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Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Dist. - Nadia, West Bengal PIN - 741 252
The university established in 1974, has completed five decades of its existence as the pioneer institute of Agricultural Education, Research and Extension. The main objective of this Viswavidyalaya is to provide facilities for the study of Agriculture, Horticulture and Agricultural Engineering. It is also to conduct researches in these sciences and undertake the educational and extension programmes in agriculture among the rural clientele base, keeping in view the requirements of the state.
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ThesisItem Open Access In vitro direct regeneration and Agrobacterium rhizogenes-mediated hairy root culture for enhanced forskolin production in Indian coleus (Coleus forskohlii Briq.)(Department of Agricultural Biotechnology, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, 2022-11-21) Mitra, Monisha; Dr. Nirmal MandalColeus forskohlii is an herbaceous and aromatic plant belonging to the family Lamiaceae and order Lamiales having multifaceted uses namely in food industry, in cosmetic as well as in pharmaceutical arenas. The major metabolite found exclusively in the cork cells of the root is forskolin. Forskolin is used commercially for the treatment of glaucoma, asthma, and heart ailments. The major shortcoming faced in traditional cultivation of this plant is slow growth and incidence of pest and diseases mainly root rot caused by Macrophomina phaseolina. Furthermore, low metabolite concentration in the plants, and lower levels of forskolin is another limitation for its commercial uses. Considering all these aspects the present research has been proposed with the following two key objectives namely; 1) Establishment of in vitro propagation of Coleus forskohlii via direct regeneration approaches as well as their genetic fidelity assessment and 2) Establishment of Agrobacterium rhizogenes-mediated hairy root culture for enhanced forskolin production. Initial culture was established following a non-toxic, simple, and effective sterilization and then inoculated in basal MS medium fortified with different types and doses of single cytokinin, out of which MS medium augmented with 1.5 mg L-1 mT (meta Topolin) turned out to be promising since it displayed higher response (100% ±0.00), maximum number of shoots (12.66±1.20) and number of leaves (20±0.66) per inoculated shoot tip and also exhibited earliest response to shoot induction (5.33±0.33 days). In shoot proliferation experiment, the maximum number of shoots (12.66±0.66) along with highest number of leaves (23.66±0.88) was obtained when MS medium was supplemented with 1 mg L-1 mT + 0.05 mg L-1 NAA. The maximum number (19.33±1.76) and the length (4.76±0.38 cm) of healthy roots per plant were achieved on control medium (MS without PGRs). The sustainability of shoot regeneration ability was checked up to five subculture passages and meagre variability in terms of abnormalities like vitrification, malformation of shoots and yellowing was detected in the 5th subculture. In acclimatization experiment, the survival rate of plantlets cultured in a combination of vermiculite + sand was (91.00±0.57%). The concentration of photosynthetic pigments in plantlets during acclimatization (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) displayed a minimal increase in the first 10 days and then it increased rapidly and its maximum concentration was noted in the 30 days of acclimatization period. The micromorphological characterization of in vitro and ex vitro hardened plantlets on the basis of stomata and trichome revealed similarities in the features and it strengthened the proof towards the trueness of mother plant and the acclimatized plantlets. DNA fingerprinting of the in vitro-regenerated plantlets via analysis of inter simple sequence repeats (ISSRs) showed clonal fidelity of the plantlets with the mother plant. A stable transgenic hairy root (HR) culture has been developed using engineered three different types of Agrobacterium rhizogenes strains namely LBA1334 harbouring pCAM:2×35S:gusA binary vector; MTCC 532 and MTCC 2364, to achieve high yield of secondary metabolites. Putative transgenic HRs were observed in all the three strains. The highest transformation frequency (56.67±1.66%) was noted in internodal explants when immersed in A. rhizogenes suspension for 15 min and co-cultivated for three days. Putative transformation was confirmed by histochemical GUS staining assay that showed blue coloration in the transgenic HRs. This was further confirmed by PCR analysis of rolA, rolB, rolC and virD genes. Time course study revealed that the transgenic HRs grew rapidly in ½MS liquid medium with highest biomass production 0.464±0.0008 g per 50 mL DW after 42 days of culture. In the elicitor mediated experiment, when 20 mg L-1 methyl jasmonate was added to the 15 days old growing culture the biomass of HRs increased by 1.06-fold (DW=0.490±0.06 g). Histochemical localization through vanillin staining revealed the presence of forskolin and related terpenoids in the hairy roots since it displayed violet coloration in the cells. The forskolin content was compared among the transgenic HRs, in vivo as well as in vitro roots with the help of HPLC. The estimation revealed that there is 2.10 and 1.14 times increase in forskolin content in HRs (2.99±0.009 mg g-1 DW) in comparison to the amount accumulated in the in vivo (1.42±0.003 mg g-1 DW) and in vitro-grown (2.63±0.006 mg g-1 DW) roots, respectively. Addition of 20 mg L-1 methyl jasmonate to the 15 days old HR culture boosted the forskolin accumulation to the level of 5.42±0.007 mg g-1 DW, which was 1.8 times of the non elicitated HR culture, respectively. In addition, the content of several important phenolic and flavonoid compounds was increased in the transgenic HRs as analysed by spectrophotometer. The outcome of the present investigation shows great potential for a systematic and efficient in vitro direct regeneration system of plantlets and transgenic HR culture mediated enhanced forskolin accumulation that can serve as a substitute of conventional extraction technologies in order to provide resources of forskolin to the pharmaceutical industries.ThesisItem Open Access Development of genetic and genomic resources for Downy mildew resistance and low ODAP content in Lathyrus sativus L.(Agricultural Biotechnology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia – 741252, 2023-12-20) Saha Pratik; Dr. Md. Nasim AliGrasspea (Lathyrus sativus L.) belongs to the family Fabaceae and the tribe Vicieae having chromosome number (2n = 2x = 14). India is the largest producer of lathyrus which is the third most important pulse crop after chickpea and pigeon pea. It is one of the most important climate resilient crops which thrive well not only in extreme weather and stressed condition but also in poor soil. It is a good source of dietary proteins and is used as food and fodder including improvement of soil fertility. The cultivation of this crop is neglected due to the presence of anti-nutritional compound β-N-oxalylamino-L-alanine (BOAA) or β-N- oxalyl-L-α, β-diaminopropionic acid (ODAP) which is considered as causative agent of the disease Lathyrism, the paralytic disorder. In recent past, many breeders started working to develop low-ODAP lines like Ratan, Prateek and BioL-212 etc. containing ~ 0.2% ODAP. Among different biotic factors, downy mildew (Peronospora lathi-palustris) poses a serious threat in case of water stagnation during winter rains. On the other hand, introgression of disease resistance gene leading to increase yield has also been initiated in grasspea using marker assisted selection. Therefore, it is necessary to develop trait linked molecular markers for marker assisted breeding for developing new Lathyrus varieties. The present work was aimed to screen out the segregants, from F2 and F3 population derived from two different cross combination, Berhampore local x Mahateora and Mahateora x BioL-212 attributed with high grain yield coupled with zero/negligible ODAP content and resistance to downy mildew resistance. In the present study, the segregating population (F2 and F3) developed from two sets of crosses (Mahateora x BIO L-212 and Berhampore Local x Mahateora) were characterized. Simple Sequence Repeats (SSR) marker-based assessment was done for parents and segregating populations in search of trait associated marker and to deduce molecular diversity. A total of 120 SSR markers including 30 EST-SSR were used for molecular diversity and screening for association with ODAP and /or Downey mildew. All morphometric traits were statistically significant in segregating populations derived from both the parental crosses. Based on the mean seed yield plant-1, the F2 segregants MBiF2-1, MBiF2-2 MBiF2-4 and three F3 Segregants MBiF3-43, MBiF3-54, and MBiF3-52 from Mahateora x BioL-212 as well as from Berhampore Local x Mahateora, F2 segregants BLMF2-23, BLMF2-37 and BLMF2-50 and F3 segregants BLMF3-46, BLMF3-143 and BLMF3-100 were found very promising. Low ODAP content (≤0.1%) was found in BLMF3- 32, BLMF3-78, BLMF3-8, BLMF3-186, BLMF3-39, BLMF3-46 and BLMF3-211. With respect to the percent disease incidence (PDI), the identified resistant segregants to Downy mildew were BLMF3-128 (2%), BLMF3-8 (4%), BLMF3-36, (12%) BLMF3-39 (12%), BLMF3-195 (14%) and BLMF3-100 (18%). The PDI was highest in BLMF3-38 (44%). Based on highest seed yield/plant (>20 g), low ODAP (≤0.1%) and PDI (<12%), the most promising segregants identified were BLMF3-39, BLMF3100, BLMF3211, BLMF3168. The seed yield/plant were found to be significantly and positively correlated at both genotypic and phenotypic level with Plant height, number of branches plant-1, number of pods plant-1, pod length, number of internodes plant-1 and number of seeds pod-1. The Regression model identified number of pod plant-1, 100 seed weight, number of seeds pod-1 and pod length in F2 of the crosses where as in the F3 population of Berhampore Local x Mahateora, number of pods plant-1, number of seeds pod-1, number of internodes plant-1, 100 seed weight, plant height and number of branches plant-1 were considered as the better explanatory variables. No significant differences between GCV and PCV were found for most of the traits, indicating lesser influences of environmental factors. High heritability coupled with high Genetic advance over mean for plant height, number of branches plant-1, number of pods plant-1, and seed yield plant-1 were recorded to be used for indirect phenotypic selection. Multivariate analysis based on morphometric parameters from the segregants from Mahateora x Bio L-212 viz, MBiF2-49, 2, 4, 55, 46, 20, 11, 19, 52, 33, 25 and MBiF3-52; from Berhampore Local X Mahateora the segregants BLMF2-27, 56, 30, 37, 50, 59 and, BLMF3-128, 143, 46, 211, 100, 168 and 39 were found as the diverged genotypes. Molecular diversity based on SSR genotyping formed 7 distinct groups where Cluster V was found to be the largest with 15 cluster members. Based on higher Polymorphic Information Content (PIC) and Marker Index (MI) value, the SSRs namely c39279_g1_i12, c46049_g2_i1, c34810_g1_i1, c35787_g1_i1, c43114_g1_i1, c46949_g2_i1, c13442_gi_i1 and S-168 could be used in molecular diversity analysis in lathyrus for future breeding programme. From the study, the EST-SSR marker c31994_g1_i1 and c35336_g1_i4 were found putatively associated with low ODAP content from both F2 and F3 population. In the F3 population c39249_g1_i1 and S-159 showed association with downy mildew resistance.ThesisItem Open Access Elucidating the role of lignin pathway genes and enzymes in determining the fibre quality traits in Tossa jute (Corchorus olitorius L.)(Department of Agricultural Biotechnology, Bidhan Chandra Krishi Viswavidyalaya , Mohanpur , Nadia-741252, 2023) Sayani Bandyopadhyay; Dr. Md. Nasim AliJute plant, belongs to genus Corchorus, is the second most important fibre crop after cotton. The fibre of jute is bio degradable, eco-friendly and renewable in nature which can be utilized in the replacement of synthetic fibre. The quality of jute fibre decreases with higher levels of lignin and lower levels of cellulose content, which needs to be addressed and improved. The fibre quality linked genes reportedly found in two different biochemical pathways, namely, lignin biosynthesis including shikimate aromatic amino acid pathway, monolignol pathway and cellulose biosynthesis pathway. In order to get the molecular insights of the fibre biogenesis, the present study was aimed to elucidate the role of the enzymes as well as the genes involved in lig jute varieties viz, JRO 524, which is a popular jute variety with comparatively coarse fibre and JBO 1, which has finer fibre were selected. The comparative yield performances were observed along with 14 other genotypes collected from AINP, J&AF. The qualities of the fibre in respect of fibre fineness and bundle strength were also assessed. estimation of biochemical parameters (acid detergent fibre, cellulose, acid detergent lignin, total protein); enzymatic assay (phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase) and histochemical assay (primary, secondary phloic fibre bundle and S lignin detection) were also carried out. The relative expression of Coumarate 3-hydroxylase (C3H), Ferulate 5-hydroxylase (F5H) and Cellulose synthase (Ces) genes at different growth stages in the bark tissues of the two tested varieties was studied using qRT-PCR. The statistical significant differences in respect of the studied traits among the selected genotypes were found. The highest fibre weight/ plant and fibre % were recorded in JRO 524 among all the genotypes. Among the tested varieties, the plant height, basal diameter and fibre weight/ plant of JRO 524 were significantly higher than JBO 1. Based on the quality parameters, JBO 1 can be considered as fine fibre and JRO 524 can be termed as coarse fibre. The fibre weight/ plant was significantly and positively correlated with plant height, green weight/ plant, dry weight/ plant, number of nodes/ plant and stick weight/ plant as evident from correlation analysis. Based on cluster analysis, the 16 genotypes were grouped into 5 clusters where the selected two varieties belong to same cluster indicating lesser genetic diversity among them. In the bark tissue of JBO 1, the acid detergent fibre and acid detergent lignin content, PAL and CAD enzyme activities were significantly lower than JRO 524 at 30 Days after germination (DAG) and 60 DAG stages whereas cellulose content of the bark tissue of JBO 1 was significantly higher than JRO 524 at 60 DAG. On the basis of histochemical analysis, variations in the fibre cell morphology were found between the two varieties where the fibre cell bundles of JRO 524 were overall more dignified than JBO 1. Four genes viz, PAL, CAD, C3H and F5H involved in lignin biosynthesis and Ces gene involved in cellulose biosynthesis of tossa jute were recognized and functionally characterized by finding the molecular properties and by constructing phylogenetic trees along with other homologous genes of other organisms. Sequencing of the partial CDS of F5H gene of JRO 524 and JBO 1 was done and the CDSs were functionally characterized and submitted at NCBI (Accession nos: OP629814.1 and OP629813.1). Based on the qRT-PCR analysis, it was observed that that C3H gene involved in monolignol pathway, had significantly lower expression in the bark tissue of JBO 1 at both 30 DAG (0.43 fold) and 60 DAG (0.84 fold) in comparison with JRO 524, whereas, F5H gene involved in monolignol pathway, was significantly overexpressed (1.29 folds) in JBO 1 at 30 DAG and under expressed (0.51 fold) at 60 DAG. The Ces gene involved cellulose biosynthetic pathway was significantly expressed more at 30 DAG (1.08 folds) and expressed lesser at 60 DAG (0.75) compared to JRO 524. Thus, from the present study it is evident that the fine fibre in jute is attributed with more bundle strength, lower lignin content and lower activity of enzymes involved in lignin biosynthetic pathway, higher cellulose content, lower expression of C3H gene at both 30, 60 DAG, lower expression of F5H at 60 DAG along with higher Ces expression at 30 DAG.ThesisItem Open Access A Study On Some Biochemical Changes In Relation To Preparation And Utilization Of Compost(Bidhan Chandra Krishi Vishwavidyalaya;Nadia, 2003) Das, Sandip Kumar; Chakravarty, Arunabha
