Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb

Arun Kumar Khajuria; Christophe Hano; Narendra Singh Bisht

doi:10.3390/plants10040761

Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb

Plants (Basel). 2021 Apr 13;10(4):761. doi: 10.3390/plants10040761.

Authors

Arun Kumar Khajuria^{1

2}, Christophe Hano^{3

4}, Narendra Singh Bisht¹

Affiliations

¹ Plant Tissue Culture Laboratory, Department of Botany, Campus Pauri, HNB Garhwal University, Pauri Garhwal 246001, Uttarakhand, India.
² Department of Botany, Cluster University of Jammu, Jammu 184001, Jammu & Kashmir, India.
³ Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), INRAE USC1328, Campus Eure et Loir, Université d'Orléans, F-28000 Chartres, France.
⁴ Bioactifs et Cosmétiques, Centre National de la Recherche Scientifique (CNRS) GDR3711, Université d'Orléans, CEDEX 2, F-45067 Orléans, France.

Abstract

Viola canescens Wall. ex. Roxb. is an important but threatened medicinal herb found at 1500-2400 m above mean sea level in the Himalayas. Overexploitation and habitat preference have put the plant under serious threat. Thus, the present study was undertaken to develop an efficient protocol for in vitro propagation via somatic embryogenesis. The results revealed that plant can be regenerated successfully through somatic embryogenesis using leaf derived calli. Regular subculturing of calli on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D)/indole-3-butyric acid (IBA)/kinetin (Kn) and varying combinations of 2,4-D+Kn induced somatic embryogenesis. The maximum average number of somatic embryos (SE) (19.15 ± 2.66) was induced on the medium with 0.15 + 0.05 mg L^-1 of 2,4-D and Kn, respectively, and this medium was used as a control. To enhance somatic embryo induction, the control MS medium was supplemented with l-glutamine (200-400 mg L^-1) and casein hydrolysate (1-4%). The maximum average number of SE (27.66 ± 2.67) and average mature SE (13.16 ± 3.48) were recorded on the medium having 2 % l-glutamine and 50 mg L^-1 casein hydrolysate. The induced SE were asynchronous, so, to foster their maturation, the culture medium (free from growth regulators) was supplemented with abscisic acid (ABA) and silver nitrate (AgNO₃). The maximum average number (35.96 ± 3.68) of mature SE was noticed on MS medium supplemented with 1.5 mg L^-1 ABA. Mature embryos had two well-developed cotyledons and an elongated hypocotyl root axis. The development of SE into plantlets was significant for embryos matured on the medium with AgNO₃ and ABA, with 86.67% and 83.33% conversion on the medium with 0.20 mg L^-1 6-benzylaminopurine (BAP). The plantlets thus produced acclimatized in a growth chamber before being transferred to the field, which showed 89.89% survival. The plants were morphologically similar to the mother plant with successful flowering.

Keywords: abscisic acid; callus; casein hydrolysate; cytokinin; silver nitrate; survival.