Techniques in genetic engineering and micro-biology to improve agriculture

Any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals or to develop microorganisms for specific uses is considered as biotechnological technique. Tissue culture, recombinant DNA technology, cloning, and genetic engineering are the important areas of biotechnology.

Techniques in genetic engineering and micro-biology to improve agriculture

(Conceived by Arbab Ahmad from Plant biotechnology by K.G.Ramawat.)

1. INTRODUCTION

Any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals or to develop microorganisms for specific uses is considered as biotechnological technique. Tissue culture, recombinant DNA technology, cloning, and genetic engineering are the important areas of biotechnology.

The potential application of biotechnological methods is of special significance in crop improvement since conventional methods involve several difficulties, including heterozygosity and a long span between successive generations; hence many investigators are devising methods whereby biotechnology could be fully exploited to improve crop varieties.

The role of biotechnology in crop improvement could be identified in four areas: (a) as an aid to conventional breeding programme; (b) as a tool of unconventional breeding programme; (c) in clonal propagation, and (d) in obtaining disease-free plants.

Before launching a large-scale programme of crop improvement through genetic engineering, it should be ensured that the method is economically viable.

II. TISSUE CULTURE AND CONVENTIONAL BREEDING

In a conventional breeding programme, a plant breeder comes across several obstacles. How plant breeder has advantageously used the techniques of tissue culture to overcome such obstacles is described in the following paragraphs.

A. Embryo Culture

Interspecific crosses may fail because of several reasons, but when the development of embryo is arrested owing to the degeneration of the endosperm, or when the embryo aborts at an early stage of development, embryo culture is the only technique to recover hybrid plants. Incidentally, embryo has been the first plant part successfully cultured in isolation. It was in 1904 that Hanning could rear seedlings from cruciferous embryo excised from immature pods.

B. Ovary and Ovule Culture

Embryos at very early stages of development have not been amenable to culture, primarily because of the problems involved in excision and the complex nutrient requirements. Where such a problem exists, it is logical to culture either the entire ovary or the ovule. This facilitates the availabil­ity of maternal tissue and eliminates the cumbersome problem of dissecting very young embryos and selecting a very exacting medium. The culture of pollinated ovaries and ovule in vitro has been successful in a number of plants.

C. Nucellus Culture

The culture of nucellar tissue has also yielded encouraging results and is now being employed in the improvement of Citrus crop. During recent years nucellus culture has been attempted in many vegetable and fruit crops towards clonal propagation of selected materials, e.g., Aegle marmelos, Cucumis melo, Luffa cylindrica, Trichosanthes anguina, Vitis vinifera. Nucellar culture also helps in the elimination of viruses, e.g., in Citrus species.

D. Control of Fertilization

In his attempts to evolve a desirable variety, the plant breeder is often confronted with several barriers. The common ones are: (a) disharmony in the flowering periods of the parents, (b) short life span of pollen, (c) failure of pollen germination, (d) slow growth of pollen tube, (e) inhibition or bursting of pollen tube in style, and (f) failure of male gametes to cause fertilization. Using the techniques of tissue culture, embryologists have successfully evolved methods for overcoming these barriers to crossability. For successful pollination and fertiliza­tion in vitro, the following considerations are important: (a) culture of pollen grains and ovules at the right stage of development, (b) a suitable nutrient medium, and (c) appropriate cultural conditions. Success has been achieved in Nicotiana tabacum, where the entire pistil is cultured and stigma polli­nated artificially. Consequent to such test-tube pollination, pollen germination, growth of pollen tubes, entry of tubes into ovules, fertilization, development of embryo and endosperm, viable seed-setting and seed germination take place in vitro. This technique has been used for cross pollination in N. rustica and Petunia violacea. In another method of ovule from un-pollinated ovaries are aseptically excised and cultured on a suitable nutrient medium. Pollen from freshly dehisced anthers is then dusted the next day on cultured ovules. The pollen grains germinate, and growth of pollen tube and fertilization are brought about followed by normal development of endosperm, embryo and seed.

E. Endosperm Culture

Endosperm, usually a triploid tissue in angiosperms, can be suitable ex-plant for raising trip­loids of cereals. The successful culture of en­dosperm tissue has opened up new avenues for plant breeders who can apply this technique to eco­nomically important crop plants, especially where conventional breeding methods prove futile.

III. TISSUE CULTURE: A TOOL OF NOVEL UNCONVENTIONAL BREEDING PROGRAMME

A. Anther Culture

In vitro gametic embryogenesis has become an efficient means of producing haploids in a growing number of species. Success in anther culture and more recently in isolated microspore culture has stimulated interest in this technology for plant breeding and applications in both monocots and dicot species of plants. The utility of haploid production in the rapid production of homozygous breeding lines from highly heterozygous parents is well recognized including reduced time requirements per cycle of plant breeding due to immediate phenotypic expression of the recessive traits in haploid derived homozygous plants.

For successful induction of androgenic haploids, the anthers should be cultured just before, or during or immediately following microspore mitosis. Most of the cultured anthers require an induc­tive phase, which depends on exposure to certain conditions of light and temperature for a particular period.

B.            Somatic Hybridization and Genetic Modification

Hybridization, involving both sexual fertilization and a variety of protoplast-protoplast and cytoplast-protoplast fusion have been successfully demonstrated in vitro in a few crop species. ‘Test-tube’ fertilization has been employed to help overcome pre- and post-fertilization cross-incompat­ibilities, while parasexual fusion have been employed to produce inter specific and intergenomic hy­brids, and recombine cytoplasmically inherited traits. Utilization of in vitro hybridization and fusion has been of a limited value in crop improvement, till date. Plant protoplasts cultures offer a potential system to achieve somatic hybridization and to cre­ate genotypically and cytoplasmically novel hybrids (hybrids and cybrids). Plant protoplasts also offer a system for genetic modification to attain the objective of crop improvement. This could be possible due to the remarkable property of plant protoplasts for the uptake of macro­molecules such as proteins, DNA, ferritin, latex, polystrene, virus, bacteria and even isolated chloro­plasts and whole nuclei.

C. Somaclonal Variation

In vitro cell and tissue cultures of plant give rise to genetic variation spontaneously. While spontaneous variation is not desired during propagule multiplication, it has been useful in providing some genetic variants among crop species. The observed frequencies of such somaclonal variants vary widely and are probably related to both time and culture and the observer’s ability to detect genetic variants. Several point mutations have been observed, the majority of analysed spontaneous genetic variants from somatic cultures appears to have resulted from induction of aneuploidy, loss or interchanges or intra-chromosomal segment duplication, deletions or structural rearrangements. Bet­ter cultivars have been produced in Citronella grass for higher biomass and oil contents.

Spontaneous or mutagen induced genetic variation in somatic cell culture coupled with in vitro selection techniques have been effective in isolating desired novel genetic variants with cellular level expression while in a heterozygous condition. Haploid cell cultures were reported to have higher frequencies of detectable variants than diploid cultures.

Among the numerous reports of in vitro variants, several may provide genetic variation useful for crop improvement. Modified tolerance to specific herbicide, altered composition, and modi­fied pest / stress tolerance if not associated with deleterious pleiotrophic effects (the production by a single gene, two or more apparently unrelated effects) will likely to emerge in commercial crop varieties in future.

IV. PROPAGATION THROUGH TISSUE CULTURE

A. Clonal Propagation

Clonal propagation by vegetative methods is a practice followed since man started cultivating plants. The main objective of clonal propagation has been to reproduce plants of selected, desirable qualities uniformly and in bulk. The tradition & propagation methods, requires long duration, whereas tissue culture helps in rapid plant multiplication.

V. PRODUCTION OF DISEASE-FREE PLANTS

A majority of the commercial crop plants propagated vegetatively through tubers, bulbs, cut­tings and grafting which may contain systemic bacteria, fungi and viruses. Such infections affect the yield and quality of the crop, unless it is readily detected and the plants made free from infection. The traditional method of eliminating viruses by heat treatment is applicable only to a few varieties. Generally, the apical meristem of a virus diseased plant contains very little virus, or is altogether free. Hence, plants obtained from apical meristem are usually free of viruses. There are a number of in­stances where meristem and/or floral bud culture has resulted in elimination of the virus and restora­tion of healthy plants e.g., Solanum tuberosum, Saccharum officinarum, Petunia spp, Allium sativum, Ananas comosus, Brassica oleracea, Musa sp and Pisum sativum.

The task of crop improvement by conventional methods is quite laborious and time-consum­ing, and poses many obstacles in the way of a plant breeder. The techniques of tissue culture offer a potential method to resolve these difficulties, and in many instances to shorten the time taken in crop improvement programmes. One of the important applications of tissue culture has been in clonal propagation, and in obtaining disease-free plants.

C. IMPACT ON HORTICULTURE

In vitro culture techniques have had numerous applications to fruit crops beginning nearly 60 years ago with embryo rescue techniques for stone fruits. Later on, the method has been applied successfully to produce commercially acceptable early-ripening peach and nectarine cultivars. The methods have been adapted to other crops as well, e.g. in breeding programmes to produce both early-ripening and seedless grapes.

Historically, the second application of in-vitro culture methods to fruit crops was to eliminate disease-causing viruses from strawberries. Meristem-tip culture has since become an integral part of virus-indexing programmes for a number of fruit crops, usually in conjunction with theromotherapy. In some cases, it has been necessary to micrograft the meristem tip into an in vitro-grown seedling as is done for citrus.

Within the past 25 years, micropropagation of fruit crops has become an important application of in vitro technology. Strawberry was the first fruit crop for which the method was developed. Now many fruit crops are being micropropagated commercially .

More recent uses of in vitro culture emphasize applications for genetic improvement of fruit crops. These applications include production of hybrid plants from fused protoplasts, somaclonal variation and mutagen-driven changes in regenerated plants, haploids plants from anther culture and transfer of specific genes via Agrobacterium-mediated transformation.

Followings methods are used to improve and multiply the horticultural crop plants.

1.  Micropropagation

2.  Virus elimination

3.  Genetic improvement

4.  Germplasm conservation

5. Haploid production

Micropropagation of ornamental plants (foliage and flower plants) has become a major plant tissue culture based industry in developed and developing countries.