Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

A SIMPLE AND EFFICIENT MICROPROPAGATION PROTOCOL FOR DEVELOPING PLANTLETS OF EXACUM BICOLOR ROXB. – AN ENDANGERED, ORNAMENTAL, AND ANTIDIABETIC HERB

Objective: Exacum bicolor Roxb. is an endangered medicinal herb due to overexploitation by humans and its inefficient vegetative reproduction. Here, we report an efficient and simple procedure for the regeneration of E. bicolor Roxb. using leaf as an explant. Methods: The optimal concentrations of the hormones needed for callus induction were determined by full factorial method using DOE (Design expert ver. 8.0). The hormones selected based on literature were kinetin, indole acetic acid, and 6-Benzylaminopurine (BAP). Multiple shoot regeneration was carried out in liquid and solid media with the optimal concentrations of the hormones obtained by DOE. Rooting was initiated using Murashige and Skoog media containing naphthalene acetic acid 0.5 mg/l, indole butyric acid (IBA) 1.0 mg/l, and gibberellic acid 3 0.5 mg/l along with 0.2% of activated charcoal. Results: Analysis of full factorial design run showed that BAP in combination with kinetin was effective for the growth of callus and multiple shoot regeneration was higher in liquid media (81.25%). The rate of rooting was observed to be 88.23% and the average number of roots was 0.26. Plantlets with budding apical region and well-established leaves and roots were observed in 30 days. Conclusion: The protocol reported here can be used for effective production of E. bicolor plants in a shorter duration compared to the conventional approach.

Micropropagation of Salacia macrosperma Wight - An Endemic Medicinal Plant of Western Ghats

Salacia macrosperma Wight. - a potent medicinal plant facing the verge of rare and endemic status in the Western Ghats region of southern India. The effective protocol has been standardized for callus induction and multiple shoot regeneration using leaf and nodal explants. The Murashige and Skoog (MS) medium fortified with various plant growth regulators like 2,4-dichlorophenoxyacetic acid (2,4-D), Benzyl amino purine (BAP), Thidiazuron (TDZ), Indole acetic acid (IAA), Kinetin (Kn), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA). The leaf explants produced more calli than nodal explants in MS medium supplemented with 2, 4-D and BAP in combination than individual hormones. Likewise, MS medium with 1.5 mg L-1of 2, 4-D, 2.5 mg L-1 of BAP and 1.5 mg L-1of TDZ along with 1% activated charcoal was apt for multiple shoot regeneration (93.33%) from nodal explants with slight embryogenic callus. Further, each developed plantlets were produced maximum rhizogenesis in liquid MS medium supplemented with 1.0 mg L-1 of IAA. Furthermore, the cytological study of embryogenic callus revealed variations in callus cells such as multinucleate, multi-nucleolate, cytodifferentiation, chromosomal bridges were noticed, besides normal dividing stages. Further, by scanning electron micrograph (SEM) analysis of embryogenic callus different stages of morphogenic developmental features were recorded.

In vitro Micropropagation of Bacopa monnieri (L.) Penn. - An Important Medicinal Plant

Investigation on in vitro multiple shoot regeneration in Bacopa monnieri (L.) Penn. using leaf and nodal explants was carried out on MS containing various concentrations and combinations of BAP, Kn, NAA and 2,4-D. Of the two explants, leaf showed the best response towards shoot regeneration and subsequent plant development on MS with 1.0 mg/l BAP and 0.25 mg/l Kn. In this combination, the mean number of shoots/explant was 10.6 ± 0.11 in leaf and 9.6 ± 0.29 in nodal explants. Maximum shoot length was recorded as 12.6 ± 0.21 and 11.20 ± 0.30 from leaf and nodal explants after six weeks of culture, respectively. Half strength of MS supplemented with 0.25 mg/l IBA was found to be the best medium for root formation. The in vitro regenerated plantlets were successfully transplanted in soil after acclimatization. Plant Tissue Cult. & Biotech. 30(1): 57-63, 2020 (June)

The Influence of Seed Viability on the Germination and In Vitro Multiple Shoot Regeneration of Soybean (Glycine max L.)

The moisture status of seeds is usually high during the period of harvest and deterioration (loss of viability) starts to occur when seeds are stored for longer periods. In the present study, soybean seeds were evaluated using a standard germination test, in vitro germination, and for efficient multiple shoot induction, following storage under ambient conditions for 0, 3, 6 and 9 months. Results showed that seeds stored for more than 3 months had reduced moisture content and decreased germination percentages in LS677, LS678, Dundee, Peking, TGx1740-2F and TGx1835-10E of the tested genotypes. In particular, seeds stored for 9 months showed significantly poor seed viability and less than 50% overall seed germination (Dundee—42%, LS678—49%, TGx 1740-2F—44%, TGx 1835-10E—48%), except for LS677 and Peking, with 52 and 55%, respectively. The efficiency of multiple shoot induction also decreased with prolonged seed storage, with all genotypes recording an overall decline from about 96% to 40% regeneration efficiency within 9 months. The results obtained clearly indicated that high germination rates and efficient in vitro shoot induction depended largely on seed viability and storage duration, and significantly differed according to genotypes.