Application Of CPPU On Ramie Plant Tissue Culture

Ramic(Boehmeria nivea L. Gaud) is one of the most important bast fiber crops in China, which has a long history of cultivation and processing. With the further study of genetic engineering, ramie transgenic technology has gradually started. The establishment of ramie in vitro regeneration system and its optimization has becoming more and more important. In this study, the regeneration system of Huazhu No.4 and Zixingma, elite ramie varieties were studied using N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) which was an efficient cytokinin. The effects of different concentrations of CPPU on regeneration of shoot apex, induction of callus from leaves and stems explants and differentiation of adventitious bud from the callus were measured. The endogenous hormonal changes were surveyed and the cytological observation were conducted on callus induced from stem explants. The main results are as follows:(1) Used in propagation of plantlets regenerated from shoot apex in ramie, CPPU was a more efficient cytokinin compared with the control 1 mg/L 6-BA. The optimal concentrations of CPPU were 0.02,0.05 mg/L on the Huazhu No.4 and Zixingma respectively, which is one-fiftieth of 6-BA concentration. The highest average number of regenerated was above 2.6 times of 6-BA; 1.0 mg/L of CPPU was the optimal concentration for Huazhu 4 and Zixing Ma in callus induction and bud differentiation, the concentrations 0.2 mg/L and 0.5 mg/L of CPPU is most suitable for Huazhu No. 4 and Zixingma respectively compared with the control 2 mg/L of 6-BA. The CPPU concentrations of 0.2 mg/L-1.0 mg/L were conducive to the formation of ramie callus and adventitious buds.(2) CPPU in the culture medium has effect on endogenous hormone levels and ratios of stem explants. With the concentrations of CPPU increasing, the content of endogenous hormones IAA, ABA, ZR increased. The content of IAA, ABA tended to be stable when CPPU were increased to a certain value, while the concentration of ZR increased with CPPU concentrations. With the elongation of the callus culture period, the content of endogenous hormones decreased without adding exogenous hormones; the concentration of IAA and ZR were increased with exogenous hormones, but ABA content increased first and then decreased. Combined the study results 1, it can be speculated that the hormone IAA/ZR=0.56, ABA/ZR=0.28 in ramie callus and the concentration of exogenous hormones CPPU was 0.5 mg/L were most conducive to the differentiation of adventitious buds.(3) Through observation of freehand section and paraffin section of callus induced from stem explants, we found that there were many dense cell clusters on the surface of callus showing highly efficient regeneration ability. These cell clusters were separated, with regular shape, dense cytoplasm, large nucleus and mitochondria abound. As the incubation time elongated, the bud primordia appear clearly.