There is an increase in the demand of fruits and vegetables as the population is increasing exponentially. In order to produce 360 mt of horticultural produce from current level of 150 mt by 2020, careful planning and application of newer tools of genetic engineering and biotechnology is required. Conventional plant breeding techniques have made considerable progress in development of improved varieties but it is not able to keep the pace with increasing demand for vegetables and fruits. Thus, there is a need to integrate biotechnology and genetic engineering to the conventional programs to speed up the crop and yield improvement of the horticultural crops by providing new varieties of plants and planting material, more efficient and selective pesticides and improved fertilizers. Developed countries have already adopted this structure of research and have already established many genetically modified crops in the market. Modern biotechnology encompasses broad areas of biology from utilization of living organisms or substances from those organisms to make or modify a product, to improve plant or animal or to develop micro-organisms for specific use. The major areas of biotechnology which can be adopted for crop and yield 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 and 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 variations are potentially useful to plant breeders.

II. Genetic Engineering of Plants

Genetic engineering primarily involves the manipulation of genetic material (DNA) to achieve the desired goal in a pre-determined way. The other terms in common use to describe genetic engineering are as follows:

Gene manipulation
Recombinant DNA technology
Gene cloning (molecular cloning)
New genetics

Genetic Engineering involves three major steps:

Identification and isolation of suitable genes for transfer
Delivery system to insert desired gene into recipient cells.
Expression of new genetic information in recipient cells.

Many molecular biology tools are used to carry out the genetic manipulation experiments. These DNA modifying molecules are as follows:

Restriction endonucleases(RE)- The DNA cutting enzymes, also called as molecular scissors. There are three major classes of REs, class I, II and class III, out of which class II REs are generally used in recombinant DNA technology.
DNA ligases- The DNA joining enzymes (T4-DNA ligase)
Linkers and adaptors- they are chemically synthesized, short, double stranded DNA molecules. Linkers possess RE cleavage sites. Adaptors contain cohesive or sticky ends.
Enzymes modifying the ends of DNA: (a) Alkaline phosphatase (That removes the terminal phosphate group) (b) Polynucleotide kinase (Involved in the addition of phosphate group (c) Terminal transferase (repeatedly adds nucleotides to any available 3′- terminal ends.
Polymerases- enzymes that catalyze the synthesis of nucleic acid molecules. These are of three types:
DNA dependent DNA polymerase
RNA dependent DNA polymerase
DNA dependent RNA polymerase

Plant genetic engineering basically deals with the transfer of desired gene (resulting in desired trait) from any source to a plant. The term transgene is used to represent the transferred gene, and the genetic transformation in plants is broadly referred as transgenic plants. Transgenic plants are developed by integrating the application of recombinant DNA technology, gene transfer methods and tissue culture technique. The ultimate goal of transgenics is to improve the crops, with the desired traits. Some of the desired traits are as follows:

Resistance to biotic stresses i.e. resistance to diseases caused by insects, viruses, fungi and bacteria.
Resistance to abiotic stresses- herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone, intense light.
Improvement of crop yield, and quality e.g. storage, longer shelf life of fruits and flowers.
Transgenic plants with improved nutrition
Transgenic plants as bioreactors for manufacture of commercial and therapeutic products e.g. proteins, vaccines and biodegradable plastics.

Transgenic plants have covered about 52.6 m hectares in the Industrial and developing countries. Genes for the following traits have been introduced to the crop plants.

Herbicide tolerance: Herbicides are chemicals used by the farmers to get the herbs removed, but it also deleteriously effects the crop plants. A transgenic plant resistant to herbicides allowing the farmers to spray crops so as to kill only weeds but not their crop will be of great help in yield improvement programs. 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 essential enzyme called EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) 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 to produce 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 packets 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 understood.  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 degrades 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): Immunological 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 finger-printing 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. Use of agriculturally important micro-organisms

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