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Professor Jayashankar Telangana State Agricultural University, Hyderabad (Telangana State)
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ThesisItem Open Access GENETIC TRANSFORMATION OF SORGHUM (Sorghum bicolor (L.) Moench) FOR ABIOTIC STRESS TOLERANCE USING ANNEXIN GENE(Professor Jayashankar Telangana State Agricultural University, 2004) PRAI3HAKAR.A RAO , K.; MAHESWARI , M.Sorghum is the major grain crop in semi arid tropics. Until recently the genetic improvement of sorghum for abiotic stresses has been carried out through traditional plant breeding methods. Non-availability of efficient transformation techniques is one of the 8 1.1imitations for the application of biotechnology for genetic improvement of this crop. - Annexins are known to be multigene family members with diversified functions. Their role in oxidative stress tolerance was implicated in E.coli (Gidrol et al., 1996). Present investigation was aimed at developing transgenic sorghum with annexin gene through microprojectile mediated gene transfer. The cDNA of annexin gene isolated fiom Brassica juncea, which is 954 bp length, was cloned into pTZ57R vector by T/A cloning for creating restriction sites, followed by cloning in pRTlOO vector to mobilize CaMV35S promotei and terminator. The expressioncassette was cloned in pCAMBIA 1300 binary vector. Bombardment parameters like pressure of the helium gas, flight distance of the microprojectile and type of explant, which influence the efficiency of bombardment, were optimized with E CAMBIA 1305.1 vector as this vector has the GUS as reporter gene. Sorghum shoot apices isolated from 3d old seedlings were used as explant to induce calli on MS medium containing 2,4-D. Thirteen day old calli were bombarded with annexin gene construct. The putative transgenics were selected and maintained till rooting on hygromycin selection pressure. PCR verification of putative transgenics was done using nptII and hpt primers. The amplification products obtained were 1050 bp and 350 bp respectively confirming the 'presence of transgene. Annexin gene specific primers also yielded the expected 954 bp product. The amplification profiles of the putative transgenic plants confirmed the integration of the transgene in the genomic DNA. The PCR positive transgenics were transferred to green house for acclimatization. Further molecular characterization of these plants to analyze the number of copies of the inserted transgene has to be carried out using Southern blotting.ThesisItem Open Access Cloning of orfH522 from CMS Sunflower (Helianthus annuzis L.) for Anther Specific Expression(Professor Jayashankar Telangana State Agricultural University, 2004) Harinath, D.; Dinesh Kumar , v.The present investigation was carried out with the aim of developing plant transformation vectors suitable for induction of male sterility in crop plants. This research intended to clone a mitochondrial, male stedhy inducing gene orfl522 from PET1 CMS line of sunflower under a tapetum specific promoter, TA29 promoter fiom tobacco in fiame with a mitochondria1 target peptide coxIV pre-sequence fiom yeast, so that ORFH522 would be targeted into mitochondria in the target plants, evethough the protein is synthesised as a nuclear encoded protein in the transgenic plants. nos terminator fiom Agrobacterium was proposed to be used as termination signal in the gene cassette. As appropriate controls gene cassettes were developed where orfH522 was cloned under a constitutive promoter, CaMV 35s in frame with or without mitochondria1 target sequence so that the effect of expressing ORFH522 constitutively could be determined in comparison to tapeturn specific expression. The development of the proposed gene cassettes was carried out using a three step strategy. In the first step, the component sequences TA29 promoter fkom tobacco, coxIV pre-sequence from yeast, 0@22 fiom sunflower and terminator sequence fiom Agrobacterium were amplified hm their respective sources using appropriate PCR techniques and cloned in TIA cloning vector. The authenticity of the protein encoding . sequences (coxIV preaequence and or-522) was confirmed by sequencing. In the second step, these sequences were cloned in plasmid vector pUC18 in appropriate/correct orientation by following serial directional cloning using the restriction enzymes sites incorporated in forward and reverse primers employed for amplifying each of the component sequences in the first step. The orientation of cloning of the component sequences in pUC18 was confirmed by appropriate restriction digestion analysis using cloning enzymes and PCR analysis using component sequence specific primers. Also, these cassettes were completely sequenced to obtain the ultimate level of confirmation of orientation of cloning and genetic 'information of the gene cassettes. In the third step, these cassettes were excised out fiom pUC18 vectors and were cloned in binary vector pCAMBIA 1305.2. The cloning was confirmed hy restriction digestions. orfH522 with or without coxIV pre-sequence was also cloned under CaMV 35s promoter. For this, orfl522, 'coxN presequ&ce-orjH522' were amplified and cloned in TIA cloning vector and later cloned in pRTlOO (which has CaMV 35s promoter and CaMV poly A signal flanking the MCS) with appropriate restriction enzymes. The two gene cassettes were excised out from pRTlOO with Hindu1 and cloned in pCAMBIA 1305.2 :. Thus, in the present investigation four gene cassettes were developed which when successfully introduced into crop plants, could induce male sterility. These constructs could unequivocally establish the role of om522 in causing male sterility in PET1 CMS line of sunflower.ThesisItem Open Access FROM RICE 'EST' RESOURCES: CONSTRUCTION OF PLANT EXPRESSION VECTORS FOR FUNCTIONAL TESTS(Professor Jayashankar Telangana State Agricultural University, 2004) SEKHAR, K.; ARJULA REDDY, R.loping transgenics for down regulation or over expression of genels of interest. In this regard, the study was taken up with the isolation of full length cDNA clones for DREBlB and Metallothionein-like protein from rice EST resources. The plant expression vectors under 35s promoter for Agi-obacterium were constructed and genetic transformation was done in indica rice. DREB protein is a stress responsive transcription factor. This protein binds to DRE/LTRE/CRT elements on promoter region and regulates the expression of stress responsive genes. Metallothioneins (MTs) are a group of proteins with low molecular mass and high cystein content, which are organized into two clusters and bind to transition metals with strong afiimty. In the lab, EST database was generated by partial sequence of cDNA clones fiom drought stress rice seedlings. Two cDNA clones represented the DREB fdy while six dfierent cDNA clones for metallothionein-like protein were in the database. The cDNA clones were individually sequenced from both sequence length obtained for DREBlB was 862bp (AccNo: BU673228) and for metallothionein-like protein was 525 bp (AccNo : CB 1 99 1 3 7 j . These genes were found to encode for DREB lB and metallothionein-like protein through in-silico analysis. DREB 1 B and metallothlonein lke protein sequence were individually aligned against public database using BLASTN algorithm. DREB 1 B showed 98 per cent sequence identity while metallotbonein-like protein revealed 90 per cent identity. The amino acid sequence of DREB 1 B and metallothionein-lke protein were independently aligned against protein database using BLASTP. DERBlB showed 61 per cent sequence identity while metallothionein-like protein revealed 66 per cent similarity. The comparative analysis of amino acid sequence deduced from the gene encoding DREB 1 B and metallothionein-like protein using CLUSTALW showed high degree of conservation among very diverse plant species. Full length DREBlB and metallothionein like protein were individually cloned into 35s promoter with a poly 'A' signal in sense orientation using pRTl00 vector and sub cloned in to PstI site of binq vector pCAMBL41301. These recombinant plasmids were mobilized into Agrobacterium tzrinefacie~zs by electroporation for transformation in rice. The transformation of rice and tobacco using these constructs for analysis of the gene function is under way.ThesisItem Open Access GENETIC TRANSFORMATION OF SORGHUM(Professor Jayashankar Telangana State Agricultural University, 2004) Aravinda Laxmi, Y.; VISARADA , K.B.R.S.Sorghum is an important dryland crop used as food, feed and fodder. Due to increasing demand towards its usage in diverse applications, it is imperative to deploy modern bioteclinological tools to improve its productivity and quality. Two principal methods viz., Agr-obacteriunz infection and particle bombardment were used for genetic transformation. In vitro response of sorghuni is low, monocalcitrant and is higlily genotype dependent. Tissue culture and regeneration response was low when compared to other cereals. During the course of study, efficiency of different expl;tnts were studied in two inbred lines AKMS 14B and 296B through transient g~ls ex, ession profile and regeneration percent. Particle bon~bardn~ent though showed high percent of regeneration in all the explants, Agrobacteritrr?~ method of transfor11i:ltion was preferred because of its cost efficacy and low copy number and transient gin expression was more through Agrobacten'ur~z infection than tl~rough pa~ticle bombardment. Shoot meristems isolated from germinated seedlings were no st suited explants in terms of irz vitr-o handling and transformation efficiency. 3d and 7d old seedlings were studied through electron microscopy to nleasure the nleristen~atic zone that correlate with the transformation parameters. Infection at two OD60O values, 1.0 and 2.0 absorbiincc were studied. Absorbance at ODboo 1.0 gave low transient grr.r; expression but controllecl Agrobacterizlnz growth after cocultivation; whereas at OD6oo 2.0 absorbancc ga1.e high transient gtls expression with overgrowth of Agr-obncteriunl. Different antibiotic treatments and culture conditions were tested that can control Agrobactc~-izlrn growth and facilitate growth of plant tissues. Host specific response towards antibiotics was evident. Addition of L-Cysteine at 400mg~-' improved the growth of shoot tips during cocultivation. The study suggests that, better transformability from direct explants than from the callus derived explants in two sorghum inbred lines using Agr.ohc[c.tc/-i~rn~ as the method of gene transfer.ThesisItem Open Access MARKER-ASSISTED BACKCROSSING OF STAY-GREEN QTLs INTO ELITE SORGHUM LINES(Professor Jayashankar Telangana State Agricultural University, 2004) RAMU , P.; JONATHAN , C.y. This C4 grass is grown in more than 80 countries mostly in tropical and sub- tropical regions. After soil nutrient deficiencies, drought &ess is the moat important abiotic constraint to sorghum production globally. Dry spells can occur at any stage of the crop growth period. In sorghum, rapid premature leaf death generally occurs when water is limiting during the grain filling stage. Therefore drought stress during the grain filling period is referred as "post-flowering drought or terminal drought". The plant charadter associated with post flowering drought tolerance is called "stay-green". Stay- green is associated with knctional green leaf area (GLA) during and after the grain filling period. Stay-green in sorghum is associated with charcoal rot, lodging resistance and superior ruminant quality. This complex trait is difficult to score. Genetic mapping of QTLs associated with stay-green is an important step towards 'developing drought tolerant hybrids. Different sources of stay-green have been identified in sorghum. The most commonly used lines in breeding program are B35 and E36-1. . Different research grotips independently developed QTL maps for stay-green using different donor parents and marker systems. After identifying the consistent Q'Ks markers. these can be tested through introgression of QTLs from their mapped sources into sorzhum elite breeding lines. This can be accomplished by cloning the genes expressing QTLs and transferring these genes to recipient breeding lines or through marker-assisted breeding (MAB) program, where QTLs are introgressed into elite breeding lines using molecular markers. MAB is the most appropriate technique when traits are complex and difficult to score/measure like yield, abiotic stress tolerance, where the genes contributing to QTLs expression have not yet been identified, and where plant transformation systems are not well established. - With the development of molecular tools and molecular genetic linkage maps for plants, marker-assisted selection (MAS) has become much more broadly applicable. From the last decade, developing ability to transfer target genomic regions using DNA markers resulted in extensive mapping experiments aimed at development of MAS. Molecular marker based genetic linkage map of sorghum has permitted the identification of six QTLs for stay-green (post-flowering drought tolerance) in sorghum line B35. This project aimed at transferlintrogression of these QTLs fiom B35 to recurrent parents, ISIAP Dorado and R16. BC3 and BC4 generations fiom each recurrent parent were genotyped with the markers linked to stay-green QTLs for foreground selection and evenly distributed unlinked markers for background selection to speed the recovery of recurrent parent genotype in genomic regions that are not associated with the target stay-green QTLs. Genotypes with desired marker allele profiles were selected and advanced to next generations. Further studies are necessary to confirm the introgression of QTLs and expression patterns ibr stay-green by phenotypic evaluation of selected genotypes.ThesisItem Open Access A DIVERSITY ANALYSIS OF EARLY-MATURTNG GROUNDNUT GERMPLASM USING SSR MARKERS(Professor Jayashankar Telangana State Agricultural University, 2004) ASHOK KUMAR , C.; JONATHAN H, C.Groundnut (Arachis hpgaea L.) is an important crop internationally for both direct human consumption and as an oilseed crop, which is being cultivated in 1.08 countries of the world. About two thirds of world groundnut production comes &om the semi-arid tropic (SAT) regions which are characterized by uncertain rainfall and frequent droughts. Groundnut yields are low and average about 0.8 t/ha in the SAT countries compared to more than 2.6 tlha of the developed world. One contribution to increasing yield is development of early maturing, high yielding cultivars that are needed for short growing seasons, multiple cropping systems and which avoid late season droughts. In most breeding programs few sources of early maturity have been used resulting in the narrow genetic base of groundnut cultivars. There is a need for broadening the genetic base to enhance groundnut breeding prospects. The high diversity detected by SSR markers is consistent with the known characteristics - that they are more variable and high expected heterozygosity than the RAPDs, or AFLPs. The high levels of polymorphism associated with SSRs are expected because of unique evolution of these genomic regions: replication slippage rather than mutations, insertions or deletions. The present study was initiated to assess diversity using SSR markers among 29 groundnut accessions belonging to two subspecies jkstigiala and hyhygaea and three botanical varieties vlrlgcrris, fasfigiafa, and lypogaea, originating fiom fifteen countries which include 25 early-maturing and 4 late-maturing accessions. Initially 7 to 10 individual plants fiom each of ten accessions were assayed for intra-accession variation using 5 SSR primer pairs. These ten accessions include ICG 3540, ICG 4558, ICG 4890, ICG 9427, ICG 1 1914, ICG 148 14, Gangapuri, JL 24, Chico and TMV2. UPGMA clustering of the SSR band profiles revealed significant variation within the accessions. A total of 22 alleles were detected by five primer pairs with an average number of 4.4 alleles per primer pair. The number of alleles ranged from 2 alleles for 2B10 to 8 alleles for 2D12R To capture this intra-accession diversity in the main study equal amounts of DNA from individual plants were pooled for each accession. Inter-accession diversity analysis of 29 accessions was pairs, which detected a total of 57 alleles with an average of 2.85 alleles per primer pair. The number of alleles per marker ranged from two to five. The PIC values, ranged from 0.53 (1 7F6) to 0.93 (1 5C 12), with an average of 0.78. The AMOVA analysis indicated 42% of variation in SSR markers used between early and late-maturing accessions. The clustering revealed significant diversity among the 29 accessions used for this study. The different botanical varieties were grouped into 3 different clusters except one accession. The MDS analysis supported the clustering obtained by UPGMA. Clustering also revealed significant diversity among accessions within a particular country. Comparison of genotypic data with phenotypic data for these accessions may project the complete picture of diversity. This analysis will assist groundnut breeding programs aimed at improving early-maturity to maximize the genetic base of their breeding populations.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF MICROSATELLITES FROM THE WILD SILKMOTH Antheraea mylitta(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD, 2004) RAVI KUMAR, A; NAGARAJU, JThe study is aimed at establishing a standard method for the isolation of Microsatellites from the wild silkmoth Antheraea mylitta, ecorace Daba trivoltine. Microsatellites were isolated by the selective hybridization method in which DNA fragments containing repeat motifs specific to repeat probe used in hybridization were captured. In the present study six biotinylated oligos of the Di-, Tri- and Tetranucleotide repeat types were used as probes in individual hybridization reactions. Genomic DNA was digested with Sau3A, dephosphorylated, size selected from 100-1000 bp to which Sau3A ds linkers were ligated. The linker ligated genomic digest was hybridized against biotinylated repeat oligo and eluted microsatellite enriched DNA fragments were subjected to linker digestion with Sau3A, purified and cloned into BamH I digested pBluescript (KS+) plasmid and transformed into INVF competent cells. Totally 110 white colonies were screened and the clones having insert size above 100 bp were selected and sequenced. Totally 81 clones were sequenced of which 18 were found to be positive for microsatellites. Out of 18 microsatellites 15 are dinucleotide repeats and 3 are trinucleotide repeats and no tetranucleotide repeats were captured.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF THE NEW CYTOPLASM DERIVED FROM Helianthus argophyllus USING SOUTHERN ANALYSIS AND rep-PCR(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD, 2006) KAVITHA, P; MANORAMA, KCytoplasmic male sterility (CMS) in higher plants is encoded by the mitochondrial genome. Cytoplasmic male sterility is the most viable system for hybrid seed production in sunflower. Cultivated hybrid sunflower (Helianthus annuus) production depends on a single male- sterile cytoplasm PET1, derived from H. petiolaris Nutt. And few restoration genes. The development of commercial sunflower hybrids based on new CMS sources is of special interest for reducing the potential risk of vulnerability to pathogens and for increasing the genetic diversity. In this regard, the present investigation was carried out with the aim of molecular characterization of the new ARG for hybrid seed production. This study was undertaken to find the genetic relatedness of the new ARG cytoplasm with conventional PET1 and other cytoplasms derived from H. argophyllus ARG 2, ARG 3 using Southern analysis and rep-PCR. For characterization of the new ARG cytoplasm, we have used DOR developed ARG and CMS lines, PET 1 was used for comparison. Here the comparison was done by rep-PCR analysis using the Box primer (BoxA1R). The primer yielded 3 to 7 amplicons of 1.9 kb with all the mitochondrial DNA samples. The banding pattern was repeatable and consistent. The new ARG cytoplasm and 2023A showed a similar banding pattern, whereas the ARG wild species had an additional unique band. Hence, rep-PCR with Box primer was able to differentiate among the new ARG cytoplasm, PET1, ARG 2 and ARG 3 cytoplasms. Rep-PCR with all the three sets of primers i.e., REP, BOX and ERIC was done for characterization of different Helianthus wild species. Though REP and ERIC showed non-polymorphic profiles, BOX was able to distinguish among few species. H. resinosus could be characterized by the absence of a prominent band where as H. strumosus showed an additional band of lower size. For hybrid sunflower development, several introgression lines have been developed and they need to be characterized at the molecular level i.e., with respect of the organelle genomes they carry. This confirmation is to be done with Southern analysis using organelle genome specific probes. In present study, Southern blot analysis was performed using genomic DNA digested with Bgl II restriction endonuclease enzyme and atp 9 mitochondrial probe. The results show that the atp 9 probe hybridized to DNA fragments of different sizes which indicates the differences in the cytoplasms of DOR developed lines with H. petiolaris though some are identical. Helianthus anuus was found to be unique and clearly different from other cytoplasms. Also ARG2, ARG3 and PET 1 cytoplasms showed clear and unique differences from other samples. ARG2 and 4011A showed a similar pattern but 2023A had a different pattern. There were no bands with respect to the new ARG cytoplasm.ThesisItem Open Access GENETIC TRANSFORMATION OF SORGHUM (Sorghum bicolor (L.) Moench) FOR ABIOTIC STRESS TOLERANCE USING GLYOXALASE I GENE(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD, 2004) SOWJANYASEEMA, A; MAHESWARI, MSorghum is the major grain crop in semi arid tropics. Until recently the genetic improvement of sorghum for abiotic stresses has been carried out through traditional plant breeding methods. Non-availability of efficient transformation techniques is one of the limitations for the application of biotechnology for genetic improvement of this crop. Present investigation was aimed at developing transgenic sorghum with glyoxalase I gene through microprojectile mediated gene transfer. The gly I gene which is 784 bp length, was mobilized along with CaMV35S promoter and terminator from pRT100 vector. The whole casette was cleaved by HindIII restriction enzyme and cloned into pCAMBIA 1300 binary vector having hygromycin resistance for plant selection. Sorghum shoot apices isolated from 3 days old seedlings were used as explant to induce calli on MS medium containing 2,4-D and kinetin. Thirteen days old calli were bombarded with gly I gene construct. The putative transgenics were selected and maintained till rooting on hygromycin selection pressure. PCR verification of putative transgenics was done using nptII, hpt and gly I primers. The amplification products obtained were 1050 bp, 350 and 558 bp, respectively, confirming the presence of nptII, hpt and gly I genes.