India has emerged as a major player in the horticulture sector. Today, India is second largest producer of fruits and vegetables in the world. However, in order to produce 360 mt of horticultural produce from current level of 150 mt by 2020 requires careful planning and application of newer tools such as biotechnology. Many important fruit crops such as mango, papaya, litchi, guava, aonla, bael and many underutilized fruit crops are being grown in subtropical parts of India. However, the production of these crops is not up to the mark. Lack of genuine planting material, lack of characterization and documentation of germplasm of important crops, lack of suitable methods for controlling major pest diseases and post harvest losses of fruits are some of the bottlenecks in expansion of these fruit crops. Biotechnology can pave the way for molecular characterization of important fruit germplasm and molecular assisted breeding, mass multiplication of elite fruit varieties through micropropagation and development of transgenic crops resistant to biotic and abiotic stresses.
Transgenic Fruit Crops
According to a report of ISAAA, India is among the top ten countries in the world growing 50,000 hectares or more under transgenic crops. In addition to India, other developing countries are China, Indonesia, Argentina, Brazil, Mexico and South Africa has commercialized transgenic crops.
It is now being realized that the genetic base on which the conventional breeding is dependent is either shrinking or is not available because of crossability barriers. Hence, search for alternate strategies has become mandatory if the pace of horticultural growth has to be matched with the ever-increasing demand for fruit & vegetables. Fortunately, advancements made in recent years in the area of recombinant DNA technology have provided an altogether new dimension to horticultural research. Research on development of transgenic fruit crops has created interest globally. However except transgenic papaya resistant to viruses which has been commercialized in Hawaii, USA, none of the fruit or vegetable crops has reached to end user. Research on transgenic vegetables (brinjal, tomato, cabbage, cauliflower) using Bt gene is going on in ICAR as well as private labs. Research on transgenic papaya and banana is already carried out at ICAR Institute and private labs. Recently, Monsanto has announced that Transgenic papaya resistant to Papaya Ring Spot Virus will be developed in four years time.
There is need to utilize r-DNA technology in few commercial fruit crops for very specific traits such as resistance against biotic (pest, viruses, fungus) & abiotic (salt, moisture) stresses, nutritional quality etc. Genetic engineering has enabled successful transfer of bacterial gene i.e. delta endotoxin gene from Bacillus thuringiensis in to crops. Bt brinjal and tomato lines have been developed and field tested at IARI, New Delhi and multilocational trial is under progress. Similar work is already in advance stage at IVRI, Varanasi. Research over past two decades has provided a better understanding of the molecular biology of stress responses in plants. This has led to identification of several gene and gene product that are induced upon exposure to the plants to various abiotic stresses viz., drought, salinity and low and high temperature Recently, transgenic tomato ectopically expressing the Arabidopsis CBF1 gene showed enhanced resistance to drought, chilling and oxidative stress. Transgenic potato and tomato with osmotin and cod A genes are also potential prospects for elevated tolerance to abiotic stresses. In the past, it was possible to increase the quantity of food grains by conventional breeding methods; however, substantial improvement in nutritional quality of fruit and vegetables could not be achieved. Another avenue where improvement could not be attained through classical breeding is the post harvest management of fruit and vegetables. A post harvest loss of 10-30 % has been reported to occur in fruits and vegetables due to physical damage, pathological decay and over-ripening. One of the notable successes in Indian context is the transfer and expression of a gene encoding a protein with a balanced composition of all eight essential amino acids, Ama1 from Amaranthus in to potato to increase the nutritive value. Transgenic tomato and many other fruit crops are being currently produced with delayed ripening to save the post harvest losses that occur primarily due to over-ripening. This technology can also be employed in mango to reduce post harvest losses.
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 (table4). 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 with desired architecture
- Transgenic plants as bioreactors for manufacture of commercial products e.g. proteins, vaccines and biodegradable plastics.
Genetic transformation has opened new vistas to combat different stress related problems (biotic and abiotic) and also in improvement of the crop produced. It has also given hopes to produce edible vaccines in different fruits. Genetic transformation offers a fast approach towards hybrid development than conventional fruit breeding which faces many problems like long juvenility period, self-incompatibility, seedlessness, nucellar embryony, sterility, and requirement of relatively large land area.
Biotic stress tolerance:
Virus resistance: Virus resistance can be obtained by using the technique of PDR (Pathogen derived resistance) conferred by incorporating the coat protein expressing genes of the viral pathogens. Incorporation of viral non-structural genes (replicase, protease, movement proteins), interferon-related proteins have also shown evidence of viral resistance.
Bacterial resistance: Several naturally occurring antibacterial genes like attacin, chicken lysozyme, T4phage lysozyme, P22 phage gene 13 and 19, lactoferrin and Cecropein B or synthetic gene –SB37 (Shiva series) are used as transgenes to confer bacterial resistance in plants. Bacterial blight diseases can be controlled by using this technology.
Insect-Pest resistance: Genes expressing the insecticidal proteins (Cry genes) have been isolated from Bacillus thuringiensis. Cry protein expressing gene CRYA(c) is generally used to transform plants to confer resistance against Lepidopteran, Dipteran and Coleopteran insect larvae. Btk-ICP gene (Bacillus thuringiensis var. kurstak gene encoding the toxin HD 73) is also being incorporated in cranberry and has conferred resistance against a wide range of insect larvae.
Abiotic stress tolerance: Several transgenes are used to confer heat tolerance and oxidative stress. Osmoprotectant hyper accumulator genes are used to obtain salt, chill and freeze resistant plants.
Ripening related genes: Several genes which regulate the ripening in fruits have been identified and they have been cloned in pTOM series vectors to be used to generate transformants which show controlled ripening.
Current scenario of transgenics technology in various fruit crops:
Apple: Successful transformation has been accomplished in apple using gus, nptII, nopaline synthase gene through Agrobacterium tumefaciens. Successful integration of following genes has been accomplished:
- Cry1A from Bacillus thuringiensis to confer resistance against insect larvae.
- ICP expressing ipt gene –encoding for iso-pentyl transferase, the first enzyme in cytokinin biosynthetic pathway.
- Als gene (acetolactate synthase gene) to confer resistance herbicide Glean.
- Ac-AMP2, Mj- AMP2, RS-2S albumin and Rs-AFP2to express various anti microbial proteins were expressed under CaMV 35-S promoter.
Pear: Successful transformation of pear has been accomplished by nptII and gus genes; up to 42 % of the inoculated explants produced transformed buds. ‘Conference’, ‘Doyenne du comice’, and ‘Passe Crassane’ cultivars has been successfully transformed and acclimatized. Success has also been achieved for quince (Cydonia oblonga L.) rootstock.
Apricot: It has been successfully accomplished with gus and PPV-CP via Agrobacterium-mediated transformation. The integration of PPV-CP gene into apricot genome has been demonstrated using gus staining assay and PCR.
Cherry: Transformation of cherry is being carried out inItaly, successful regeneration of transformants has been accomplished from somatic tissues of Vittoria cherry (Prunus avium) and ‘Colt’ root stock. In Russia, scientists have obtained high frequency (50-60%) of transgenic sour cherry calli with a high activity of nptII and nopaline synthesis.
Peach: Transformation experiments are going on in peach via Agrobacterium tumefaciens. The regeneration of transformants was a limiting step which was overcome by using a shooty mutant strain of A.tumefaciens. microprojectile mediated transformation protocol has also been established in peach, which has been confirmed by PCR and gus assays.
Plum:Protocols have been established for successful integration and constitutive expression of the transgene using Agrobacterium tumefaciens. Prunus domesticus has been transformed with CP gene (Coat Protein gene) of plum pox virus (PPV) using Agrobacterium . The expression of the PPV-CP immunoreactive protein and western blotting.
Walnut and Pecan: Somatic embryos of walnut has been transformed by using Agrobacterium. Integration of gus, nptII and the Cry1A protein into the walnut genome has been confirmed by using Southern blot. The genetic transformation system used in walnut has been successfully applied to pecan plants, confirmed by S.blot and PCR, but the success was genotype dependent.
Tamarillo: Atkinson and Gardner (1993) reported transformation of Tamarillo using Agrobacterium. The integration of nptII, gus and als genes were confirmed by PCR, Southern hybridization and the inheritance of kanamycin resistance.
Mango: Co-cultivation of embryogenic cultures with Agrobacterium resulted in formation of transgenic mango (Mathews et.al., 1992). A prolonged selection protocol is required to eliminate the chimeric clumps. Kanamycin resistant mango embryos expressing the gus gene were developed. The plants failed to be acclimatized due to non-functional root formation.
Grapes: studies to develop a successful transformation protocol in grapes started way back in 1985 with the efforts of Hemstad and Reisch. First transgenic plant in grapevine root stock Vitis rupestris was made by Mullins et.al. (1990) with confirmation by Gus, nptII and southern blot analysis. Later on following genetic manipulations have been successfully achieved in grapevine:
- Incorporation of GCMV-CP (Coat protein encoding gene from Grapevine chrome mosaic virus) using Agrobacterium to confer resistance to GCM virus. Expression was confirmed by ELISA and W.Blot.
- Successful integration and expression of GFLV-CP (coat protein encoding gene from Grapevine fan leaf virus) using Arobacterium tumefaciens.
Citrus: transformation in citrus was achieved by using PEG (polyethylene glycol) mediated direct DNA transfer protocol. Vardi et.al.(1990) has successfully produced rooted transgenic plants of citrus expressing nptII. Transgenic plants were confirmed by nptII activity measurement. Agrobacterium mediated genetic transformation was established when Hidaka et.al. (1990) produced transformants expressing nptII gene. Transgenic calli were reported to be produced using PEG mediated direct transfer of DNA. Following these experiments, CTV-CP gene (Coat protein gene from Citrus tristeza virus) have been successfully integrated and transgenic plants were produced. Kobayashi et al. (1996) obtained transgenic plants of trifoliate orange expressing the gene encoding Human epidermal growth factor (HEGF) under the control of 35S-CAMV promoter. Cultivar specific regeneration and gene transfer protocols are required in citrus.
Papaya: Transformation studies in papaya began in late 80’s when Pang and Sanford (1988) produced stable transformed calli using Agrobacterium infection in leaf discs. In 1990, Fitch et.al. produced the first transgenic papaya plant using biolistics (microprojectile particles coated with the gene of interest). Transgenic papaya plants were developed to confer resistance against PRSV (papaya ring spot virus) using PRSV-CP as the transgene . Tennant et.al. (1992) reported that transgenic papaya plants expressing the PRSV-CP exhibited differential protection against PRSV. Transgenic papay plants expressing bar, gus and nptII geges have been produced using zygotic embryos and embryogenic callus as target cells for particle bombardment (Cabrera-Ponce et. al., 1995). In 1996, transgenic papaya plants were obtained by using Agrobacterium rhizogenes mediated genetic transformation (Cabrera-Ponce et.al.,1996). Yang et.al.(1996) obtained transgenic papaya plants using Agrobacterium tumefaciens. Cheng et.al. (1996) obtained transformed plants by wounding the explant using Carborendum prior to Agrobacterium infection. This protocol increased the transformation efficiency to 15.9%. Currently in India at Central Institute for Subtropical Horticulture (CISH), attempts are being made to produce transgenic papaya plants expressing PRSV-CP and PaLCuV-Rep genes (Replicase gene from Papaya leaf curl virus) to combat PRSV and PaLCuV (papaya Leaf Curl Virus) infections.
Guava: Attempts are being made to standardize the transformation protocol for guava plants. In 2007, Biswas et.al. reported the need for development of transgenic guava to ensure cold hardiness enhancement in guava. In India, at CISH, Chandra et.al. has developed a technique to transform guava shoot buds using Agrobacterium tumefaciens mediated genetic transformation. Transformation efficiency was increased when wounding was done by bombarding tungsten (0.6-1.0 µ) microprojectile particles using gene gun HE-GenePro2000.
Cervera, M., Pina, J.A., Juárez, J., Navarro, L. and Peña, L.. (1998) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Reports 18: 271-278. DOI: 10.1007/s002990050570
Cheng, Y.H., Yang, J., and Yeh, S. (1996). Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with caborundum. Plant Cell Reports 16:127-132. DOI: 10.1007/BF01890852
James, D.J., Passey, A.J., Webster, A.D.,Barbara, D.J., Dandekar, A.M., Uratsu, S.L. and Viss, P. (1993). Transgenic apples and strawberries: advances in transformation, introduction of genes for insect resistance and field studies of tissue cultured plants. Acta Horticulturae, 336:179-182
Mathews, H., Wagoner, W., Cohen, C., Kellogg, J. and Bestwick, R. (1995). Efficient genetic transformation of red raspberry, Rubus ideaus L. Plant Cell Reports 14:471-476. DOI: 10.1007/BF00232777
McGranahan, G.H., Leslie, C.A., Dandekar, A.M., Uratsu, S.L., and Yates, I.E. (1993). Transformation of pecan and regeneration of transgenic plants. Plant Cell Reports 12 :634-638. DOI: 10.1007/BF00232814
Peña, L., Cervera, M., Juárez, J., Navarro, A., Pina, J.A., Durán-Vila, N. and Navarro L. (1995). Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Reports 14:616-619. DOI: 10.1007/BF00232724
Scorza, R., Cordts, J.M., Ramming, D.W. and Emershad, R.L. (1995). Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants. Plant Cell Reports 14: 589-592. DOI: 10.1007/BF00231944
Yao, J.L., Cohen, D., Atkinson, R., Richardson, K. and Morris, B. (1995). Regeneration of transgenic plants from the commercial apple cultivar Royal Gala. Plant Cell Reports 14:407-412. ISSN: 0721-7714 (Print) 1432-203X (Online)