Regulation of antioxidant enzymes and osmo-protectant molecules by salt and drought responsive genes in Bambusa balcooa.

Bio-energy crops need to be grown on marginal salt and drought lands in India as per policy. Understanding environmental stress response in bio-energy crops might help in promoting cultivation of bio-energy feedstock on marginal salty and drought land. This is one of the first report for vegetative propagation of Bamboo (Bambusa balcooa) under salt and drought stress to understand antioxidant enzymes' gene regulations to combat stress through activation of antioxidant enzymes and osmo-protectant molecules to scavenge reactive oxygen species as measured by physiological changes. Morphological, physiological, and biochemical traits were noted as indicators of plant health upon different sodium chloride (NaCl) salt-stress while various drought conditions with correlation analysis. A significant up-regulation of genes related to most of the antioxidant enzymes was observed up to salinity of 14 mS cm- 1 electric conductivity (EC) at 150 mM NaCl experimental salt stress which declined with higher salt-stress. While in the case of drought-stress, all genes remained up-regulated while proline dehydrogenase (PDH) remained down-regulated up-to 100% drought-stress having 4% soil moisture. The gene expressions of antioxidant enzymes were significantly correlated with their corresponding gene-products namely super-oxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) activities. Biochemical parameters such as, soluble sugar, proline, malondialdehyde (MDA), total amino acids, hydrogen peroxide and electrolyte leakage ratio also showed positive correlation (p = 0.001) with salt condition. Genetic and biochemical test parameters were significantly correlated with physiological attributes of plant health at soil EC of 14 mS cm- 1 shown as 150 mM NaCl salt stress and 60% drought-stress having 17% soil moisture content, were the optimum stress tolerance limits observed. Application of these data would be useful to cultivate 0.63 million ha of salinity affected land and 10.05 million ha of drought affected land among wastelands in India to meet biofuel need.

Analysis of toxic, antidiabetic and antioxidant potential of Bambusa balcooa Roxb. leaf extracts in alloxan-induced diabetic rats.

Bambusa balcooa (Poaceae) is native to India and has been used traditionally by the tribes of Northeast India to treat diabetes. The present investigation was aimed to evaluate the toxicity, anti-diabetic activity along with in vitro antioxidant activity of the leaf of B. balcooa in alloxan-induced diabetic rats and also identify active compounds by using HPLC. The acute toxicity test of aqueous extract of B. balcooa leaf revealed that the median lethal dose (LD50) of B. balcooa aqueous extract (BAQE) was 5.18 g/kg body weight in mice. Administration of BAQE at 100 and 200 mg/kg in alloxan-induced diabetic rats showed significant reduction in fasting blood glucose and glycated hemoglobin while plasma insulin level was elevated compared to diabetic control. Both the doses were effective when compared to diabetic glibenclamide rats. The BAQE treated diabetic rats showed significant increase in the endogenous antioxidant enzymes superoxide dismutase, glutathione peroxidase and decrease in malondialdehyde levels. HPLC analysis of BAQE showed the presence of rutin, gallic acid and ß sitosterol. Thus, it can be inferred from this study that BAQE possess antidiabetic and in vivo antioxidant activity. The overall activity might be possibly due to the presence of potential antioxidants.

Elimination and molecular identification of endophytic bacterial contaminants during in vitro propagation of Bambusa balcooa.

Bambusa balcooa is an economically important, multipurpose bamboo species, decidedly used in construction industry. Availability of natural bamboo is depleting very rapidly due to accelerated deforestation and its unrestrained use. The large number and timely supply of saplings are the need of the hour for the restoration of bamboo stands. Micropropagation, being the potent alternative for season independent rapid regeneration, is restricted in bamboo because of endophytic contamination. An in vitro attempt has been taken to overcome the endophytic contamination by using broad spectrum antibiotics as surface sterilant as well as a media component. Ampicillin sodium salt (5 mg/ml for 30 min) as a surface sterilant was found as the best treatment for high bud breaking (80%) coupled with high branching and low contamination (20%) but it was found ineffective to control the contamination during multiplication stage. Then, two endophytes were isolated and minimum inhibitory concentration was determined through antibiotic susceptibility test for successful eradication at multiplication stage. Finally, contamination free cultures were obtained when streptocycline (100 µg/ml) and gentamicin sulphate (75 µg/ml) were added into the medium. The two isolated endophytes, BB1 and BB2, were identified through 16S rDNA techniques and NCBI-BLAST algorithm with 99% sequence similarity with those of Janibacter sp. (KX423734) and Serratia marcescens strain (KX423735). To our knowledge, this is the first report for B. balcooa where antibiotics were used as surface sterilant as well as medium component, to control endophytic bacterial contaminants, followed by their identification.

Isolation of total RNA from hard bamboo tissue rich in polyphenols and polysaccharides for gene expression studies

RNA isolation from hard and woody internodal bamboo (Bambusa balcooa) tissue is very difficult due to the presence of secondary metabolites, polysaccharides, and polyphenolics. These compounds often co-precipitate with isolated RNA and hinder downstream applications. We have developed an efficient, cost effective and reproducible RNA isolation method from hard tissue of bamboo internode. This protocol includes an additional organic solvent refinement steps to remove endogenous phenolic compounds and acidic phenol (pH 4.2) to critically stabilize RNA in extraction buffer. In addition to these, two 2M Lithium chloride washing steps were introduced to eliminate DNA and polysaccharides contamination. The RNA isolated from the present protocol was found to be superior, when compared to total RNA extracted by other available protocols. The A260/A280 absorption ratio of the isolated RNA was found ranging between 1.89-1.97. The integrity of 28S and 18S rRNA was highly satisfactory when analyzed in agarose denaturing gel. RNA was further used for RT PCR, northern hybridization, cDNA library and subtractive hybridization without any further refinement.