| In this experiment, the leaves, shoot tips and stem nodes of adult trees and the seedings fromseeds were used as explants for the studies on the in vitro culture and the vitrification in Acaciacrassicarpa.The main contents of the experiment were as follows:①Optimization of the conditions ofin vitro germination of seeds;②establishment of the in vitro regenerating system from the leaves,shoot-tips and stem nodes and the discovery of the vitreous phenomenon in Acacia crassicarpa;③thefactors influencing vitrification and the preventive measures in Acacia crassicarpa;④the histiocyticobservation by paraffin section and the tests of physiology and biochemistry between the normal andthe vitreous shoots in Acacia crassicarpa.1．The establishment of the regenerating system and the discovery of thevitreous phenomenon in Acacia crassicarpa.①The optimization of the in-vitro germinating conditions from the seedsAfter the treatment of the boiled water for 20 min combined with the thick vitriol for 25 minwithout HgCl sterilization, the seeds of Acacia crassicarpa germinated at high frequency and at lowcontamination rate, which could avoid the latent harm to the seeds from HgCl.②The induction of callus and the regeneration of plantlets from the leaves of adult treesThe effects of the different treatments of collecting time, hormones and sterilization time on theleaves culture from the adult trees were carried out, and the results showed that the quality of thecallus was related to the collecting time, and the leaves collected in November produced the goodcallus which was yellowy, loose and wet at high frequency; the high concentration of 2,4-D combinedwith the high concentration of BA also produced the high quality of callus, but the treatment of 2,4-Dcombined KT was opposite; After sterilization with 0．1% HgCl for 6 min, the leaves was easy toproduce callus on the 1/2MS medium adding 1．0mg.L BA and 1．0 mg. L 2．4-D, from which theplantlets were induced.③The shoot tips and stem nodes culture from adult trees and the phenomenon ofvitrification in Acacia crassicarpaThe obtained results showed that after the pretreatment of washing for 2 h for the adult stemnodes, the contamination rate was low and the germinating rate was high; the sterilization time alsoaffect the germination rate, and the longer the time was, the lower the germination rate would be. Inthe culture of stem nodes, the vitrification occurred popularly, and the rate of vitrification almostreached above 50 % .2．The influencing factors and the preventive measures for the vitrification①The effect of the explants on the vitrification The effects of both the part and the length of explants on the vitrification were carried out, andthe obtained results indicated that the length of the explant was the major factor affecting theproliferation rate and the vitrification rate, the shorter the explant was, and the higher the proliferationrate and the vitrification rate would be, but the part of the explant affected the vitrification rate slightly.Therefore , the proper length of the explant was between 0．8 cm and 1．0 cm.②The effects of the hormones on the vitrificationThe obtained results showed that BA affected the proliferation rate and the vitrification rateobviously, and the suitable concentration for BA was about 1．0 mg.L, but the suitable concentrationfor KT ranged widely from 0．01 m g.L to 4．0 m g.L.The auxins of IAA, 1BA and NAA affected thevitrification slightly. However, adding 0．1 m g.L 2,4-D or 0．5 m g.L GA was beneficial toreducing the vitrification rate.③The effect s of the sucrose, gels and AC on the vitrificationThe results indicated that the high concentration of sucrose was better to the improvement ofmultiplication and to the decrease of the vitreous rate; the low concentration of the gels resulted in theimprovement of the multiplication and the vitrieous rate; and AC could improve rooting and reducethe vitreous rate obviously, but the high concentration of AC (>6g.L ) suppressed the growth ofplantlets.④The effects of the elements of the MS medium on the vitrification and the optimizationof the combination of the mediumThe results showed that MgS0．7HO, KHPO, MnSO.4HO, Nicotinic acid, HBO, NHNO,Glycin improved proliferation; MgSO.7HO, KHPO, Glycin, NHNO, VB were beneficial to thegermination of adventitious buds; and the excessive quantity of NHNO, KNO, CaCI.2HO,KHPO (especially NH4NO3) promoted vitrifying. Through summarizing the experiment results,three components of the revised media were screened, and then compared with the media of MS,1/2MS and B5, the obtained results indicated that all the three revised media and B greatly promotedmultiplication and suppressed vitrification. Therefore, eliminating or decreasing NHNO couldsuppressed vitrifying effectively.⑤The effect of tissue culture conditions on the vitrificationThe results showed that the containers and sealing materials had little influence to thevitrification, but the light intensity and the pH value affected the vitrification remarkably, and the highlight intensity or acid condition suppressed vitrification.⑥The preventive measures to overcome vitrificationAccording to the experiment above, the general measures to overcome the vitrification weresummarized as follows: (a) choosing the explants with the length between 0．8 and 1．0 cm; (b)adding 1．0 m g.L BA or 0．01～4．0 m g.L KT, together with 0．01 mg.L 2, 4-D or 0．5 mg.L GA; (c) Using the hgih concentrations of sucrose from 30．0 to 50．0 g.L; (d) adding 8．0 g.L agar or cala glue; (e) adding activated carbon (<6g.L) ; (f) adopting B5 medium or revised medium, from which NH4NO3 was removed or reduced: (g) increasing the light intensity, or controlling the pH value at 5．4．3．The histiocytic mechanism of the vitreous occurrenceThe difference of the histiocytic observation by paraffin section between the normal shoots and vitreous shoots was carried in Acacia crassicarpa. The results indicated that by microscopical observation there was little difference in stem nodes between the normal shoots and the vitreous shoots, but compared with the leaf of normal shoots, the leaf of the vitreous shoots had been changed as follows: the number of the palisade tissue decreased; the arrangement of the palisade tissue and spongy tissue was irregular, the intercellular space was more broader, the vascular bundle sheath also arranged loose, and the cytoplasm was more washy.4．The physiological and biochemical changes of the vitreous shootsThe comparisons of the physiologicl and biochemical difference between the normal shoots and vitreous shoots were carried out in Acacia crassicarpa. The results showed that as the vitreous degree increased, the water content, total chlorophall, protein content and catalase(CAT) activity decreased, but peroxidase (POD) activity increased ,and the content of deoxyribonucleic acid(DNA) almost kept fixed.As a whole, in this experiment the major factors of the establishment of regenerating system was discussed in Acacia crassicarpa; the influencing factors and preventive measures for the vitrification were found, which would offer the effective technical means for industrialization and commercialization in Acacia crassicarpa; Simultaneity, the histiocytic observation by paraffin section and examination of the physiological and biochemical difference between the normal shoots and vitreous shoots vitrification, would provide the important scientific evidence for exploring the vitreous mechanism and finally solving the vitreous problems roundly and thoroughly, and promote the commercialization of micropropagation and the application to biotechnological breeding in Acacia crassicarpa .
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