Abstract:

Objective: This study was aimed to explore a method for in vitro conservation of callus by selecting osmotic regulators and growth inhibitor on the basis of the optimization of plant hormones in order to providing a theoretical foundation and technical support for in vitro conservation of germplasm resources in Cyclocarya paliurus. Method: The callus of C. paliurus was used for in vitro conservation, the contents of 6-BA (0.4, 0.6, 0.8, 1.0 mg·L^-1) +IBA (0.2, 0.4 mg·L^-1) in the medium were optimized by using two-factor complete randomization. Sugar contents (30, 45, 50, 55, 60 g·L^-1) and number of days for conservation (60, 90, 120, 150, 180 days), contents of osmoregulators (mannitol:0, 10, 20, 30, 40, 50 g·L^-1; inositol:0, 10, 20, 30, 40, 50 g·L^-1), contents of growth inhibitors (paclobutrazol (PP₃₃₃):0, 2, 4, 6, 8, 10 mg·L^-1; daminozide (B₉):0, 2, 4, 6, 8, 10 mg·L^-1) were tested in the in vitro conservation tests by using single-factor complete randomization. Cell ploidy and genetic stability of the callus at different conservation times were detected by flow cytometry and SSR markers, and the callus at the fifteenth generation (T15) was used for re-growth test. Result: The results showed that the optimization of 6-BA+IBA content combination, and the selection of osmoregulator, growth inhibitor and number of conservation days, the improved MS medium with 0.8 mg·L^-16-BA+0.2 mg·L^-1IBA +45 g·L^-1sugar+6.0 g·L^-1agar (pH5.8) was suitable for in vitro conservation of the callus, effectively inhibiting the growth of callus. The suitable duration for callus subculture was 90 days, with a callus survival rate of 100%, and the callus was yellowish green in color, granular in shape, small and porous. Compared with the leaves used as control, no changes were found in cell ploidy and genetic stability in different generations of subculture. The calluses in vitro conservation were all able to regrow and differentiate in the medium, displaying good performance in morphology, viability and growth vigor. Conclusion: Our results demonstrated that the appropriate medium for callus in vitro conservation of C. paliurus was the improved MS medium with 0.8 mg·L^-16-BA+0.2 mg·L^-1IBA +45 g·L^-1sugar+6.0 g·L^-1agar (pH5.8), and the optimal subculture is once every 90 days under (20±1)℃. No variation was found in the callus in vitro conservation indicating good genetic stability. Use of additives of mannitol, inositol, PP₃₃₃ and B₉ was harmful to in vitro callus conservation of C. paliurus.

Key words: Cyclocarya paliurus, callus, in vitro conservation, osmoregulator, growth inhibitor