Optimization Of Aconitum Coreanum(Levl.) Rapaics Hairy Roots Culture Conditions And Researches On Plant Regeneration

The hairy root system A0489 of Aconitum coreanum(Levl.) Rapaics could cultured in liquid medium growth fast, short growth cycle(about 40 days). The content of Guanfu base A(GFA) in hairy roots is only 0.06%(DW) lower than five-year old root tubers. It has a broad prospect of application and use value if we can realize industrial intensive production of hairy roots. Experiments were done to optimize culture conditions of A. coreanum hairy roots. To realize large-scale culture of A. coreanum hairy roots, experiments were did to explore and optimize the large-scale culture conditions. Hairy root of A. coreanum were used as explants to induce regenerated plant. We obtained micrographs of different phases during regenerative process and got the skill of making paraffin sections.1. The growth statue and growth rate of A. coreanum hairy root were affected by culture method,p H, medium contents and elicitors. Hairy roots cultured on solid medium grew slowly but had a high drying rate, and always be used to keep materials. Hairy roots cultured in liquid medium grew fast and always be used to expanding propagation. The p H of the culture medium was degraded and the clarity of medium had changed after high sterilization. The hairy roots grew better at p H 5.80. After enlarging10, 20, 30 times of major elements of the medium, high osmotic pressure leaded to worse growth of hairy roots. The trace elements had little toxic to hairy root. When Zn2+ was 20 times bigger, the growth accumulation of hairy roots was biggest. SA, YE, Me JA and CH were added in the medium on the beginning. SA and YE had little effection on the growth, but Me JA and CH would inhibit the growth of hairy roots.2. The growth of hairy roots were effected by culture vessel, statue of inoculate hairy roots,inoculum size and so on. The length and biomass of hairy roots were increasing as the culture vessel becoming bigger, which proved that it’s possible to enlarge cultivation hairy roots. Hairy roots had a high drying rate but a lower growth rate in smaller vessel. It’s suitable to place 2 L medium in 5 L bioreactor, and put 20 g hairy roots cultured on solid medium in the bioreactor directly. For intolerance of shear force, the hairy roots always grew slowly, autolysis and turned brown by using airlift bioreactors. The coordination of ventilation volume and hairy roots density was crucial to the enlargement culture of the hairy roots. It has a great prospect to find a new, suitable bioreactor and a new way to culture A. coreanum hairy roots.3. During inducing regenerated plant from hairy root of A. coreanum, hairy roots became enlargement and dedifferentiated to form embryonic callus. After redifferentiation, adventitious buds and plants were formed. A whole plant was got after inducing roots. The optimal culture medium for inducing callus and adventitious buds was 1/2MS+BA 2.0 mg/L+NAA 0.2 mg/L+sugar 3%. The best root inducing medium was 1/2 MS+BA 0.5 mg/L +NAA 0.1 mg/L+GA 0.5 mg/L+ sugar 3%. Through observation of the paraffin sections, we found the histological structure of tissues were effect by culture methods, tissues place, plant types. The paraffin sections showed that hairy root had a different structure to regenerated root on cell layer, size, nucleus numbers. Callus had diversity morphological structuresand could divide into embryonic callus and non-embryogenic callus. Embryonic callus had small cell size, relatively regular shape, arranged tightly, dense cytoplasm, big cell nucleus, non-embryogenic callus were in contrast. The structures of adventitious buds and roots, stem, leaf of regenerated plant were similar to routine dicotyledon’s but had differences on cell size, layer, thickness.