MSc

Abstract

Coscinium fenestratum (Gaertn.) Colebr. is a medicinally important, perennial woody climber belonging to the family Menispermaceae. It is commonly known as Tree turmeric in English and locally as Maramanjal in Kerala and Tamil Nadu. Berberine, a yellow crystalline isoquinoline alkaloid is the main active principle compound present in the plant. The plant is a high volume traded one and its only source now is wild vegetation. Due to the combined impacts of high demand and over exploitation, the existence of this plant is under threat. Long pre-bearing age, seed dormancy, viability and regeneration problems also led to the extinction of this species and now the plant is listed as a critically endangered species in the IUCN red list of threatened species.
The present study entitled “Plant regeneration of Coscinium fenestratum (Gaertn.) Colebr. through axenic seed culture and axillary bud culture” was undertaken at tissue culture laboratory of Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara during the academic year 2018 - 2020. The objective of the study was to develop a feasible and reproducible in vitro protocol for mass propagation of Coscinium fenestratum. Experiments included, identification of best seed surface sterilization procedure, identification of best in vitro seed germination medium, standardization of shoot regeneration medium for nodal explants and cotyledonary nodal explants and also standardization of embryo culture method.
Study revealed the presence of fungal and bacterial endophytes in the seeds of Coscinium fenestratum. Among the different sterilants tested for surface sterilization of seeds, 0.1% HgCl₂ (15 min) was found to be the best agent for the culture establishment with minimum contamination. Among the different media tested for in vitro seed germination, the sterilized sand: coir pith (1:1) media soaked with distilled water was found to be better with highest germination percentage (22.95 %) and lowest germination time (55.68 days).
An efficient shoot initiation and multiplication protocol was developed using seedling cotyledonary nodal explant. MS media supplemented with 0.06 mg L-1 2, 4 - D along with different concentrations of cytokinin, 0.2 mg L-1 BA, 0.2 mg L-1 kinetin



and 0.4 mg L-1 kinetin responded to shoot initiation. On an average bud initiation was observed within 14 days after culture establishment. Significantly highest number of shoots were produced in MS media supplemented with 0.2 mg L-1 BA and 0.06 mg L-1 2, 4 - D (4.5 shoots/culture), followed by MS media supplemented with 0.2 mg L-1 kinetin and 0.06 mg L-1 2, 4 - D (3.83 shoots/culture) and MS media supplemented with
0.4 mg L-1 kinetin and 0.06 mg L-1 2, 4 - D (2 shoots/culture). The shoots produced in MS media supplemented with BA were stout and bigger than that obtained from kinetin supplemented media and they produced large and broad leaves.
Among the basal media tried for nodal explants, WPM was found to be better than the MS medium. Among the growth regulators, kinetin at 0.4 mg L-1 was found to be superior for shoot induction (91.63 %) with WPM basal medium. The period of morphogenic response for shoot induction was faster in the WPM medium (10 days). Even though shoot initiation was noticed in the WPM medium, all the treatments failed to give multiple shoot production.
Mature embryo of Coscinium fenestratum excised from the GA₃ pre-treated seeds could be easily cultured on the MS basal media. Zygotic embryo excised from GA₃ pre-treated seeds (4000 mg L-1 GA₃ solution for 72 hours) when cultured on MS medium in dark condition for 2 weeks followed by exposure to the light condition showed faster development of the embryo, radicle emergence (100 %), plumule emergence (77.78 %) and seedling development (44.44 %). These axenic seedlings without microbial contamination could be used as an explants for further micropropagation studies.
The study resulted in developing a feasible in vitro shoot regeneration protocol using seedling explant and axillary bud culture. The research results can be used as the stepping stone for further development of a high frequency plant regeneration protocol for the critically endangered medicinal plant Coscinium fenestratum and its conservation.