Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2019, Vol. 40 ›› Issue (2): 314-322.DOI: 10.3969/j.issn.1000-2561.2019.02.015

• Biotechnology and Tissue Culture • Previous Articles     Next Articles

Establishment of New Protocorm-like Bodies System of Anoectochilus roxburghii by ‘Double Two-steps’ Method

JIAO Zhan1,AN Shengjun2,*(),SHAO Tiemei1   

  1. 1. Hebei Chemical & Pharmaceutical College / Center of Bioreactor and Protein Drug Research and Development of Hebei Universities, Shijiazhuang, Hebei 050026, China;
    2. Hebei University of Chinese Medicine, Shijiazhuang, Heibei 050091, China
  • Received:2018-08-13 Revised:2018-10-21 Online:2019-02-25 Published:2019-05-16
  • Contact: AN Shengjun

Abstract:

In this study, the stem sections of Anoectochilus roxburghii in vitro were used as the material, and the method of ‘double two-step’ was discussed. The ‘two-step-induction’ had the etiolation stem sections induced by dark culture and the protocorm-like bodies induced by etiolation stem sections, and the ‘two-step-proliferation’ had the new PLBs cultured on induction medium for 3 weeks and proliferative culture program optimized by the orthogonal experimental design of L16(4 5). The effects of source and treatment method of explants, dark culture time, medium and additional elements on induction of PLBs, and the effects of culture time, light intensity, and UV irradiation time on proliferation culture were studied. The results showed that, medium which was suitable for the etiolation stem sections to induce PLBs was MS +2.5 mg/L 6-BA +0.2 mg/L NAA +1.0 mg/L S3307. The best explant was the etiolation stem sections from leaf axil in dark culture, and most suitable treat method was the whole stem sections horizontally onto the medium. Three weeks dark treatment could improve the induction frequency of PLBs from the tiolation stem sections significantly. The order of effects of the five factors was NH4 +/NO3 ->concentration of NAA>concentration of 6-BA> concentration of S3307>concentration of sucrose. The optional experimental conditions were MS+0.5 mg/L S3307+4.0 mg/L 6-BA+0.4 mg/L NAA+ NH4 +/NO3 -(15 mmol∶45 mmol)+ 40 g/L sucrose, which could get the proliferation fresh weight 246.90 g/L. The optimal proliferation culturing time was 6 weeks. Light intensity 500 lx, and UV irradiation 253.7 nm for 6 h per day for 6 weeks were suitable for PLBs proliferation and accumulation of total flavonoids. New protocorm-like bodies system of A. roxburghii was established by the ‘double two-step’ method. The inducing rate, amplification of initial materials, and proliferation multiple of the method were higher than the index of the traditional single step method. 92% of the PLBs could keep the state of steady proliferation, and not to differentiate nor to formate callus.

Key words: Anoectochilus roxburghii, protocorm-like bodies (PLBs), efficient system, induction, total flavonoids

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