Abstract

For the first time, an in vitro protocol for efficient shoot multiplication has been developed from shoot tip explants of Anisodus tanguticus, an endangered medicinal plant in the Qinghai-Tibet Plateau. Multiple shoots were induced from the shoot tip explants on Murashige and Skoog (MS) medium supplemented with various concentrations of N6-benzylaminopurine (BA), kinetin (KT) or in combination with α-naphthalene acetic acid (NAA). The presence of BA was more effective than KT on shoot multiplication. A maximum of 3.2 shoots were obtained per explant on MS medium supplemented with 2 mg L^-1 BA. Addition of 0.5 or 1 mg L^-1 NAA to the BA-containing medium promoted callus formation and reduced shoot multiplication. Shoots treated with 0.5 mg L^-1 indole-3-acetic acid (IAA) showed the highest average root number (4.08) and the highest percentage of rooting (75.2%). Micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Genetic stability of the regenerated plants was assessed by 25 inter simple sequence repeat (ISSR) markers. Out of 25 ISSR markers, 18 markers produced clear, reproducible bands with a mean of 6.7 bands per marker. The results confirmed that the regenerants maintained high genetic fidelity. This in vitro technique may help in the conservation and propagation of Anisodus tanguticus.

Key words: Anisodus tanguticus, micropropagation, shoot multiplication, genetic stability, inter simple sequence repeat (ISSR).

Abbreviation

AFLP, Amplified fragment length polymorphism; BA, N6-benzylaminopurine;CTAB, cetyltrimethylammonium bromide; dNTPs, deoxyribonucleotide triphosphate; 2,4-D,2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; ISSR,inter simple sequence repeat; KT, kinetin; MS, Murashige and Skoog; NAA, α-naphthaleneacetic acid; PCR, polymarase chain reaction; RAPD, randomly amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; UBC, University of British Columbia.