An efficient and reproducible indirect shoot regeneration from female leaf explants of Simmondsia chinensis, a liquid-wax producing shrub

Abstract

Simmondsia chinensis (Link) Schneider is a perennial, dioecious, drought resistant and multipurpose seed oil crop grown in arid and semi-arid conditions throughout the world. A reproducible and more efficient method for indirect shoot organogenesis from female leaf explants has been standardized. The leaf explants cultured on Murashige and Skoog (MS) medium with 1.0 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) alone produced the highest frequency of callus compared with 1.5 mg l(-1) IBA. Maximum proliferation of callus was observed on MS medium containing a combination of 1.0 mg l(-1) 2,4-D with 0.5 mg l(-1) BAP. For shoot differentiation, the proliferated callus was subcultured on MS medium supplemented with 6-benzylaminopurine (BAP) (1.0-4.0 mg l(-1)) along with 40 mg l(-1) adenine sulphate as additive or in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA). Optimum shoots differentiated from callus was obtained on MS medium supplemented with 2.0 mg l(-1) BAP and 0.2 mg l(-1) NAA. On this medium, 100 % cultures were responded with an average number of 14.44 shoots per explant with their mean length of 4.78 cm. In vitro rooting (6.22 roots per explant) was achieved on half strength MS medium containing 2 % sucrose with 3.0 mg l(-1) IBA and 300 mg l(-1) activated charcoal (AC). Rooted plantlets were successfully hardened under control conditions and acclimatized under field conditions with 90 % success rate. The present protocol is highly efficient, reproducible and economically viable for large scale production of female plants.

Keywords: 2,4-D; Callus; Differentiation; Liquid-wax; Shoot; Simmondsia chinensis.

Fig. 1

Plant regeneration through callus induction from female leaf explants of S. chinensis. a Establishment of leaf explant. b Callus initiation on leaf explant. c Callus induction on MS + 1.0 mgl⁻¹ 2,4-D. d Callus proliferation on MS + 1.0 mgl⁻¹ 2–4, D + 0.5 mgl⁻¹ BAP. e Shoot differentiation from callus on MS + 2.0 mgl⁻¹ BAP. f Multiple shoots induction on MS + 2.0 mgl⁻¹ BAP + 0.2 mgl⁻¹ NAA. g In vitro root induction on half strength MS + 3 mgl⁻¹ IBA + 300 mgl⁻¹ AC. h Hardened plantlet in vermiculite medium. (Scale bar = 1.0 cm)