猫须草无菌短枝组织培养与快速繁殖体系的建立

Establishment of a Sterile Short Shoot Tissue Culture and Rapid Propagation System for Clerodendranthus spicatus

猫须草又称作“肾茶”,是一种生长在热带和亚热带地区的多年生草本植物,有一定的药用价值和观赏价值。在东南亚,猫须草作为一种传统的茶饮而深受人们喜爱,在我国则更多是作为一种中草药用于治疗肾脏疾病。然而猫须草野生药材资源日趋枯竭,传统生产繁殖方式难以满足市场需求,因此采用植物组织培养快速繁殖技术,为猫须草大规模种植提供种苗已成为急需解决的问题。为了研究适合猫须草的无菌短枝组织培养快速繁殖体系,本研究以带一对腋芽的猫须草幼嫩茎段为外植体,探究不同消毒方法、激素类型与浓度及培养基配方对猫须草无菌短枝组织培养的影响。结果表明,猫须草无菌短枝组织培养的最佳消毒方法为75%酒精浸泡10 s或15 s+0.1%升汞浸泡6 min,当0.1%升汞消毒时间为8 min时,猫须草无菌短枝的萌芽率显著下降,当0.1%升汞消毒时间为4 min时,猫须草无菌短枝的污染率显著提高。最佳初代培养基配方为MS+6-BA 1.0 mg/L+NAA 0.5 mg/L或MS+6-BA 0.5 mg/L+NAA 0.2 mg/L;但当6-BA的浓度达2.0 mg/L时,组培苗长势细弱,叶片发黄,并有玻璃化发生。最佳继代培养基配方为MS+TDZ 0.05mg/L+IBA 0.2 mg/L;与添加6-BA相比,添加TDZ更有利于猫须草无菌短枝继代组培苗的生长。最佳生根培养基配方为1/2MS+NAA 1.0 mg/L+IBA 1.0 mg/L+活性炭3 g/L,组培苗生根率可达95%。培养基配方及不同激素组合均会影响猫须草无菌短枝组培苗的生根,1/2MS培养基明显优于MS培养基,同时添加NAA和IBA比单独添加NAA的效果好。将组培苗移栽至珍珠岩、细河沙、泥炭土的体积比为1∶1∶1的混合基质中,植株生长良好,成活率高。研究结果可为猫须草大规模工厂化生产提供科学可行的技术支持。

Clerodendranthus spicatus, also known as Clerodendranthus spicatus(Thunb.) C. Y. Wu, is a perennial herb growing in tropical and subtropical regions, with certain medicinal and ornamental value. In Southeast Asia, C. spicatus is deeply loved by people as a traditional tea, but in China, it is more used as a Chinese herbal medicine to treat kidney diseases. However, the wild medicinal material resources of C. spicatus are increasingly exhausted, and the traditional production and reproduction methods are difficult to meet the market demand. Therefore, it has become an urgent problem to adopt the rapid propagation technology of plant tissue culture to provide the seedlings needed for large-scale cultivation of C. spicatus. In order to study the suitable rapid propagation system of sterile short shoot tissue culture for C. spicatus, the tender stem segment with a pair of axillary buds was used as the explants to explore the effects of different disinfection methods, hormone types and concentrations and medium types on the sterile short shoot tissue culture of C. spicatus. The results showed that the best disinfection method for the sterile short shoot tissue culture of C. spicatus was 75% alcohol immersion for 10 s or 15 s +0.1% mercury chloride immersion for 6 min. When the disinfection time of 0.1% mercuric chloride was 8 min, the germination rate of the sterile short shoot of C. spicatus decreased significantly, and when the disinfection time of 0.1% mercuric chloride was 4 min, the pollution rate of the sterile short shoot of C. spicatus increased significantly. The best initial medium tissue culture was MS+6-BA 1.0 mg/L+NAA 0.5mg/L or MS+6-BA 0.5 mg/L+NAA 0.2 mg/L. However, when the concentration of 6-BA was 2.0 mg/L, the growth of the tissue cultured seedlings was weak, the leaves turned yellow and vitrification occurred. The optimal subculture medium was MS+TDZ 0.05 mg/L+IBA 0.2mg/L. Compared with the addition of 6-BA, the addition of TDZ was more beneficial to the growth of subcultured seedlings. The optimal rooting medium was 1/2MS+NAA 1.0 mg/L+IBA 1.0mg/L+ activated carbon 3 g/L, and the rooting rate of tissue culture seedlings was 95%. The type of culture medium and different hormone combinations would affect the rooting of the sterile short shoot tissue culture seedlings of C. spicatus.1/2MS medium was obviously better than MS medium, and the effect of adding NAA and IBA at the same time was better than adding NAA alone. When the tissue culture seedlings were transplanted in the mixed matrix with the volume ratio of perlite, river sand and peat soil with a volume ratio of 1∶1∶1, the plants grew well and the survival rate was high. The results of this study could provide a scientific and feasible technical basis for the large-scale industrialized production of C. spicatus.