TITLE:
Thin Cell Layer (TCL) Culture System for Herbal Biomass Production and Genetic Transformation of Bacopa monnieri L. Wettst.
AUTHORS:
LaMont Alexander Croom, Carissa L. Jackson, Brajesh Nanda Vaidya, Prahlad Parajuli, Nirmal Joshee
KEYWORDS:
Bacopa, Plant Tissue Culture, Total Polyphenols, Transformation, Transverse Thin Cell Layer
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.7 No.8,
June
17,
2016
ABSTRACT: Bacopa monnieri (L.) Wettst. (Scrophulariaceae) is a highly sought after medicinal plant with therapeutic
properties as cognition enhancer as well as for other brain and body functions.
Research was conducted to optimize a thin cell layer explant based micropropagation
system to assist mass propagation. Thin cell layers (TCL) derived from leaf and
internode segments were used as explants. Murashige and Skoog medium was used to
formulate shoot induction, elongation, and rooting media. Shoot induction media
were prepared by supplementing three concentrations (0.1, 1.0, and 10.0 μM) of four
cytokinins 6-benzylaminopurine, 2-isopentenyl-adenine, 6-3-Hydroxybenzylaminopurine,
and thidiazuron to study adventitious shoot bud induction response. An optimum shoot
bud induction response was observed on MS medium supplemented with 10.0 μM 6-benzylaminopurine
for both leaf and stem transverse thin cell layer (tTCL) explants. The average number
of shoot buds from leaf tTCL explants was 59, whereas, on an average, 33 shoot buds
were regenerated from internode tTCL explants. Elongation of adventitious shoot
buds was achieved best in a liquid medium using
Liquid Lab Rocker® system. Elongated shoots recorded 100% rooting in MS medium supplemented with 5 μM indole butyric acid. Bacopa micropropagation employing tTCL explants
for initial shoot bud induction and using LLR® boxes in subsequent
elongation step can achieve cost effective way to regenerate high volume of plantlets
and biomass required for herbal industry. Leaf and stem tTCL explants both were
suitable for Agrobacterium tumefaciens (EHA105) mediated genetic transformation. Successful transformation was scored within
three days of co-cultivation with Agrobacterium suspension on the basis of Enhanced Green Fluorescent Protein (EGFP) expression
as an early and non-destructible screening device. Transformation frequencies of
83% and 76% were accomplished for leaf and stem tTCL explants, respectively. Greenhouse
grown Bacopa plants were analyzed as fresh
and dry methanolic extracts for total polyphenol content (811.93 ± 7.98 and 814
± 17.64 GAE mg g-1) and the Trolox Equivalent Antioxidant Capacity values were 1918.25
± 173.12 and 3163.14 ± 403.25 μmol/g, respectively.