Author: Vartika Srivastava
Scientist, Tissue Culture and Cryopreservation Unit
ICAR-NBPGR, Pusa Campus, New Delhi
The requirement of fruits and vegetables is increasing proportionally with the increasing population in the country. Although conventional plant breeding techniques have made considerable progress in the development of improved varieties, they have not been able to keep pace with the increasing demand for vegetables and fruits in the developing countries. Therefore an immediate need is felt to integrate biotechnology to speed up the crop improvement programmes. Biotechnological tools have revolutionised the entire crop improvement programmes by providing new strains of plants, supply of planting material, more efficient and selective pesticides and improved fertilisers. Many genetically modified fruits and vegetables are already in the market in developed countries. Modern biotechnology encompasses broad areas of biology from utilisation of living organisms or substances from those organisms to make or to modify a product, to improve plant or animal or to develop micro-organisms for specific use. It is a new aspect of biological and agricultural science which provides new tools and strategies in the struggle against world’s food production problem. The major areas of biotechnology which can be adopted for improvement of horticultural crops are –
- Tissue Culture
- Genetic Engineering
- Molecular markers
(a) Micropropagation:
One of the widest applications of biotechnology has been in the area of tissue culture and micropropagation in particular. It is one of the most widely used techniques for rapid asexual in vitro propagation. This technique is economical in time and space affords greater output and provides disease free and elite propagules. It also facilitates safer and quarantined movements of germplasm across nations. When the traditional methods are unable to meet the demand for propagation material this technique can produce millions of uniformly flowering and yielding plants. Micropropagation of almost all the fruit crops and vegetables is possible now. Production of virus free planting material using meristem culture has been made possible in many horticultural crops.
Example: banana, papaya, date palm mulberry, passion fruit, grape vine etc.
(b) Embro rescue:
Embryo rescue is another area where plant breeders are able to rescue distant crosses which would otherwise abort. Culture of excised embryos of suitable stages of development can circumvent problems encountered in post zygotic incompatibility. This technique is highly significant in intractable and long duration horticultural species. Application of embryo rescue can overcome can overcome some of the pre and post-fertilization barriers in fruit crops. This technique has important role in haploid production, shortning of breeding cycle, rapid seed viability test and propagation of rare plants.
Example: grape, apple etc.
(c) Anther culture:
Haploid generation through anther/pollen culture is recognized as another important area in crop improvement. It is useful in being rapid and economically feasible. The purpose of anther and pollen culture is to produce haploid plants by the induction of embryogenesis from repeated division of monoploid spores, either microspore or immature pollen grains.
Example: pineapple, papaya, litchi, apple, citrus etc.
(d) Conservation of germplasm:
In vitro germplasm conservation is of great significance in providing solutions and alternative approaches to overcoming constrains in management of genetic resources. In crops which are propagated vegetatively and which produce recalcitrant seeds and perennial crops which are highly heterozygous seed storage is not suitable. In such crops especially, in vitro storage is of great practical importance. These techniques have successfully been demonstrated in a number of horticultural crops and there are now various germplasm collection centers. In vitro germplasm also assures the exchange of pest and disease free material and helps in better quarantine.
(e) Somaclonal variation:
Plant breeders are continually searching for new genetic variability that is potentially useful in cultivar improvement. A portion of plants regenerated by tissue culture often exhibits phenotypic variation atypical of the original phenotype. Such variation, termed somaclonal variation may be heritable i.e. genetically stable and passed on to the next generation. Alternatively, the variation may be epigenetic and disappear following sexual reproduction. These heritable variations are potentially useful to plant breeders. These variations are due to variation in chromosomal number, structural variation of chromosomes due to deletions, duplication, translocation, genetic and cytoplasmic mutation etc.
Example:
Hwang and Ko(1987) identified somaclonal variations in the cultivars Giant Cavendish with putative field resistance to Fusarium wilt (race 4) but inferior in agronomic characters.
(f) Somatic hybridization:
Somatic hybridization provides a method where sexual incompatibility in the plants can be by-passed. Protoplast culture includes a series of operations such as isolation of the protoplasts from cells, culturing them in a suitable medium, inducing them to divide and then regenerating plantlets from them. Fusion of protoplasts may occur spontaneously or they may be induced to fuse in the presence of fusigenic agents. The polyethylene glycol (PEG) is the most widely used fusigenic agent (Chadha et al ., 2000).
Example:
Citrus (Tangelo) + Murraya paniculata {electrofusion}
(C. reticulata × C. paradisi) + C. jambhiri {electrofusion}
C. sinensis + C. reticulate {commercial method-PEG}
C. sinensis + Clausena lansium {electrofusion}
II. Genetic Engineering of Plants
The advent of recombinant DNA technology has opened tremendous possibilities for transforming almost any plant by transferring any gene from any organism across, taxonomic barriers.
Genetic Engineering involves three major steps:
i) Identification and isolation of suitable genes for transfer
ii) Delivery system to insert desired gene into recipient cells.
iii) Expression of new genetic information in recipient cells.
Using techniques of genetic engineering many useful genes have been introduced into plants and many transgenic plants have been developed in which the foreign DNA has been stably integrated and resulted in the synthesis of appropriate gene product. Transgenic plants have covered about 52.6 m hectares in the Industrial and developing countries up to 2001.
Gene transfer technology:
Important gene transfer methods used for production of transgenic plants are as under:
- Agrobacrterium -mediated transformation
- Microprojectile bombardment- mediated transformation
- Protoplast- mediated transformation
- In-planta electroporation
- Intact tissue electroporation
- Silicon carbide fibres
- Electrophoresis
- Microinjection
- Sonication
- Laser-mediated gene transfer Important fruit crops in which transgenic plants are reported: Kiwi fruit var. Hayward, Papaya var. Sunrise Solo, Lime, Mandarin (C. reticulata), Walnut, Banana var. Grand Nain etc.
Role of Genetic engineering
(i) Improving shelf life of fruits (Name of Crop and Gene)
Apple: ACC synthase, Attacin esterase, Polygalacturonase
Banana: ACC synthase
Strawberry: Acetolactate synthase, Chitinase, Glucanase, PG inhibitor protein,SAM transferase
(ii) Edible vaccines Transgenic banana is one of the most recent examples, where genes of antigenic proteins of many deadly disease causing pathogens have been expressed in banana fruits. Thus, children can be immunized only by feeding with these transgenic fruits, instead of painful injections for immunization.
(iii) Biodegradable plastic Polyhydroxy butyrate (PHB) is used as a substrate for the production of biodegradable plastic. By transforming the PHB genes into the plants, this could be produced on a large scale and at a very low cost, which may reduce the threat imposed by polyethylene to the ecosystem.
(iv) Pest management: Fruit crops suffer from a variety of insect pests. It is possible to implement biotechnological approaches to manage insect pests in a rational, durable and ecofriendly manner. Therefore, novel insecticidal proteins and their respective genes need to be identified and used in conjugation with Bt to prevent development of resistant insect. In addition, IPM will have to play a central role in sustainable horticulture. Disease resistance, herbicide resistance, abiotic resistance etc. are the other areas where genetic engineering can play an important role in imparting resistance in fruit crops. Example: In apple gene attacin (from Hyalophora cecropia) lysozyme (farm chicken) and cecropin B (from H. cecropia) can be used for disease resistance against Eriwinia amylovora.
III. Molecular Markers
The possibility of using gene tags of molecular makers for selecting agronomic traits has made the job of breeder easier. It has been possible to score the plants for different traits or disease resistance at the seedling stage itself.
Uses
- Characterization of germplasm
- Varietal identification and clonal fidelity testing
- Assessment of genetic diversity
- Validation of genetic relationship
- Marker assisted selection
- Developing linkage map in fruit crops.
The use of RFLP (Restriction Fragment Length polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) and isozyme markers in plant breeding are numerous. RFLPs are advantageous over morphological and isozyme markers primarily because their number is limited only by genome size and they are not environmentally or developmentally influenced. RFLPs have wide ranging applications including cultivar finger printing, identification of quantitative trait loci, analysis of genome organization, germplasm introgression and map-based cloning. AFLP is becoming the tool of choice for fingerprinting because of its reproducibility compared to RAPD. Microsatellile or simple sequence repeats (SSRS) markers have also become the choice for a wide range of applications in genotyping, genome mapping and genome analysis.
Examples:
1. Bochemical marker In mango enzyme polymorphism has been used to distinguish nucellar from zygotic seedlings, in citrus for classification, in grapes for root stock identification isozyme pattern was used.
2. DNA based markers
(i) In the genetic diversity assessment Avocado, banana, blueberry, citrus, grapes, mango, strawberry, walnut etc.
(ii) In varietal identification Annona sp.(RAPD), Apple (minisatellite, RAPD), Grapes (SSR), Grape root stock (RAPD), Lemon (RAPD).
(iii) Marker assisted selection (MAS)
Apple – columnar growth habit, scab resistance.
Avocado – skin colour
Citrus – differentiation of zygotic and nucellar progeny
Papaya – sex determination
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