Studies On Seed Dormancy Mechanism And Methods To Break Dormancy In Cyclocarya Paliurus

Cyclocarya paliurus (Batal.) Iljinskaja is the native to China and is the sole species in its genus. Because of the multi- value including as a high value medicinal plant, fine timeber and ornamental, it is regarded as a rare tree in China. However, the seeds remain deeply dormant for 2 years in the natural environment. In order to study the dormancy mechanism of the Cyclocarya paliurus seeds, seed coat structure, endogenous inhibitors, the changes of the storage substances, some enzymes activities and endogenous hormones were examined by synthetical approaches. Through the researches of the dormancy mechanism, the optimal techniques of breaking dormancy were advised. The main results were as follows.1) The causes of seed dormancy were studied with isolated embryos and embryos with whole or partial seed coat treated by different means. The results indicated that the germination percentage of isolated embryos cultured on agar-solidified media for 30d was 90%, and seedling rate was 85%. From these results we inferred that embryo of C. paliurus was be out of physiological dormancy. Although the mechanical restraint and permeability barrier of seed coat were relieved by slitting seed coat and removing a half of seed coat, the germination percentage of these embryos were only 2% and 26% respectively, and that the seedling rate of them was almost zero. It was deduced that the mechanical restraint and permeability barrier of seed coat influenced the germination to some extent, but the main reason resulted in the seed dormancy may be the strong endogenous inhibitors in the seed coats. Furthermore, embryos with half of seed coat were cultured on agar media containing different concentration of GA₃, exogenous application could significantly promote germination. The germination rate of embryos with half of seed coat was up to 88%. Experimental results have validated the above conclusion preliminarily.2) The untreated seed coat was scanned under electic scanning microscope. Result showed that the seed coat of C. paliurus was cornified and the mid seed is lignified and hard, and this type structure may be prevent the embryo from protruding and the water and gas from exchanging. The experiments of seed water-absorbing indicated that the seed coat was permeable, nevertheless existed the permeability obstacle to a certain extent. Compared with the untreated seeds, the chemical scarification immersed in concentrated sulphuric acid and the stratification can effectively destroy the seed coat structure. After soaking in sulphuric acid, the seeds became smaller, the seed coat became thinner, the germ pore and germinal slit became more obvious At the same time, water absorption capacity of these seeds also enhanced. Scanning Electro-microscope photos showed that stratified seeds under integrated treatments changed greatly, and the permeability of water and air was improved. In conclusion, the integrated treatments consisting of chemical scarification and stratification could relieve the mechanical restraint and promote the exchange of gas and water, thus accelerating dormancy breaking and increasing the germination rate.3) The results showed that the inhibitors were distributed in all the parts of seeds, and that the order of inhibitory effect was: testa＞endopleura＞embryo.Because of the stronger inhibitor activity, testa was selected as further experimental materials. With the methods of Systemic solvents, the methanol extract of testae were separated elementarily. Bioassay showed that the inhibitory activitiy of ether phase was strongest, and the result of GC-MS revealed that the maximal component of 35.09% was Vanillic acid.On the basis of the above results, two potent substances were isolated and purified from the ether phase of seed coat by TLC(Thin Layer Chromatography) and Column Chromatography, and identified as Vanillic acid and Protocatechuic acid by UV spectrum and the ¹H and ¹³C NMR spectrum. Moreover, Vanillic acid and Protocatechuic acid were identified by TLC. Then, standards of two substances and ether phase of testa were developed on the same thin layer plate by TLC, and Vanillic acid was the main component of R_f value 0.6 and Protocatechuic acid was the main component of R_f value 0.23. Finally, the contents of Vanillic acid and Protocatechuic acid in the methanol extract of testa were determined by HPLC, 0.120mg.g^-1 and 0.025 mg.g^-1 respectively.Furthermore, the isolated embryos were inoculated on the agar media containing different concentration of Vanillic acid, and the germination rate and root length were measured. The result of calculations showed that IC₅₀ (50% inhibitory concentration) of germination rate and root length were 82.33 mg.L_-1 and 33.95 mg.L_-1 respectively. All these results suggested that the endogenous inhibitors including Vanillic acid existing in testa may be the main reason induced to be dormant.4) Some important physiology indices correlated with seed dormancy and germination physiology were measurated. The results suggested that the relieving degree of dormancy was close with the metabolism of the storage substances in embryos. During the release of dormancy, the water content of embryos increased and the speed of inspiration were enhanced. At the same time, three main storage substances including fat, protein and amylum were successively degraded, and the activity of some interrelated enzymes became stronger, and the ratios of promoting germination hormones and inhibitory hormones increased accordingly.5) It was concluded that C. paliurus seeds exhibit combinational dormancy which included forcing dormancy caused by seed coat structure and physiological dormancy caused by endogenous inhibitors. In accordance with its dormancy mechanism, corresponding treatments were examined by the orthogonal design. The preferable treatment to break dormancy of seeds, i. e. S9, and 84.41% germination rate was achieved after 90 days. The processes of treatment were as follows: to beginwith, the seeds were soaked in concentrated sulphuric acid for 5h; then, soaked in 300 mg.kg^-1 GA₃ for 48h and moistened stratification media with 600 mg.kg^-1 GA₃; finally, they are stratified in the low temperature(5℃) followed by the warm temperature(18℃). The best medium to promote embryo germination was E6 medium of WPM + GA₃ 10 mg·L^-1+ sucrose 30 g·L^-1, and all the embryos could germinate after 30days. A high frequency of seedling formation reached 87.5% on the E5 medium of WPM + GA₃ 10 mg·L^-1+ maltose 30 g·L^-1.