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Contents

Topic Name

Page No.

Introduction .. 02 Meristem-Definition . 03 Specific features of meristems .. 03-04 Meristematic region .. 04 Characteristics of Meristematic Tissue Functions . 04 Classification of meristems: 1. Types of meristems based on position .. 04-06 2. Types of meristems based on origin and development ... 06-07 3. Types of meristems based on planes of division .. 07-08 Indeterminate growth of meristems .. 08 Role of Hormones in Shoot Apical Meristem Functions ... 08-10 Meristem Tip Culture .... 10-11 Meristem-tip culture for propagation and virus elimination ... 11-12 Advantages of Meristem-tip culture .... 12 Disadvantages of Meristem-tip culture .. 13 Applications of Meristem-tip culture .. 13 Conclusion ... 14 References . 15

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Introduction:
In the early stages of development of the plant embryo all the cells undergo division, but with further growth and development cell division and multiplication become restricted to special parts of the plant which exhibit very little differentiation and in which tissues remain embryonic in character and the cells retain the ability to divide. These embryonic tissues in the mature plant body are called Meristems. Meristematic cells are usually thin-walled, more isodiametric in shape than the cells of mature tissues, and relatively richer in protoplasm.

Fig.: A typical meristematic region

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Meristerm--Definition:
A meristem is a group of undifferentiated plant cells (found at growth tips) which can undergo divisions to form all types of tissues. Generally explant used is shiny dome shaped structure of length less than 0.1mm with one or two pairs of youngest leaf primordia. A meristem is the tissue in most plants consisting of undifferentiated cells (meristematic cells), found in zones of the plant where growth can take place. The meristematic cells give rise to various organs of the plant, and keep the plant growing. The Shoot Apical Meristem (SAM) gives rise to organs like the leaves and flowers. The cells of the apical meristems - SAM and RAM (Root Apical Meristem) - divide rapidly and are considered to be indeterminate, in that they do not possess any defined end fate. In that sense, the meristematic cells are frequently compared to the stem cells in animals that have an analogous behavior and function. The term meristem was first used in 1858 by Karl Wilhelm von Ngeli (1817 1891) in his book Beitrge zur Wissenschaftlichen Botanik. It is derived from the Greek word merizein, meaning to divide, in recognition of its inherent function.

Fig. : Meristematic Tissue

Specific Features of Meristems:

Some specific features of meristematic tissues are given below The term meristem (Greek meristos meaning divisible) emphasizes the cell division activity characteristic of the tissue which bears this name. Meristematic cells are usually isodiametric in shape. They are composed of immature cells which are in a state of division and growth. They have very high metabolic rates. They are compactly set without distinct intracellular spaces. The cell wall, made of cellulose, is thin and homogenous.

Page |4 They are living with dense cytoplasm and one or more prominent nuclei. The plastids are in proplastid stages. The vacuoles in the cells may be quite small or altogether absent. Ergastic matters are absent. It has been stated that meristems are the formative regions where new cells are added to the body. Though in general, meristematic cells possess the features discussed above or before, but departures cant be ruled out. The meristematic cells of vascular cambium are fusiform in shape often with prominent vacuoles. Ergastic matters (tannins and starch) may also be present. Considering these departures some anatomists are in favor of using the term eumeristem for those having the features mentioned before.

Meristematic Region:
Meristematic tissue is found in the following locations:

near tips of roots and stems. This is called apical meristems. in the buds and nodes of stems. in the cambium between the xylem and phloem in dicotyledonous trees and shrubs. under the epidermis of dicotyledonous trees and shrubs (cork cambium). in the pericycle of roots, producing branch roots.

Characteristics of Meristematic Tissue Functions:

Meristematic cells are analogous in function to stem cells in animals, are incompletely or not at all differentiated, and are capable of continued cellular division (youthful). Furthermore, the cells are small and protoplasm fills the cell completely. The vacuoles are extremely small. The cytoplasm does not contain differentiated plastids (chloroplasts or chromoplasts), although they are present in rudimentary form (proplastids). Meristematic cells are packed closely together without intercellular cavities. The cell wall is a very thin primary cell wall. Some of the common characteristic functions of Meristematic tissue are as follows The cells of meristematic tissue are similar in structure & have thin cellulose cell walls. The meristematic cells may be spherical, oval, polygonal or rectangular in shape. The meristematic cells are compactly arranged & do not contain any intercellular space between them. Each meristematic cell contains dense or abundant cytoplasm & a single large nucleus. The meristematic cells contain few vacuoles or no vacuoles at all.

Classification of Meristems:
1. Types of meristems based on position:
There are two types of meristems are present in plants based on position. These are as follows-

i. Apical Meristem: Apical meristems are found at the tips of the stems and roots of
vascular plants. The derivatives of apical meristems differentiate in course of time into permanent tissues which together constitute the primary body. Growth in length of the plant axis is entirely due to their activities (growing points). This meristem generally exhibits a dome shaped structure

Page |5 and clear demarcation in the outer layer (tunica) and the inner mass (corpus). The growth begins with one or more cells situated at the apex of the organ. These cells always maintain their individuality and position and are called apical cells or apical initials. In higher vascular plants these cells occur in groups which may be terminal or sub-terminal in position, whereas in pteridophytes solitary apical cells are found.

Fig.: Apical Meristem

ii. Lateral Meristem: In dicotyledons and gymnosperms, lateral meristems occur laterally in
the axis, parallel to the side of the organs. Such meristems are composed of initials which divide periclinally. The derivatives gradually differentiate into permanent tissues called secondary tissues. These tissues are responsible for increase in thickness. The cambium of the vascular bundles and the phellogen or cork cambium are lateral meristems.

Fig.: (A) diagram to show positions of meristems in the I.s. of a shoot, (B) T.s. of A at the point shown by dotted lines.

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iii. Intercalary Meristem: Intercalary meristems are found in the stems and leafsheaths of
several monocotyledons, particularly in grasses and in the horse-tails. These meristems are portions of apical meristems which are separated from the apex during the growth of the axis and remain intercalated between permanent cells. This meristem is intermodal in its position. This

meristem is ephemeral (short-lived).

Fig.: Intercalary Meristem

2. Types of meristems based on origin and development:

Meristems are classified into three types based on their origin and development. These types are as followsi. Promeristem or Primordial Meristem: Promeristem or primordial meristem is the foundation stage of new growth region. It may be called the earliest embryonic condition consisting of young initials and their derivatives. All the cells possess the characteristics of the true meristematic cells. As soon as the cells begin to show tendency of differentiation, they have passed the earliest promeristematic condition. Promeristem, therefore, is undoubtedly of limited duration and differentiation; as they differentiate into primary meristem later on.

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Fig.: Diagrammatic representation of meristems in a stem and their gradual differentiation in longitudinal view (left) with corresponding transverse views (right).

ii. Primary Meristem: Primary meristems are those that build up the primary part of the plant and consist in part of promeristem. This meristem is composed of cells which are direct descendants of embryonic cells. So, this meristem is also called a later developmental stage. Primary meristems are chiefly located at the tips of root, stem and other appendages. iii. Secondary Meristem: At a later stage of plant body development, the secondary meristems appear. It is always lateral in position, and the cork cambium (phellogen) arising from epidermis, cortical and other cells during secondary increase in thickness, is the example of secondary meristem. Some living permanent cells which regain the power of cell division constitute the secondary meristem, as they originate from permanent cells.

3. Types of meristems based on planes of division:

Meristems are classified into three types based on planes of division. These three types of meristems are given belowi. Plate Meristem: This meristem divides chiefly anticlinally into two planes, so that the new cells are formed but number of layers does not increase. This meristem is found in uniseriate epidermis and multiseriate flat blade of leaf. ii. Mass Meristem: This meristem grows by means of dividing in all planes; consequently the bodies formed are either isodiametric or have no definite shape. This pattern of growth is found in spores, young embryo and endosperm. iii. Rib Meristem: This meristem divides anticlinally to the long axis and gives rise to

Page |8 longitudinal files or rows of cells. Such pattern of division is clearly seen in the development of cortex and pith. This meristem is also called file meristem.

Indeterminate Growth of Meristems:

Though each plant grows according to a certain set of rules, each new root and shoot meristem can go on growing for as long as it is alive. In many plants, meristematic growth is potentially indeterminate, making the overall shape of the plant not determinate in advance. This is the primary growth. Primary growth leads to lengthening of the plant body and organ formation. All plant organs arise ultimately from cell divisions in the apical meristems, followed by cell expansion and differentiation. Primary growth gives rise to the apical part of many plants. The growth of nitrogen fixing nodules on legume plants such as soybean and pea is either determinate or indeterminate. Thus, soybean (or bean and Lotus japonicus) produce determinate nodules (spherical), with a branched vascular system surrounding the central infected zone. Often, Rhizobium infected cells have only small vacuoles. In contrast, nodules on pea, clovers, and Medicago truncatula are indeterminate; to maintain (at least for some time) an active meristem that yields new cells for Rhizobium infection. Thus zones of maturity exist in the nodule. Infected cells usually possess a large vacuole. The plant vascular system is branched and peripheral.

Role of Hormones in Shoot Apical Meristem Function:

Plant organs are produced in meristems in a continuous and predictable but nevertheless flexible manner. Phytohormones and transcription factors cooperate to balance meristem maintenance and organ production. Recent research has provided clues to the mechanisms underlying this cooperation. KNOTTED1-like homeobox (KNOX) and WUSCHEL (WUS) transcription factors facilitate high cytokinin activity in the shoot apical meristem (SAM), whereas high gibberellin and auxin activities promote the initiation of lateral organs at specific sites in the SAM flanks.

Regulators of SAM (Shoot Apical Meristem) function:

Several groups of transcription factors have been shown to take part in SAM maintenance. Class I KNOTTED1-like homeobox (KNOXI) proteins are expressed in many plant species in specific patterns in the SAM. Loss- and gain-of-function mutations have implicated KNOXI proteins in the maintenance of the indeterminate nature of the meristem and in the formation of organ boundaries. In vegetative apices, members of the NAC gene family are expressed in narrow strips in the SAM, which correspond to the future organorgan and meristemorgan boundaries. WUSCHEL (WUS) encodes a homeodomain protein, which is expressed in a defined group of cells below the CZ and is involved in the maintenance of meristem indeterminacy. WUS activities are non-cell autonomous, as this protein is expressed below the CZ but is required to maintain the CZ. Recent studies have revealed some of the relationships between these transcription factors and the hormones cytokinin (CK), gibberellin (GA) and auxin in the SAM. Cytokinins are involved in meristem maintenance: CKs positively regulate cell division. Recent advances in understanding the CKsignaling cascade in Arabidopsis have enabled an assessment of the role of CK in SAM function

Page |9 through the analysis of triple mutants of the three genes encoding CK receptors: Arabidopsis HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4/CRE1/WOODEN LEG (WOL). ahk2 ahk3 ahk4 triple mutants displayed pleiotropic phenotypes including a dramatic reduction in meristem size and leaf-initiation rate, as well as impaired leaf development.

Fig.: Interactions between hormones and transcription factors in the shoot apical meristem (SAM). Top row: the expression patterns of the Arabidopsis genes STM (a KNOXI gene), CUC and WUS are shown on a schematically drawn SAM. Middle row: predicted distribution of auxin, gibberellin (GA) and cytokinin (CK).

Arabidopsis type A response regulators (ARRs) are CKinduced negative regulators

of CK signaling. Several ARRs, including ARR5 and ARR7, have recently been shown to be negatively regulated by WUS and positively regulated by CLV3 in Arabidopsis. It might appear contradictory that STM activation results in an increase in ARR5 expression, whereas WUS activation downregulates its expression. However, the induction of ARR5 by STM is secondary to its effect on CK accumulation, and the effect of WUS is direct. It thus emerges that meristem maintenance requires CK action. However, CK function in the SAM does not seem to follow an all-or-none scenario, as CK is also involved in leaf development. Rather, local gradients of CK content and response might be facilitated by the very specific expression patterns of the KNOXI and WUS transcription factors, the ARR7 and ABPH1 response regulators, and additional yet-unknown factors, resulting in a range of differential CK effects. Gibberellin production and activity are downregulated in the SAM: GA affects numerous developmental processes in plants, including leaf initiation and morphogenesis. It has emerged that tight spatial restriction of GA accumulation is crucial for

P a g e | 10 the specification of the boundary between the meristem and the incipient leaf primordium. KNOXI proteins have been shown to negatively regulate GA biosynthesis in several plant species, through direct transcriptional repression of the GA-biosynthesis gene GA 20-oxidase. The combination of reduced CK and increased GA in the meristem, achieved by constitutive mis-expression of CKX in the background of the spy mutant (which displays constitutive GA signaling), resulted in a range of phenotypes similar to those of strong stm alleles, including meristem abortion and fused cotyledons. Thus, KNOXI mediated meristem maintenance probably involves a delicate balance of the levels of several hormones. In addition to the opposite effects of KNOXI proteins on CK and GA biosynthesis, these hormones have recently been shown to negatively affect each others activity. CK responses were enhanced by the activity of the GA negative regulator SPINDLY (SPY). Thus, CK and GA activities are antagonistic, further refining local gradients and boundaries. Auxin is a positive regulator of leaf initiation: Another hormone classically implicated in antagonistic interactions with CK is auxin. In recent years, there have been some major breakthroughs in our understanding of the auxin response. Auxin is emerging as a major coordinator of plant development, starting as early as the initial stages of embryo patterning. Several recent studies have suggested that high relative auxin concentrations in the P0 region downregulate the expression of CUC and KNOXI genes, allowing primordium initiation. Analysis of the dynamics of gene expression in the Arabidopsis inflorescence meristem during the development of lateral primordia suggested that domains of auxin maxima are nearly complementary to the domains of STM and CUC2 expression. High auxin concentrations in P0 seem to facilitate lateral organ initiation through downregulation of CUC and KNOXI. Conversely, some evidence suggests that KNOXI proteins might also inhibit auxin transport, indicating a possible feedback relationship between auxin and KNOXI proteins. NAC1 is involved in auxin-mediated lateral root development. NAC1 expression was rapidly induced by auxin and downregulated after a longer auxin application, possibly through a desensitization mechanism. Auxin might similarly downregulate CUC levels in initiating lateral primordia by inducing miR164. Auxin application induces primordium initiation only in the PZ and not in the CZ. Indeed, although auxin was recently shown to accumulate in the CZ of the meristem, lateral organ primordia only initiate from the PZ. Thus, additional factors are likely to control auxin receptivity with respect to lateral organ initiation. A natural candidate is CK. Low CK:Auxin ratios, rather than absolute auxin concentrations, might be necessary for the specification of P0. Ethylene: Ethylene signaling has been shown to act antagonistically with the Arabidopsis KNOXI gene KNAT2 in the SAM. A constitutive triple response1 (ctr1) mutant, with constitutive ethylene response, showed disrupted SAM structure and reduced KNAT2 expression. Application of an ethylene precursor had a similar effect, which was restored by the induction of KNAT2 activity.

Meristem Tip Culture:

In vitro culture of a generally shiny, dome-like structure measuring less than 0.1 mm in length when excised, most often excised from the shoot apex. The essence of meristem-tip

P a g e | 11 culture is the excision of the organized apex of the shoot from a selected donor plant for subsequent in vitro culture.

Meristem-Tip Culture for Propagation and Virus Elimination:

The conditions of meristem-tip culture are regulated to allow only for organized outgrowth of the apex directly into a shoot, without the intervention of any adventitious organs. The excised meristem tip is typically small (often Cl mm in length) and is removed by sterile dissection under the microscope. The explant comprises the apical dome and a limited number of the youngest leaf primordia, and excludes any differentiated provascular or vascular tissues. A major advantage of working with such a small explant is the potential that this holds for excluding pathogenic organisms that may have been present in the donor plants from the in vitro culture. A second advantage 1s the genetic stability inherent m the technique, since plantlet production 1s from an already differentiated apical meristem and propagation from adventitious meristems can be avoided. Shoot development directly from the meristem avoids callus tissue formation and adventitious organogenesis, ensuring that genetic instability and somaclonal variation are minimized. If there is no requirement for virus elimination, then the less demanding, related technique of shoot-tip culture may be more expedient for plant propagation. In this related procedure the explant is still a dissected shoot apex, but a much larger one that is easier to remove and contains a relatively large number of developing leaf primordia. Typically, this explant is between 3 and 20 mm in length, and development in vitro can still be regulated to allow for direct outgrowth of the organized apex. The axillary buds of in vitro plantlets derived from meristem-tip culture may also be used as a secondary propagule. This technique adds a high propagation rate to the original meristem-tip culture technique, and together the techniques form the basis of micropropagation, which is so important to the horticulture industry. It is possible; however, that callus tissue may develop on certain portions of the growing explant, particularly at the surface damaged by excision . The only acceptable situation under such circumstances is where the callus is slow-growing and localized, and the callus mass and any organized development on it can be excised at the first available opportunity. A further advantage of meristem culture is that the technique preserves the precise arrangement of cell layers necessary if a chimera1 genetic structure is to be maintained. The technique of meristem culture may be exploited m situations where the donor plant is infected with viral, bacterial, or fungal pathogens; whether or not symptoms of the infection are expressed the basis of eradication is that the terminal region of the shoot meristem, above the zone of vascular differentiation, is unlikely to contain pathogenic particles. If a sufficiently small explant can be taken from an infected donor and raised in vitro, then there is a real possibility of the derived culture being pathogen-free. Such cultures, once screened and certified, can form the basis of a guaranteed disease-free stock for further propagation. The meristem-tip technique can be linked with heat therapy to improve the efficacy of disease elimination, or antiviral, chemotherapeutic agents may be investigated. Whatever variants of technique are employed for virus eradication, the key to success is undoubtedly the size of the explant. The smallest explants are those that typically, will be the least successful during in vitro culture, but will produce the highest proportion of virus-free material when entire plants are reared in the glasshouse or field. If meristem culture alone is not successful m producing any virus-free plants, then temperature stress treatment of donor plants and/or the use of antiviral agents will have to be considered. Heat therapy relies on the growth of the donor plants at elevated temperatures,

P a g e | 12 typically 30-37C and may involve a treatment of several weeks. It is useful to note that extended, low-temperature treatment of donor material may also be effective (14). In attempts to eliminate hop latent viroid, the low-temperature (2--4C) treatment of parent plants was only effective. Antiviral chemicals can be used as additives in the culture medium, and one of the most widely used is ribavirin, also known as virazole. Increasing concentrations of ribavirin and increasing length of culture incubation m the presence of the compound typically increase the effectiveness of virus elimination, but slowed growth and phytotoxity may be evident at high concentrations.

Fig.: Strategies for obtaining virus-free plants by meristem culture

Advantages of Meristem-Tip Culture:

The major advantages of meristem culture are that it provides: 1. Clonal propagation in vitro with maximal genetic stability; 2. The potential for removal of viral, bacterial, and fungal pathogens from donor plants; 3. The meristem tip as a practical propagule for cryopreservation and other techniques of culture storage, 4. A technique for accurate micropropagation of chimeric material; and 5. Cultures that is often acceptable for international transport with respect to quarantine regulations. 6. A technique for production of virus free germplasm. 7. A technique for mass production of desirable genotypes.

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Disadvantages of Meristem-Tip Culture:

There have some disadvantages of using meristem-tip culture for propagation and for the production of virus free plants. These disadvantages are as follows1. The cost and availability of man power for this technique can be a limiting factor. The type of condition and the skill of the labor force have a marked effect on this output. 2. The small plantlets produced by this tissue culture method are obviously more difficult to handle than the conventional material. 3. A number of handling stages are required and all of these operations are time consuming. 4. During culture, the removal of meristem causes temporary set-back in the growth of the mother plants.

Applications of Meristem Culture:

Some of the common applications of meristem culture are given below Virus and parasite elimination The resulting plantlets are often free of viruses and parasites. Meristems are devoid of viruses. There are certain reasons behind this. Vascular system is absent in meristem. Vascular system has prime importance in the travelling of viruses through the body. Meristematic cells are actively dividing and have high metabolic activity. These factors have negative impact on virus multiplication. Shoot apex has high endogenous auxin level, which act as inhibitory factor for virus multiplication. Used in propagation of haploid plants. Storing genetic resources of seed producing plants of heterozygous nature. Used in micropropagation

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Conclusion:
Meristem is a formative plant tissue made up of small cells capable of dividing indefinitely and giving rise to similar cells or to cells that differentiate to produce the definitive tissues and organs; an undifferentiated cellular region in plants characterized by repeated cell division. Meristematic cells are analogous in function to stem cells in animals, are incompletely or not at all differentiated, and are capable of continued cellular division (youthful). Meristem tip from different region of a plant (i.e. shoot etc.) are used to do meristem-tip culture for the production of virus free plant and for propagation. There are large number advantages of using meristem as explant for the production of disease free and excellent quality of plants. Meristems are also used for the production of haploid plants. Meristems are better than other region and organs of plants for tissue culture technique due to its rich hormonal concentrations and metabolic activity. But there are also have some disadvantages. In spite of these disadvantages, meristems are widely used in plant tissue culture for the production of economically beneficial and high qualified plant materials via tissue culture techniques.

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References:
i. ii. iii. iv. v. vi. vii. viii. ix. x. xi. xii. xiii. xiv. xv. xvi. Introduction to Plant Tissue Culture- M. K. Razdan Competition Science Vision- Mahendra Jain Introduction to Plant Biotechnology- H. S. Chawla Wikipedia, http://en.wikipedia.org/wiki/Tissue_(biology)#Meristematic_tissues theagricos, http://theagricos.com/tissue-culture/tissue-culture-techniques/ Molecular-plant-biotechnology,http://www.molecular-plant-biotechnology.info/plantbiotechnology/ Eilon Shani, Osnat Yanai and Naomi Ori: The role of hormones in shoot apical meristem function. Current Opinion in Plant Biology 2006, 9:484489. Scribd, http://www.scribd.com/doc/48266162/Meristem-culture/ Tutorvista, http://www.tutorvista.com/biology/meristem-tissue-culture/ http://vannocke.hrt.msu.edu/plb865/Meristem%20dynamics/meristems.html http://www-plb.ucdavis.edu/labs/rost/rice/stems/meristem.html Le Bui Van: PartII- Plant Tissue Culture (Overview). Plant Biotechnology, Vietnam OpenCourseWare, April 2009. Brian W. W. Grout: Meristem-Tip Culture for Propagation and Virus Elimination. Methods in Molecular Biology, Vol. 7 11 Plant Cell Culture Protocols. Hu, C. Y. and Wang, P. J (1984) Meristem, shoot-tip and bud culture, in Handbook of Plant Cell Culture (Evans, D. A., Sharp, W R , Ammirato, P V , and Yamada, Y., eds ), Macmillan, New York, pp. 177-277. http://www.botany.uwc.ac.za/sci_ed/grade10/plant_tissues/meristematic.htm http://www.daylilies.org/ahs_dictionary/meristem.html