| Zizania latlfolia(Zizania latlfolia Turcz.)is one of the traditional Chinese aquatic vegetables.Ustilago esculenta(Ustilago esculenta)is an important factor that cause the enlargement of swollen stem gall.The influence of locust formation,the reasons for the production of easily-developed sporulating galls and unpregnant water bamboo are not yet clear,these problems have severely constrained the development of Zizania latlfolia cultivation with high quality and high yields.In this study,"YD-3" was used as a test material to explore the entire growth and developmental changes of Ustilago esculenta,isolation and identification methods,and the suitable conditions for Ustilago esculenta culture.The tissue culture regeneration system of unpregnant water bamboo and artificial inoculation technique tries to provide some clues for the mechanism of Ustilago esculenta influencing the formation of Zizania latlfolia,and provides a certain technical basis for the production to the formation mechanism of single and double season Zizania latlfolia and sporulating galls and unpregnant water bamboo.The result showed that:1.In the process of growth and development of the plant,the suitable dehydration time is 5/12h,1h,2h,4h,8h and 12h,in order of bud and seedling growth point,initial,middle,middle and late stage of fleshy stem enlargement,commodity period and mature aging period.the change process of Ustilago esculenta in the tissue can be clearly observed;the bud stage is the initial stage of growth of Ustilago esculenta,and the initial stage of swollen stem enlargement is the period with the sharpest morphological change of Ustilago esculenta,resulting in indigo stem swelling.The commercial and mature aging phase respectively are the stable growth and spore formation phase of the swollen stem.These two phases are suitable material for the separation of Ustilago esculenta.There is no hyphal in the unpregnant water bamboo leaf sheaths,whereas the swollen stem leaf sheaths contains hyphal.It can be used as one of the verification methods for the successful inoculation of unpregnant water bamboo by Ustilago esculenta,2.3%NaClO was used to disinfect the whole Zizania latlfolia for 4 min.Then the pellets and spore suspension separation methods were used to separate Ustilago esculenta.In the identification process,the universal primers ITS1 and ITS4 were used to amplify the ITS-5.8S rDNA could not be specifically identified to Ustilago esculenta.Two pairs of specific primers were designed to select the two gene fragments,Lam16A and Pra2 of Zizania latlfolia,which could be specifically identify to Ustilago esculenta.The most suitable medium for Ustilago esculenta culture was PDA,YEB is not suitable for culturing Ustilago esculenta.3.The successful establishment of the regeneration system for unpregnant water bamboo scorpion tissue culture has explored the appropriate disinfection methods and optimal plant growth regulator formulations for explants during tissue culture.After explant buds were washed with water for 2 hours and then treated with 0.1%HgCl2 for 8 minutes,the optimal hormone concentration for induction of buds growth was 1.0 mg/L IAA+1.0 mg/L 6-BA;the optimal concentration of hormones for the regenerating buds.The formulation was 1.0 mg/L 2,4-D+1.0 mg/L 6-BA with the proliferation coefficient of 3.5±0.5,the rooting hormone concentration was 0.5 mg/LNAA.4.A preliminary study of the technical system of artificial inoculation of unpregnant water bamboo with turfgrass found that morphological and molecular identification methods can successfully inoculate bacteriophage injection.The specific procedure is to wipe off the base of the root seedlings with 75%alcohol.After that,use a syringe with a needle to suck up the base of the turmeric bacteria injection to overflow the broth,and then put it into a plastic basin containing 6 cm of matrix,keep the shallow layer of 2-3 cm in the plant growth chamber,and set the temperature to 15 ? and 25 ? during the night and day,the photoperiod was 12h/d.When the OD600 of the fungus was 2.5,the successful rate of inoculation was 40%. |
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