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.
Highlights
Viola canescens Wall. ex, Roxb., is an important endangered Himalayan herb as reported by the International Union for Conservation of Nature and Natural Resources
These 8-week-old embryogenic calli were inoculated on Murashige and Skoog (MS) medium for somatic embryogenesis responses, and 2,4-D, indole-3-butyric acid (IBA) and Kn were used to induce somatic embryogenesis
The superiority of 2,4-D for somatic embryogenesis has been reported for Foeniculum vulgae [27], Bunium persicum [28], Chlorophytum borivilianum [29] and Sapindus mukorossi [30]
Summary
Viola canescens Wall. ex, Roxb., (family Violaceae) is an important endangered Himalayan herb as reported by the International Union for Conservation of Nature and Natural Resources. Ex, Roxb., (family Violaceae) is an important endangered Himalayan herb as reported by the International Union for Conservation of Nature and Natural Resources. In India, it is known by different names in different regions: Banfsha or Vanaksha in Jammu and Kashmir; Kauru, Vanafsha or Banfasha in Uttarakhand; and Ratmundi, Gugluphul and Banfsaha in Himachal Pradesh [1,2,3]. The plant is one of the preferred herbs and used in both codified (Ayurveda, Unani, Siddha, homeopathy) and non-codified (folk) medicinal systems. The plant is known to have different pharmacological activities such as antimalarial [11], antifungal [12] and antibacterial activities [13,14,15,16]. Solvent extracts of V. canescens showed relevant antioxidant and hepatoprotective activities [17]
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