Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid

Abstract

(Lindl.) Deb, a small, perennial, monopodial, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000 m asl. The pseudobulbs are important ingredients of century old drug 'Ashtavarga' and a polyherbal energetic tonic 'Chyavanprash'. An efficient genetically stable in vitro propagation protocol using transverse thin cell layer culture system was established for . In the present report, meta-topolin alone proved to be three times more beneficial compared to other routinely used cytokinins in inducing highest number of shoot buds, plant height and growth of regenerated shoots. The highest regeneration frequency (89%) along with maximum number of adventitious shoots per explant (22.5 ± 0.6) was observed in MS medium supplemented with 1.0 mg/l meta-topolin and 0.5 mg/l α-naphthalene acetic acid. Highest rooting frequency with highest number of roots (8.66 ± 0.3) was achieved in half-strength MS medium fortified with 1.0 mg/l indole acetic acid. Clonal stability of in vitro-derived plantlets was evaluated and compared to donor plant using intron splice junction (ISJ) markers and flow cytometry. ISJ markers revealed 4.76% clonal variability indicating high degree of genetic stability amongst the in vitro-derived regenerants. The nuclear DNA content of (2n) was found to be 2C = 2.760 ± 0.02 pg and therefore, 1349.64 Mbp (1C). Flow cytometry analysis of actively growing young and mature leaves from donor as well as in vitro-derived plantlets revealed presence of three peaks corresponding to 2C, 4C and 8C, while 2C was the most abundant. In the present investigation, there was no significant difference in the 2C DNA content between the mother and in vitro-derived plants; however, the frequency of endopolyploid cells varied in young and adult plants. An increased HO content as well as lipid peroxidation activities were observed during early stages of acclimatization which declined afterwards. The enhanced activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in acclimatized plantlets as compared to in vitro-grown ones revealed their active involvement in growth and development against oxidative stress under external adverse environment.

Recent advances and novelties in the thin cell layer-based plant biotechnology – a mini-review

“…In at least two cases, tTCLs served as the donor explants for genetic transformation experiments (Croom et al, 2016;Vaidya et al, 2016), and root-derived TCLs continue to serve as basic tools in assessing axillary root development in A. thaliana (Della Rovere et al, 2016;Fattorini et al, 2017Fattorini et al, , 2018. Except for three studies (Bose et al, 2017;Bhattacharyya et al, 2018;Parthibhan et al, 2018), none of the other new studies, similar to the vast majority of older studies on TCLs (Teixeira da Silva and Dobránszki, 2015), assessed the genetic stability relative to plants derived from the conventional culture, and such genetic testing needs to be implemented in future research on TCLs. Parthibhan et al (2018) used inter-simple sequence repeats (ISSR) to confirm the monomorphic nature of the resulting somatic embryos.…”

“…Parthibhan et al (2018) used inter-simple sequence repeats (ISSR) to confirm the monomorphic nature of the resulting somatic embryos. Bose et al (2017) tested genetic fidelity by flow cytometry and intron splice junction (ISJ) markers, finding some polyploidy (5-11% 8C) in the leaves of the donor and acclimatized plants when the former was used, and 5-17% polymorphism in tissue cultured plants when five of the 15 ISJ primers were used. Bhattacharyya et al 2018, on the other hand, applied interretrotransposon-targeted amplified region (IRAP) and ISSR separately and in combination to evaluate the genetic fidelity of regenerated shoots.…”

“…In a study by Bose et al (2017), adventitious shoots were induced from stem tTCLs in 89% of explants, forming 22.5 shoots per tTCL when plated in MS basal medium with 4.1 μM meta-topolin and 2.7 μM NAA. Shoots Recent advances and novelties in the thin cell layer-based plant biotechnology -a mini-review 93 were then rooted in a half-strength MS medium with 5.7 μM indole-3-acetic acid.…”

Structural Adaptation and Antioxidant Response of Guarianthe bowringiana (O’Brien) Dressler & W. E. Higgins (Orchidaceae Juss.) Seedlings During Ex Vitro Acclimatization

“…SOD converts the superoxide anion into hydrogen peroxide. erefore, this enzyme is used as a biomarker of both oxidative damage and antioxidant defense enzymes in plants (Bose et al, 2017).…”

“…e exhibited rate of increase of SOD activity allows us to suggest that the activity of this enzyme reached the highest value on the seventh day of acclimation stage. e further decline in the SOD activity may be correlated with the reduced oxidative stress and membrane peroxidation, which signifies adjustment of plants to the external stress during the latter phase of the acclimatization period (Bose et al, 2017).…”