Establishment And Developmental Mechanism Of In Vitro Shoots Of Manchurian Ash (Fraxinus Mandshurica Rupr.)

Manchurian ash(Fraxinus mandshurica Rupr.)is a rare broadleaf tree species in Northeast China. With high quality timber, elegant form of crown and relative high resistanc to stress, this species is of great economic value and landscape value.In this work, embryo hypocotyls of Manchurian ash were used as explants, and the in vitro shoot regeneration of this species was systematically studied.By using microscopy, HPLC and differential display PCR techniques, we tried to study the mechanism of shoot regeneration of Manchurian ash in cellular and molecular levels, which covered the aspects of variation of cells and tissue structure, physiological processes and differential expression of gene. The results were as follows:Micropropagation system was established through in vitro culture of hypocotyl of mature seed. Adventitious shoots were obtained on both MS and WPM medim,with regeneration rate of 83.3% and 80.0% respectively. High mortality occurred on MS medium, while mortality on WPM medium remained very low during the whole culture period.On MS medium,TDZ was more effective than BA to induce adventitious shoots,while on WPM medium BA was better than TDZ. The results shown that WPM medium supplemented with 10μM BA is suitable for induction of adventitious shoots, and WPM medium supplemented 10μM BA and 0.05μM IBA is suitable for elongation of shoots,then WPM medium supplemented with 0.5μM IBA was used for rooting of the shoots.The rooting rate of shoots were 81.6%, and plantlets were successfully acclimated to greenhouse.In 3 days of culture, parenchyma cells at the cut ends of hypocotyls were observed to started divission. In 6 days of culture, cells go into a state of vigorous division. These cells were similar to cells at apical meristem, with the arrangement in order, thick cytoplasm, larger nuclei and thinner cell wall.In 9 days of culture, bud primordia was observed, with cells that has tightly arrangement, thicker dyeing and clear nuclei.The changes of soluble sugar, SOD, CAT, POD, soluble protein, overall phenol, PPO and nucleic acid during shoot regeneration reflected the relationship between these substance and the vigorous division of cells, initiation of shoot regeneration as well as browning of explants. The content of soluble sugar and soluble protein kept increasing with the initiation of shoot regeneration, indicated that these substance were both material basis and energetic basis of shoot regeneration. The level of POD, overall phenol and PPO were showed to be higher in browning explants, suggesting that these substance were related to the stress reaction of plant tissue in browning explants.The peak value of ZT occured in 6 days of culture. The ratio between ZT and IAA play an important role in the induction of adventitious shoot. ABA level increased with the increasing of browning of explants.The dynamics of hormones indicated that the balance between endogenous hormone and plant regulators has important effect on the initiation of shoots of Manchurian ash, suggested that the "co-effects" of media, cytokinins and auxins, especially the balance of hormones play an important role in the regeneration of Manchurian ash and development of regenerated buds.The acceptable reaction system of DDRT-PCR for the shoot regeneration of Manchurian ash were as follows:one 5'random primer,40℃of annealing temperature and 100μmol/L dNTP. mRNA differential display indicated that gene expression during shoot regeneration of Manchurian ash was in a pattern of time sequencing expression, and the variation in the transcriptional level occured at the early stage of shoot initiation.