| Dioscorea zingiberensis C. H. Wright enriched with Dioscin in rhizome is an important steroid medicine resources plant that is endemic to China. However, low survival rate of plantlets in vitro culture blocked the rapid-propagation of high quality varieties of Dioscorea zingiberensis. So an idea of induction and producing propagule as reproduce materials was occurred and put into practice, which was proved to be an effective method of resolving problems existed in fast propagations. By using of nodal with axillary buds as explants , procedure and micropropagation system of PLB (Protocorm-like body ) was established. Based on the system, physical and chemical mechanism of propagule development were studied systematically at morphology, cytology , photosynthesis , endogenous polyamines and phytohormones levels. Differential display of mRNA in PLB related to different development period was also studied. The results are as follows:1. Establishment PLB micropropagation systemEmploying orthogonal design, an optimal PLB micropropagation system was established. And effect of the system was testes by using optimum combination of different factors. The survival rate of PLB plantlets was over 90%. Induction medium for dense callus was : MS+6-BA 2.0 mg·L-1+NAA0.4 mg·L-1+ KT0.6 mg·L-1 +Sucrose3%; Media for PLB induction and multiplication: MS+6-BA 4.0 mg·L-1+ KT1.0 mg·L-1+ Sucrose %; Medium for rooting: 1/2MS+NAA 0.3 mg·L-1+ IAA0.8 mg·L-1 +AC 0.3%+sucrose1.5%.2. Morphological, Anatomical and ultra structure analysis of PLBAnatomical structure observation and analysis on transverse section of PLB of D. zingiberensis indicated that the PLB is composed of periderm, ground tissue and vascular bundle scattered among the ground tissue. PLB obtained from in vitro culture had the same anatomical structure of monocotyledonous stem , and was similar tostructure of swelling corm at seedling hypocotyl, which verified that the propagule is a kind of stem in nature , and it was given a proper name as PLB.The PLB was a dense corm wrapped with several layers of squamous, and cells in the squamous rich in chloroplast and had high capacity of photosynthesis; there were lot of starch granule, and vacuole in chloroplast, which were the nutrition for development of PLB. The silicon and keratose layer covered on the squamous played protection function.The morphogenesis of PLB in D. zingeberensis is much different from organogenesis and embryogenesis. It is derived from embryogenic cell clusters, however no radicle was formed during its late development period, and its shoot was unilateralism polarity. Adventitious roots formed following with shoot elongating, and the squamous leaves became brown, the ability of acclimation enhanced.PTM consist of comparatively small cells that arranged in several lines. They were active in periclinal division, which contribute to the PLB thickening. Procambium was developed under the shoot apical and then formed vascular bundle. The ultra structure changes of procambium cell organelle showed that the xylem molecules are suffering in Programmed Cell Death.The root primordium originates from the phellogen and developed into adventitious root, meanwhile vascular system of root developed to connect with that of the PLB, which contributed a lot to the excellent acclimation of PLB. On the contrary, plantlet with shoot developed from adventitious buds and roots developed from callus is hardly survived, due to the disconnection of vascular systems in shoot and root.Histochemical test on callus, PLB, swelling corn bearing at the base of axillary bud displayed light red compared with that on tissue from 2-year old rhizome (used as CK ),which indicated that Dioscin existed in all the tested propagules. 3. Soluble protein contents and AOEs activity changed with the development PLBSoluble protein contents and AOEs activity were closely related with PLB development progress. The protein synthesis were high at the stages of embryogenic callus and then decreased with the development of PLB, however at the rooting stage soluble protein content increased. Activities of POD,SOD, AMY and theirisoenzyme had the same change curve a soluble did. But CAT activity has reverse change of soluble.For POD and AMY isoenxyme ,there were special bands corresponded to different development stage of PLB, which can be used as biochemical index of morphological development. Regarding to POD PI was the special band at stage II, III, and IV, while P5, P6 and P7 was the special band displayed at stage V. Special bands of Al and A2 displayed at stage II for MAY, which was the marker of PLB development, A3 was displayed at stage V, while A4 was closed in stage V.Results of IEF/SDS-PAGE showed there was great difference quantitatively and qualitatively of protein from PLB at different development stage. There are 43 protein newly expressed in embryogenic callus at the 2nd stage of PLB development. Among those, there are about five new 40KD proteins. Whether they were related to proteins in potato, still need to be verified , as well as its function in the development of PLB in D. ingiberensis. 5 special proteins expressed in the non-embryogenic callus. The different protein expression system probably play different roles in PLB development. Up-regulated and down-regulated proteins also induced along with PLB development. 4. Endogenous hormonal and their balance within endogenous polyamines playeda key role in PLB development regulation.The contents of four endogenous hormones such as IAA^ GA3^ ZRn Z in embryogenic calli were obviously higher than that in non- embryogenic calli, which were related to the callus embryogenic capability and cells division. Along with the PLB development, the concentration levels change differently, but all increase at stage V. That low level contents of ABA was detected would suggest that it has a little function for PLB development, though there's a increase during the rooting stage of PLB, which may contribute to drought resist of PLB. The balance of IAA^ ZR+Z and GA3 was necessary for the embryonic cells initiation and differentiation in PLB.There were close relationship between the level of endogenous polyamines and PLB induction and development. The concentration of spermine(Spm) is very low in the PLB, but putrescine(Put) and spermidine(Spd) is increasing along with the PLB development. At early stages of PLB embryogenesis, high Put content promoted cell division and differentiation, which was benefit to the formation of PLB. Spd increasing all thedevelopment time, but the increasing rate decline at the ripe stage. The changes indicate that PAs closely related to PLB development and growth. 5. Gene expression during PLB development orderly.Based on the in vitro system ofD. zingiberensis, the fluoro DDRT-PCR was employed to investigate gene expression during PLB development. Total RNA was isolated from vegetative PLB, there are 62 cDNA bands differentially displayed in total. Reverse northern blot analysis confirmed that 20 cDNA clones(among 40 bands selected) differently expressed at different stages of PLB development. Sequence analysis suggested that 2 clones are likely to be transcription initial factors, and some other clones showed homology with those appeared special in the embryo or the seed development, such as F-box protein and caleosin, etc. |
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