| Aquilaria sinensis (Lour.) Gilg., the sole plant sources of agilawood in China’s mainland, whose officinal parts is the resin containing heartwood. Agilawood is with lukewarm property and pungent bitter taste, activating Qi to relieve pain, warm innards to stop vomit, nourish the kidney Qi and relieve asthma, etc. A. sinensis can produce eaglewood resin when encounter external injury or stress, which is low probability in natural environment. Because of long time predatory exploitation, the wild resources of A. sinensis were almost depleted. It has been listed as Level3protected rare and endangered plants in China since1987, and included in the national key protected wild plants by the State Council since1999.Therefor, it is important to conduct on artificial cultivation, varieties breeding and tissue culture of A. sinensis for protecting wild resources.1ObjectiveTry to develop asexual reproduction system of A. sinensis using tissue culture technology and provide a foundation to solve the problem of the scarcity of resource; and expect to establish an in vitro regeneration system for study on mechanism of resin production and germplasm innovation of eaglewood. At the meantime, study the polyploidy induction using seed, root tip, and adventitious bud as experimental materials, to obtain new germplasm with ploidy variation.2Methods2.1Study on tissue culture of Aquilaria sinensisUsing leaf, root tip and stem segments as explants to study the influence of exogenous hormones on callus induction, adventitious buds differentiation, axillary buds germination, multiple shoots induction, and rooting induction. 2.2Polyploidy induction of Aquilaria sinensisDifferent doses and processing time of colchicines solutions were applied on A. sinensis seeds, seedling root tip and adventitious buds to induce ploidy variation. Flow cytometry was used to identify the chromosome ploidy of variant plants and roots.3Progresses3.1Study on tissue culture of Aquilaria sinensis3.1.1The sterilization conditions optimizationBy optimization of sterilization agent and sterilization time, optimal condition was obtained as:0.1%HgCl2, for10min for leaf explants, and,0.01%HgCl2for3min for root explants.3.1.2Callus inductionFor callus induction from leaf, different leaf age, explants size, explants inoculation methods, light condition and hormone concentration and combinations were studied. The results indicated that optimal callus induction rate up to100%was obtained using12-day-stage leaf cut into0.5cm2, and cultured in dark on MS+2,4-D (0.5～1.5) mg·L-1+ZT (0.5～1.5) mg·L-1with leaf adaxially positioned.For callus induction from root explants, the influence of hormone concentration and combinations were studied. Results showed that2,4-D combined with6-BA had a higher callus induction rate (up to83.3%) than NAA (53.3%), the optimal medium was MS+2,4-D0.1mg·L-1+6-BA0.1mg·L-1.3.1.3Adventitious bud inductionCallus induced from leaf and root explants were cultured on a variety of hormone combinations medium for induction of adventitious bud, no progress has been achieved to date in current study.3.1.4Multiple shoots inductionAxillary buds germination had preliminary succeed form stem explants containing buds, and the optimal medium used was1/2MS+6-BA0.5mg·L-1+NAA0.2mg·L-1.Germinated axillary buds were used to study the effect of hormone combinations on multiple shoots induction. Result showed that multiple shoots can be induced on MS medium with6-BA, but not on MS medium with KT. With increased concentration of6-BA from0.1mg·L-1to2.0mg·L-1, the proliferation rate of shoots first rose and then fell, the highest proliferation rate on medium MS+6-BA0.5mg·L-1with4.07. With6-BA concentration higher than1.0mg·L-1stem segments swelled and formed callus. Adding 0.01mg·L-1NAA to the medium could increase proliferation rate, but higher concentration of NAA (0.1mg·L-1) declined the proliferation rate.Added activated carbon to MS medium avoided etiolated seedlings, however the multiple shoots proliferation rate was taken down to0.3.1.5Rooting inductionFor rooting induction, Two-step rooting method showed higher rooting rate than One-step rooting method. The specific approach to obtain roots was pre-culturing the adventitious buds on1/2MS medium containing1.0mg·L-1NAA for7d, before transferring to hormone-free1/2MS medium for one month induction, which achieved a rooting rate of75%.3.2Polyploidy induction of Aquilaria sinensis3.2.1Polyploidy induction using seedsFor polyploidy induction from seeds, different experimental conditions used could all arose growing point bulge of seeds when treated with1.2%colchicines for36h, with a germination rate up to10.5%. In the two seedlings acquired, results of chromosome ploidy analyzed by flow cytometry (FCM) showed that the chromosome of multi-branching plant wasn’t mutated; the slow growing plant was chimeras that consisted of diploid cells and tetraploid cells. No significant morphological difference was observed between chimeras plant and normal plant.3.2.2Polyploidy induction using root tipsSeedling root tip arose growing point bulge and continued to grow after treated with0.2%colchicines for12h; chromosome ploidy of treated root was analyzed by FCM. Results indicated that swelled root area and expands root elongation area both had chromosome variations.3.2.3Polyploidy induction using adventitious budsSuspected polyploidy adventitious buds were found when adventitious buds were treated with colchicines (0.4%and0.8%for1d、2d and3d), and the morphological changes including low plant growth, thick and yellowish leaves, thicker stems.4Achievements(1) A rapid propagation system from axillary buds explants of A. sinensis was established and optimized.(2) A chimeras plant that consisted of diploid cells and tetraploid cells, and a normal chromosome plant with multi branches from seeds were obtained in polyploidy induction experiment. And chimeras root was obtained from live plant root after treated with colchicines; Morphological suspected polyploidy adventitious buds in vitro was also obtained after colchicines treatment. |
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