The stem of Ginkgo biloba were cultured on MS,modified MS and DCR basal medium,supplemented with various concentration of hormones—NAA,ZT (zeatin) and two types of nurture-CH (casein hydrolysate) and Ade (adenine). The aim was to study the effects of various basal medium,different hormones and nurtures on the growth and development of Ginkgo biloba stem. The result showed that the modified MS with CH 500 mg·L -1 was the best medium for inducing the development of axillary buds;the medium with NAA,ZT and Ade promoted axillary buds to be callus and inhibited them developing into shoots. The shoot rate was 89.3%. After cultured for 40 days,the clustered buds were emerged from axillary buds. The detached buds from clustered buds were cultured on modified MS basal medium or supplemented with CH 500 mg·L -1 . The shoots grew well,and the reproduction coefficient was 3. The shoots from apical buds were also reproduced,and the reproduction coefficient was 4. The shoots were induced to root on MS+NAA 0.1 mg·L -1 medium,and the rooting rate was 33.3%.
The rDNA internal transcribed spacer 1(ITS-1) regions of two Chinese fir provanences was amplified and cloned by PCR reaction.The PCR reaction was following:97℃ 5 minutes→95℃ 5 minhtes→Adding the Tag polymerase→94℃ l?min 56℃ 1?min,72℃ 2?min;thirty\|six cycles→72 ℃ 10?min.High quality DNA template is necessary for the amplification of ITS-1 sequence,during the PCR reaction,ten minutes denaturation time and 56℃ annealing temperature are beneficial to amplification.The ITS-1 fragment was ligated to PUC19 plasmid,digested with Hind Ⅱ and transformed into competence cells of E.coli JM83 strain,the cloned strains harboring recombinant plasmid were obtained,those recombinant plasmids were used to sequence for ITS-1 fragment.Sequence analysis indicated that the sequence length is 273 bp,the using percentage of A\,T\,C\,G within ITS1 sequence of Chinese fir were 27.5%\,23%\,21.6%\,27.9% respectively and the G/C content of ITS1 sequence was 48.35%.Comparing with other plants,the G/C content of Chinese fir was less than other plants,whose ITS1 regions have been sequenced.As to ITSI sequence,there was no difference among two Chinese fir provenances,sequence analysis disclosed there were two repeat sequences [AAAG] n and [TTG] nappeared within ITS1 sequence of Chinese fir.
