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2016 - 20174
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Subject: Agricultural Biotechnology × End date: 2017 × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin
    (AAU, Jorhat, 2017-07) Das, Panchashree; Sen, Priyabrata
    Citrus 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.
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    ThesisItemOpen 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.
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    ThesisItemOpen Access
    SCREENING OF HYDROGENASE ACCESSORY GENES hypA AND hypB IN FEW ACID TOLERANT BACILLUS SP
    (AAU, Jorhat, 2017-07) POOJA, S; Barooah, Madhumita
    The 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.
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    ThesisItemOpen Access
    Characterization and Identification of Potential Probiotic Lactic Acid Bacteria (LAB) Isolated from Fermented Dairy Product of Assam (Doi)
    (AAU, Jorhat, 2016-07) FATMA, JAIBA; Barooah, M.
    Probiotics are the health promoting viable microorganisms that exhibit a beneficial effect on the health of human being by improving the intestinal microbial balance. Lactic acid bacteria (LAB) are important and well known agents. Fermented milk products like Dahi are known source of probiotic organisms. In the present study, LAB was isolated from 25 different ‘Doi’ samples collected from 8 different districts of Assam viz, Tinsukia, Dibrugarh, Sivsagar, Jorhat, Lakhimpur, Udalguri, Morigaon and Bongaigaon. A total of 30 bacteria were isolated initially but only 20 bacteria isolated from Doi meet the classification of LAB as Gram-positive and catalase negative (Salminen et al., 1993). These 20 LAB isolates were characterized based on their phenotypic, biochemical and molecular characteristics. Further, the isolates were screened for their potential probiotic properties based on conditions simulating human gastrointestinal tract (GI). These isolates were able to survive at low pH 2.5 which is similar to the pH of the human GI tract and able to convert lactose into lactic acid. Further studies revealed that these isolates were able to grow in a temperature ranging from 15-40ºC and grow at 9 % NaCl concentration. The antimicrobial activity of these isolates were tested against six pathogenic bacteria viz, Escherichia coli, Pseudomonas putida, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella enterica and Bacillus cereus. Four isolates (ABT-Z2, ABT-Z3, ABT-Z4 and ABT-Z22) were able to inhibit the growth of all the tested six pathogenic bacteria. All the isolates produced Exopolysaccharide (EPS) whose production was maximum at pH-6. Seven best LAB isolates were characterized by 16S rRNA gene sequencing and sequence analysis using nBLAST revealed that five of the seven isolates belonged to Lactobacillus plantarum and two belonged to L. brevis. The results of the present analysis revealed that strain L. plantarum is the predominant species of LAB found in Assamese Doi. The strain also displayed the best probiotic potential among the tested isolates. In future, this strain of bacteria can be used in preparing probiotic milk based fermented product and can be developed as an efficient starter culture to make Doi.