BIOTECHNOLOGICAL APPROCHES ON ONION & BRINJAL

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Onion & Brinjal:

Onion & Brinjal

Establishment of DH line;:

Establishment of DH line; Plant Development Ploidy Level of Gynogenic Plants Colchicine Treatment

Plant Development:

Plant Development There are three distinquished steps of haploid production ; Culture of unpollinated ovules, ovaries or flower the product of which we called embryos; Development of gynogenic embryos into rooted plants 8 to 10 cm high; Micropropagation and transplant to soil of gynogenic clones.

Policy Level of Gynogenic Plants:

Policy Level of Gynogenic Plants The examination of ploidy level carried out in the gynogenic plants of onion, led to discordant results. Keller (1990b) obtained 62% of haploids, Muren (1989) 70%. Whereas Campion et al. (1994) contained 90%. This could be due to the different age of flowers collected since no difference was so far found between ovary and flower culture on n/2n plant production.

Colchicine Treatment:

Colchicine Treatment Resently, the study of fertility restoration with the in vitro induction of chromosome doubling and a new technique has been set up. The diploidization of the shoot apices was achived in onion by culturing haploid bulb scales on M3 solid medium contaning 10mg/l of colchicines for 72 to 80 hours followed by the standard procedure of micropropagation. This technique is not to exceed 80 hours of treatment in order to avoid the formation of polyploidy cells. Field data concerning the fertility of gynogenic plants treated with colchicines are not yet available.

(4).Embryo Rescue:

(4).Embryo Rescue There are about 500 species within the geneus Allium that represents are enoroums potential source of useful germplasm for the genetic improvement (the transfer of genes between different though close) releted specises of Allium is not an easy task, and less so between more distantly releted species. In order to overcome failure of interspecific hybridization, embryo rescue and ovule culture have been successfully employed. In Allium hybrid embryos from crosses between A.cepa and A. fistulocum have been successfully rescued.

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The rate of success has been up to 70%. Success has also been achived through embryo rescue among following crosses A.cepa X A.galanthum A.cepa X A.vailovii A.cepa X A.altaicum A.fistulosum X A.schoenoprasum Embryo rescue was not successful in following crosses, A.cepa X A.schoensprasum A.cepa X A.angulosum A.cepa X A.senescens

(5).Protoplast culture:

(5).Protoplast culture Hansen et al (1995) initiated cell suspensions from callus from preselected regenerating lines of A.cepa and A.fistulosum . Small culture of callus selected for greter friability were placed into BDS liquid medium contaning 2,4-D and BA and subcultured biweekly. Rapidly growing, finely dispersed lines were used for protoplast isolation. Protoplast were cultured in modified K8P liquid medium. Microcalli recovery depended on the number of weeks the cell suspension had been in culture with higest recovery from4-to5-month-old cell suspensions.

(6).Somatic embryo genesis :

(6).Somatic embryo genesis Plant regeneration Regenerated onion From callus plants

Somatic embryogenesis:

Somatic embryogenesis Phillips and Hubstenberger(1987)standerdized procedures for micropropagation and plant regeneration from callus(based on piclorm as the main growth regulator) for A.fistulosum,A.altaicum,A.galanthum,A,roylei and selected progeny from the interspecific crosses A.cepa x A.fistulosum, A.cepa x A.galanthum and A.cepa x A.oschaninii. All the genotypes studied gave rise to shoots and most to regenerated plants. The methods described are recommended for rapid propagation and for use in various breeding studies.

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Bio-technology

Marker Assisted Breeding in Allium:

Marker Assisted Breeding in Allium As we understand, markers can either be based on the phenotype (morphological marker) or on the genotype (biochemical,cytological,and moleculer) Phenotypic markera are less effective for the purpose of mapping and indirect selection compared to the genotypic markers. The reason for this is, phenotypic markers can cause undersirable effects on the phenotype,whereas, genotypic markers act as natural manner. Apart from this there is shortage of phenotypic markers as compared to genotypic markers.

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it is cler that isozymea are useful markers in Aillum , however, their number is very low as only twelve isozymes system have been found to show polymorphism. This clearly indicates that this type of marker covers a small size of Aillum genome. Similarly, the number of polymorphism detected by cytogenetical marker such as Giemsa-C and Giemsa-G bands is also low,except in few instance where it is unusually higy (Peffley and Vries,1993). Thus the uses of both biochemical and cytogenetical markers in Aillum research though valuable but their use is restricted.

Molecular Markers:

Molecular Markers RFLP Marker Studies in Allium Marker Based on PCR AFLP Marker Studies in Allium

1.RFLP Marker Studies in Allium:

1.RFLP Marker Studies in Allium Genome size of Aillum is large, onion has a genome size of 15500 megabasepairs (mbp) per 1C nucleous . Most of the other plant genomes are smaller.e.g. tomato and cauliflower are 1000 mbp per 1C nucleus and 650mbp per 1C respectively. This large genome size has been an obstacle to the application of the RPLF markers in Aillum In comperision to nucler genome,organellar genomes are easily analyzed by means of southern hybridization as number of mitochondrial(mt) and chloroplast (cp) genomes per cell is in the order of several hundred genome copies,whereas the nucler genome in diploid plant cell is only two copies.

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Analysis of Mt genome of onion have shown that the N and T cytoplasms resemble each other to a large extant, where as the S cytoplasm deviates significantly(Havey,1993) For the chloroplast genome of onion a physical map is available(katayama et al 1991).Phylogenetic research of the genus of cpDNA resterction enzyme analysis has ben carried out by Havey (1992). Thus the RPLF marker is promising tool to the study the nucler genome of Aillum , in organeller genomes the RFLP markers have already proven to be very valuable in biosystematic studies.

2.Marker Based on polymerase chain Research (PCR):

2.Marker Based on polymerase chain Research (PCR) A new category of moleculer markers based on PCR technology is available in Aillum, Which is the Randomly Amplified Polymorphic DNA (RAPD) marker. RAPD marker were identified with close linkage to downy mildew (peronospora destructor) Which orignatd from Aiuum roylei (kofoet et al ,1990). The major advantage of marker-aided selection in this specific case is that the downy mildew fungus is not needed any more for the resistance screening. This help in developing a quick and reliable screening test, which can evern be performed at seedling stage.

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RAPD are having less importance for the construction of molecular marker maps ,in cross fertilizing species. A heterozygote can not be distinguished from a homozygote in RAPDs. RAPD technique has been found useful and efficient tool in the classification and identification of garlic genetic resources (Xu.Peiwen et al 1997).

3.Amplifid Fregment Length Polymorphism (AFLP):

3.Amplifid Fregment Length Polymorphism (AFLP) More recently Amplified Fragment Length Polymorphism (AFLP) has proven to be good source of genetic markers (Sjaak Van et al 1997) The AFLP method has been used to develop an AFLP linkage map in AILLUM by using (F2) population from an interspecific cross between A.cepa X A.roylei . A SCAR marker was identified which is tightly linked to downy mildew resistance gene and is placed in the AFLP linkage map. With the use of AFLPs, it is possible to generate within reasonable shorttime genetic linkage mapss in Aillum with hundreds of marker.

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For molecler assisted breeding purpose, it is then possible to identify linked markers for disease resistance gene and other economically important traits. RFLPs and AFLPs were identified which were flanking the nuclear locus (Ms) conditioning restoration of male fertility in plants possessing cytoplasmic-gene male sterility (CMS). Polymorphism has been identified distinguishing normal fertile (N) and sterile (S) cytoplasms of onion. This has helped in reducing the number of test crosses required to identify individual maintainer (N ms ms ) plants.

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Attempts are now being made to identify AFLPs more closely liked to the Ms locus. Incresed- knowledge of Aillum genetic will start to payoff in breeding firstly through the use of marker-assisted breeding to speed up back-cross programmes then eventually through intervention at the gene level to tailor enzymes for flovur chemistry, and storage carbohydrates to consumer requirements.

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Tear Free Onion

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SCIENTISTS claim to have created a "tear-free" onion using Australian-developed biotechnology to switch off the gene behind the enzyme that makes us cry. New Zealand research institute Crop and Food used gene-silencing technology to make the breakthrough Colin Eady, the institute's senior scientist, said the project started in 2002 after Japanese scientists located the gene responsible for producing the agent behind the tears. “ We previously thought the tearing agent was produced spontaneously by cutting onions, but they proved it was controlled by an enzyme,” he said from his home outside Christchurch.

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“Here in New Zealand we had the ability to insert DNA into onions, using gene-silencing technology developed by Australian scientists. The technology creates a sequence that switches off the tear-inducing gene in the onion so it doesn't produce the enzyme. So when you slice the vegetable, it doesn't produce tears.”

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“Dr Eady said that by stopping sulphur compounds from being converted to the tearing agent and redirecting them into compounds responsible for flavour and health, the process could even improve the taste of the onion. “We anticipate that the health and flavour profiles will actually be enhanced by what we've done,” he said. “What we're hoping is that we'll essentially have a lot of the nice, sweet aromas associated with onions without that associated bitter, pungent, tear-producing factor.”

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The breakthrough has caused ripples overseas, following an international symposium in the Netherlands and after the trade journal Onion World featured Eady's work on the front cover of its December issue. Dr Eady, who has several model onion plants at the institute, said despite the excitement about the prospect of “no tears” onions in every home, it would be 10 to 15 years before this happened. “This is an exciting project because it's consumer orientated and everyone sees this as a good biotechnology story,” he said.

Conclusion:

Conclusion Use of biotechnological techniques in to onion breeding programs has lead to new opportunities. The development of brodly applicable moleculer marker in onion can leads to significant increase in the selection efficiency and to minimize the amount of labour required to carry out a breeding program and shortning of breeding cycle. Production of gynogenic lines for induction of haploidy from femeal reproductive organs has helped in development of genetically uniform pure line. It is expected that the progress in these area will eventually lead to the use of wide rang of onion germplasm for breeding purpose and improved onion variety in future.

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Brinjal

Introduction:

Introduction Brinjal is one of the common and popular vegetable grown in India and in other parts of the world including Bangladesh, China, Philippines, France, Italy and the united states. Brinjal belongs to family solanaceae. Brinjal is normally self-fertilized perennial but cultivated as annual crop and are usually transplanted. The plant is a warm-season crop and mature within 100-140 days with high average day night temperatures.Brinjal has nutritive value(protine 1.4%, fat 0.3%, carbohydrates 4%) (Aykroyd.1963).

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Tissue culture

(1).Micropropagation :

( 1).Micropropagation Polisetty et al (1994) described a protocol for micropropagating brinjal from leaf explants taken from plants grown in the field or in sterile conditions. The culture was initiated using 1 mg IAA + 2.9mg Kin + 0.10 mg IBA/l.Low sugar concentration was important for shoot regeneration. It took 3-4 months from initiation to establishment in pots. 2).Anther culture Production of haploid plant through anther culture of brinjal has been reported (Isouard et al,1979 ; Damus de Vaulx and Chambounet,1982).These studies suggested that the anthers required pretreatment with high temperature (35’C). A temperature of 25 ‘C produced 3.4 plants per 100 anthers while 8-day pretreatment at 35’C yielded an average of 12 plants per 100 anthers.

(3).Protoplast fusion:

(3).Protoplast fusion Bletsos et al. (1998) also reported the production of interspecific hybrids between three brinjal cultivars and two wild species.( S.torvum and S.sisymbriifolium ).A wild relative of brinjal , S.torvum was found to be tolerant to Verticillium. (4).Somatic embryogenesis Magioli et al (1998) described efficient method of organogenesis from calli of brinjal by application of thidiazuron. Leaves and cotyledons were found to be the most responsive explants. Optimal rates of shoot bud induction (75-100 buds/explant) were achived in the presence of 0.2uM TDZ.

(5).Artificial seeds :

(5).Artificial seeds Mariani(1992) discussed somatic embryogenesis and development of artificial seed from the somatic embryos. Bipolar somatic embryos developed in 10 days and regenerated on solid MS medium.Embryos of 3-5mm were mixed in 2%Na-alginate solution and dropped individually into calcium .Best germination rate (67%) of artificial seed was obtained using an Na-alginate solution of pH 6 GA3 and sucrose had negative effects on artificial seed technology. (6).Somaclonal Variation It was observed among the plants regenerated through somatic embryogenesis induced by NAA or 2,4-D (Hitomi et al ,1998). Variation including leaf shape, plant height, flower number per cluster, fruit shape, anther number per flower and pollen fertility were studied. It was observed that the frequencies in leaf shape, plant height,fruit shape and pollen fertility were higher in the somaclonal variants obtained by treatment with NAA than those with 2,4-D.

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Biotechnology

Isozymes:

Isozymes Zhou (1992) analyzed seventy one BC1 progeny from the interspecific cross.( S.melongena XS.incanum ) and F1 hybrids using isohyets electrophorosis. Isohyets profiles of shikimate dehydrogenase (SDH), peroxidase and malate dehydrogenase (MDH) were differented among the BC1 progenies, F1 hybrids and parents. Genetic linkage was found between leaf spines and stem spines, flower and stem spines, SDH isohyets and leaf base angle and SDH isohyets and style length.

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Molecular markers RFLP RAPD

1.RFLP:

1 . RFLP RFLP analysis of polymerase chain reaction (PCR) amplified 3.2 kb regione of ct DNA bounded by the conserved sequences in rbcL and ORF 106 was performed in brinjal and its releted Solanum spp. (Isshiki et al 1988). 2. RAPD The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and brinjal were studied by Samoylov and Sink (1996). The asymmetric somatic hybrid plants were morphologically similar to brinjal and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average size tomato chromosomes.

Genetic transformation:

Genetic transformation Szasz et al. (1998) reported a novel time and labour saving protocol for the genetic transformation and regeneration of brinjal. Parthenocarpy Resistance to Colorado potato beetle

1 .Parthenocarpy:

1 .Parthenocarpy For the parthenocarpic development of fruit, the heterologous gene defH9-iaaM was used in transformation experiments to increase the concentration of IAA in the ovule.Lines transformed with this gene give normal- sized parthenocarpic fruits even from emasculated flowers. 2.Resistance to Colorado potato beetle To develop Colorado potato beete (CPB) resistant brinjal, the coding region of a wild type Bacillus thurenginesis cryIIIb gene under the transcriptional control of different promoters and terminators was introduced into S. integrifolium and S. melongena .

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Bt- Brinjal

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FSB-resistant brinjal or Bt brinjal was developed using a transformation process similar to the one used in the development of Bt cotton. Bt brinjal incorporates the cry1Ac gene expressing insecticidal protein to confer resistance against FSB.

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The cry1Ac gene is sourced from the soil bacterium Bacillus thuringiensis (Bt). When ingested by the FSB larvae, the Bt protein is activated in the insect’s alkaline gut and binds to the gut wall, which breaks down, allowing the Bt spores to invade the insect’s body cavity. The FSB larvae die a few days later. Bt Brinjal was developed by the Maharashtra Hybrid Seeds Company (Mahyco).

Conclusion:

Conclusion Biotechnological tools in brinjal are being used rightly to solved majore problems facing its cultivation. Bt brinjal has enormous potential to benefit both farmers and consumers Which is out- come of these technology.Use of biotechnological methods is ,to supplement and complement conventional breeding method of brinjal with valuable breeding lines carriying traits of resistance.