38. Impact of phyto-hormone concentrations in optimizing cell suspension culture of flue-cured tobacco (Nicotiana tabaccum L.) cultivars

Abstract

Plant cell suspension cultures are mostly used for the biochemical investigation of cell physiology, growth, metabolism and production of secondary metabolites. The study was undertaken to optimize protocol for callus induction and cell suspension in K-326 and Honghuadajinyuan (HD) cultivars of tobacco (Nicotiana tabaccum). The experiment was conducted at the Key Laboratory of Tobacco Genetic Improvement and Biotechnology, Chinese Academy of Agricultural Sciences (CAAS), Qingdao, P.R. China. In this study explants leaf were inoculated on the (Murashig and Skoog) media with supplementation of exogenous growth regulators of plant. For the morphogenic callus proliferation and indication form explants, (MS) medium was used with (NAA, 6-BA and 2, 4-D phytohormones. Callus derived leaves were stabilized for 4 week of period.  The tobacco cell suspension culture was established initially through the culture of friable leaf derived callus in the medium of liquid callus induction. The results of growth regulator combination significantly (P<.0.05) affected on the calli growth. In this study between different treatments, the highest frequency of callus induction was recorded in the level (1) (i.e. NAA) (.33mg/l) and 2, 4-D (0.5mg/L, followed by the level of 03 (i.e. 0.75mg/LNAA, 2.4mg/L 6-BA and 0.5mg/L2, 4-D), respectively. The subsequently sub-culturing of friable callus on callus induction media enhanced callus biomass subculture cycle. The callus induction obtained from HD and K-326 leaf explants was 82.5% and 80%, respectively. The cell growth curve showed that, cells of HD and K-326 produced highest fresh cell mass of 38.07 and 37.67 g, respectively. The production of callus biomass became stable after three subculture cycles. The cells subsequently grew healthy and maintained well in MS liquid medium supplement with the optimal hormone. The browning occurred when the cultures reached the highest fresh cell mass. Therefore, in order to maintain healthy cultures, sub-culturing should be done before browning occurs.

Keywords: Callus induction; Cell culture; Tobacco; Growth regulators

http://dx.doi.org/10.19045/bspab.2020.90275