Theses

Browse

Search Results

Now showing 1 - 9 of 10

Open Access
GENETIC STUDIES FOR IMPROVING YIELD UNDER DROUGHT STRESS ENVIRONMENTS IN RICE OF ASSAM
(2019-07) PARRAY, ROUF AHMAD; Baruah, Akhil Ranjan
Drought is a major limiting factor for rice under rainfed ecosystem in Assam. In this context, thirteen rice cultivars with varied level of drought tolerance were chosen from a set of 272 different rice genotypes based on a field experiment conducted during 2014-15 season under drought. The thirty days old seedlings of 13 cultivars were tested for extensive morpho-physiological, biochemical parameters, relative transcript accumulation and global gene expression using next generation sequencing (NGS) method, and data were recorded at fifth, tenth and fifteenth day of withholding water (DWW) in order to obtain detail trait based gene architecture and to improve high yielding variety of Assam using transcript dynamics. Among the physiological traits studied, stomatal conductance decreased as the dehydration stress increased but the effect was minimum in Apo, Dumai and Tepi Dumai compared to others. Photosynthetic rate decreased with increasing water deficit, but the effect was less pronounced in Apo, Dumai and Tepi Dumai. The rate of transpiration decreased upto 5DWW but gradual increase was observed in later stage. Moreover, the fall in transpiration rate was less in Apo. Water use efficiency (WUE) of rice plants was enhanced significantly under moisture stress at all the three periods of stress (5DWW, 10DWW, 15DWW) in Apo, Tepi Dumai and Dumai. Reduction in RWC was experienced across all genotypes but the decrease was less prominent in Apo, Dumai and Tepi Dumai. Drought stress condition led to increased proline content across genotypes as compared to irrigated condition. Apo, Tepi Dumai, Dumai and Kali Murali showed rapid increase compared to others. Increase in root length was observed across all cultivars with Apo being the longest followed by Dumai and Ranjit. Then, five drought responsive pathway genes (OsDREB2, OsNAC1, bZIP16, OsbZIP 23, OsbZIP72) were chosen to check the differential expression patern in the cultivars at the same data point as mentioned above. Expression profiling of OsDREB2 showed significant increase in gene expression with increase in drought stress in the case of Apo and Dumai. Significant expression of the OsNAC1 was found in Apo, Dumai at different time points of dehydration stress whereas expression of ARC 10372 was prominent in 15DWW. Apo showed significant difference in expression of bZIP16 under all the three stages of water stress whereas Dumai and Ranjit showed enhanced expression compared to other cultivars. Expression profile of OsbZIP23 showed significant accumulation of transcripts in Apo in all stages followed by Dumai. Significant expression of OsbZIP72 was observed in Apo at 10DWW and 15DWW followed by Ranjit and Dumai. Based on the results of morpho-physiological, biochemical and expression analysis, three cultivars, viz., Ranjit, Apo and Dumai were chosen to study the detailed transcriptome at only 10DWW. Transcriptome profile revealed highest mapped genes in Dumai followed by Ranjit and Apo, however, only 14.5% genes were in common. Ranjit was found to be more responsive to abiotic stimulus including water stress. Gene ontology (GO) suggested no significant change of pathway genes upto 10 DWW among the three cultivars. The transcriptome data were validated using five differentially expressed genes in these three cultivars along with a F4 mapping population. It revealed similar trend, suggesting the present transcriptome data set was in good fit. However, detail transcriptome study in vital plant parts at different stages under drought stress will throw more light about the interaction of pathway genes to adress the problem better.
Open Access
Molecular characterization and nutritional equivalence evaluation of transgenic chickpea expressing either a cry1Ac or cry2Aa gene
(AAU, Jorhat, 2019-10) Gupta, Rubi; Sarmah, Bidyut Kumar
Biosafety assessment of transgenic chickpeas having B.thuringiensis genes for resistance to pod borers is a regulatory requirement and mandatory to document before releasing in the field. Therefore, Bt chickpea lines harbouring either a cry1Ac or cry2Aa gene were characterized for the presence and expression of the transgene in their advanced generations, biosafety assessments and transcript profile were studied. The homozygous lines were selected for comparative nutritional equivalence assay. Biochemical estimations of major nutritional components such as proximates, vitamins, minerals, fatty acids and anti nutrients confirmed that the Bt chickpeas lines are nutritionally equivalent to their non-transgenic counterparts and the seed composition is similar or within the range reported, previously. Seed protein quality was investigated by separating the proteins in PAGE and eluted proteins after mass spectrometry (MS) showed expected fractions of 11S legumin, 7S vicilin, and 2S albumin of chickpea storage proteins in the transgenic lines. The protein digestibility was assayed using the multi-enzyme system and transient pepsin hydrolysis to mimic simulated gastric fluid followed by trypsin hydrolysis to mimic simulated intestinal fluid. Total seed proteins of both the transgenic and nontransgenic lines were digested at a similar rate and enzyme-resistant peptides were not observed in transgenic Bt chickpea lines. The unintended changes in the whole transcriptome profile of Bt chickpeas were surveyed using a homozygous transgenic line expressing a cry1Ac gene. The differentially expressed genes (DEGs) profiling confirmed a low (0.69%, log2fold change≥2) frequency of differentially expressed in the transgenic chickpea line. Only a small (34 upregulated) proportion of genes showed > 2 fold (P-value of 0.05, FDR of 0.05) change in their expression, while only 23 genes down-regulated by >2 fold. Furthermore, no transcripts for potential allergenic proteins were represented in the DEGs. Most of these genes appeared to be developmentally regulated or stress-related which was expected because absolute synchronous growth and development even under a controlled environment are challenging. A few upregulated (AT-hook motif nuclear-localized protein 17-like, probable inactive 2-oxoglutaratedependent dioxygenase AOP2, protein EXORDIUM-like) and down-regulated (histone H2B, gonadal, embryonic abundant protein VF30.1-like, fasciclin-like arabinogalactan protein 12) genes were subjected to qPCR. The qPCR data validated the fold change of the up-regulated (>2) and down-regulated (>-2) genes. Thus, the above data revealed no potential alterations in the nutritional equivalence or transcript profile of transgenic Bt chickpeas.
Open Access
STUDY OF MITOCHONDRIAL MORPHOLOGY AND MEMBRANE POTENTIAL DURING ELECTRON TRANSPORT CHAIN DYSFUNCTION IN THE MODEL ORGANISM C. elegans
(AAU, Jorhat, 2019-07) GADI SRI HARSHA VARDHAN; Baruah, Aiswarya
Mitochondria are highly dynamic, double membrane-bound cell organelles ubiquitously found in most eukaryotic cells. The major source of cellular energy in the form of ATP is generated through oxidative phosphorylation through Electron Transport Chain (ETC) of innermitochondrial membrane. Mutations in ETC complex proteins and mitochondrial toxicants will lead to ETC dysfunction which results in the disruption of mitochondrial membrane potential (ΔΨm). Mitochondria undergo coordinated cycles of fission, fusion, biogenesis, and degradation (mitophagy) and these process are linked with ΔΨm. Disruption of ETC is directly associated with many mitochondrial, metabolic, neuronal diseases and also with aging. So, it is important to understand the mitochondria morphological adaptations in maintaining cellular function and cellular homeostasis. Using Caenorhabditis elegans as a model, we studied the effect of ETC dysfunction on mitochondrial morphology and membrane potential. We analyzed the pattern of change in mitochondrial shape, distribution along with ΔΨm during ETC dysfunction caused by mutations in mitochondrial proteins and paraquat (produce superoxide) induced oxidative stress. The C. elegans mitochondrial mutant strain isp-1(qm150) with a defective complex- III (Reiske iron-sulfur protein) is used to study mutation induced ETC dysfunction. A consistent significant difference in mitochondrial morphology and membrane potential between ETC mutant isp-1(qm150), paraquat treated and control (wild type) has been observed using confocal microscopy. We observed consistent decrease of mitochondrial quantity, ΔΨm and increase in circularity of mitochondria in worms treated with different concentrations of paraquat compared to control. Even though we didn’t found significant difference of mitochondria morphological between wild type and isp-1(qm150) but there is a notable difference in ΔΨm, indicating decreasing in the rate of respiration. As mitochondrial function is conserved from worm to human and plants, understanding these basic process will helps in future intervention of diseases to improve human health and also crop productivity.
Open Access
Study on effects of qDTY3.1 contributing yield under drought in Ranjit x Apo derived F4 populations
(AAU, Jorhat, 2019-07) PARBIN, SHAHNAJ; Baruah, Akhil R.
Tolerance to low land drought stress in rice is under polygenic control and numerous quantitative trait loci(QTLs) have been identified, out of which a major QTL for yield under drought (qDTY3.1) derived from ‘Apo’ has been detected with significant additive effects. Intermittent drought profoundly affect the yield of ‘Ranjit’, a predominant high yielding variety for sali season in Assam. Therefore, a study was conducted to improve drought tolerance in ‘Ranjit’ and to understand the genetics of qDTY3.1 in the F4 lines of Ranjit x Apo. The screening of F4 populations for qDTY3.1 using the nearest marker RM520 revealed 45 Ranjit type, 69 Apo type and 66 with heterozygous alleles, respectively. Although indicated a slight bias towards Apo type allele yet the Chi square test showed that the data were in good fit, and were not significantly deviated from the expected values. Further, screening with markers far from the qDTY3.1 revealed a total of 35 Ranjit/Apo recombinants having qDTY3.1 intact, indicating suitability of these lines for advanced breeding materials. To check the nucleotide diversity and informative sites within and near the QTL region, sequencing analysis was performed in coding sequences (CDS) for some of the proteins expressed under drought/abiotic stress. Out of the CDSs sequenced, two CDSs within the QTL region did not show any polymorphism. The one CDS for NAC family detected with one non synonymous substitution in which the Apo type (with qDTY3.1) was encoded with ‘Proline’ and Ranjit type with ‘Arginine’, warrants detail study to validate the data along with inclusion of more regions to be sequenced. The effect of the qDTY3.1 allele was also estimated. Mean values showed the better yield performance of ‘Apo’ types than the ‘Ranjit’ types in drought condition. Although symptomatically the leaf rolling scores of ‘Ranjit’ is comparable to ‘Apo’, however the yield penalty for both these types was evident. Significant correlation was observed for panicle length and number of grains, effective booting tiller and tiller number, effective booting tiller and grain yield in ‘Ranjit’ and panicle length and number of grains, spikelet fertility with number of grains in ‘Apo’ under drought stress. The effects of qDTY3.1 in Ranjit x Apo imparting drought tolerance in F4 lines was observed. However, the role of other minor effect QTLs with the change of other background may not be ruled out. The study will help in generating promising breeding lines for low land drought tolerance in Assam rice.
Open Access
OPTIMIZATION OF AN EFFICIENT REGENERATION AND TRANSFORMATION PROTOCOL FOR BLACK RICE CHAKHAO POIREITON
(AAU, Jorhat, 2019-07) GEORGE, REYNOLD; Baruah, Aiswarya
The black rice germplasm suffers from inherent low productivity due to inefficient performance of yield attributing characters. Besides, the crop is also susceptible to biotic and abiotic stress. Gene technology can provide an effective approach for improvement of black rice. The availability of a robust and reproducible plant regeneration system that is amenable to transformation plays a major role in achieving this objective. Therefore a study was conducted to establish a stable and reproducible regeneration system in black rice Chakhao poireiton. The main plant hormone responsible for callus induction 2,4-D was used in combination with vitamin assay casamino acid in 2N6 medium with varying concentrations to establish a better callus inducing medium. It was revealed that a combination of 2mg/l 2,4-D and 1g/l vitamin assay casamino acid stood better in terms of callus induction frequency, callus weight and relative growth rate compared to the already established protocol by Hiei et al., (2008). The best shoot inducing hormone combination was found out by using kinetin in varying concentration and kinetin in combination with BAP. Kinetin at 4.5mg/l proved to have the highest regeneration percentage. Agrobacterium strain LBA4404 harbouring pCAMBIA1304 binary vector containing hptII gene (selectable marker gene) and gusA gene (reporter gene) was used for genetic transformation. GUS histochemical Assay showed visual confirmation of genetic transformation. Varying concentrations of hygromycin 30mg, 40mg, 50mg and 60mg/l were tried for screening transformants, but gave 100% mortality in all the concentrations. In future, better transformation protocols for Chakhao poireiton need to be established.
Open Access
CHARACTERIZATION OF XYLANASE ENZYME FROM FUNGAL STRAINS OF NORTH-EASTERN REGION OF INDIA
(AAU, Jorhat, 2019) SAHA, DIPANKAR; Boro, R.C.
Xylanases are hydrolytic enzymes produced by a variety of microorganisms including fungi. The enzyme hydrolyzes the main hemicelluloses component by cleaving the β-1, 4 backbone of the complex plant cell wall polysaccharide xylan. Fungi are reported to produce a wide variety of xylanases that are not only capable of degrading xylan to renewable fuels and chemicals but have also found industrial application in food, paper and pulp industries. In recent years, there has been growing awareness in applying green biotechnology to industrial processes to decrease pollution as well as improve the quality of the product produced. Scouring for readily available and cost-effective source of this enzyme is important in the context of environmental sustainability. The Northeastern region is known for its biodiversity harbors. Several species of fungi whose industrial application or as a source of important products has been reported scantly. The present study focuses on the isolation and characterization of xylanase enzymes from selected fungal isolates of this region. Twenty five (25) previously isolated fungal isolates were taken from the Microbial Biotechnology Lab, Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat for the study.. Preliminaryplate screening of the isolates revealed xylanase activity in four isolates viz. Lentinus squarrosulus, Fusarium oxysporum, Lentinussajor-caju and Fusariumfujikuroi. TheXylanase positive isolates were grownin liquid media and enzyme activity was assayed up to 15 days at a 2 days interval. Highest xylanase activity was recorded on the 7thday of inoculationin Lentinussquarrosulus, Fusarium oxysporum, whereas Fusariumfujikuroiand Lentinus sajor-cajushowed the highest xylanaseactivity on the 5th and 9th day of inoculation respectively. Xylanase enzyme was partially purified from culture supernatant by precipitation using ammonium sulphate (80% saturation). The precipitated crude enzyme was dialyzed against 1mM sodium acetate buffer (pH 5.3).Fusarium oxysporum showed the highest xylanase activity (28.71±0.11 U/mL) in the partially purified extract. Zymogram analysis of partially purified enzymes suggested the presence of single active xylanase in each sample. However, electrophoretic mobility of the xylanase of each sample was different. SDS PAGE analysis indicated the presence of multiple subunits in the xylanase enzyme. Xylanase activity and stability were optimized in different pH and temperatures. All the samples are pH and temperature specific and highly stable at 5.0-6.0 pH and 20oC-50oC. Further studies with improved purification techniques will pave the way for purified xylanase enzyme that may be useful in different industrial purposes.
Open Access
In vitro regeneration of Musa spp. cultivars Malbhog and Bhimkol, and assessment of genetic fidelity of regenerated plantlets using molecular markers
(AAU, Jorhat, 2019-07) Kataria, Raghav; Bhorali, Priyadarshini
Banana (Musa spp.), belonging to the plant family Musaceae, is one of the major fruit crops of the world. Banana cultivars Bhimkol (Musa balbisiana, BB) and Malbhog (Musa paradisiaca, AAB) are popularly grown in the North-Eastern parts of India. Both cultivars are popular and important from the evolutionary and commercial point of view and, particularly Bhimkol banana is associated with improved vigor and tolerance to biotic and abiotic stresses. Malbhog is highly valued and the most desired for its excellent fruit qualities particularly marvelous taste, appealing aroma and a greater number of fruits per hand and higher post-harvest life. Since ancient times, people have been consuming banana fruit as dessert, dietary supplement, and for nutrition as it is rich in carbohydrates, vitamins and proteins. However, conventional banana propagation is time-consuming and gives lower yields due to various constraints such as biotic/abiotic stresses and lack of availability of healthy suckers. Moreover, the production by conventional means is not able to meet the demands of the growing market. So, the use of tissue culture technique and development of in vitro regeneration protocol for popular cultivars such as Malbhog and Bhimkol will not only lead to mass multiplication with better yields but also help in conservation of local genotypes. In the present investigation, an attempt has been made to optimize an efficient in vitro shoot regeneration system for Bhimkol and Malbhog. The study was initiated by standardizing shoot regeneration protocol, using banana shoot tips as explants in modified MS medium by using different concentrations of BAP (6-Benzylaminopurine) (2, 3, 4, 5, 6, 7, 8, 9 and 10 mg/l). In Bhimkol, although shoot regeneration was observed in 7 mg/l BAP, but the regeneration was much slower and no multiple shoot formation was seen. Since, it is a slow growing genotype and also the level of phenolic compounds is very high, regeneration was not found to be satisfactory even on treating the explant with ascorbic acid. Hence, for Bhimkol the in vitro regeneration protocol needs further standardization. In Malbhog, MS medium supplemented with various combinations of BAP and NAA (1-Naphthaleneacetic acid) were used for the optimization of multiple shoot regeneration. The results indicate that shoot regeneration and proliferation was significantly higher in the presence of 10 mg/l BAP and 0.2 mg/l NAA as compared to other concentrations in terms of the number of days required for shoot initiation, number of shoots developed per explant and length of shoots. For assessment of genetic fidelity of the multiple shoots, DNA was isolated from leaves of in vitro regenerated shoots and the field grown mother plant. Thirteen SSR primer pairs (already reported) were used in the analysis which resulted in banding patterns that were uniform and monomorphic for all the clones tested and comparable to the mother plant from which the cultures had been established. This indicated that all the regenerated shoots are true to type with no variation in their genetic makeup. Thus, the optimized shoot regeneration protocol could be potentially used for mass multiplication of Malbhog, once the in vitro rooting and hardening of regenerated plantlets is standardized.
Open Access
Molecular characterization and nutritional equivalence evaluation of transgenic chickpea expressing either a cry1Ac or cry2Aa gen
(AAU, Jorhat, 2019-10) Gupta, Rubi; Sarmah, Bidyut Kumar
iosafety assessment of transgenic chickpeas having B.thuringiensis genes for resistance to pod borers is a regulatory requirement and mandatory to document before releasing in the field. Therefore, Bt chickpea lines harbouring either a cry1Ac or cry2Aa gene were characterized for the presence and expression of the transgene in their advanced generations, biosafety assessments and transcript profile were studied. The homozygous lines were selected for comparative nutritional equivalence assay. Biochemical estimations of major nutritional components such as proximates, vitamins, minerals, fatty acids and anti nutrients confirmed that the Bt chickpeas lines are nutritionally equivalent to their non-transgenic counterparts and the seed composition is similar or within the range reported, previously. Seed protein quality was investigated by separating the proteins in PAGE and eluted proteins after mass spectrometry (MS) showed expected fractions of 11S legumin, 7S vicilin, and 2S albumin of chickpea storage proteins in the transgenic lines. The protein digestibility was assayed using the multi-enzyme system and transient pepsin hydrolysis to mimic simulated gastric fluid followed by trypsin hydrolysis to mimic simulated intestinal fluid. Total seed proteins of both the transgenic and nontransgenic lines were digested at a similar rate and enzyme-resistant peptides were not observed in transgenic Bt chickpea lines. The unintended changes in the whole transcriptome profile of Bt chickpeas were surveyed using a homozygous transgenic line expressing a cry1Ac gene. The differentially expressed genes (DEGs) profiling confirmed a low (0.69%, log2fold change≥2) frequency of differentially expressed in the transgenic chickpea line. Only a small (34 upregulated) proportion of genes showed > 2 fold (P-value of 0.05, FDR of 0.05) change in their expression, while only 23 genes down-regulated by >2 fold. Furthermore, no transcripts for potential allergenic proteins were represented in the DEGs. Most of these genes appeared to be developmentally regulated or stress-related which was expected because absolute synchronous growth and development even under a controlled environment are challenging. A few upregulated (AT-hook motif nuclear-localized protein 17-like, probable inactive 2-oxoglutaratedependent dioxygenase AOP2, protein EXORDIUM-like) and down-regulated (histone H2B, gonadal, embryonic abundant protein VF30.1-like, fasciclin-like arabinogalactan protein 12) genes were subjected to qPCR. The qPCR data validated the fold change of the up-regulated (>2) and down-regulated (>-2) genes. Thus, the above data revealed no potential alterations in the nutritional equivalence or transcript profile of transgenic Bt chickpeas.
Open Access
Cloning, Characterization and RNAi mediated silencing of gene encoding 1-deoxy-D-xylulose 5-phosphatereductoisome rase (DXR) in Centella asiatica
(AAU, Jorhat, 2019-01) Sharma, Richa; Sen, Priyabrata
Centella asiatica (L.) is one of the most valuable medicinal plants which belong to the family Apiaceae. The medicinal importance of this green leafy vegetable is known since prehistoric times. The pharmaceutical importance of this herb is due to the accumulation of large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids synthesized by the isoprenoid biosynthesis pathway. Biosynthesis of triterpenoid in the plants proceeds via either of the two pathways, viz. Mevalonate (MVA) pathway (in the cytosol) or 2-C-methyl-Derythritol 4-phosphate (MEP) pathway (in plastid). In Centella, the pathway leading to the accumulation of triterpenoid is still not known. Thus, to know whether the MVA or MEP pathway or contribution of both has a role in the biosynthesis of triterpenoid, silencing the key regulatory enzyme gene using RNAi tool, of each of the pathway and then to analyze a metabolite is an efficient approach. In our lab, HMGR (a key enzyme of MVA pathway) RNAi construct has already been designed, confirmed by RT-PCR and validated by Agro-infiltration. 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) play a role in catalyzing the first committed step of the MEP pathway. The present study is the first step aimed to delineate the MEP pathway using RNAi silencing approach to knock down rate limiting 1-deoxy-Dxylulose- 5-phosphate reductoisomerase (DXR) enzyme. The full-length DXR gene sequence (JQ965955) of Centella has been characterized using in silico approach. CaDXR is a 1425bp ORF encoding a peptide of 474 amino acids and of molecular weight of 51.5 KDa. Multiple sequence comparison using MEGA tool showed the presence of two NADPH binding motif, two substrates binding motif, and one cleavage site motif. In this study, the 3-D structure of CaDXR was identified and validated along with this molecular dynamics simulation and finally docking with cofactor NADPH was done. The expression analysis suggests that CaDXR is differentially expressed in different tissues (with maximal expression in node and lowest in the roots). Our result suggests that nodes may be crucial to terpenoid biosynthesis in Centella asiatica. The RNAi-DXR construct was designed using the pHANNIBAL vector and subsequently cloned into a binary vector pART27. The binary vector pART27 containing RNAi-CaDXR construct was transformed into Agrobacterium strain AGL1. The transient analysis of the RNAi-CaDXR using semiquantitative RT-PCR confirmed the silencing of the endogenous DXR gene in Nicotiana and further confirmed in Centella asiatica. Thus, further incorporation of both the RNAi construct (HMGR and DXR) in transformed Centella shall shed light into the pathway that leads to the synthesis of principal secondary metabolites i.e centelloids.

Theses

Browse

Thesis Type

Type

Author

Date

Subject

Search Results