RAPID IN VITRO PROPAGATION OF WESTERN GHATS CULTIVAR-COLOCASIA ESCULENTA FOR THE HIGH FREQUENCY OF PLANTLETS

Article Info: Received 23 January 2019; Accepted 16 February. 2019 Cite this article as: R, J., K M, S. M., ., H., & ., V. (2019). RAPID IN VITRO PROPAGATION OF WESTERN GHATS CULTIVAR COLOCASIA ESCULENTA FOR THE HIGH FREQUENCY OF PLANTLETS. Journal of Drug Discovery And Therapeutics, 7(2). DOI: https://doi.org/10.32553/jddt.v7i2.447 Address for Correspondence: Jyothi R, Research scholar, Department of Biotechnology and Microbiology, Bangalore University, Bengaluru Disclosure statement: The authors have no conflicts of interest.


INTRODUCTION
C. esculenta is commonly referred to as taro and it belongs to araceae family. The various parts are cherished for their rich taste and nutritional properties. Every 100 g of taro corms possess 112 kcal, 26.46 g carbohydrate, 1.50 g protein, 0.20 total fat and 4.1g fiber (USDA National Nutrient Data Base,1998).Besides its nutritional value, taro is used as a medical plant and provides bioactive compounds used for therapeutic purposes (Prajapati et al.,2011). Traditionally, taro is vegetatively propagated from tuber fragments, a practice that encourages pathogen distribution. C. esculenta is a widely distributed food crop in the humid tropics and subtropics. Despite of its wide distribution, Taro plants are commonly infected by many pathogens. The virus induces conspicuous mosaic, malformation, dwarfing or feathering on leaves in taro. As the results of infection, it reduces the quality and yield of taro production greatly. This virus is thus considered as a major limiting factor in the production of taro. Hence in vitro propagation plays a major role in producing the disease resistant plants round the year with high quality. The tissue culture technology offers promising alternative compared to the traditional production methods for rapid multiplication and production of quality planting materials. (Alfred et al., 2015).

Media preparation
Optimized Murashige and Skoog (MS) medium was used as the basal medium and was prepared according to the following composition:  The basal medium was supplemented with various plant growth regulators such as Kinetin, 2, 4dichlorophenoxyacetic acid (2, 4-D), Naphthalene acetic acid in different concentrations to verify their callus induction properties. Initiation was commenced using the various media trials after the surface sterilization of explants and incubated in the growth room and their observance was noted every day in order to record the changes in their growth and to identify the contamination percentage.

Collection and sterilization of Explant
The fresh leaves from healthy plant of Colocasia esculenta were collected from the western ghats of Karnataka and the sterilization was performed and the survival of explants were observed for callus induction. The explants were then subjected to treatment with washing surfactant (Tween-20), which was added in drops and explants in the solution were agitated constantly for 10 minutes. Thereafter, the detergent was completely drained out from the explants by rinsing it with sterilized water to expel the microbial load and dust particles. Consequently, the explants were taken to laminar airflow cabinet for further sterilization.
After transmission to laminar airflow chamber, the explants were further sterilized with 0.1% mercuric chloride ( Ritu et al., 2013). The different trials with series of three, five and seven minutes time of exposure to the 0.1% mercuric chloride was carried out. After this treatment, explants were thoroughly washed 2 times with sterilized water to evacuate any trace of the surface sterilants under aseptic conditions. The sterilized explants were inoculated on MS media and kept for incubation at 37 o C up to 30-45 days with regular monitoring in every 2 -3 days.

Shoot proliferation
After incubation, the growth of shoots was monitored. The individual shoots, which formed at the end of establishment stage, were re-cultured on MS media having 0, 1.0, 2.0, 3.0, 4.0, 5.0 mg/l of benzylamino purine (BAP). Each bottle was containing 35-40 ml of medium. The pH was adjusted at 5.6-5.8, while they were autoclaved at 121ᵒC under a pressure of 1.5 kg/cm 2 for 20 min. The duration of multiplication stage was four subcultures. Each subculture period ranged from 30-45 days. Number of shoots at the end of each multiplication subculture was recorded.

ROOTING:
The proliferated shoots were induced in the rooting media with various growth regulators for the emergence of roots. After 4 weeks, the percentage of rooting, number of roots and root length were recorded. The well-developed, fully elongated roots and shoots were then acclimatized for Hardening.

CULTURE CONDITIONS:
The culture bottles were subjected to light intensity for 10-12 h in the growth room for rapid multiplication. Photoperiod provided by cool white fluorescent lamps of 1500-3000 lux, temperature of about 25 ± 2 °C and humidity of 35 -40%.

RESULTS AND DISCUSSION: STERILIZATION SURVIVAL:
The explants were found to survive at 7 min treatment of Mercuric chloride.

Figure 1: Survived Explants
Majority of the explants survived after treatment when they were exposed to mercuric chloride for about 7 min. Treatment with 3 min were found with more mortality of the explants whereas treatment at 5 min found to be non-responsive. INITIATION MEDIA TRIALS:  Using 6BAP, the responded explants were transferred to the proliferation media where the response was recorded at 5 mg/l 6BAP that led to 76% response followed by 1.58 multiplication ratio unlike the other concentrations. At lower concentration, there was least response with increase in the response rate as the hormone cytokinin concentration increased.    This protocol offers potential in vitro system that can be used for improvement, conservation and efficient mass multiplication of clones of Colocasia esculenta to prevent from an endangered and endemic species. Further, In vitro mass propagation reduces cost and completion of life cycle can be attained within reasonable time, reducing risk of extinction to the endangered wild population.