Seed Health Management for Better Productivity - Govind Ballabh ...

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(Seed Health Management for Better Productivity)viewed on a sheet attached to electrical current in the form of bands (tiny spots, Fig. 1 & 2).However the quantity of DNA extracted from seed or leaf or root tissue is too small and it isdifficult to view such tiny spots. Hence the tissue extracted DNA needs to be amplified manytimes to obtain required quantity using PCR. Thermal cycler is used for running the PCR reactionand amplifieng DNA in sufficient quantities (Fig.3). To run a PCR the essential ingredients are-High quality plant tissue extracted DNA, Primers( starter DNA for initiating amplification),enzymes and chemical budffers to amplify DNA. DNA will only duplicate itself, if there is a primeror “starter DNA” in the mix, which is compatible with the DNA. The primer sets (for duplicatingboth forward and reverse DNA strands) specific to our Gene of interest can be designed insillicousing Bioinformatics tools such as PRIMER3 and DNASTAR.Fig. 2. Thermal CyclerFig.3. Gel Electrophoresis apparatus forfor DNA amplification. viewing DNA polymorphisms.Two important commonly used techniques viz. RFLP and RAPD will be discussed in detailbelow in order to explain their importance in discrimination of the crop varieties:Various PCR or non PCR based molecular markers which are used for varietalidentification of crop plants are also used for determination of hybridity of crop plants. The use ofRFLP and RAPD in determination of hybridity is discussed below :RFLP: RFLP markers behave as codominant markers. RFLP’s can successfully be employed todetermine genetic contribution of each parents and can be used to determine the extent ofheterozygosity. Two selected varieties ( say A and B) are crossed to produce F1 and F2/backcrossgenerations. DNA is isolated from the parental varieties, the F1 and F2 generation and used todetermine RFLP’s with various probes for which they show polymorphism. For example, probe 1detects a relatively slower moving band in variety A and a faster moving band in variety B. Thesebands may be regarded as two different alleles of a single gene, say allele A for slow moving bandand allele a for fast moving band. Similarly, probe 2 detects a fast moving band in strain A ( thiswe may denote as allele b) and a slow moving one in strain B ( designated as allele B). The F1hybrid between varieties A and B will show both slow and fast moving bands for each of the twoprobes.- 174 -
(Seed Health Management for Better Productivity)RAPD:RAPD’s are generated by using random sequence, ordinarily, 10 base long oligonucleotides asprimers for PCR amplification of genomic DNA extracted from different varieties. Polymorphism isproduced due to complementary sequence for the primer used being used in one strain (variety A)but not in the other ( variety B). As aresult an amplification product will be detectable as a band instrain A while strain B will not show the product. The F1 hybrid will two strains will show the bandwhile in F2 a 3:1 ratio will be obtained. Thus RAPD’s behave as dominant markers. Further thepresence of amplification product can be regarded as dominant allele and it’s absence asrecessive allele.RAPds have similar applications to those of RFLP. However they are faster andmore convenient to perform than RFLP.Biotechnological tool for maintenance of hybridityBarnase-barstar system : Gene barnase encodes an RNAse which kills the cells in which it isexpressed by degrading RNA. The expression of barnase was confined to tapetal cells by fusing itwith the promoter of tobacco tapetum specific gene TA29 ( gene construct : pTA29- barnase; ppromoter ). When the chimaeric gene construct was transferred and expressed in tobacco andoilseed rape, the tapetal cells of anthers were destroyed and there was no pollen development.However there was no effect on female fertility. Since the male sterility due to barnase isdominant, the male sterile lines are always heterozygous (barnase/- ; the – sign indicates absenceof barnase gene in homologous chromosome) and they have to be maintained by crossing to anynormal, non transformed male fertile line (-/-; barnase gene absent). Thus male sterile lines (barnase/-) will have to be crossed to be normal fertile lines (-/-), and only 50% of the progeny fromsuch crosses will be male sterile while rest 50% will be male fertile (-/-). In a hybrid seedproduction programme the male fertile plants present in male sterile line must be identifiedandeasily eliminated. This has been done by linking the barnase gene with the bar gene fromStreptomyces: bar gene confers resistance to herbicide phosphinothricin.When such male sterile(barnase-bar/-) plants are maintained by crossing with normal male fertile (-/-), all the male sterileprogeny ( barnase-bar/- ) are resistant to the herbicide, while all the male fertile plants (-/-) areherbicide susceptible. The male fertile plants are therefore eliminated by a herbicide spray at anearly stage of plant growth.The male fertility of barnase male steriles is restored by another gene, barstar, of thebacterium B.amyloliquefaciens. The gene barstar encodes a specific inhibitor of barnase encodedRNase. The barstar product forms a higly stable 1:1 noncovalently bound complex with thebarnase RNase; this reaction provides protection to the bacterial cells from their own RNaseproduct. Transgenic plants expressing barstar are male fertile without any phenotypic effect, andare easily maintained in the homozygous state. When a homozygous barstar male fertile line iscrossed with a barnase male sterile all the progeny plants are male fertile since barstar geneproduct effectively inhibits the barnase RNAse in barnase-bar/barstar plants. This male- 175 -
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Page 221 and 222: 9. Session Arrangement CommitteeDr.
Page 223 and 224: 9. Dr. Pradip Kumar BorahSr. Scient
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TRAININGONANNEXURE-IIISEED HEALTH M
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ANNEXURE-IVCENTRE OF ADVANCED STUDI
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SundayApril 6MondayApril 7TuesdayAp
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TuesdayApril 15WednesdayApril 16Thu
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