The influence of silver nitrate (AgNO 3 ) and cobalt chloride (CoCl 2 ) on shoot multiplication and in vitro flowering in Capsicum frutescens Mill. was investigated. Exogenous administration of AgNO 3 and CoCl 2 at a concentration of 30 µM resulted in the maximum tissue response in terms of shoot length and number of shoots after 45 days culturing on MS medium. Both silver nitrate (40 µM) and cobalt chloride (30 µM) influenced in vitro flowering after 25 and 45 days respectively. This is the first report on in vitro flowering in C. frutescens. The study also demonstrated successful transformation of pollen obtained from the in vitro flowers. Since capsicum is highly recalcitrant to in vitro plant regeneration, the results of the study may be highly useful in transformation of capsicum using germ free in vitro flowers.
Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472-497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 lM indole-3-acetic acid, 10 lM silver nitrate and either of 13.31-89.77 lM benzyl adenine (BA), 9.29-23.23 lM kinetin, 0.91-9.12 lM zeatin, 2.46-9.84 lM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 lM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 lM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.
Basella spp. a perennial vine of Basellaceae family used as a leafy vegetable. Phytonutrient of Basella spp. is being exploited in Indian medicinal system since antiquity for its antifungal, anticonvulsant, antiin ammatory, antipyretic, antiulcer and analgesic properties. Propagation of Basella through seeds have limitations for germination and owering, in vitro regeneration was studied on MS (Murashige& Skoog) media supplemented with various plant growth regulators, indole acetic acid (IAA), Indole butyric acid (IBA), 1-naphthalene acetic acid (NAA), N,6-benzyladenine (BA), kinetin (KIN), zeatin (ZEA), gibberillic acid (GA 3 ), and adenine sulphate (ADS), silver nitrate (AgNO 3 ) as additives. The shoot bud initiation was observed in all the combinations studied showing a good response for direct regeneration. Shooting (84%) was observed in 12 days after inoculation in 1mg l − 1 BA + 0.1 mg l − 1 NAA. Liquid media containing 0.1mg l − 1 BA + 0.5 mg l − 1 KIN + 0.1 mg l − 1 IAA was preeminent in multiple shoots (22 ± 0.13) production with average shoot length (5.81 ± 0.19) in 5 weeks (wk). Supplementation of 40 mg l − 1 AgNO 3 and 40 mg l − 1 ADS to media containing 1mg l − 1 BA + 0.1 mg l − 1 NAA resulted in enhanced number of elongated shoots with number of leaves. In vitro owering was obtained on MS media containing 0.5mg l − 1 BA + 0.5 mg l − 1 GA 3 concentrations. The survival rate of hardened plants was 90% after transferring to soil. This protocol can be e ciently used for mass production for regeneration of genetically transformed Basella spp. in studying its metabolite pro le specially betalains and transformation of Basella.
Since ages, plants continue to provide new remedies to mankind. Hemidesmus indicus L. R. Br. is one such plant belonging to family Apocynaceae, showing potent medicinal properties known through traditional knowledge. Hemidesmus is also explored for the presence of flavoring compound namely 2-hydroxy-4-methoxybenzaldehyde (HMB) which is used in pharmaceutical and nutraceutical industries. Due to anthropogenic activities, the plant has been exploited till the ridge for its ethnobotanical properties for mankind. Biotechnological intervention to conserve this endangered sps through in vitro plant cultures, micropropogation and genetic transformation studies is the pre-requite to maintain it from extinction. The objective of the study is to improve the regeneration potential and optimize the genetic transformation in Hemidesmus indicus. The direct regeneration of Hemidesmus indicus through leaf explants, nodal explants with subsequent plant regeneration using Murashige and Skoog (MS) medium supplemented with various plant growth regulators (auxins, cytokinins, and gibberellic acid), adenine sulphate, TRIA. The Agrobacterium tumefaciens mediated genetic transformation studies in Hemidesmus indicus was carried out in callus cultures using the plant expression vector pCAMBIA 1301. The caulogenic response of 78.8%, 73.3% and 71.4% was observed when the leaf explant was inoculated on MS media containing 2.3 mgL− 1 BAP + 0.2 mgL− 1 2,4-D, 0.02 mgL− 1 TRIA + 2 mgL− 1 BAP, 1 mgL− 1 KIN + 1 mgL− 1 NAA respectively with creamish yellow nodular friable callus by the 4 weeks. The initiation of shoot bud was observed within three days after inoculation of nodal explant on media supplemented with 1 mgL− 1 BAP + 0.1 mgL− 1 NAA, 1 mgL− 1 BAP + 0.1 mgL− 1 NAA + 40 mgL− 1 AgNO3, 1 mgL− 1 BAP + 0.1 mgL− 1 NAA + 40 mgL− 1 AgNO3 + 40 mgL− 1 adenine sulphate respectively and incubated in the dark for 2 weeks. Shoot regeneration from the leaf explants was also observed within 4 weeks after inoculation in MS medium with 1 mgL− 1 BAP + 0.1 mgL− 1 NAA. Agrobacterium mediated genetic transformation was carried out successfully in callus cultures of H. indicus. The transformation efficiency was found to be 26%. The efficient shoot regeneration was observed within 4 weeks and transformation study can be further applied for over expression of biosynthetic genes to enhance the bioactive components that have immense significance in pharmaceutical, nutraceutical and cosmetic industries.
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