| Isatis indigotica Forture is biennial medicinal and dye plant belonging to the family of Cruciferae.lt is the origin plant of the important traditional Chinese medicines Radix Isatidis and Folium Isatidis.The existing varieties of cultivable Lindigoticaim China is often invaded by many kinds of insects and pathogens,which would induce a decline of the medicine yield and quality.In order to cultivate fine breed of genetic improvement,it is necessary to establish efficient methods of regeneration of this plant.This study was to develop an efficient regeneration in TDZ for different explants of I.indigotica.Based on this system,an efficient agrobacterium-mediated transformation was established and Cry1C gene was successfully transformed into I.indigotica.The main results were as follows:1 Hypocotyls explants of Isatis indigotica were cultured on Murashige and Skoog(MS)medium with different concentrations of thidiazuron(TDZ),6-BA,NAA individually or 6-BA combination with NAA or TDZ combination with NAA.The highest frequency of shoot regeneration(97.44±4.44%)and mean number of shoots per explant(19.88±2.07)were obtained on medium containing 0.05 mg/L TDZ individually.Leaf,cotyledon,and stem tip explants of I.indigotica were also all observed effective shoot organogenesis on medium containing the proper concentration of TDZ.The most appropriate concentration was 0.1 mg/L,0.05mg/L and 0.05mg/L of TDZ for leaf,cotyledon and stem tip explants,respectively.The highest frequency regeneration was 97.04±3.39%,72.22±5.09%and 92.22±1.92%,respectively.2 Elongated shoots were capable of developing roots in the 1/2MS medium with different concentrations of NAA or IBA,but the highest frequency of rooting occurred in 0.5mg/L NAA(93.54±3.03%).3 Our experiments showed serious inhibition of regeneration of adventitious shoots at 5 mg/L Basta and the differentiation rate were dropped to 23.20%.Cb was the least toxic of the antibiotics to the differentiation of the hypocotyls explants.4 Chosen lamina and hypocotyl as transformation receptor,under 30d cultivated.,the Basta resistance rate is 17.19±5.65%and 84.12±1.86%,respectively.Preculture 5 days led an better transformation efficiency.The shoot buds were subjected to repeated selection on 5 mg/l Basta for 90 days to 120 days,the positive rate of resistant material was 96.67%to 100%.5 An efficient agrobacterium-mediated transformation was established by pBar-gus carrier.Transformation efficiency was 27.06%.Leaves,flower organ and pods were expressed the GUS positive.6 Cry1C gene was successfully transformed into I.indigotica by PBar-Cry1C carrier,PCR analysis confirmed 9 transformant plants were obtained and apparently morphologically normal and flowered normally and set seeds. |
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