| Michelia alba DC.is an evergreen arbor species widely planted in the southern China,whose seeds are rare in the subtropical region and sextual reproductive rate was low.The study had attempted to research on its bud introduction and the mechanism and technology of its subculture,then determined the variation law of axillary buds and tissue cultured buds by HPLC,in order to fit the requirements of the key technology for large scale seedlings in the future,and provided consultation as well as theory evidence of the foundation of Michelia alba DC.tissue culture system and the high efficient bud introduction and breed of other species.The main results were:1.Determination of endogenous hormones in natural buds of Michelia alba DC..The axillary buds germinated on the plants grew to 3 cm after bud amputated for 42 d.Endogenous ZT and IAA contents were increased from 164.78 ?g·gFW-1 and 8.76 ?g·gFW-1 to 175.43 ?g·gFW-1 and 13.43 ?g·gFW-1,the ratio of endogenous hormone ZT and IAA decreased from 18.81 to 13.06.2.Explants disinfection in tissue culture.When 0.1%mercuric chloride disinfection was 4 min,the highest survival rate can reach 48.89%.3.Callus induction.Select the terminal buds or tender stems of plants as explants.The optimized formula of medium was MS + NAA 0.5 mg·L-1 + IBA 1.5mg·L-1 + 6-BA 1.5mg·L-1+ sucrose 30 g·L-1+agar 7 g·L-1,and the induction rate up to 86.67%.4.Callus proliferation culture.The results showed that the best concentration of sucrose was 30 g·L-1.The optimized formula of medium was MS + NAA 0.5 mg·L-1 + 6-BA 1.0 mg·L-1 +KT 1.0 mg·L-1+ sucrose 30 g·L-1+agar 7 g·L-1.After 35 d of callus culture,the weight reached to 3.70 g,the increment was 3.35 g.and the quality changed significantly in 0-21 d,at the same time,ZT,GA3 and IAA showed an upward trend,and after 21 d,the growth rate slowed down.5.Bud induction.The optimized formula of medium was MS+ NAA 0.5 mg·L-1 + 6-BA 1.0 mg·L-1 + sucrose 30 g·L-1+agar 7 g·L-1,which the bud induction rate was 72.22%.Analysis of the effect of exogenous hormones on endogenous hormones in bud induction by HPLC.Fixed 6-BA concentration of 0.5 mg·L-1 changed NAA concentration,it was found that the addition of exogenous NAA could promote endogenous ZT and IAA.When the NAA concentration was 0.5 mg·L-1,the endogenous ZT content reached a peak of 175.91 ?g·gFW-1 at 28 d,and the bud induction rate was 70.37%.When the concentration of NAA was 1.0 mg·L-1,the increment of IAA was the highest of 2.90 ?g·gFW-1.Fixed NAA concentration of 0.5 mg·L-1 changed 6-BA concentration,when the concentration of 6-BA was 2 mg·L-1,the content of endogenous ZT in 28 d reached a maximum of 178.36?g gFW-1,increment of 13.51?g gFW-1,and the bud induction rate was 59.26%.However,the promotion of 6-BA to IAA is not strong.6.Bud multiplication culture.The optimized formula of medium was B5 + NAA 0.05 mg·L-1 + TDZ 0.05 mg·L-1+ sucrose 30 g·L-1+agar 7 g·L-1,which the induction rate was 72.22%,which the maximum multiplication was 2.78.The primary and secondary relation of the factors affecting the multiplication of buds was TDZ>NAA>medium.When cultured 21 d,the content of endogenous hormone ZT reached a peak of 180.96 ?g·gFW-1,the content of endogenous IAA was 11.76?g·gFW-1 after culture for 35 d,showed a stable performance,the ratio of the two increased from 14.83 to 15.18.During proliferation,new buds began from 7 d,and then growth rapidly after 21 d,at 35 d can reach to 1.0 cm.Compared with the same size of the natural axillary bud,the content of endogenous ZT in the proliferating buds was 6.16 ?g·gFW-1 higher than that of natural buds,and the content of IAA was 1.17?g·gFW-1 higher,and the ratio of ZT to IAA was 1.1 lower.At this time,the bud growth was weak,illustrated that the proliferation of a small bud growth weak than natural bud,it needed further training to adjust endogenous hormone content and ratio to get the buds stronger.7.Strong seedling culture.The optimized formula of medium was MS + 6-BA 0.01 mg·L-1 +NAA 0.1 mg·L-1+sucrose 30 g·L-1+agar 7 g·L-1,which can obtain the best results and the seedlings reached 3 cm.After 35 d of the seedling cultivation endogenous ZT content reached 180.22?g·gFW-1,increment of 1.68?g·gFW-1,and the endogenous IAA content reached 14.40 ?g·gFW-1,increment of 2.68 ?g gFW-1.And the ratio of the two hormones decreased from 15.18 to 12.52.Compared with natural axillary buds of the same size,the content of endogenous ZT in tissue culture seedling was4.79 ?g·gFW-1 higher,the content of endogenous IAA was 0.97 ?g gFW-1 higher,but the ratio of ZT to IAA was 0.53 lower than that of natural axillary buds.At this time,the growth trend of tissue culture seedling was better and faster,showed the significance of cultivating seedling.8.Divide the growth process of tissue culture buds and natural buds into 1 cm,2 cm and 3 cm and compare their growth time and endogenous hormones.From budding to 3 cm,the tissue culture buds need 49 d and natural buds need 42 d.but tissue culture buds was 7 d shorter in 1 cm to 3 cm than natural buds.So the advantages of tissue culture can showed when mass propagation.From 1 cm to 3 cm,the content of endogenous ZT and IAA in the tissue culture buds and natural buds were both increased,The content of endogenous ZT in tissue culture buds and natural buds from 1 cm to 3 cm were 179.69 ?g·gFW-1 and 175.43?g·gFW-1,natural buds increment is greater of 3.05 ?g·gFW-1,The content of endogenous IAA were 11.76 ?g·gFW-1 and 13.43 ?g·gFW-1,natural buds increment is greater of 2.84 ?g·gFW-1.The ratio of endogenous ZT and IAA showed downward trend when the bud from 1 cm to 3 cm,the ZT/IAA of tissue culture buds and natural buds was similar of 14.88 and 14.29 when 2 cm,the tissue culture buds ZT/IAA was higher than that of natural buds 1.29 when it was 3 cm,it showed that the growth rate and the trend of tissue culture buds were higher and better than that of natural buds. |
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