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.

Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal.

Withania somnifera (L.) Dunal, is an important medicinal plant being the source of extremely important compounds like withanolides and withaferin. Influence of different plant growth regulators (PGRs) were evaluated for induction of callus, callus mediated regeneration and production of secondary metabolites in them. Explants for callusing were collected from plants grown in vitro and maximum callusing (98 %) was obtained on MS medium supplemented with a combination of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 mg l(-1)) and kinetin (KN) (0.2 mg l(-1)). Among different types of calli, best shoot regeneration was observed on green, compact calli produced on MS medium with a combination of 6-benzylamino purine (BAP) and indole butyric acid (IBA). MS medium supplemented with BAP (2 mg l(-1)) showed highest frequency (98 %) of shoot bud regeneration. The micro-shoots were efficiently rooted on MS media supplemented with 0.5 mg l(-1) IBA. Rooted plants were transferred to soil-vermi-compost (1:3; w/w) medium in greenhouse for acclimatization. Presence of withanolide A and withaferin A in calli was validated through high performance thin layer chromatography (HPTLC). It was interesting to observe that the PGRs showed significant influence on the secondary metabolites production in callus and 2,4-D having the least effect. Histological studies revealed the origin of shoot tip in the callus during regeneration.

A two step method for accelerated mass propagation of Dendrocalamus asper and their evaluation in field.

An accelerated protocol for large-scale propagation of Dendrocalamus asper, an edible bamboo, has been described. Seven axillary shoots were induced in vitro from each excised tender node (15-20 mm in length) containing single axillary bud when nodal segments were inoculated in semisolid Murashige and Skoog (MS) medium fortified with 5 mg/l 6-benzylaminopurine (BAP). Maximum multiple shoot formation (14) was observed when in vitro generated axillary shoots were transferred to liquid MS medium containing 5 mg/l BAP and 40 mg/l adenine sulphate. A maximum of 93.33 % shoots were effectively rooted when transferred to liquid MS medium supplemented with 1 mg/l indole-3-biutyric acid (IBA). A simple acclimatization procedure of 55 days, primarily in cocopeat for 20 days and finally in a blend of sand, soil and farm yard manure (1:1:1 v/v), ensured a very high survival rate within next 35 days. After acclimatization, rooted plantlets were further multiplied by splitting of rhizomes, formed in vivo within 90 days of growth. After 90 and 180 days of acclimatization, plants were successfully transferred to the field and maintained in an unirrigated condition with the initial supplementation of farm yard manure @ 10 kg/pit; where around 85 % survivability with 25 culms per bush attaining an average height of 4.5 m was recorded up to four years.