MSc

The study entitled “Development of multiple shoot culture of ashwagandha (Withania somnifera) for in vitro alkaloid stimulation studies” was conducted at the Department of Plant Biotechnology and Department of Plant Physiology, College of Agriculture, Vellayani, Thiruvananthapuram, during 2019-2020. The primary objective of the study was to establish multiple shoot cultures of ashwagandha (Withania somnifera) for in vitro alkaloid stimulation studies. The seeds of Withania somnifera, variety Arka ashwagandha procured from IIHR Bangalore were used for the study. The seeds were subjected to various treatments to enhance seed germination under in vitro condition.
Among the different treatments studied, S5 (the treatment in which the seeds were soaked in water overnight, surface sterilized with 5% Tween 20 for 5 minutes followed by 0.1% HgCl2 treatment for 5 minutes and then inoculated in half strength MS media with 0.3% GA3) was found to be the best for seed germination (100%). But the treatment S1 (seeds soaked in water overnight, surface sterilized with 5% Tween 20 for 5 minutes followed by 0.1% HgCl2 treatment for 10 minutes and then inoculated to half strength MS media without hormones) failed to induce germination. Treatment S2 (seeds soaked in 250ppm GA3 overnight, surface sterilized with 5% Tween 20 for 5 minutes followed by 0.1% HgCl2 treatment for 10 minutes and then inoculated in half strength MS media without hormones) also failed to induce any germination. But in the treatment S3, 40% of germination was found on 14th day of inoculation and the seedlings showed an average shoot length of 3.75 cm in 28 days. In this treatment S3, the seeds were soaked in tender coconut water for 1hour and then soaked in 250ppm GA3 overnight and the next day surface sterilized with 5% Tween 20 for 5 minutes followed by 0.1% HgCl2 treatment for 10 minutes and then inoculated in half strength MS media with 0.3 ppm GA3.The growth of the seedlings was very slow and the yield was also very poor in the treatment S4 (in which the seeds were soaked in water overnight and the next day surface sterilized with 5% Tween 20 for 5 minutes followed by 0.1%
HgCl2 treatment for 10 minutes and then inoculated in half strength MS media with 0.3 ppm GA3).In treatment S5, the time of exposure of the seeds to HgCl2 treatments was reduced to 5 minutes and the rest was the same as above. In S6, the concentration of HgCl2 was reduced to 0.05% but the time of exposure of the seeds to HgCl2 treatments was 10 minutes. In S7, the concentration of HgCl2 and time of exposure of the seeds to HgCl2 treatments were reduced as 0.05% and 5 minutes respectively. The treatments S4, S6 and S7 failed to give any response. Among these treatments, treatment S5 has shown maximum germination rate (100%) with an average shoot length of 5.87 cm. Germination was found within 6 days of inoculation. Compared to the rest of the treatments, S5 has shown not only maximum germination rate but also faster development of the seedlings.
Nodal, leaf and shoot tip explants were taken from one month old in vitro grown seedlings and subjected to different shoot multiplication media for shoot induction. The explants were inoculated in plain MS media without any hormones in treatment T0 and this treatment failed to show any response. In the treatment T1 (MS+1mgNAA+3mgKn) shoot multiplication response of different explants viz. shoot tip (10%), leaf (10%) and nodal explants (16%) were very poor. Similarly in treatment T2 (MS+1.5mgNAA+1mgKn) also the response of shoot tip (10%), leaf (10%) and nodal (16%) explants were not satisfactory. In the treatment T3 (MS+1mgNAA+1.5mgKn), leaf explants showed shoot induction (10%) but there was no response in both shoot tip and nodal explants. But in the treatments T4 (MS+0.5mgBA+0.1mgIAA) and T7 (MS+3mgBA+1mgKn), 33% of shoot induction was found in both cases of shoot tip and nodal explants and callus development was found in case of leaf explants. Leaf explants showed 12% shoot induction in treatment T6 (MS+2.5mgBA) and no shoot induction was found in both shoot tip and nodal explants. Maximum response was found in treatment T5 (MS+2mgBA) with 80% shoot induction in nodal explants, 33% shoot induction in shoot tip explants and callus development was found in case of leaf explants.
Among the treatments, T5 (MS+2mgBA) was found to exhibit both maximum shoot induction and faster development of the multiple shoots. Shoot induction was found after 30 days of inoculation. Among the three explants used, nodal explants showed maximum shoot induction (80%; the number of cultures showing multiplication-20±0.570, number of shoots per explant-15±0.670) with an average shoot length of 1.85±0.750cm in treatment T5 (MS+2mgBA). Hence in the present study, the use of in vitro derived nodal explants and subjecting them to treatment T5 (MS+2mgBA) is adjudged as the best protocol for establishing multiple shoots which can be used for in vitro propagation and alkaloid stimulation studies of Withania somnifera.