India has emerged for a major player in the horticulture sector. Today, India is definitely second largest producer involving fruits and vegetables in the world. However, in an effort to produce 360 mt of gardening produce from current amount of 150 mt by 2020 requires meticulous planning and application of newer gear such as biotechnology. Many essential fruit crops such as mango, papaya, litchi, guava, aonla, bael and many underutilized fruit harvest are being grown in subtropical aspects of India. However, the production of these crops is not up to the mark. Deficiency of genuine planting material, deficiency of characterization and documentation associated with germplasm of important crops, deficit of suitable methods for controlling major pest diseases and submit harvest losses of fruits and veggies are some of the bottlenecks in expansion of them fruit crops. Biotechnology can certainly pave the way for molecular characterization of essential fruit germplasm and molecular assisted mating, mass multiplication of elite fruit varieties through micropropagation and continuing development of transgenic crops resistant to biotic and abiotic strains.

Transgenic Fruit Crops

According to a study of ISAAA, India is among the top countries in the world growing 60,000 hectares or more under transgenic herbs. In addition to India, other building countries are China, Australia, Argentina, Brazil, Mexico and Nigeria has commercialized transgenic crops.

It is now being realized that the genetic basic on which the conventional breeding would rely is either shrinking or possibly is not available because of crossability barriers. For this reason, search for alternate strategies is becoming mandatory if the pace with horticultural growth has to be coordinated with the ever-increasing demand for fruit & fresh vegetables. Fortunately, advancements made in recent times in the area of recombinant DNA technologies have provided an altogether new element to horticultural research. Study on development of transgenic fruit plants has created interest globally. Nevertheless except transgenic papaya resistant to worms which has been commercialized in Hawaii, U . s ., none of the fruit or vegetable crops has got reached to end user. Study on transgenic vegetables (brinjal, tomato, cabbage, cauliflower) employing Bt gene is going on in ICAR as well as private labs. Research on transgenic papaya and banana is already completed at ICAR Institute and private labs. Recently, Monsanto has announced in which Transgenic papaya resistant to Papaya Wedding ring Spot Virus will be coded in four years time.

There is must utilize r-DNA technology in handful of commercial fruit crops regarding very specific traits like resistance against biotic (pest, viruses, candida) & abiotic (salt, moisture) stresses, health quality etc. Genetic anatomist has enabled successful transfer of bacterial gene i.e. delta endotoxin gene out of Bacillus thuringiensis in to crops. Bt brinjal and tomato strains have been developed and subject tested at IARI, New Delhi in addition to multilocational trial is under improvement. Similar work is already beforehand stage at IVRI, Varanasi. Research over past two decades has offered a better understanding of the molecular chemistry and biology of stress responses inside plants. This has led to identity of several gene and gene product that are induced upon exposure to the plants to various abiotic stresses viz., drought, salinity and low and temperature Recently, transgenic tomato ectopically expressing the Arabidopsis CBF1 gene proved enhanced resistance to drought, relaxing and oxidative stress. Transgenic potato and tomato with osmotin and cod A new genes are also potential prospects regarding elevated tolerance to abiotic stresses. Previously, it was possible to increase the amount of food grains by regular breeding methods; however, substantial improvement in nutritional good quality of fruit and vegetables could not be exercised. Another avenue where development could not be attained via classical breeding is the write-up harvest management of fruit and vegetables. A post harvest loss of 10-30 % may be reported to occur in vegatables and fruits due to physical damage, pathological weathering and over-ripening.  One of the notable achievements in Indian context is definitely the transfer and expression of a gene encoding a protein that has a balanced composition of all 8 essential amino acids, Ama1 from Amaranthus in potato to increase the nutritive cost. Transgenic tomato and many other fruit crops are being currently produced together with delayed ripening to save a post harvest losses in which occur primarily due to over-ripening.  This technology can also be employed in mango to scale back post harvest losses.

Plant genetic engineering basically works with the transfer of sought after gene (resulting in desired trait) from any source to a plant. The word transgene is used to represent this transferred gene, and the genetic alteration in plants is greatly referred as transgenic plants. Transgenic vegetation is developed by integrating the application of recombinant Genetic make-up technology, gene transfer methods along with tissue culture technique. The supreme goal of transgenics is to enhance the crops, with the desired qualities (table4). Some of the desired traits are highlighted below:

Resistance to biotic stresses i.at the. resistance to diseases caused by insects, viruses, fungi and microbes.

Resistance to abiotic stresses- herbicides, temperature (warmth, chilling, freezing), drought, salinity, ozone, rigorous light.

Improvement of bounty yield, and quality at the.g. storage, longer lifespan of fruits and roses.

Transgenic plants with improved healthy eating plan

Transgenic plants with desired design

Transgenic plants as bioreactors for output of commercial products e.grams. proteins, vaccines and recyclable plastics.

Genetic transformation features opened new vistas to combat different stress related challenges (biotic and abiotic) and also in improvement on the crop produced.  It has as well given hopes to produce delicious vaccines in different fruits. Anatomical transformation offers a fast method towards hybrid development when compared with conventional fruit breeding that faces many problems including long juvenility period, self-incompatibility, seedlessness, nucellar embryony, sterility, and feature relatively large land area.

Biotic stress tolerance:

Virus weight: 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 infections. Incorporation of viral non-structural passed dow genes (replicase, protease, movement proteins), interferon-related proteins have likewise shown evidence of viral resistance.

Bacterial resistance: Several natural antibacterial genes like attacin, fowl lysozyme, T4phage lysozyme, P22 phage gene 13 and 19, lactoferrin along with Cecropein B or synthetic gene –SB37 (Shiva collection) are used as transgenes to consult bacterial resistance in crops. Bacterial blight diseases could be controlled by using this technology.

Insect-Pest weight: Genes expressing the insecticidal proteins (Shout genes) have been isolated coming from Bacillus thuringiensis. Cry protein expressing gene CRYA(g) is generally used to transform flowers to confer resistance against Lepidopteran, Dipteran in addition to Coleopteran insect larvae.  Btk-ICP gene (Bacillus thuringiensis var. kurstak gene encoding a toxin HD 73) is also being incorporated in cranberry and has now conferred resistance against a wide range of insect larvae.

Abiotic stress tolerance: Several transgenes are employed confer heat tolerance and also oxidative stress.  Osmoprotectant hyper accumulator genes are employed obtain salt, chill plus freeze resistant plants.

Ripening related genes: Several genes which will regulate the ripening throughout fruits have been identified and they’ve got been cloned in pTOM series vectors to be used to generate transformants which present controlled ripening.

 

Current case of transgenics technology in various fruit crops:

Apple: Successful transformation continues to be accomplished in apple working with gus, nptII, nopaline synthase gene through Agrobacterium tumefaciens. Successful integration of following genes has been achieved:

Cry1A from Bacillus thuringiensis to confer immune to insect larvae.

ICP expressing ipt gene –encoding to get iso-pentyl transferase, the first enzyme in cytokinin biosynthetic pathway.

Als gene (acetolactate synthase gene) to confer resistance herbicide Discover.

Ac-AMP2, Mj- AMP2, RS-2S albumin and Rs-AFP2to express various stop microbial proteins were expressed under CaMV 35-S promoter.

Pear: Successful transformation of pear has been accomplished by nptII and gus genes; up to 45 % of the inoculated explants produced modified buds. ‘Conference’, ‘Doyenne du comice’, and ‘Passe Crassane’ cultivars has been effectively transformed and acclimatized. Success has been specifically achieved for quince (Cydonia oblonga L.) rootstock.

Apricot: It is often successfully accomplished with gus and also PPV-CP via Agrobacterium-mediated transformation. The intergrated , of PPV-CP gene into apricot genome has been revealed using gus staining assay plus PCR.

Cherry: Transformation of cherry is being carried out inItaly, successful regeneration of transformants may be accomplished from somatic tissues of Vittoria cherry (Prunus avium) and ‘Colt’ root stock. Inside Russia, scientists have obtained substantial frequency (50-60%) of transgenic sour cherry calli by using a high activity of nptII and nopaline synthesis.

Peach: Transformation findings are going on in apple via Agrobacterium tumefaciens. The regeneration connected with transformants was a limiting step which has been overcome by using a shooty mutant strain of your.tumefaciens. microprojectile mediated transformation protocol has also been recognized in peach, which has been confirmed by PCR and gus assays.

Plum:Protocols have been in existence for successful integration as well as constitutive expression of the transgene  using Agrobacterium tumefaciens. Prunus domesticus has become transformed with CP gene (Layer Protein gene) of plum pox virus (Pay per view) using Agrobacterium . The expression of the PPV-CP immunoreactive protein and western blotting.

Walnut and Pecan: Somatic embryos of walnut has become transformed by using Agrobacterium. Integration involving gus, nptII and the Cry1A protein into the maple genome has been confirmed by using Southern blot. The genetic change for better system used in walnut is successfully applied to pecan plants, confirmed by S.blot in addition to PCR, but the success was genotype reliant.

Tamarillo: Atkinson and Gardner (1993) reported change for better of Tamarillo using Agrobacterium. The incorporation of nptII, gus and als genes have been confirmed by PCR, Southern hybridization and the inheritance of kanamycin resistance.

Pear: Co-cultivation of embryogenic cultures with Agrobacterium ended in formation of transgenic mango (Mathews et.alabama., 1992). A prolonged selection project is required to eliminate the chimeric clumps.  Kanamycin repellent mango embryos expressing the gus gene were being developed. The plants still did not be acclimatized due to non-functional root creation.

Grapes: studies to develop a successful transformation protocol in grapes started way back in 1985 with the efforts of Hemstad and also Reisch. First transgenic plant in grape vine root stock Vitis rupestris was made by means of Mullins et.al. (1990) with confirmation by Gus, nptII and southern blot evaluation. Later on following genetic manipulations happen to be successfully achieved in grape vine:

Incorporation of GCMV-CP (Coat health proteins encoding gene from Grapevine stainless- mosaic virus) using Agrobacterium in order to confer resistance to GCM virus. Manifestation was confirmed by ELISA in addition to W.Blot.

Successful is intergrated and expression of GFLV-CP (jacket protein encoding gene from Grapevine fan leaf virus) using Arobacterium tumefaciens.

Citrus fruit: transformation in citrus was achieved by using PEG (polyethylene glycol) mediated direct Genetic transfer protocol. Vardi et.al.(1990) has successfully produced rooted transgenic plants of citrus expressing nptII. Transgenic flowers were confirmed by nptII action measurement. Agrobacterium mediated genetic transformation was established when Hidaka et.al. (1990) made transformants expressing nptII gene. Transgenic calli were reported to be developed using PEG mediated  direct transfer involving DNA. Following these tests, CTV-CP gene (Coat protein gene from Lemon or lime tristeza virus) have been successfully built-in and transgenic plants were developed. Kobayashi et al. (1996) obtained transgenic flowers of trifoliate orange expressing this gene encoding Human epidermal development factor (HEGF) under the control of 35S-CAMV promoter. Cultivar specific  regeneration and gene transfer methodologies are required in citrus.

Pawpaw: Transformation studies in papaya began in late 80′s when Pang and Sanford (1988) produced stable transformed calli using Agrobacterium infection in leaf backbone. In 1990, Fitch et.al. produced the very first transgenic papaya plant using biolistics (microprojectile particles coated with the gene of interest). Transgenic pawpaw plants were developed to consult resistance against PRSV (papaya ring spot virus) using PRSV-CP as the transgene .  Tennant et.. (1992) reported that transgenic papaya plants expressing the PRSV-CP demonstrated differential protection against PRSV. Transgenic  papay plants expressing bar, gus and nptII geges have been developed using zygotic embryos and embryogenic callus as  target tissues for particle bombardment (Cabrera-Ponce et. al., 1997). In 1996, transgenic papaya vegetation were obtained by using Agrobacterium rhizogenes mediated ancestral transformation (Cabrera-Ponce et.al.,1996). Yang et.ing.(1996) obtained transgenic papaya facilities using Agrobacterium tumefaciens. Cheng et.al. (1996) acquired transformed plants by wounding the explant using Carborendum prior to Agrobacterium infection. This particular protocol increased the modification efficiency to 15.9%.  Currently within India at Central Company for Subtropical Horticulture (CISH),  attempts are increasingly being made to produce transgenic papaya vegetation expressing PRSV-CP and PaLCuV-Rep genes (Replicase gene out of Papaya leaf curl virus) to be able to combat PRSV and PaLCuV (papaya Leaf Snuggle Virus) infections.

Guava: Attempts are now being made to standardize the transformation project for guava plants. In 3 years ago, Biswas et.al. reported the need for continuing development of transgenic guava to ensure cold hardiness enhancement within guava. In India, at CISH, Chandra et.ing. has developed a technique to transform guava blast buds using Agrobacterium tumefaciens mediated genetic alteration. Transformation efficiency was enhanced when wounding was done by junk mail tungsten (0.6-1.0 µ) microprojectile particles employing gene gun HE-GenePro2000.

 

 

Reference:

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Cheng, Y.., Yang, J., and Yeh, S. (96). Efficient transformation of papaya by coat protein gene regarding papaya ringspot virus mediated by Agrobacterium next liquid-phase wounding of embryogenic tissues with caborundum. Flower Cell Reports 16:127-132. DOI: 13.1007/BF01890852

 

James, D.J., Passey, Your.J., Webster, A.D.,Barbara, Deb.J., Dandekar, A.M., Uratsu, Ohydrates.L. and Viss, P. (1993). Transgenic apples and strawberries: breakthroughs in transformation, introduction regarding genes for insect resistance and field studies of tissue cultured plants. Acta Horticulturae, 336:179-182

 

Mathews, K., Wagoner, W., Cohen, C., Kellogg, J. and Bestwick, R. (1995). Efficient genetic transformation of red raspberry, Rubus ideaus L. Plant Cell Records 14:471-476. DOI: 10.1007/BF00232777

 

McGranahan, G.L., Leslie, C.A., Dandekar, Any.M., Uratsu, S.L., plus Yates, I.E. (1993). Transformation of pecan and regeneration regarding transgenic plants. Plant Cell Reviews 12 :634-638. DOI: 10.1007/BF00232814

 

Peña, L., Cervera, M., Juárez, J., Navarro, A., Pina, J.A new., Durán-Vila, N. and Navarro L. (1996). Agrobacterium-mediated transformation of sweet fruit and regeneration of transgenic crops. Plant Cell Reports Fourteen:616-619. DOI: 10.1007/BF00232724

 

Scorza, R., Cordts, J.L., Ramming, D.W. and Emershad, S.L. (1995). Transformation associated with grape (Vitis vinifera L.) zygotic-derived somatic embryos and renewal of transgenic plants. Plant Cell Reports 14: 589-592. DOI: 10.1007/BF00231944

Yao, N.L., Cohen, D., Atkinson, R., Richardson, Nited kingdom. and Morris, B. (1995). Regeneration regarding transgenic plants from the commercial apple company cultivar Royal Gala.  Plant Cell Reviews 14:407-412. ISSN: 0721-7714 (Print) 1432-203X (Online)