmissible pathogens it is necessary to
use healthy and high quality trees in the new plantings. The production of these trees
requires the establishment of three different but related programs: sanitation, quarantine
and certification (Navarro, 1993). Sanitation programs have the objective to recover
healthy plants from local varieties and quarantine programs have the objective of
importing foreign varieties avoiding introduction of new pests and diseases. STG plays a
key role in these two programs that produce healthy plants for the certification programs,
which have the objective to guarantee that the sanitary status of the initial material is
maintained during the process of commercial propagation at the nurseries. STG is being
used in most citrus growing countries to recover healthy plants. Also the in vitro
quarantine procedure is being legally used in several countries to import citrus genotypes.
In Spain we have used this quarantine procedure to import more than 250 genotypes from
other countries.

Research Applications
STG is becoming a very useful technique for production, propagation and
regeneration of elite genotypes in several research areas. In vitro grafting for this purpose
may be done by using larger shoots (at least up to 1 cm) and different types of incisions
(Figs. 2a-c) with close to 100% grafting success. Following are the description of some
research applications of STG (Fig. 2).
1. Regeneration of Somatic Hybrids. In protoplast fusion experiments, abnormal
embryos are very often produced. These include multiple fasciated cotyledons,
germinating embryos that only produce shoots or roots, or without a good vascular
connection among shoot and root, and abnormal shoot proliferation, among other
alterations (Figs. 2d-e) (Olivares-Fuster, 1988). These embryos do not produce viable
plants that can be established in the greenhouse, thus reducing the efficiency of recovery
of somatic hybrids and loosing potentially valuable genotypes. In our laboratory, we
routinely graft in vitro shoots and roots produced by these embryos to produce plants that
are established in the greenhouse with high efficiency (Figs. 2f-h).
2. Regeneration of Plants from Irradiated Shoots. Irradiation is used in citrus
improvement programs in attempts to reduce the number of seeds produced by high
quality genotypes, particularly with mandarins. However, in many cases unstable
chimeras are produced that revert to the original variety after some cycles of propagation.
In assays to produce seedless clementines, shoot tips were isolated, placed upwards in a
Petri dish with agar media and irradiated. After irradiation, whole plants were recovered
by STG, transplanted to soil and evaluated (Figs. 2i-k). A relatively high number of
apparently stable mutants have been produced and the new variety ‘Nulessín’ with reduce
fertility was selected, protected and released to growers (Asins et al., 2002).
3. Regeneration of Haploid Plants. Haploid plants have interesting applications in citrus
genetics. They can be obtained by in situ parthenogenesis after pollination with irradiated
pollen, which produce aborted seeds that in some cases contain haploid embryos. These
embryos can regenerate into plants after in vitro culture, but haploid plants were
established in the greenhouse with very low efficiency. We have used this approach and
haploid embryos produced abnormal clusters of proliferating tissues that did not allow the
regeneration of plants. However, grafting in vitro some of these shoot-like tissues allowed
us to regenerate several ‘Clemenules’ clementine plants that were successfully established
in the greenhouse, where they even flowered (Figs. 2l-o). One of these haploid plants
regenerated by STG has been used by the International Citrus Genome Consortium to
sequence the whole citrus genome (Aleza et al., 2009a).

639
 

Fig. 2. a-c. Different types of STG in vitro for propagation of elite genotypes. d-f.
Abnormal embryos produced after protoplast fusion experiments and grafted plant
in vitro. g-h. STG from embryos with only root development and recovered from
protoplast fusion. i-j. Regeneration of plants from irradiated shoot tips. k. Original
‘Clemenules’ clementine and selected irradiated ‘Nulessin’ clementine with

640
 

reduced fertility. l-m. Abnormal proliferation of embryos from aborted seeds

produced after in situ parthenogenesis. n-o. Regeneration of haploid ‘Clemenules’
clementine plant selected for whole citrus genome sequencing. p. Transgenic
shoot grafted on an untransformed ‘Troyer’ citrange rootstock. q. ‘Pineapple’
sweet orange fruits from adult control (up) and transformed plants (down). r-u.
Production of stable tetraploid plants of non-apomictic genotypes. r. Window
incision in a rootstock on which a ‘Clemenules’ clementine shoot-tip was
deposited. s. Drop of a colchicine solution applied seven days after micrografting.
t-u. Successful grafts of tetraploid plants.

4. Production of Stable Tetraploid Plants of Apomictic and Non-Apomictic

Genotypes. Tetraploid plants of apomictic and non-apomictic genotypes are very
important to be used as male or female parents in triploid mandarin breeding programs.
However, these types of genotypes are not easily available. We have attempted the
production of stable tetraploid plants by STG in vitro combined with treatment with
colchicine or oryzalin to achieve chromosome doubling and ploidy level analysis by flow
cytometry (Figs. 2r-u) (Aleza et al., 2009b). We were able to recover stable tetraploid
plants of ‘Clemenules’, ‘Fina’ and ‘Marisol’ clementines, ‘Moncada’, ‘Fortune’,
‘Wilking’, ‘Orri’, ‘Temple’ and ‘Encore’ mandarins that are now the most widely used
male and female parents in our triploid breeding program (Navarro et al., 2003; Aleza et
al., 2012).
5. Regeneration of Transgenic Plants. One of the major limitations of citrus
transformation was the difficulty to recover plants from transgenic shoots (Peña et al.,
2008). With many genotypes, rooting efficiency of transgenic shoots is very low and
rooting of shoots from adult material is almost impossible. The use of STG to regenerate
plants from transformed shoots or buds has become a routine procedure in most
laboratories, allowing significant increases of the efficiency of genetic transformation
protocols (Figs. 2p-q).

Literature Cited
Aleza, P., Juárez, J., Hernández, M., Ollitrault, P. and Navarro, L. 2009a. Recovery and
characterization of a Citrus clementina Hort. ex Tan. ‘Clemenules’ haploid plant
selected to establish the reference whole Citrus genome sequence. BMC Plant Biology
9:110, doi:10.1186/1471-2229-9-110.
Aleza, P., Juárez, J., Ollitrault, P. and Navarro, L. 2009b. Production of tetraploid plants
of non apomictic citrus genotypes. Plant Cell Reports 28:1837-1846, DOI
10.1007/s299-009-0783-2.
Aleza, P., Juárez, J., Hernández, M., Ollitrault, P. and Navarro, L. 2012. Implementation
of extensive citrus triploid breeding programs based on 4x × 2x sexual hybridisations.
Tree Genetics and Genomes DOI 10.1007/s11295-012-0515-6.
Asins, M.J., Juárez, J., Pina, J.A., Puchades, J., Carbonell, E.A. and Navarro, L. 2002.
Nulessín, una nueva clementina. Levante Agrícola 359:36-40.
De Lange, J.H. 1978. Shoot tip grafting -a modified procedure. Citrus Subtropical Fruit
Journal 539:13-15.
Li, X. 1997. Cultivo de tejidos in vitro de especies relativas de los cítricos. PhD thesis,
Universidad Politécnica de Valencia, Valencia, Spain.
Murashige, T., Bitters, W.P., Rangan, T.S., Nauer, E.M., Roistacher, C.N. and Holliday,
B.P. 1972. A technique of shoot apex grafting and its utilization towards recovering
virus-free citrus clones. HortScience 7:118-119.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays
with tobacco tissue cultures. Physiolgia Plantarum 15:473-497.
Navarro, L., Roistacher, C.N. and Murashige, T. 1975. Improvement of shoot tip grafting
in vitro for virus-free citrus. J. Am. Soc. Hort. Sci. 100:471-479.

641
  

Navarro, L. 1976. The citrus variety improvement program in Spain. p.198-202. In: E.C.
Calavan E.C. (ed.), Proceedings 7th Conference International Organization Citrus
Virologists. IOCV, Riverside.
Navarro, L., Juarez, J., Pina, J.A. and Ballester, J.F. 1984. The citrus quarantine station in
Spain. p.365-370. In: S.M. Garnsey, L.W. Timmer and J.A. Dodds (eds.), Proceedings
9th Conference International Organization Citrus Virologists. IOCV, Riverside.
Navarro, L. 1993. Citrus sanitation, quarantine and certification programs. p.383-391. In:
P. Moreno, J.V. da Graca and L.W. Timmer (eds.), Proceedings 12th Conference
International Organization Citrus Virologists. IOCV, Riverside.
Navarro, L., Pina, J.A., Juárez, J., Ballester-Olmos, J.F., Arregui, J.M., Ortega, C.,
Navarro, A, Duran-Vila, N., Guerri, J., Moreno, P., Cambra, M. and Zaragoza, S.
2002. The Citrus Variety Improvement Program in Spain in the period 1975-2001.
p.306-316. In: N. Duran-Vila, R.G. Milne and J.V. da Graça (eds.), Proceedings 15th
Conference International Organization Citrus Virologists. IOCV, Riverside.
Navarro, L., Juárez, J., Aleza, P. and Pina, J.A. 2003. Recovery of triploid seedless
mandarin hybrids from 2n x 2n and 2n x 4n crosses by embryo rescue and flow
cytometry. p.541-544. In: I.K. Vasil (ed.), Plant Biotechnology 2002 and Beyond.
Kluwer Acad. Pu., Dordrecht.
Navarro, L. and Juárez, J. 2007. Shoot-tip grafting in vitro. p.353-364. In: I.A. Khan (ed.),
Citrus Genetics, Breeding and Biotechnology. Wallingford: CABI.
Olivares-Fuster, O. 1988. Fusión de protoplastos de cítricos. PhD Thesis, Universidad
Politécnica de Valencia, Valencia, Spain.
Peña, L., Cervera, M., Fagoaga, C., Romero, J., Ballester, A., Soler, N., Pons, E.,
Rodríguez, A., Peris, J., Juárez, J. Navarro, L. 2008. Citrus. p.1-62. In: C. Kole and
T.C. Hall (eds.), Compendium of Transgenic Crop Plants: Vol. 5, Transgenic Tropical
and Subtropical Fruits and Nuts, Blackwell Publishing, Oxford, UK.

642

View publication stats