Applications of Biotechnology in Horticulture

Introduction

The requirement of fruits and vegetables is increasing proportionally with the increasing population in the country. How do we keep horticultural production on par with the burgeoning population? 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 revolutionized the entire crop improvement programmes by providing new strains of plants, supply of planting material, more efficient and selective pesticides and improved fertilizers. Many genetically modified fruits and vegetables are already in the market in developed countries. Modern biotechnology encompasses broad areas of biology from utilization 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 diagnostics and
Molecular markers
Development of Beneficial microbes

I. Tissue Culture:

One of the widest applications of biotechnology has been in the area of tissue culture and micro propagation 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. Embryo rescue is another area where plant breeders are able to rescue their 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. Many of the dry land legume species have been successfully regenerated from cotyledons, hypocotyls, leaf, ovary, protoplast, petiole root, anthers, etc., Haploid generation through anther/pollen culture is recognized as another important area in crop improvement. It is useful in being rapid and economically feasible. Complete homozygosity of the offspring helps in phenotype selection for quantitative characters and particularly for qualitatively inherited characters making breeding much easier successful isolation, culture and fusion of plant protoplasts has been very useful in transferring cytoplasmic male sterility for obtaining hybrid vigour through mitochondrial recombination and for genetic transformation in plants.

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.

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 variation are potentially useful to plant breeders.

II. Genetic Engineering of Plants

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 upto 2001. Genes for the following traits have been introduced to the crop plants.

Herbicide tolerance: Transgenic plants are developed that are resistant to herbicides allowing farmers to spray crops so as to kill only weeds but not their crops. Many herbicide tolerant plants have been developed in tomato, tobacco, potato, soybean, cotton, corn oilseed rape, petunia, etc. Glyphosate is one of the most potent broad spectrum environment friendly herbicide known, it is marketed under the trade name Round up. Glyphosate kills plants by blocking the action of an enzyme (5-enolpyruvyl shikimate-3-phosphate synthase) (EPSPS) an essential enzyme in the biosynthesis of aromatic amino acids, tyrosine, phenylalanine and tryptophan. Amino acids are building blocks of protein. Transgenic plants resistant to Glyphosate have been developed by transferring gene of EPSPS that over prodoce this enzyme thus inhibiting the effect of Glyphosate. A number of detoxifying enzymes have been identified in plants as well as in microbes. Some of these include glutahthione-s-transferase or GST in maize and other plants which detoxifies the herbicide bromoxynil and phosphinothricin acetyl transferase (PAT) which detoxifies the herbiside PPT (L-phosphinothricine). Transgenic plants using bxn gene from Klebsiella and bar gene from Strepotomyces have been obtained in potato, oilseed, sugarbeet, soybean, cotton and corn and are found to be herbicide resistance. These transgenic plants reduce the use of weeding labour, farmers cost and increase yield.

Engineering pathogen resistance:   Viruses are the major pests of crop plants which cause considerable yield losses. Many strategies have been applied to control virus infection using coat protein and satellite RNA. Viruses are submicroscopic pockets of nucleic acid (DNA or RNA) enclosed in a protein coat and can multiply within a host cell. Use of viral coat protein as a transgene for producing virus resistant plants is one of the most spectacular successes achieved in plant biotechnology. Coat protein gene from tobacco mosaic virus (TMV) classified as a positive strand RNA virus has been transferred to tobacco, making it nearly resistant against TMV. Using gene for nucelocapsid protein resistance has been introduced in crops like tomato, tobacco, lettuce, groundnut, pepper and in ornaments like Impatiens, Ageratum and Crysnathemum against tomato spotted wilt virus. Use of satellite RNA (SATRNA) makes many transgenic plants resistant to Cucumber Mosaic Virus (CMV). Transgenic resistant plants have also been developed against alfalfa mosaic virus, potato virus X, Rice tungro virus, tobacco rattle virus and Papaya ring spot virus.

During the last decade many resistance genes whose products are involved in recognizing the invading pathogens have been identified and cloned. A number of signaling pathways which follow the pathogen infection have been dissected. Many of the antifungal compounds synthesized by plants which combat fungal infections have been identified. The major strategies for developing fungal resistance have been production of transgenic plants with antifungal molecules like proteins and toxins, and generation of hypersensitive response through R genes or by manipulating genes of SAR pathway. A chitinase gene from bean plants in tobacco and Brassica napus showed enhanced resistance to Rhizoctonia solani. In another case chitinase gene obtained from Serratia marcescens (soil bacterium) is introduced in tobacco making it resistant to Alternaria longipes which causes brown spot diseases.    Acetyl transferase gene is introduced in tobacco making it resistant to Pseudomonas syringea, a causal agent of wild fire disease.

Stress resistance : A number of genes responsible for providing resistance against stresses such as to water stress heat, cold, salt, heavy metals and phytohormones have been identified. Studies are also being conducted on metabolites like proteins and betains that have been implicated in stress tolerance.   Resistance against chilling was introduced into tobacco plants by introducing gene for glycerol-1-phosphate acyl-transferase enzyme from Arabidopsis. Many plants respond to drought stress by synthesizing a group of sugar derivatives called polyols (Mannitol, Sorbitol and Sion) . Plants that have more polyols are more resistant to stress. Using a bacterial gene capable of synthesizing mannitols it is possible to raise the level of mannitol very high making plants resistant to drought.

Fruit Quality: Tomatoes which ripen slowly are helpful in transportation process. Transgenic tomato with reduced pectin methyl esterase activity and increased level of soluble solids and higher pH increases processing quality. Tomatoes exhibiting delayed ripening have been produced either by using antisense RNA against enzymes involved in ethylene production (Eg ACC synthase) or by using gene for deaminase which degraded l-aminocyclopropane-l-carboxylic acid (ACC) an immediate precursor of ethylene. This increases the shelf life of tomatoes. These tomatoes can also stay on the plant long giving more time for accumulation of sugars and acids for improving flavour. It is produced at commercial level in European and American countries. Tomatoes with elevated sucrose and reduced starch could also be produced using sucrose phosphate synthase gene. Starch content in potatoes has been increased by 20-40% by using a bacterial ADP glucose pyrophosphorylase gene.

Pest resistance : The insecticidal beta endotoxin gene (bt gene) has been isolated from Bacillus thuringiensis the commonly occurring soil bacteria and transferred to number of plants like cotton, tobacco, tomato, soybean, potato, etc. to make them resistant to attack by insects. These genes produce insecticidal crystal proteins which affect a range of lepidopteran, coleopteran , dipteran insects. These crystals upon ingestion by the insect larva are solubilised in the highly alkaline midgut into individual protoxins which vary from 133 to 136 kDa in molecular weight. Insecticidal crystal protein produced during vegetative growth of the cells (VIP)are also found to be highly effective against insect control. Bt resistant plants are already in the market.

Male sterility and Fertility restoration: This is helpful in hybrid seed production. Transgenic plants with male sterility and fertility restoration genes have become available in Brassica napus. It facilitates production of hybrid seed without manual emasculation and controlled pollination as often done in maize. In 1990, Mariani and others from Belgium have successfully used a gene construct having another specific promoter from TA29 gene of tobacco and bacterial coding sequence for a ribonuclease gene from Bacillus Sp. (barnase gene) for production of transgenic plants in Brassica napus. Here the translated gene prevented normal pollen development leading to male sterilily.

III. Molecular Diagnostics

Nucleic acid probes:- It is now possible to detect the plant diseases even before onset of symptoms by using cDNA probes. Probes are nucleic acid sequences of pathogen causing organisms labeled with certain markers. cDNA probes corresponding to specific regions of the pathogens can be generated using standard recombinant DNA technique.

Monoclonal antibodies (McAb): Immunochemical techniques are extremely useful for the rapid and accurate routine detection of plant pathogens and ultimately the diagnosis of plant disease and their relatedness, The introduction of hybridoma technology has provided methods for the production of homologous and biochemically defined immunological reagents of identical specificity which are produced by a single cell line and are directed against a unique epitope of the immunizing antigen. The great potential of McAbs in phytopathological diagnostics is essential because of homogeneous antibody preparations with defined activity and specificity can be produced in large quantities over long periods. Even though hybridoma technology is a laborious and expensive enterprise compared to standard immunization procedures it is going to be widely used for large scale diagnosis.

IV. Molecular Markers

The possibilities 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. 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. Molecular maps now exist for a number of crop plants including corn, tomato, potato, rice, lettuce, wheat, Brassica species and barley. 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.
V. Development of Microbial Inoculan

Indiscriminate and injudicious use of chemical fertilizers and pesticides for the crop production and control of insect-pests has resulted in pollution of the environment deterioration of soil health and development of resistance by many insects and residue problems. Hence there is a great concern world wide to use safer biofertilisers and biopesticdies in the integrated nutrient management and pest management systems.

Biofertilizers are micro-organisms which fix atmospheric nitrogen or solubilise fixed phosphorus in the soil and make more nutrients available to the plant. Some of the organisms providing major inputs are the biological nitrogen fixing organisms like Rhizobium, Azotobacter, Azospirillum and phosphate solubilising organisms like Bacillus polymyxa, B. magaterium, Pseudomonas striata and certain fungal species of Aspergillus and Penicillium.
The benefits of using micro-organisms as fertilizers are many fold. They are less expensive, nontoxic to plants, do not pollute the ground water nor render the soil acidic and unfit for growth of plants. Rhizobium forms nodules on the roots of leguminous plants and help in fixing nitrogen from the atmosphere to ammonium irons which get converted to amino acids in the plant system. Inoculation with this bacteria helps in reducing addition of nitrogenous fertilizers to the soil. Azospirillum is also found colonizing inter cellular spaces inside the root system. These bacteria also contribute substantially to the nitrogen requirement of the plant.

Phosphate solubilising bacteria are another group of micro-organisms which solubilise the insoluble phosphorus in the soil and make them readily available to the crop.

Mycorrhiza is the symbiotic association of the roots of crop plants with non-pathogenic fungus. They provide nutrients absorbed from deeper layers of soil to the plants. They help the plants in better plant establishment and growth when inoculated. Many fruit crops like papaya, mango, banana, citrus, pomegranate are found to be dependent on this association and are greatly benefited by its inoculation in procuring higher phosphate and other nutrient from the soil. These mycorrhizal associations help the plants in overcoming pathogen attack also. They improve soil characters too.

Genetic modification of microbes: By using DNA recombination technique it has been possible to genetically manipulate different strains of these bacteria suitable to different environmental conditions and to develop strains with traits with capacity for better competitiveness and nodulation.

Biopesticides are biological organisms which can be formulated as that of the pesticides for the control of pests. Biopesticides are gaining importance in agriculture, horticulture and in public heatlh programmes for the control of pests. The advantages of using biopesticides are many. They are specific to target pests and do not harm the non target organisms such as bees, butterflies and are safe to humans and live stocks, they do not disturb the food-chain nor leave behind toxic residues.

Some of the microbial pesticides used to control insect pests are Bacillus thuringiensis species to control various insect pests. Insecticidal property of these bacteria are due to crystals of insecticidal proteins produced during sporulation. These proteins are stomach poisons and are highly insect specific. Bt toxins could kill plant parasitic nematode too. Number of baculoviruses (BV) nuclear polyhedrosis virus (NPV) is being developed as microbial pesticides both nationally and internationally, A few examples of these are Heliothis, Spodoptera, Plusia, Agrotis, Trichoplusia, etc.

Biocontrol agents : These are other microbes which are antagonistic to several pathogenic fungus and are good substitutes to fungicides or insecticide. These are Bacillus sps. Pseudomonas fluorescens, Trichoderma, Verticillium sp., Streptromyces sps. etc. These organisms are commercially available.

The extent of commercial application of plant biotechnology is the important mark for measuring the vitality of this newly emerging technology. Small and marginal farmers can adopt less expensive technologies like the use of biofertilizers and biopesticides while capital intensive technologies can be adopted by rich farmers

Dr Sukhada Mohandas, Indian Institute of Horticultural Research, Hessaraghatta,
Bangalore 560 089.