Plant tissue culture:It can be defined as a culture of cell, tissue, organ or whole plant in medium under aseptic and controlled conditions. The importance of disease-free and quality planting material in agriculture cannot be overemphasized. In many vegetatively propagated crops, continuous clonal propagation leads to accumulation of pests and pathogens leading to decline in vigour and quality.
PLANT TISSUE CULTURE
Hence, large-scale production of disease-free planting materials pose serious challenge in sugarcane, potato, banana and several other horticultural crops. Moreover, in many of these crops the rate of multiplication by conventional methods is very low. Thus alternative methods of rapid multiplication using plant tissue culture are desirable.
Totipotency and application of plant tissue culture
The capability of isolated single cells to multiply and differentiate into whole organism is called totipotency. Although this concept is inherent in the cell theory propounded in 1838-39 by Schleiden and Schwann, practical demonstration of totipotency in plants was achieved only in 1964 by Vasil and Hildebrandt. Similar demonstration with animal cells is still to be achieved.
The ability to direct growth and differentiation in-vitro by manipulating nutrition and physical culture conditions, provides several opportunities for both basic and applied researches. As a result plant tissue culture is now not only a technical tool but also a major technology. Application plant tissue culture includes the following.
- investigation of growth and morphogenesis and other basic studies
- micropropagation for large-scale clonal multiplication of plants
- doubled haploid production for obtaining homozygous lines
- creation of genetic variations
- production of secondary metabolites of pharmaceutical and commercial value
- transfer of genes from wild related species into crop plant species
- genetic engineering of plants collection, conservation and exchange of germplasm.
Micropropagation refers to in-vitro multiplication of plants starting from a small tissue explant. This explant may be leaf, root, nodal-cutting or seedling. Successful micropropagation involves establishment of aseptic culture, shoot multiplication, rooting, hardening etc. In general, organized shoot meristem or nodal cutting is used as a starting material as it ensures clonal fidelity.
Under the cytokinin influence, the shoot meristems are forced to proliferate into multiple shoots, which are subcultured to produce either more shoots or transferred to root induction medium for obtaining plantlets. The nutritional and in-vitro environmental conditions required for micropropagation of a variety of plant species have been worked out.
In particular, media formulations of Murashige and Skoog, Mccoven, Gamborg etc. have proved highly useful. The micropropagation technique, first devised for orchids, has now been adopted worldwide for the multiplication of orchids. Similarly, micropropagation is used for large-scale multiplication of several important crops, and has been established as a major global industry. The chief advantages of micropropagation are as follows:
- rapid multiplication (literally thousands of plants can be produced within a year starting from a single explant)
- freedom from pests and pathogens (healthy plants can be recovered even from the infected mother plant variety)
- round-the-year multiplication as per demand
- economy of space and resources (thousands of plants can be raised in a few square metres of laboratory space)
- easy transport due to miniaturization
- selective multiplication of desired plants (e.g. female trees in dioecious crops like papaya or plus trees in forest species).
The significance of micropropagation in transforming agriculture is best illustrated by the case of oilpalm in Malaysia. In the 1980s, Malaysia adopted tissue culture technology to clonally multiply selected high yielding oilpalm genotypes. Large-scale planting of such tissue-cultured materials has led to a major increase in productivity. This has helped Malaysia to be the world leader in edible oil trade. Similar opportunities exist in other crops and countries.
For example, in India, propagation of coconut, mango, citrus, papaya, date-palm and several forest plant species can benefit greatly by micropropagation. But micropropagation methods are not well standardized for all crops (mango, coconut are some examples). Further, plants obtained Via tissue culture are expensive than those derived by conventional methods. Thus cost considerations limit its wider use in crops where micropropagation is technically feasible.
Meristem culture and production of virus-free plants
The observation of Morel and Martin that plants regenerated from excised shoot apical meristems (< 0.5 mm) of virus-infected dahlia plants were free from virus has paved the way for the production of disease-free “plants tissue culture“. Since a small explant is to be used for initiating culture, the media requirements for meristem culture are more complex than that of simple shoot culture.
Also, the size of the meristem is critical for successful eradication of viruses. For example, elimination of Potato Virus S (PVS) can be possible using meristems of 0.3-0.5 mm size, and a much smaller meristem size (0.12 mm) is necessary to obtain PVX-free plants. The meristem culture technique is now well established and is routinely used for obtaining Virus-free plants of potato, sugarcane, citrus etc. Many old varieties of cassava, which had declined in vigour, could be rejuvenated following meristem culture. This suggests involvement of latent infections leading to varietial decline in such vegetatively propagated crops.
Plant tissue culture industry in India
Commercial tissue culture is widely practised in India. Department of Biotechnology, Government of India, has been playing a proactive role in this regard. It has set up Micropropagation Technology Parks in Haryana (under The Energy Research Institute, New Delhi) and Pune (under the National Chemical Laboratory, Pune), and Virus Diagnosis and Quality Control Laboratory at the Indian Agricultural Research Institute, New Delhi. And six hardening units have been set up in dlfferent parts of the country for acclimatization and successful establishment of plant tissue culture raised in soil.
There are about 100 private enterprises producing about 50 million plantlets peroyear. While a majonty of the produce is targeted for the internal market, about 25% is exported. The crops being marketed include banana, cardamom, sugarcane, potato, ornamental plants, bamboo, forest tree species etc.
Production of haploids and doubled haploids
Doubled haploids are individuals derived from haploid gametes and carry two sets of identical chromosomes. Thus doubled haploids are 100% homozygous and breed true. Such homozygous lines serve as varieties in self-pollinated crops and as inbred lines of hybrids. Traditionally homozygous lines are produced by repeated selfmg (6-7 generations) of selected individuals. Severe loss of vigour (inbreeding depression) and self-incompatibility (i.e. genetic failure to set seed upon self-pollination), however, limit production of inbred lines in many crops.
Haploid plants could be regenerated for the first time in 1964 from in-vitro culture of anthers containing developing microspores. Subsequently, haploids have been produced in more than 200 plant species through in-vitro culture. Haploids fail to undergo normal meiosis as they lack homologous partner chromosome for pairing and hence are sterile. By treating them with colchicine, chromosome number could be doubled, yielding fertile doubled haploids. Thus in a single step homozygous lines can be established. Doubled haploids can thus cut down time required for breeding of improved varieties and hybrids.
Not only anthers but also isolated microspores can be cultured to obtain haploids. Such microspore cultures were widely used in Brassica napus, B. rapa, B. juncea and B. oleracea (cabbage and cauliflower) for obtaining varieties and inbred lines in Europe and Canada.
Haploids can also be derived from ovules plant tissue culture. For example, in wheat pollination with maize triggers embryo development from unfertilized eggs. In-vitro culture of such immature embryos yields haploids. In sugarbeet, gynogenesis offers a convenient method for the production of doubled haploids.
China has made use of anther culture technology for breeding rice varieties. A number of improved rice varieties were released for commercial cultivation in China. Some promising anther culture derived lines of rice were identifred for commercial release in North-East India. Commercial varieties have been developed via doubled haploid technique in tobacco, wheat, eggplant, capsicum, sugarbeet etc.
Doubled haploids are also of great value in basic studies. In particular, genome Inapplng and identification of molecular markers linked to various traits can be greatly simplified and speeded up using doubled haploids. For example, a high density Brassica juncea map has been prepared using doubled haploid lines, tissue culture and wide hybridization.
Wild relatives of crop species are a treasure trove of useful genes. In particular, wild species harbour genes for resistance/tolerance against pests, pathogens and environmental stresses such as drought, water logging, salinity, high/low temperature etc. However, strong barriers to hybridization hamper exploitation of wild species resources for crop improvement. Plant tissue culture has been extremely useful in overcoming these interspecific barriers and to recover hybrids.
Hybrid incompatibility may operate at pre or post-fertilization stages. In-vitro pollination and culture of excised ovules can overcome pre-fertilization barriers, operating in the style or stigma. Embryo culture has been widely used for obtaining hybrids which fail to develop normally owing to improper nourishment of the young embryos.
By optimizing conditions such as age of the embryo, nutritional milieu and culture conditions, a large number of interspecific hybrids have been produced in wheat, rice, Brassica etc. From such hybrids important genes have been introgressed into cultivated species through backcross breeding. Examples of introgression include rust resistance in wheat, blackleg resistance in Brassica napus, nematode resistance in sugarbeet etc.
Embryo culture is also resorted to in grapes and banana that produce seedless fruits. Breeding of these crops is severely limited by scedlcssness, which is a desirable trait from the consumer viewpoint. However, even in these genotypes viable embryos, are initiated after fertilization.
Thus by early excision and culture of embryos, novel genetic recombinants can be obtained. Banana improvement has witnessed spectacular progress at the INIBAP (International Network for the Improvement of Banana and Plantains) in the past two decades. New plantain varieties with resistance to black sigatoka disease were developed through interspecific hybridization with Musa balbisiana.
Techniques of isolation and culture of plant protoplasts and their fusion in in-vitro devised in 1960s and 1970s have broken all barriers to hybridization including failure of flowering or meiosis. Although hybrid cells can be produced by fusing any two protoplasts, regeneration of hybrid individuals is problematic in very distant cross combinations. Nevertheless more than 100 somatic hybrids have been reported.
Nuclear and organelle genomes of both the parents are brought together in a somatic hybrid. Thus somatic hybrids have for the first time permitted genetic analysis of organelle genomes and have thus proved invaluable in basic studies. In particular, mitochondrial genome manipulation to obtain cytoplasmic male sterile lines has been successfully used in Brassica.
Similarly, a number of useful gene introgressions via somatic hybridization have been reported in Brassica, potato, tomato etc. The recent transfer, identification and ultimate cloning of a gene for late-blight disease resistance from Solanum bulbocastanum into potato is noteworthy. Similarly, development of a number of cytoplasmic male sterile lines and introgression of fertility restorer genes in Brassica by somatic hybridization, is a major achievement.
Plant tissue culture and their transformation
Current methods of plant transformation can deliver DNA only into individual cells rather than transform all cells of an organism. Transgenic technology relies on totipotency of plant cells to recover whole plants from a single such transformed cell. Thus “plant tissue culture” regeneration is key to plant genetic engineering. Since only a few cells out of the several thousands exposed to transformation treatment actually get transformed, novel selection schemes are employed to selectively multiply transformed cells.
For example, most transformation vectors carry, besides the gene (s) of interest, a linked constitutively expressed selectable marker gene (s) conferring either herbicide or antibiotic resistance to transformed cells. By culturing cells subjected to transformation on a medium fortified with appropriate herbicide or antibiotic, the non-transformed cells/plants are detected or eliminated.
Using these strategies, transformation has now been achieved in over 100 plant species. Various critical factors that influence transformation have been identified to improve transformation efficiency. Still, some important crop species, Viz. cotton and grain-legumes, are not routinely amenable to transformation primarily because of difficulties in plant regeneration.
Significant genotypic differences in transformation efficiency among cultivars suggest involvement of host genetic factors. A better understanding of molecular biology of plant regeneration will be needed to crack problem of recalcitrancy (i.e. failure to regenerate plants in response to in vitro treatments) of plant tissue culture.
Plant tissue culture and production of secondary metabolites
Plants are a major source of pharmaceuticals and other biologicals, imparting flavour, colour, aroma etc. Since most secondary metabolites are produced in small quantities in specialized organs/tissues, and after attainment of certain growth, sometimes lasting years, their production from natural sources is often difficult and expensive.
Further, many pharmaceuticals are extracted from plants collected from the wild, leading to severe genetic erosion and pushing them to near extinction. It has now been demonstrated that many secondary metabolites, which are produced in specialized organs in plants, can be produced in cell cultures in-vitro.
For example, capsaicin, the pungent principle of capsicum, which is produced in the placental tissues of fruits, can be obtained in cell cultures. Furthermore, higher levels ofexpression than observed in nature can be realised in cell cultures. Production of secondary metabolites through cell culture offers several other advantages such as quality control, scheduling of production.
Therefore, production of useful compounds from plant cell cultures in large bioreactors is considered most desirable. However, plant cells, unlike bacteria, grow in clusters and are shear sensitive. Therefore, large-scale culture ofplant cells in bioreactors for the production ofsecondary metabolites has been proved to be a challenge both from technical and cost point ofview. Nevertheless, a plant dye shikonin is commercially produced in Japan from cell cultures of Lithospermum erythrorhizon, which demonstrates that the problems are not insurmountable. Considering the impossibility of sourcing some very potent products from nature (e.g. antitumor agent taxol from Taxus yew tree), cell cultures offer best alternative for production of such compounds.
In-vitro conservation of germplasm
Gerrnplasm of vegetatively propagated plants and plants bearing recalcitrant seeds, is maintained as clonal repositories in field. This entails huge costs of labour and resources and poses several challenges. For example, germplasm in the field is always under threat from environmental vagaries, pests and pathogens. Plant tissue culture has been found extremely useful in germplasm conservation.
The cost and space requirements for conservation are greatly reduced and the cultures are attained under aseptic conditions. Moreover, whenever required, the samples can be easily multiplied and readily exchanged with minimum quarantine restrictions. Large in-vitro banks holding important horticultural and forest tree species are already operating in developed countries.
In India, the National Bureau of Plant Genetic Resources is maintaining over 1,000 accessions of important plant species, Viz. banana, ginger, pepper, sweet-potato, yarns and some endangered medicinal plants in the form of shoot cultures in-vitro. Likewise, collections of potato, sweet-potato, yams, banana etc. are held in plant tissue culture banks in several CGIAR centres.
Plant tissue culture is also relevant for long-term storage of germplasm in liquid nitrogen at ultra low (-196°C) temperature. Plant tissues contain more than 80% moisture and hence are not amenable to storage below freezing point. F urther, large mass of tissues cannot be desiccated without losing Viability. However, small cell clumps and meristems can be desiccated under controlled conditions to permit cryopreservation.
Complete plants can be regenerated from such cryopreserved cells or shoot apices following standard tissue culture techniques. Cryopreservation of cells and meristems has now been standardized for several plant species including potato, banana, yarns, and several temperate fruit species. Cryopreservation is highly cost-effective and entails minimum genetic change. Therefore, in future, most gennplasm collections ofclonally propagated plants will be maintained in cryobanks for long-term preservation.
Protein and DNA-based disease diagnosis kits
Pathogen infections particularly those of viruses are not readily identifiable at seedling stage and hence ensuring disease-free status of the planting material poses problems. In vegetatively propagated crops like potato, banana, citrus etc. such infected planting materials lead to heavy yield losses. Modern biotechnological tools have greatly facilitated accurate, quick and efficient identification of hidden infections.
These diagnostic tools have been developed in the form of simple kits that could be used either at the field level or in laboratories. A small sample of leaf or other tissue is used for diagnosis. These kits are specific and each kit usually detects one or a limited group of infectious agents.
In developing protein-based kits, a protein specifically produced by the infectious agent is isolated, purified and injected into mice or rabbits. Such injected mice or rabbits produce antibodies (proteins that recognise and destroy the foreign protein). These antibodies are extracted and used for diagnosis of the pathogen.
For DNA-based diagnosis, a specific DNA or RNA fragment of the infectious agent is cloned and sequenced. Based on the sequence information, single stranded DNA sequences (primers) are synthesized and used in polymerase chain reaction (PCR) along with DNA extracted from the test plant sample. If the test sample is infected, there will be amplification of a known size of DNA fragment in the reaction. These kits are specially useful in seed/plant propagule certification.