| Guangcigu, chinese medicinal material, is the dried bulb of Tulipa edulis (Miq.) Baker, belonging to Tulipa genus under Liliaceae family. The flavor of Guangcigu is sweet, coldness is the characteristic, poisonous. It has effects in detoxication, lump dissipation and removing blood stasis to promote blood circulation. Guangcigu has been used for treatment of throat-swelling, scrofula, carbuncle-abscess, sore and stasis after childbirth. Along with the constant growing demand for Guangcigu of traditional Chinese medicine industry, the contradiction between Guangcigu supply and demand becomes more and more serious.The source mainly comes from wild herbs presently, research results about T. edulis or Guangcigu were poor. The artificial cultivation has such problem as low propagation coefficient. Tissue culture can produce large amount of high quality seedlings in short term, tube seedlings induced bulblet in vitro can effectively improve production efficiency.Bulbs of T. edulis treated with sand to break dormancy,screening suitable explants,plant growth substances and culture conditions to set up test-tube plantlet, after that though plantlet to induce test-tube bulblets, establish the culture system of tissue culture and in vitro bulblets induction of T. edulis. The main conclusions were as following:(1)Bulbs of T. edulis hided in sand for 120 days under 0～2℃,sterilized with 75% alcohol 30 seconds and soaked in 0.1% HgCl2 for 12-15 minutes or soaked in HgCl2 6+6 minutes continuously. Bulbs induced bud stem(shortening bud stem)and bulbil in hormonc-free MS medium.Mercuric choridc soaking time in accordance with the size of bulbs.Bud stem and bulbil were used as primary explants.(2)Bud stem(divided into bud stem segments and bud stem apex) and shortening bud stem was suitable for callus induction, bulbil was suitable for adventitious bud induction. In the medium of MS+6-BA 0.5 mg·L-1+NAA 2.0 mg·L-1,callus induction rate of bud stem apex was 78.54%, cultured in MS+6-BA 1.0 mg·L-1+NAA 0.01 mg·L-1 medium, shortening bud stem had a high rate of callus induction 85.46%,bulbil inoculated in MS+6-BA 1.0 mg·L-1+NAA 0.01 mg·L-1 medium had a adventitious bud induction rate of 89.53%.The optimal medium for callus proliferation was MS+6-BA 0.5 mg·L-1+NAA 2.0 mg·L-1. The type of yellow, compact and smoothly surface callus was suitable for bud differentiation and the optimal medium for callus differentiation was MS+6-BA 2.0 mg·L-1+NAA 0.2 mg·L-1 with a shoot differentiation rate of 66.21% and the number of average bud was 1.73.The optimal medium for the shoot multiplication was MS+6-BA 0.5 mg·L-1+NAA 0.2 mg·L-1, the proliferation coefficient was 2.44.(3)Daughter bulb and core bud of T. edulis used as explants could induce adventitious bud directly. The optimal medium for bud inducted form core bud and daughter bulb were MS+TDZ 2.0 mg·L-1+NAA 4.0 mg·L-1 and MS+TDZ 2.0 mg·L-1+NAA 2.0 mg·L-1, both of them had a bud induction rate of 72.92% and 79.22%. The optimal medium for cluster buds multiplication was MS+TDZ 0.2 mg·L-1+NAA 0.2 mg·L-1, and proliferation coefficient was 2.23.(4)Multiple shoots differentiated form callus that induced form bud stem of T. edulis,after subcultured in multiplication medium turned out to be test-tube plantlets without roots which can be induced in vitro bulbs.Multiple shoots must be treated with low temperature to induce test-tube bulbs.Sucrose,6-BA and macro-elements were major factor in test-tube bulbs induction and the effect of NAA on bulbs induction was no significant.Multiple shoots(2-5) inoculated on MS+6-BA 0.1 mg·L-1+NAA 0.01 mg·L-1 medium and with a sucrose concentration of 80.0 g·L-1 had the most number of bulbs and culture condition was low temperature(5±1)℃ for 60 days followed by cultured in high temperature(23±2)℃. Medium added in paclobutrazol had no acceleration on bulb induction. Medium added in high concentration of salicylic acid(3.0 mg·L-1) could promote bulbs induction. Bulb inducted in high temperature stage,the optimal culture condition was (23±2)℃ and darkness. |
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