Establishment of Artificial Rapid Propagation System of Fritillaria crassicaulis

Abstract

Fritillaria crassicaulis S. C. Chen is a precious traditional Chinese medicine, but the number of populations has declined rapidly due to overexploitation. An artificial rapid propagation system was established to screen the suitable plant regeneration method and to explore the efficient propagation method, useful for propagation technology or for further research and development of F. crassicaulis. This study selected scale as the experimental material, set Murashige and Skoog (MS) medium as the basic medium, and optimized the types and proportions of plant growth regulator (PGR) suitable for callus induction, bulblet differentiation and proliferation, and plant regeneration by means of single-factor, full-factorial, and L9 (3)4 orthogonal experiments. Results demonstrate that in the experiment with single exogenous PGR, the high concentration of 6-benzylaminopurine (6-BA) was significantly better than kinetin (KT) to induce bulblets, 2, 4-dichloroacetic acid (2, 4-D) had a significant effect on callus induction, and a higher concentration of naphthaleneacetic acid (NAA) was beneficial to the occurrence and growth of bulbs, but the rooting effect promoted by indole butyric acid (IBA) was preferable to that by NAA. In MS medium with 0.5 mg/L 2, 4-D and 1.5 mg/L 6-BA, a large number of yellowish-green compact calli could be induced from the scales with the calli induction frequency at 93.3%, and about 11.4% materials directly differentiated bulblets. In the subsequent orthogonal experiment, after the scales were cultured in MS medium with 2.0 mg/L 6-BA, 0.5 mg/L 2, 4-D, and 0.1 mg/L NAA for 20 days, the small yellow and white globular protuberances formed near the incision, but no callus appeared, and many protuberances appeared on the surface of the scales. After 60 days, the protuberances at the incision developed into bulblets directly, while protuberances on the surface of the scales developed into few bulblets but crowded “leaf spines,” which gradually died and disappeared in the later culture; the proliferation coefficient was ∼6.30 then. Experimental results indicate that the optimal rooting medium for bulblets was 1/2MS medium with 2.0 mg/L IBA and 1.0 mg/L activated carbon (AC), with the rooting rate at 95.6%. This study identifies bulblet regeneration of F. crassicaulis, and an efficient direct organogenesis method was established: regenerated bulblets could be induced from scales in one step, so a large number of regenerated plants with the same genotype could be obtained in a short time.