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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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Subject: Agricultural Biotechnology × Author: Mitra, Monisha × End date: 2023 × Start date: 2020 × Type: Thesis ×
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    ThesisItemOpen 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 Mandal
    Coleus 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.