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ThesisItem Open Access Optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin(AAU, Jorhat, 2017-07) Das, Panchashree; Sen, PriyabrataCitrus species are the most widely grown fruit crops within the whole world. India is the fourth largest producer of orange in the world. North-Eastern India is considered as one of the centres of origin of many citrus species. Among them Khasi Mandarin is the most widely grown citrus species. According to Ministry of Agriculture and Irrigation, Govt. Of India, the yield of Khasi Mandarin is declining day by day drastically due to biotic and abiotic stress. Conventional breeding for overcoming this problem is limiting due to non-availability of resistant sources. Recent advances in genetic engineering have made it possible to incorporate desirable genes from alien sources to elite genotype mainly through Agrobacterium-mediated genetic transformation. Citrus cultivars vary in their response to in vitro organogenesis and genetic transformation. This results in need for a cultivarspecific optimization of an in vitro regeneration and transformation protocol. Most of the plant regeneration processes in citrus, through tissue culture, involve use of Cotyledon, epicotyl segment, shoot-tip, internode, root meristem as explants. A study was conducted to develop a regeneration and Agrobactetrium mediated transformation protocol for Khasi Mandarin using zygotic seedling as explants obtained from six-week-old in vitro grown seedlings. Modified MS media containing 1 mg/l BAP, 0.5mg/l NAA and 0.4mg/l Kinetin shows the best result for multiple shoot induction with an efficiency of 68%. The number of multiple shoots developed was on an average 5. The modified MS medium containing containing 0.25 mg/l BAP,0.5 mg/l NAA, 0.5 mg/l IBA shows best result for rooting with an efficiency of 82% with an average root length 2 cm. Zygotic explants with injured shoot tip were used as explant for transformation with Agrobacterium strain AGL1, harbouring plasmid pCAMBIA1301 containing hpt as selectable marker gene and gus as a reporter gene. Modified MS media containing 100mM Acetosyringone was fund to be most effective medium for co-cultivation. Regeneration and selection media containing 1 mg/l BAP, 0.5mg/l NAA, 0.4mg/l Kinetin and 30mg/l hygromycin and 250mg/l timentin shows the best result. In vitro regenerated shoots that survived upto 3rd selection cycle were considered as putative transformants. Some of the putative transformed shoots showed positive result for gus in PCR analysis. The present investigation is a preliminary study on optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin. More and more concentrated effort is needed to establish a most efficient regeneration and transformation protocol considering various factors affecting genetic transformation and regeneration efficiency.ThesisItem Open Access Validation of selected SSR markers of QTLs associated with anaerobic germination trait in deepwater rice (Bao) of Assam(AAU, Jorhat, 2017-01) Rajpoot, Sachin; Das, P.K.India is one of the hot spots of vast genetic resources as well as secondary centre of origin of rice in the world. Exploiting genetic diversity could aid in providing useful information in the selection of material for breeding. Flood in rice field is due to heavy rainfall after seeding and early seedling emergence results in partial to complete crop failure. Primarily reason is high sensitivity of rice to the anaerobic conditions during germination (Yamauchi et al. 1993).Thus, tolerance to flood at early seedling growth improves crop establishment.The present study was conducted to evaluate anaerobic germination (AG) tolerance in Bao (deep water) rice germplasms of Assam compared to positive controls (Khao Hlan On, Ma Zhan Red) and negative control (IR-64). Under present study, genetic diversity was evaluated amongst 31 Bao rice germplasms of Assam using 38 SSR markers linked to Anaerobic Germination(AG). Assessment of allelic distribution showed three major groups in which, Rangdha Kekua Bao clustered with other four cultivars under cluster I while MaZhanRed and Khao Hlan On were grouped together with 12 of 38 Bao cultivars under major cluster II. Another half of Bao cultivars grouped together under cluster III separately indicating their distinctiveness. Regression test, validated only two markers viz.RM 553_170 linked to qAG 9, RM 5378_160 linked to qAG 2 that are significantly linked to the AG trait. These markers may be employed to evaluate the molecular diversity that can be used in aspects of plant genetics concerning germination studies under hypoxia. Present study also indicated that more markers are needed for lucid understanding of AG associated QTL and Marker assisted selection.ThesisItem Open Access SCREENING OF HYDROGENASE ACCESSORY GENES hypA AND hypB IN FEW ACID TOLERANT BACILLUS SP(AAU, Jorhat, 2017-07) POOJA, S; Barooah, MadhumitaThe present investigation reports on the screening of hydrogenase accessory genes viz., hypA and hypB in previously isolated acid tolerant Bacillus sp. followed by differential expression study (pH 7.0 and pH 4.5) of the both genes in three selected strains and subsequent cloning of hypA and hypB gene of Bacillus megaterium into pET28b+ vector. A total of 50 previously isolated acid tolerant Bacillus strains were considered for screening of hypA and hypB genes. Among the 50 isolates, 10 isolates tested positive for hypA & hypB genes. Three isolates viz., B. megaterium (GG1), B. cereus (GG2) and Lysinibacillus fusiformis (GG11) were considered for further study due to their ubiquitousness and higher acid tolerance. Bacterial RNA was isolated followed by synthesis of cDNA. A q-RT-PCR was performed in the above mentioned isolates to analyse the expression level of the said genes in acidic condition. The expression of hypA and hypB genes were significantly (P ≤ 0.05) up-regulated at pH 4.5 than in pH 7.0 in all the 3 isolates viz., GG1, GG2, GG11. An 18 fold increase in the expression of hypA and a 6.1 fold increase in expression of hypB were observed in the isolate GG1 at pH 4.5 compared to pH 7.0. The expression of hypA and hypB genes in GG2 was found to be 4.6 and 3.8 fold higher respectively at pH 4.5 than pH 7.0. In GG11, hypA showed 5.3 fold and hypB showed 2.8 fold increased expression at pH 4.5 compared to pH 7.0. Maximum expression of both the genes was observed in Bacillus megaterium. These genes were further cloned into pET28b+ vector. The transformation frequency obtained for the hypA and hypB was 1.5 x 106 and 3.2 x 104 CFU/μg respectively. Successfully transformed colonies were further validated through colony PCR as well as PCR on isolated plasmid DNA. The cloned hypA gene was sequenced and BLAST analysis was performed. BLAST analysis revealed that the genes belonged to the respective genus. The results of the present study revealed that hypA and hypB genes may have role in conferring acid tolerance to soil bacteria.