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Plant Tissue Culture : A Promising Tool Of Quality Material Production With Special Reference To Micropropagation Of Banana

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INTRODUCTION

The inception of science of plant tissue culture takes its roots from the discovery of cell followed by propounding of cell theory. In 1838, Schleiden and Schwann proposed that cell is the basic structural unit of all living organisms. They visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plant. Based on this premise, in 1902, a German physiologist, Gottlieb Haberlandt for the first time attempted to culture isolated single palisade cells from leaves in knop’s salt solution enriched with sucrose. The cells remained alive for up to one month, increased in size, accumulated starch but failed to divide. Though he was unsuccessful but laid down the foundation of tissue culture technology for which he is regarded as the “father” of plant tissue culture. Plant tissue culture is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions often to produce the clones of plants (Thorpe, 2007). The resultant clones are true-to type of the selected genotype. The controlled conditions provide the culture an environment conducive for their growth and multiplication. These conditions include proper supply of nutrients, pH medium, adequate temperature and proper gaseous and liquid environment. Plant tissue culture technology is being widely used for large scale plant multiplication. Apart from their use as a tool of research, plant tissue culture techniques have in recent years, played an important role in the area of plant propagation, disease elimination, plant improvement and production of secondary metabolites, using small pieces of tissue (named explants) to produce hundreds and thousands of plants in a continuous process. A single explant can be multiplied into several thousand plants in relatively short time period and space under controlled conditions, irrespective of the season and weather on a year round basis (Akin-Idowu et al, 2009). Endangered, threatened and rare species have successfully been grown and conserved by micropropagation because of high coefficient of multiplication within less space. In addition, it is also considered to be the most viable technology for crop improvement by the production of somaclonal and gametoclonal variants. The micropropagation technology has a vast potential to produce plants of superior quality, isolation of useful variants in well-adapted high yielding genotypes with better disease resistance and stress tolerance capacities (Brown and Thorpe, 1995; Suman et al, 2012). Certain type of callus cultures give rise to clones that have inheritable characteristics different from those of parent plants due to the possibility of occurrence of somaclonal variability

(George, 1993; Suman et al, 2015) which leads to the development of commercially important improved varieties. Commercial production of plants through micropropagation techniques has several advantages over the traditional methods of propagation through seed, cutting, grafting and air-layering etc. It is rapid propagation processes that can lead to the production of virus free plants (Garcia-Gonzales et al, 2010). Meristem tip culture of banana plants devoid from banana bunchy top virus (BBTV) and brome mosaic virus (BMV) were produced (El-Dougdoug and El-Shamy, 2011). Higher yields have been obtained by culturing pathogen free germplasm in vitro. Increase in yield up to 150% of virus-free potatoes was obtained in controlled conditions (Singh, 1992).

Agricultural diversification to meet our future needs call for the adoption of new technologies in agriculture. Utilization of the best cultural practices, fertilization, pest control measures will not give the necessary results without the use of best planting material. Now a day, tissue culture is adopted as a viable horticultural propagation method which has revolutionized the horticultural industry. Mass propagation and the establishment of disease free stock material is accomplished by use of this technique (Bachraz, 1998). Tissue culture is a method of vegetative propagation based on biotechnology. The plants are derived from stem, root or leaf tissues and the technology generally aids in mass production of desired crop varieties. Tissue culture is also useful in regeneration of genetically modified cells into whole plants as well as in embryo rescue techniques (Bio Vision, 2008). Ilan and Workafes (2011) have put on record the advantages and disadvantage of this method as that controlled environment and controlled development of the plants enable very rapid multiplication rate and clean conditions for plant development that produce micro-plants free of many pests and diseases. The small size of the propagated plants saves nursery space and plant transport costs. Reproducing the planting materials of vegetatively propagated crops presents complex problems because of lack of knowledge of phyto-sanitary measures and quarantine issues related to safe movement of germplasm, plants and planting material across national borders, lack of consistent supplies of good quality planting material, variable demand for clean planting material, bulkiness and perishability of planting materials and use of traditional varietal mixtures, including local varieties (Wambugu and Kiome, 2001). The prime basic objective of this review is to describe the tissue culture techniques, various developments, present and future trends and its application in various fields with particular reference of banana.

TECHNOLOGICAL INNOVATIONS IN PLANT TISSUE CULTURE

Know-How

In the process of plant cell culture, plant tissues and organs are grown in vitro on artificial media, under aseptic and controlled environment. The technique depends mainly on the concept of totipotentiality of plant cells which refers to the ability of a single cell to express the full genome by cell division (Haberlandt, 1902). Along with the totipotent potential of plant cell, the capacity of cells to alter their metabolism, growth and development is also equally important and crucial to regenerate the entire plant (Thorpe, 2007). Plant tissue culture medium contains all the nutrients required for the normal growth and development of plants. It is mainly composed of macronutrients, micronutrients, vitamins, other organic components, plant growth regulators, carbon source and some gelling agents in case of solid medium (Murashige and Skoog, 1962). Murashige and Skoog medium (MS medium) is most extensively used for the vegetative propagation of many plant species in vitro. The pH of the media is also important that affects both the growth of plants and activity of plant growth regulators. It is adjusted to the value between 5.4 – 5.8. Both the solid and liquid medium can be used for culturing. The composition of the medium, particularly the plant hormones and the nitrogen source has profound effects on the response of the initial explant. Plant growth regulators (PGRs) play an essential role in determining the development pathway of plant cells and tissues in culture medium. The auxins, cytokinins and gibberellins are most commonly used plant growth regulators. The type and the concentration of hormones used depend mainly on the species of the plant, the tissue or organ cultured and the objective of the experiment (Ting, 1982). The high concentration of auxins generally favors root formation, whereas the high concentration of cytokinins promotes shoot regeneration. A balance of both auxin and cytokinin leads to the development of mass of undifferentiated cells known as callus.

The propagation from meristematic tissue generally provides a method of cleaning up material from viruses and other systemic pathogen infections. Micropropagation is a tissue culture (in vitro) method used for rapid and true to type multiplication of plants on artificial nutrient media under controlled environment and it is the most commercially exploited area of plant tissue culture, being widely used for production of quality planting material in vegetative propagated species. The most significant advantages offered by micropropagation are production of large of disease free propagules from a single plant in

a short period, propagation can be carried out throughout the year and the propagating material can be accommodated in a small space, reduction of labour costs for germplasm maintenance, avoidance of field inspections & environmental hazards, easy availability of material for micro propagation and rapid multiplication (Mtui, 2011). According to Chadha and Choudhary (2010) various prerequisite steps involved in production of pathogen free plantlets by meristem tip culture are: testing of parent material for the presence of viruses and similar pathogens (viroids and phytoplasmas), thermotherapy/chemotherapy of parent material if disease-free material is not available, excision of meristem tip under aseptic conditions, culture of apical dome plus one or two leaf primordia on suitable medium to produce plantlets, indexing of plantlets for presence or absence of viruses, plantlets transferred to soil, maintenance of pathogen free nuclear plant stocks and meristem culture is then followed by in vitro mass propagation of the virus-free plants thus obtained.

Technological Protocols

In recent past very fast developments have been taken placed in the area of plant cell culture-cum- microprogation with view of its viable commercial applications. For the purpose of micropropagation of planting materials the procedure of it starts with the selection of plant tissues (explant) from a disease free healthy, vigorous mother plant (Murashige, 1974). Any part of the plant (leaf, apical meristem, bud and root) can be used as explant. All the steps can be summarized into the following stages as shown in Figure 1 as documented by Hussain et at (2012).

method of propagation.

RECENT ADVANCES IN PLANT TISSUE CULTURE

Processes of somatic embryogenesis and organogenesis are the recent development in the area application of plant tissue culture by which plant regeneration takes place.

Somatic Embryogenesis

It is an in vitro method of plant regeneration widely used as an important biotechnological tool for sustained clonal propagation (Park et al, 1998). It is a process by which somatic cells or tissues develop into differentiated embryos. These somatic embryos can develop into whole plants without undergoing the process of sexual fertilization as done by zygotic embryos. The somatic embryogenesis can be initiated directly from the explants or indirectly by the establishment of mass of unorganized cells named callus (Suman and Kumar, 2016). Plant regeneration via somatic embryogenesis occurs by the induction of embryogenic cultures from zygotic seed, leaf or stem segment and further multiplication of embryos. Mature embryos are then cultured for germination and plantlet development, and finally transferred to soil. Somatic embryogenesis has been reported in many plants including trees and ornamental plants of different families. There are various factors that affect the induction and development of somatic embryos in cultured cells. A highly efficient protocol has been reported for somatic embryogenesis on grapevine (Jayasankar et al, 1999) that showed higher plant regeneration sufficiently when the tissues were cultured in liquid medium. Plant growth regulators play an important role in the regeneration and

proliferation of somatic embryos. Highest efficiency of embryonic callus was induced by culturing nodal stem segments of rose hybrids on medium supplemented with various PGRs alone or in combination (Xiangqian et al, 2002). The embryonic callus showed high germination rate of somatic embryos when grown on abscisic acid (ABA) alone. Somatic embryogenesis is not only a process of regenerating the plants for mass propagation but also regarded as a valuable tool for genetic manipulation. The process can also be used to develop the plants that are resistant to various kinds of stresses (Bouquet and Terregosa, 2003) and to introduce the genes by genetic transformation (Maynard et al, 2003). A successful protocol has been developed by using this tool for regeneration of cotton cultivars with resistance to Fusarium and Verticillium wilts (Han et al, 2009).

Organogenesis

It refers to the production of plant organs i.e. roots, shoots and leaves that may arise directly from the meristem or indirectly from the undifferentiated cell masses (callus). Plant regeneration via organogenesis involves the callus production and differentiation of adventitious meristems into organs by altering the concentration of plant growth hormones in nutrient medium. Skoog and Miller (1957) were the first who demonstrated that high ratio of cytokinin to auxin stimulated the formation of shoots in tobacco callus while high auxin to cytokinin ratio induced root regeneration.

  1. CRITICAL FACTORS INFLUENCING IN VITRO GROWTH

Choice of explant

The tissue which is obtained from the plant to culture is called an explant. Based on work with certain model systems, particularly tobacco, it has often been claimed that a totipotent explant can be grown from any part of the plant. In many species, explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. Thus it is very important that an appropriate choice of explant be made prior to tissue culture. The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the

Fig. 1 : Step-wise summary of micropropagation via tissue culture.

Fig. 2 : Steps involved in hybrid plant production via protoplast fusion.

stem tip, auxiliary bud tip and root tip because these tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins. Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil micro-flora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots, a circumstance that then allows microbial attack. These associated microfloras will generally overgrow the tissue culture medium before there is significant growth of plant tissue. Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing and the remainder usually can be killed by surface sterilization. Most of the surface microflora does not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant. An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.

Explant Size and Thin Section Culture System

The induction of a desired morphogenic event in vegetative tissues by appropriate in vitro manipulations would probably be the most significant advancement in plant tissue culture. The success in achieving such directed morphogenic events are largely determined by the cultured tissue itself. Several explant-related factors appear to influence the organogenic potential of the cultured tissue (Benson, 2000). These include growth conditions, whole plant physiology and genotype of the source plant. In addition, a negative correlation between the explants size and the number of cells potentially available for organogenesis has also been recognized (Lakshmanan et al, 1995 & 1996, ). In an earlier study, Lakshmanan et al (1995) have shown that the production of orchid protocorms in vitro can be substantially improved by manipulating the size of the explant alone. For example, the number of protocorms produced by thin transverse sections (0.6 mm thick) derived from a single shoot tip (6-7 mm long) was 5 times greater than that produced by an intact shoot tip (6-7 mm long) cultured under identical conditions. A similar observation was also made recently in sugarcane. In this crop, leaf explants

produced numerous plants (> 50 per explant) when the thickness of the explant was reduced to 1 to 2 mm. This finding clearly indicates that the explant size plays a key role in the expression of organogenic potential of the cultured tissue. This explants size-based difference in organogenic capacity has since been successfully utilized to develop thin section culture system, a novel approach in plant regeneration

4.3 In vitro Environmental Conditions

Some interesting points of basic research that could improve our understanding and hence our ability to control in vitro plant regeneration and development remain under- explored such as by recirculating the liquid culture systems it will be feasible to monitor and continuously regulate the medium composition. We need to know more about the dynamics of mineral nutrition in vitro (Williams, 1995). Light quality is one of the potentially important environmental factors, as it has been shown to affect the direction of plant morphogenesis in vitro (Morini et al, 2000) and it also plays role in between gametophytic and sporophytic pathways. Tissue culture processes often involves extensive cutting and stress injury of tissues which may cause physiological changes in plants affecting the success response (Leon et al, 2001).

SCIENTIFIC-CUM-COMMERCIAL APPLICATIONS OF PLANT TISSUE CULTURE

TECHNOLOGY

Today plant tissue culture applications encompass much more than clonal propagation and micropropagation. The range of routine technologies has expanded to include somatic embryogenesis, somatic hybridization, virus elimination as well as the application of bioreactors to mass propagation and production of secondary metabolites. The applications of plant tissue culture may be categorized into two areas of common interest:

In Agricultural Science

Plant tissue culture is used widely in plant science with its commercial applications as- screening cells rather than plants for advantageous characters, e.g. herbicide resistance/ tolerance; large scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals; to cross distantly related species by protoplast fusion and regeneration of the novel hybrid; embryo rescue (the resulting embryo as a result of cross pollination which would otherwise normally die is cultured in a medium to rescue it); for production of doubled monoploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programs, usually being done by treatment with colchicine which

causes doubling of the chromosome number; as a tissue for transformation, followed by either short term testing of genetic constructs or regeneration of transgenic plants; in vitro conservation of germplasm and so on. This technique is mainly used to conserve plant which do not produce seeds or which have recalcitrant seeds which cannot be stored under normal storage conditions in seed gene banks. Hence, vegetative propagated crops such as root and tubers, ornamentals, medicinal plants and many other tropical fruits have to be conserved by using tissue culture methods (Singh and Shetty, 2011).

Tissue culture has been introduced into agricultural practice at a rate without precedent which allows the production and propagation of genetically homogeneous, disease-free plant material by micropropagation methodology (Chatenet et al, 2001). Cell and tissue in vitro culture is a useful tool for the induction of somaclonal variation (Marino and Battistini, 1990). Genetic variability induced by tissue culture could be used as a source of variability to obtain new stable genotypes. Interventions of biotechnological approaches in in vitro regeneration, mass micropropagation techniques and gene transfer studies in tree species have been encouraging. In vitro cultures of mature and/or immature zygotic embryos are applied to recover plants obtained from inter- generic crosses that do not produce fertile seeds (Ahmadi et al, 2010). Genetic engineering can make possible a number of improved crop varieties with high yield potential and resistance against pests. Genetic transformation technology relies on the technical aspects of plant tissue culture and molecular biology for production of improved crop varieties, production of disease (virus)-free plants, genetic transformation, production of secondary metabolites, and production of varieties tolerant to salinity, drought and heat stresses and so on. Details of some prime applications of tissue culture in agricultural development are given as-

(sterile plants) or which have ‘recalcitrant’ seeds that cannot be stored for long period of time can successfully be preserved via in vitro techniques for the maintenance of gene banks. Cryopreservation plays a vital role in the long-term in vitro conservation of essential biological material and genetic resources. It involves the storage of in vitro cells or tissues in liquid nitrogen that results in cryo-injury on the exposure of tissues to physical and chemical stresses. Successful cryopreservation is often ascertained by cell and tissue survival and the ability to re-grow or regenerate into complete plants or form new colonies (Harding, 2004). It is desirable to assess the genetic integrity of recovered germplasm to determine whether it is ‘true-to-type’ following cryopreservation (Day, 2004). The fidelity of recovered plants can be assessed at phenotypic, histological, cytological, biochemical and molecular levels, although, there are advantages and limitations of the various approaches used to assess genetic stability (Harding et al, 2005; Suman et al, 2015). Cryobionomics is a new approach to study genetic stability in the cryopreserved plant materials Harding, 2010). The embryonic tissues produced by tissue culture are cryopreserved for future use or for germplasm conservation (Corredoira et al, 2004).

Table 1 : Some of secondary metabolites produced in plant cell suspension culture and being used in pharmaceutical industries.

Secondary metabolite

Plant name

Reference

Vasine

Adhatoda vasica

Shalaka and Sandhya (2009)

Artemisinin

Artemisia annua

Baldi and Dixit (2008)

Azadirachtin

Azadirachta indica

Sujanya et al (2008)

Cathin

Brucea javanica

Wagiah et al (2008)

Capsiacin

Capsicum annum

Umamaheswai and Lalitha (2007)

Sennosides

Cassia senna

Shrivastava et al (2006)

Ajmalicine

Catharanthus roseus

Zhao et al (2001)

Secologanin

  

Contin et al (1999),

Indole alkaloids

  

Moreno et al (1993)

Vincristine

  

Lee-Parsons and Rogce (2006)

Stilbenes

Cayratia trifoliata

Roat and Ramawat (2009)

Berberin

Coscinium fenustratum

Khan et al (2008)

Sterols

Hyssopus officinalis

Skrzypek and Wysokinsku (2003)

Shikonin

Lithospermum erythrorhizon

Tabata and Fujita (1985)

Ginseng saponin

Panax notoginseng

Zhong et al (1999)

Podophyllotoxin

Podophyllum hexandrum

Chattopadhyay et al (2002)

Taxane Paclitaxel

Taxus chinensis

Wang et al (1999)

of natural population is not feasible and viable too.

In the recent years, in vitro protoplast fusion tool has opened a way of developing unique hybrid plants by overcoming the barriers of sexual incompatibility. It has been applicable in horticultural industry to create new hybrids with increased fruit yield and better resistance to diseases. Successful viable hybrid plants were obtained when protoplasts from citrus were fused with other related Citrinae species (Motomura et al, 1997). The potential of somatic hybridization in important crop plants is best illustrated by the production of intergeneric hybrid plants among the members of Brassicaceae (Toriyama, 1987). In wheat crop for the purpose of gene pool recovery and improvement by resolving the problem of loss of chromosomes and decreased regeneration capacity,

Fig. 3 : Shoot tip culture for banana micropropagation: a. sword sucker and explant; b. shooting after apical disabling; c. proliferation; d. multiple shooting; e. rooting; f. nursery hardening (Source: Singh et al (2011).

successful protocol of protoplasm fusion has been established and used for the production of somatic hybrid plants by using two types of wheat protoplast as recipient and protoplast of Haynaldia villosa as a fusion donor (Liu et al, 1988). Steps involved in protoplast fusion are represented by figure 2.

androgenesis refers to the production of haploid plants from young pollen cells without undergoing fertilization. Sudherson et al. (2008) reported haploid plant production of sturt’s desert pea by using pollen grains as primary explants via tissue culture. Now a day the haploidy technology has become an integral part of crop improvement programmes through plant breeding by speeding up the production of inbred lines (Bajaj, 1990) and overcoming the constraints of seed dormancy and embryo non-viability (Yeung et al, 1981). The technique has a remarkable use in genetic transformation by the production of haploid plants with induced resistance to various biotic and abiotic stresses. Introduction of genes with desired trait at haploid state followed by chromosome doubling led to the production of double haploids inbred wheat and drought tolerant plants were attained successfully (Chauhan and Khurana, 2011).

Table 2 : Secondary metabolites of commercial importance produced in plant hairy root culture and being used in pharmaceutical industries.

Secondary metabolite

Plant name

Reference

Rosmarinic acid

Agastache rugosa

Lee et al (2007)

Deoursin

Angelica gigas

Xu et al (2008)

Resveratol

Arachys hypogaea

Kim et al (2008)

Tropane

Brugmansia candida

Marconi et al (2008)

Asiaticoside

Centella asiatica

Kim et al (2007)

Rutin

Fagopyrum esculentum

Lee et al (2007)

Glucoside

Gentiana macrophylla

Tiwari et al (2007)

Glycyrrhizin

Glycyrrhiza glabra

Mehrotra et al (2008)

Shikonin

Lithospermum erythrorhizon

Fukui et al (1998)

Glycoside

Panax ginseng

Jeong and Park (2007)

Plumbagin

Plumbago zeylanica

Verma et al (2002)

Anthraquinone

Rubia akane

Park and Lee (2009)

Silymarin

Silybium marianum

Rahnama et al (2008)

Flavonolignan

Silybium mariyanm

Alikaridis et al (2000)

Vincamine

Vinca major

Tanaka et al (2004)

Withanoloid A

Withania somnifera

Murthy et al (2008)

In Pharmaceutical Science

standard phytochemicals in large volumes but also eliminate the presence of interfering compounds that occur in the field-grown plants (Lila, 2005). The major advantage of the cell cultures include synthesis of bioactive secondary metabolites, running in controlled environment, independently from climate and soil conditions (Karuppusamy, 2009). For the industrial production point of view, a number of different types of bioreactors have been in use for mass cultivation of plant cells. The first commercial application of large scale cultivation of plant cells was carried out in stirred tank reactors of 200 liter and 750 liter capacities to produce shikonin by cell culture of Lithospermum erythrorhizo (Payne et al, 1987). A number of medicinally important alkaloids, anticancer drugs, recombinant proteins and food additives are produced in various cultures of plant cell and tissues in bioreactors. Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavanoids and amino acids

In the era of ever demanding development of pharmaceutical industry, plant cell tissue culture holds great promise for controlled production of myriad of useful secondary metabolites (Vijayasree et al, 2010). Plant cell cultures have provided an efficient platform for the production of valuable therapeutic secondary metabolites by utilizing the merits of whole-plant systems with those of microbial and animal cell cultures (Hellwig et al, 2004). In the want for alternatives to production of medicinal compounds from plants, biotechnological approaches, specifically plant tissue cultures, are found to have potential as a supplement to traditional agriculture in the industrial production of bioactive plant metabolites (Rao and Ravishankar, 2002). During the last decade exploration of the biosynthetic capabilities of various cell cultures has been carried out by a group of plant scientists and microbiologists in several countries (Siahsar et al, 2011) particularly adopting either of one of following culture modules.

(Vijayasree et al, 2010; Yesil-Celiktas et al, 2010).

Advances in scale up approaches and immobilization techniques contribute to a considerable increase in the number of applications of plant cell cultures for the production of compounds with a high added value. Some of the secondary plant products obtained from cell suspension culture of various plants are given in Table 1 (Source: Hussain et al, 2012).

point to gain a high production of secondary metabolites (Hu and Du, 2006). Some of the secondary plant products obtained from hairy root culture of various plants are shown in Table 2 (Source: Hussain et al, 2012).

Innovative Applications

The greatest value of the plant cell/tissue culture lies not so much in their application to mass clonal propagation (micropropagation) only but rather in its role underpinning development and application in plant improvement, molecular biology and bio-processing, as well as, its importance in research. The applications of it go well beyond the any bound in agriculture and other allied fields of food productions as follows-

  1. Aeration-agitation bioreactor
  2. Spin filter bioreactor
  3. Gaseous phase bioreactor
  4. Rotating drum bioreactor
  5. Air-driven bioreactor

These basic systems have already been used for the mass production of over 80 crops (Takayama, 1991) and are now being evaluated for production of several other plant species (Paek et al, 2001). As a plant production technique, bioreactors are far superior to traditional in vitro methods for all the species thus far tested. It is worth noting that with bioreactors, even the difficult-to- propagate woody and tree species can be produced relatively easily at high frequency. For instance, an efficient, somatic embryo-based mass propagation system

for the recalcitrant species Coffea arabica was recently developed using a bioreactor (Etienne-Barry et al, 1999). The normal and uniform development of coffee embryos achieved with the use of a bioreactor allowed direct sowing of embryos in the field, resulting in rapid crop establishment. In brief, bioreactors have the potential to improve product quality and substantially reduce the cost of micropropagation, but further development of technology is required to realize any commercial benefit from this system.

BANANA AS A PRIME-POTENTIAL HORTICULTURAL CROP AND ITS

STANDARDIZED MICROPROPAGATION PROTOCOLS

Banana evolved in the humid tropical regions of S.E. Asia with India as one of its centres of origin. Modern edible varieties have evolved from the two species – Musa acuminata and Musa balbisiana and their natural hybrids, originally found in the rain forests of S.E. Asia. During the seventh century AD its cultivation spread to Egypt and Africa. At present banana is being cultivated throughout the warm tropical regions of the world between 300 N and 300 S of the equator. Banana and Plantains (Musa spp.) are some of the earliest crop plants having been domesticated by humans. Bananas are consumed as ripe fruit, whereas plantains, which remain starchy even when fully ripe, need cooking for palatability and

Table 3 : Commercial cultivars of banana popularly propagated in India.

State

Varieties grown

Andhra Pradesh

Dwarf Cavendish, Robusta, Rasthali, Amritpant, Thellachakrakeli, Karpoora Poovan, Chakrakeli, Monthan and Yenagu Bontha

Assam

Jahaji (Dwarf Cavendish), Chini Champa, Malbhog, Borjahaji (Robusta), Honda, Manjahaji, Chinia (Manohar), Kanchkol, Bhimkol, Jatikol, Digjowa, Kulpait, Bharat Moni

Bihar

Dwarf Cavendish, Alpon, Chinia , Chini Champa, Malbhig, Muthia, Kothia , Gauria

Gujarat

Dwarf Cavendish, Lacatan, Harichal (Lokhandi), Gandevi Selection, Basrai, Robusta, G-9, Harichal, Shrimati

Jharkhand

Basrai, Singapuri

Karnataka

Dwarf Cavendish, Robusta, Rasthali, Poovan, Monthan, Elakkibale

Kerala

Nendran (Plantain), Palayankodan (Poovan), Rasthali, Monthan, Red Banana, Robusta

Madhya Pradesh

Basrai

Maharashtra

Dwarf Cavendish, Basrai, Robusta, Lal Velchi, Safed Velchi, Rajeli Nendran, Grand Naine, Shreemanti, Red Banana

Orissa

Dwarf Cavendish, Robusta, Champa, Patkapura (Rasthali)

Tamil Nadu

Virupakshi, Robusta, Rad Banana, Poovan, Rasthali, Nendran, Monthan, Karpuravalli, Sakkai, Peyan, Matti

West Bengal

Champa, Mortman , Dwarf Cavendish, Giant Governor, Kanthali, Singapuri

consumption. Originally crops from humid tropics, they have acclimatized to a broad range of climatic conditions. While bananas have come to occupy the status of a high value, commercial crop, plantains have remained a staple food of many ethnic groups. Irrespective of their commercial status, banana and plantains are referred as ‘Poor man’s apple’.

Banana is globally ranked fourth, next to rice, wheat and maize in terms of gross value of production. It is a major staple food crop for millions of people as well as provides income through local and international trade. Among the starchy staple food crops, banana ranks third with respect to the total production. Though cassava and sweet potato are positioned as first and second, banana and plantain have almost equal importance in all the tropical regions of the world. Traditional bananas and other species of family Musaceae have been the major calorie source of many ethnic tribes of Africa and Pacific Islands. High consumption of bananas has been reported in small countries of Pacific Islands like Samoa (132 Kcal) and Vanuatu (92 Kcal). Bananas also find importance in the diet of Caribbean (Haiti and Dominican Republic) and Latin American countries like Ecuador and Brazil (Singh et al, 2011). Banana (Musa sp.) is the second most important fruit crop in India next to mango. Its year round availability, affordability, varietal range, taste, nutritive and medicinal value makes it the favorite fruit among all classes of people. It has also good export potential.

Commercial Cultivars

Commercially, bananas are classified as dessert types and culinary types. The culinary types have starchy fruits and are used in the mature unripe form as vegetables.

Important cultivars include Dwarf Cavendish, Champa, Malbhog, Robusta, Monthan, Poovan, Nendran, Red banana, Nyali, Safed Velchi, Basrai, Ardhapuri, Rasthali, Karpurvalli, Karthali and Grand Naine etc. Among all these cultivars, Grand Naine, an imported variety from Israel is gaining popularity and may soon become the most preferred variety due to its tolerance to abiotic stresses and good quality bunches. Fruit develops attractive uniform yellow colour with better shelf life & quality than other cultivars. Important banana varieties cultivated in different states of India are given in table 3.

Micropropagation Protocols

Traditionally, banana is grown as a perennial crop where the plant is allowed to produce continuous shoots from a subterranean stem. But, the yields fall after three to five years and decline rapidly after ten to fifteen years. The need to shift to cyclic replacement with a new plantation comprising cycles of one crop and one ratoon has been realized only recently in most Asian countries. Natural calamities such as typhoons, floods, droughts and occasional volcanic eruptions cause devastating losses in banana production. The consequent need for fresh seedlings at regular intervals has led to very large increase in the demand for clean planting material. Banana is vulnerable to a number of biotic and abiotic stresses which limit its production, particularly among small and marginal farmers with limited resources. BBTV (Banana bunchy top virus), CMV (Cucumber mosaic virus), BSV (Banana streak virus) and BBMV (Banana bract mosaic virus) are the four most important virus diseases affecting bananas. In addition, banana is an attractive host for nematodes, particularly Pratylenchus coffeae,

Meloidogyne incognita, Helicotylenchus multicinctus and Radopholus similis. The pests also spread through transportation of non-quarantined planting material. Insect pests like, banana weevils (Odoiporus longicollis, and Cosmpolites sordidus) which till recently had a limited presence in some states of India have now spread to Bangladesh and beyond. Disease and pest pressure on Asian bananas is unlikely to lessen in the foreseeable future (Singh et al, 2011). Tissue culture technology has been the foundation of high quality, disease free planting material production of banana at a mass scale.

Banana is a crop with dual propagation abilities, sexual through seeds and asexual through suckers. Seed propagation is common in wild species which are diploid and undergo normal meiosis, fertilization and seed set. All cultivated commercial bananas are triploid and sterile, excepting a few parthenocarpic AA and AB diploids. Sucker propagation is the only natural means of their perpetuation; artificial methods of propagation include macropropagation and micropropagation: most commonly by using shoot tips. Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants under aseptic conditions using modern plant tissue culture methods. A common protocol by use of shoot tip as explant has been well documented by Singh et al (2011) and given below-

The earliest reports of in vitro culture of bananas came from Taiwan in the 70’s (Ma and Shii, 1974; Ma et al, 1978). Most commonly shoot tips can be extracted from the pseudostem, suckers, peepers, lateral buds or even small eyes which contain a shoot meristem (Jarret et al, 1985; Vuylsteke and De Langhe, 1985). Although all of them behave similarly under in vitro conditions, peepers and sword suckers are preferred because of their ease of handling and the minimum damage caused to the parent stool during their removal. It is always better to collect the explants from flowering plants so as to ascertain their trueness to type.

The critical steps followed for production of micropropagation based banana planting material are:

SH (Schenk and Hildebrant, 1972), N6 (Chu et al, 1975),

and LS (Linsmaier and Skoog, 1975) media. The culture media vary in both type and concentration of the components, but all have similar basic components of growth regulators, nitrogen, carbohydrates, inorganic macro and micronutrients, vitamins and organic additives (table 4). Generally, the cultures are established on a separate initiation medium, which has a lower concentration of cytokinin than the multiplication medium to which the cultures are subsequently transferred (Jarret et al, 1985; Novak et al, 1989).

After autoclave sterilization, the culture medium is stored in a clean dust free chamber for 1-2 days before use in order to check for any contamination. Bacterial contamination may be observed, particularly during the rainy season. Use of Cefotaxime in the initiation and subsequent subcultures helps to overcome even latent bacterial contaminations.

Table 4 : The composition of initiation, multiplication and rooting media used at the National Research Centre for Banana, India (NRCB). Source: Singh et al (2011)

Culture media used at different stages of banana micropropagation (for one litre)

Ingredients

Initiation medium

Shoot multiplication medium

Rooting medium

Activated charcoal

–

–

2.50 mg

Agar

–

7.5 g

7.5 g

Ammonium nitrate

1.65 g

1.65 g

1.65 g

L-ascorbic acid

10.0 mg

10.0 mg

–

6-bezyl amino purine

4.0 mg

4.0 mg

–

Boric acid

6.2 mg

6.2 mg

6.2

Calcium chloride

440.0 mg

440.0

440.0

Cobalt chloride

0.025 mg

0.025

0.025

Copper sulphate

0.025 mg

0.025

0.025

Ferric EDTA

36.7 mg

36.7

36.7

Glycine

2.0 mg

2.0 mg

2.0 mg

Indole-3-acatic acid

1.0 mg

1.0 mg

–

Indole-3-butyric acid

–

–

1.0 mg

Magnese sulphate

22.3 mg

22.3

22.3

Magnesium sulphate

370.0 mg

370.0

370.0

Myo-inositol

100.0 mg

100.0 mg

100.0 mg

-Naphaleneacetic acid

–

–

2.0 mg

Nicotinic acid

0.5 mg

0.5 mg

0.5 mg

Potassium dihydrogen ortho phosphate

170.0 mg

170.0

170.0

Phytal gel

2.0 g

–

–

Potassium iodide

0.83 mg

0.83

0.83

Potassium nitrate

1.9 g

1.9 g

1.9 g

Pyridoxine hydro chloride

0.5 mg

0.5 mg

0.5 mg

Sodium molybdate

0.25 mg

0.25 mg

0.25 mg

Sucrose

30.0 g

30.0 g

30.0 g

Thiamine hydrochloride

0.1 mg

0.1 mg

0.1 mg

Zinc sulphate

8.6 mg

8.6

8.6

Cefotaxime (0.1%) in the initiation medium is also advised.

Surface sterilized shoot tips are washed three times using sterile water. The outer surface of explant exposed to sterilizing agent is removed and the explants trimmed using surgical blade (No. 22) to bring the final size to about 3-4 cm length and 1-2 cm diameter (Fig. 3a). The explants are inoculated under sterile conditions in 30 ml of initiation medium in a 250 ml glass jar container. pH is

usually maintained at 5.8, which is prone to changes over culture duration. The optimum incubation temperature should be in the range of 24-26°C. Generally the light intensity is maintained at 1,500-3,000 lux. Higher levels of 3,000-10,000 lux during later stages improve the survival rate of plantlets upon transfer to soil. Initially, the cultures are maintained at 16 h light/8 h dark cycle and once after rooting they are shifted 14 h light/10 h dark cycle.

Decapitation and wounding of shoot tips are carried out to overcome apical dominance and to encourage axillary bud proliferation. But injuring the apical bud through transverse sections, either four or eight cuts, is a much preferred method. Injuring the explant encourages more production of phenols, but it can be kept at minimum using antioxidants like ascorbic acid.

Subsequent subculture is done by trimming the tip of emerging axillary buds and removal of dead tissue at the base of explant by gentle scratching. Clusters of proliferating buds develop during third and fourth subculture (Fig. 3c). For further subculturing, the explant is cut into three to four pieces and each slice with two to three proliferating clusters is inoculated to individual culture bottles. This subculture cycle is repeated at 3-4 weeks interval to increase the proliferation rate. During fourth and fifth subcultures, a single clump contains about

15-25 proliferating shoots. After 5-6 subculture cycles, the proliferated buds (Fig. 3d) are transferred to rooting medium containing IBA and activated charcoal. After a month, the rooted plantlets are ready for hardening (Fig. 3e). To minimize somatic variation, the subculturing is restricted to a maximum of seven cycles when each bottle contains 25-30 plantlets with well developed shoots and roots. Experimentally it has been demonstrated that proliferating shoots can be transferred to polybags (10- 20 cm size) having rooting media under green house. This reduces cost and enhances better establishment. Polybag provides enough space for plant growth and natural light enhances the process of hardening.

The plantlets from culture vessels/bottles are moved from the laboratory to a room at ambient temperature and kept open for 4-6 days. Later they are shifted to green house for primary hardening where they are first gently washed free of agar medium. This is important as sucrose in agar encourages microorganisms. 8 cm shoots with 3-4 ramified roots are planted in individual micropots in a protray. In places where weather is conducive (24- 26°C temperature and more than 80% humidity), the plantlets are hardened for 4-6 weeks in mini-sand beds. During this period, 90-95% humidity is maintained for the initial 6-8 days under diffused light. The humidity is slowly reduced to 70%, light intensity raised to normal and temperatures brought to 26°C by the end of 6 weeks.

Structures used for primary hardening vary with the climatic conditions. These can be highly sophisticated with UV-stabilized polysheet covering, multiple misting options, thermal shade net and auto-monitoring of light intensity, temperature and humidity. On the other hand, the structures can be simple with polycarbonate roofing, shade net on all sides with fogger facilities. Temperature, RH and light intensities are monitored manually using thermometer, hygrometer and lux meter, respectively.

Planting media for primary hardening range from sieved sand augmented with nutrition to mixtures of cocopeat and Soilrite with fine sand in equal proportions. NPK is provided in liquid form on weekly basis.

plantlets are transferred from micropots to polybags. Base substrate is generally soil and sand along with low cost materials like coir pith, sawdust or rice husk. Organic manure is either in the form of farm yard manure or poultry manure. Even pressed mud, a by-product of sugar factories, has been found to provide best substrate for secondary hardening along with soil (Vasane et al, 2006). Plantlets from micropots are, dipped in fungicide solution (0.1% bavistin) and planted in polybags containing suitable substrate. Initially, these are maintained in low light intensity shade nets and 70% RH. The plants are hardened by gradually increasing the light intensity and reducing RH (40%). After 5-6 weeks, the plants become ready for field planting having 3-5 well developed leaves and a good mass of fibrous roots. During both primary and secondary hardening, the stocks should be rouged for variants at weekly intervals. These could include vegetative deformities like dwarfism, leaf variegation, and rosette foliage and leaf crinkiness. Other precautions to be followed are:

chloride). Recommended package of practices is strictly followed to achieve successful field establishment and subsequent vigorous growth (Figs. 4 & 5 & Flow chart Fig. 6).

Shoot tip cultures preserve genetic stability much better than callus or cell suspension cultures, yet somaclonal variation seems to be widespread among plants regenerated from banana shoot tip cultures. Commercial varieties like Robusta, Grand Naine, Dwarf Cavendish, Shrimanthi and Madhukar are highly susceptible to these variations and the off-types number up to 74%. High level of variation is not desirable as it defeats the purpose of clonal reproduction and majority of the off-types are agronomically inferior to the parental clone. Banana and plantains have a flexible genetic make and its genetic stability under cultive is strongly influenced by external factors like growth regulators, duration of culture etc. Mild stress under in vitro results in reverting of the clone to its parental type. For example, Robusta, a Cavendish clone frequently reverts to its original type Dwarf Cavendish which is not acceptable for its low stature and poor yield. Hence, genetic fidelity testing using preferably molecular marker techniques is essential to ensure the supply of true to type quality planting material. At NRCB, besides phenotype PCR technique with ISSR markers is being successfully used for screening off-types in banana as in table 5. Flow chart figure 6 summarizes the various stages in micropropagation of banana. Using the protocol, more than 10,000 plants are expected from a single explant at the end of 320 days as per table 6.

WORK DONE BY AUTHOR IN THE FIELD OF MICROPROPAGATION OF BANANA

Author has in depth research experience in the field of development of protocol of micropropagation of banana cultivars of commercial importance and also on the influence of ploidy and genome on culture responses which is evident by her under given research publications.

Sugandh S., Rajak K. K. and Kumar H. (2012) Diversity of genome and ploidy in banana and their effect on tissue culture responses. Res. Environ. Life Sci. 5(4), 181- 183.

Banana is one of the most important fruit crop having a wide range of ploidy and genome constitution. Most of the banana cultivars have originated from inter and intra specific hybridization of two wild diploid (2n=2x=22)

species, M. accuminata (‘A’ genome) and M. balbisiana (‘B’ genome) resulting into different genomic and ploidy levels namely AA, AAA, AAB, ABB, AAAB, AABB and ABBB. Tissue culture studies in shoot tips cultures of banana comprising different ploidy levels and having different genome structures, resulted in different forms of organogenesis. The shoot tips were cultured on MS medium supplemented with different concentrations and combinations of 2, 4-D, IAA, KIN and BAP. The effect of ploidy and genome on tissue culture responses have been found very significant. Triploids gave the best response followed by tetraploid and diploids for all tissue culture responses except somatic embryogenesis for which triploids were followed by diploids only. The genotype with more ‘A’ genomes gave better response than those with ‘B’ genome for all tissue culture responses except somatic embryogenesis as represented in figures 7(A) & 7(B).

Sugandh S., Kumari R., Sharma V. K. and Kumar

H. (2015) Isozyme analysis based genetic fidelity assessment of micropropagated banana plants. J. Applied Natural Sci. 7(2), 579 – 584.

Isozyme studies of micropropagated and mother plants of banana cvs. Matti, Ney Poovan, Kechulepa, Dwarf Cavendish, Malbhog, Champa, B.B. Battisa and FHIA-1 were done to test their genetic fidelity. The banding patterns as revealed by electrophoretic variations were evaluated with respect to isozymes of acid phosphatase, catalase, esterase and peroxidase as markers. The genetic fidelity of micropropagated plants and the relationship of the different cultivars were determined by dendrogram using numerical taxonomy and multivariate analysis system (NTSYS). A clustered dendrogram was prepared by unweighted pair group method using averages (UPGAMA) method. At 87% similarity, the micropropagated and mother plants were clustered in four groups reflecting their genomic constitution. Cvs. Matti (AA) and Dwarf Cavendish (AAA) with similar ‘A’ genome were categorized in Cluster I. Cluster II comprised of cvs. Ney Poovan (AB),

B.B. Battisa (ABB) and FHIA-1(AAAB) with genomic constitution of both ‘A’ and ‘B’ type. Cvs. Champa (AAB) and Malbhog (AAB) with similar genome were grouped in Cluster III. Cluster IV contained the cv. Kechulepa (BB) having only ‘B’ genome. However, there was no somaclonal variation among the micropropagated plants and they showed 100% genetic similarity. Thus, the isozyme studies could be a reliable marker for testing the genetic fidelity of micropropagated plants and for evaluating the diversity among the banana germplasm.

Source: Singh et al (2011)

Fig. 4: Tissue cultured plantlets at growing stage. Fig. 5 : Tissue cultured plants at fruiting stage.

Fig. 6 : Summary of steps involved in production of quality plantlets of banana by microprogation (tissue culture).

  1. Sugandh S. and Kumar H. (2015) Micropropagation of Banana cv. Malbhog. The Bioscan 10(2), 647-650.

Malbhog, one of the most important and delicious local cultivar of banana in Bihar, is on verge of becoming extinct because of panama wilt and non-availability of disease free quality propagules. The culture of shoot tips taken from suckers on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of indole acetic acid (IAA) and benzyl amino purine (BAP) resulted in differentiation of

adventitious shoots. The maximum differentiation of shoots (92.05%) was observed on MS medium with 0.57 µM IAA and 17.74 ìM BAP. The number of shoots per culture was 16.75. The subculture of differentiated shoots on the same medium resulted in further differentiation (91.97%) of more than 15 shoots per culture. The in vitro developed shoots showed 100% rooting on MS medium supplemented with 4.92 ìM Indole butyric acid (IBA). The plantlets were acclimatized and field transferred. A suitable and efficient protocol for micropropagation of Malbhog cultivar of banana was

Table 5 : List of ISSR primers used for genetic fidelity testing of banana at National Research Centre for Banana (NRCB), Tiruchirapalli 620102, Tamil Nadu, India (Source: Singh et al, 2011).

ISSR Primer

Nucleotide sequence

UBC 807

5’-AGA GAG AGA GAG AGA GT-3’

UBC 808

5’-AGA GAG AGA GAG AGA GC-3’

UBC 811

5’-GAG AGA GAG AGA GAG AC-3’

UBC 812

5’-GAG AGA GAG AGA GAG AA-3’

UBC 818

5’-CAC ACA CAC ACA CAC AG-3’

UBC 830

5’-TGT GTG TGT GTG TGT GG-3’

UBC 834

5’-AGA GAG AGA GAG AGA GYT-3’

UBC 836

5’-AGA GAG AGA GAG AGA GYA-3’

UBC 840

5’-GAG AGA GAG AGA GAG AYT-3’

UBC 841

5’-GAG AGA GAG AGA GAG AYC-3’

UBC 842

5’-GAG AGA GAG AGA GAG AYG-3’

UBC 850

5’-GTG TGT GTG TGT GTG TYC-3’

UBC 868

5’-GAA GAA GAA GAA GAA GAA-3’

developed: figure 8.

  1. Sugandh S., Rajak K. K. and Kumar H. (2013) Micropropagation of Banana cv. B. B. Battisa. Biochem. Cell. Arch., 13(2), 349-354.

For banana being the most important fruit of India as whole particularly more popular in Bihar, the requirement of large numbers of quality propagules for plantation rely on the development of micropropagation protocol for important local cultivars. B.B. Battisa (ABB) is an important cultivar used as vegetable and in culinary and wine making. Shoot tips were cultured on MS medium supplemented with different concentrations of IAA (5.71, 11.42 and 22.83µM), BAP (4.43, 8.87, 17.74 and 26.61

µM) and combination of IAA (0.57, 5.71 and 11.42 µM) and BAP (8.87, 13.30 and 17.74 µM). Cultured shoot tips differentiated multiple shoots from their bases and the rate of shoot multiplication was quite high. The subculture of differentiated shoots on MS medium with

0.57 µM IAA and 17.74 µM BAP resulted in further differentiation of more than 7 shoots per culture. The in vitro developed shoots showed 100% rooting on MS medium with 4.92 µM IBA. The regenerated plantlets were transferred to a mixture of sand and farm yard manure (1:1) for acclimatization and field transfer. Thus, the present work led to the development of a suitable micropropagation protocol for cv. B.B. Battisa: figure 9.

Sugandh S., Rajak K. K., Kishore C. and Kumar

H. (2013) Micropropagation of Banana cv. Champa. Biochem. Cell. Arch. 13(2), 291-295.

Champa (AAB) is another important cultivar known for its delicious fruit used as a dessert and making pastries. Shoot tips were cultured on MS medium supplemented with different concentrations of IAA (5.71, 11.42 and

Table 6 : Success summary table of micropropagation protocol of banana (Source: Singh et al, 2011).

Particulars stage

Duration (days)

No. of plants

Initiation

25

1

Subculture stages 1st

50

3

2nd

75-80

12

3rd

100-110

48

4th

125-130

192

5th

175-180

760

6th

200-210

3,040

7th

225-230

12,160

Rooting

255-260

11-12,000

Primary hardening

270-280

11,500-11,000*

Secondary hardening

310-320

10,000-10,500**

*Success depends on the sophistication of the hardening structure

**Somaclones and off-types constitute the major discards.

22.83µM), BAP (4.43, 8.87, 17.74 and 26.61 µM) and

combination of IAA (0.57, 5.71 and 11.42 µM) and BAP (8.87, 13.30 and 17.74 µM). Cultured shoot tips differentiated multiple shoots from their bases and the rate of shoot multiplication was quite high. The subculture of differentiated shoots on MS medium with 0.57 µM IAA and 17.74 µM BAP resulted in further differentiation of more than 4 shoots per culture. The in vitro developed shoots showed 100% rooting on MS medium with 4.92 ìM IBA. The regenerated plantlets were transferred to a mixture of sand and farm yard manure (1:1) for acclimatization and field transfer. Thus, the present work led to the development of a suitable micropropagation protocol for cv. Champa: figure 10.

Sugandh S., Kumar M. and Kumar H. (2016) Plant regeneration via somatic embryogenesis from the cultured immature male floral buds of banana genotype ‘Dwarf Cavendish’. Indian J. Life Sci. 13 (2), 000-000.

Immature male floral buds of banana cv. Dwarf Cavendish were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-D (4.52, 9.05 and 18.10 µM), IAA

(5.71, 11.42 and 22.83 µM), Kin (4.65, 9.30 and 18.60

µM) and BAP (4.43, 8.87 and 17.74 µM). The culture of the explants resulted in the generation of small spherical, friable calli. These calli showed embryogenic characteristics and sub-cultured on the same medium for further maturation. After 5 months of culture on MS basal medium, somatic embryos started to regenerate. The somatic embryos were transferred to modified MS medium for regeneration into plantlets. Among the different combinations tried, MS +9.05µM 2,4-D + 18.60µM Kin was found to be the most suitable medium for somatic embryogenesis. Thus, an efficient protocol

Fig. 7(A) : Effect of ploidy on different tissue culture responses from shoot tip culture.

Fig. 7(B) : Effect of genome (A & B) on different tissue culture response from shoot tip culture.

Fig. 8 : (a-e) : Explant showing existing

shoot elongation after 15 days of culture

on medium BM (a), Multiple shoot

8

formation after 20 days of culture on

medium BM3(b), Multiple shoot formation after 25 days of culture on medium BM11(b), Root formation after 20 days of culture on medium BM10(d), Two month-old planelet (e).

for plant regeneration via indirect somatic embryogenesis have been developed: figure 11.

CRITICAL PROBLEMS AND RESEARCHABLE ISSUES

It is evident by the literatures that the development of tissue culture protocols is a rigorous procedure that involves optimization of the various chemical, physical and environmental factors of cell and tissue growth being accomplished by growing plants outside their natural environment and growth conditions. Despite the

meticulous efforts involved in growing plants in vitro and the advances made in plant tissue culture, the application of this technique is still hampered by various physiological and developmental problems. The problems are summarized by Bairu and Kane (2011) and range from abnormal growth to increased genetic variability/instability. Of these, the points of critical attention are:

Fig. 11 : Plant regeneration from callus culture in ‘Dwarf Cavendish (a) Explant showing swelling after 20 days of culture on medium

(b) Explant showing callus formation after 40 days of culture on medium BM (c) Explant showing embryogenic callus

21

BM

14

formation after 125 days of culture on medium BM (d) & (e) Regeneration of somatic embryos into plantlets cultured on modified

17

MS medium (f) Hardening of regenerated plants in green house.

dieback or shoot-tip necrosis of in vitro cultures has been associated with various culture conditions including auxin- cytokinin interactions (Bairu and Kane, 2011).

Some describe fasciation as fusion of organs due to deviation from normal meristematic processes while others suggest that it is the result of transformation of a single growing point into a line (Clark et al, 1993). Tissue proliferation is a disorder primarily found in micropropagated rhododendrons that is characterized by

the formation of abnormal callus-like growths, or tumors at or near the crown or base of the plant (Brand and Kiyomoto, 1992).

More concerted research to ameliorate these upcoming problems of plant tissue culture is highly warranted in the coming years.

CONCLUSIONS

Advances in molecular biology allow for the genetic engineering of plants through the precise insertion of foreign genes from diverse biological systems. Three major breakthroughs have played major roles in the development of this transformation technology i.e. the development of shuttle vectors for harnessing the natural gene transfer capability of Agrobacterium, the methods to use these vectors for the direct transformation of re- generable explants obtained from plant organs and the development of selectable markers. The next five to ten years could be an exciting time for the banana industry as biotechnology is applied to this crop, which is highly important to the food security of so many people in developing countries and which creates income for a long chain of people engaged in the production and trade in the product. Breakthroughs can be anticipated, and hopefully these will lead to: reduced environmental impacts by chemicals, now necessarily applied in production areas; lower costs of production; improved

worker safety; and higher productivity. Moreover, it is also anticipated that whereas genetic engineering- molecular technologies offer great opportunity to develop a world that is truly food secure, we must not forget that we all – the scientific community, the international community, the multinational life science companies and the donor community together with national governments

– bear a fundamental responsibility to ensure that the developing countries can equitably share in these exciting advances that science offers in a way that is safe for their population and environment. This calls for a more open, integrated and collaborative involvement of all the stakeholders of developing country engaged in agriculture and food production. Bananas are very much a part of this opportunity.

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