The Research On Protoplast Culture And Regenerate System Of Medicago Sativa L. For Southern China

Alfalfa, the queen of forage, for its wide adaptability, pertinacious resistance and high nutrition,is better than any others. Recently, following the rapid development of stockbreeding in our country,continuously adjustment of agricultural industrial framework and requirement of alfalfa market bothat home and abroad, the alfalfa industry has developed unprecedently. The cultivate area whichcontinuously expands to the south has been increasing constantly while keeping the development innorthern area. Yet, acidic soil, hot and moist climate and the threats of weeds, disease and insectshave badly affected the popularization in the south of our country. To solve the problems thoroughly,Cultivate and apply new variety which adapt to southern area is one of the most efficient routes,while traditional breeding methods, for its time-consuming and low-efficiency, was not the bestmethod. So, later of 80th, developed forage biotechnology, which provide more efficient ways forthe improvement and popularization of forage, was been becoming the main technology ofcharacters improvement. Among those technologies, the cellular improved methods were theimportant parts, such as the protoplast (in-between medium). This paper, on the basis of thisbackground, with the research of optimal dissociate conditions, cultivate methods and identificationof regenerate plantlet from protoplast on southern alfalfa, the integrate biotechnology platform wasestablished, which was very important to guide the efficient production of new idioplasmic resourceand apply into the breeding practice.Alfalfa of line 15 was used as the main material, adopting the methods of one-stepdissociation by enzyme and purification by sedimente, the key factors on protoplast dissociation andcultivate were researched in this study, which key factors of dissociation includes material type,enzyme type and concentration, enzyme time, centrffugate condition, kind and concentration ofinfiltrate-pressure stabilizers, pre-prepared conditions, type and concentration of cytomembranestabilizers and so on, and key factors in cultivation, such as cultivate methods, cultivate density,fundamental cultivate medium, combination and concentration of hormonesome in medium and keyfactors in medium like Ca²⁺ and NH₄⁺/NO₃^- were filtrated, furthermore, comparative identificationwere carried out through cytology, physiology and biochemistry on regenerated plantlet. The mainresults of the research were shown as follows:1. Among the alfalfa materials used in the research, the cotyledon's Integrity Production Ratereached the highest as 9.60×10⁶ units/g, but resource finitely, instead leaf was the much better onefor its moderate output(7.74×10⁶ units/g) and universality, furthermore, the second time re-producedcallus was the best one in the material of callus, but compare with cotyledon and leaf, the production rate was on the low side as 3.86×10⁶ units/g, but integrity rate reached 89.1%.2. The optimal combination of enzyme was Cellulase Onozuka R-10 2%+Macemzyme R-100.5%, the optimal enzyme time was 8h. In the process of protoplast purification, the key affectingfactors in part of physics, such as centrifugal speed, 500rpm was the optimal, and centrifugal time,6rain was the best. Generally speaking, the process of centrifugal should be carried out in cooledcentrifugal machine, and be kept stable as 4℃to decrease the damage of protoplast on the largescale.3. In the precede of protoplast dissociation, among all the pre-prepared methods, CPW13g/Lsolution pre-prepared 1h was the best efficient one, for it could distinctly increased the protoplastintegrity, reached 14.02×106 units/g, the best cytomembrane stabilizer in the enzyme solution was0.1% MES. In addition, in the various infiltrate-pressure stabilizers, at the 0.6mol/L of glucosereached the best.4. In the protopast cultivation, the solid-liquid cultivate method was the effect one, for thatcould reach the highest dissociate frequency, furthermore, among these methods, nutritiousnurse-cultivation was the most efficient, the dissociate frequency reaching 5.33%; the optimalcultivate density of protoplast was 1.5×10⁶ units/g, for after 2～3 days the celluar dissociationcould be observed, and after 25～30 days callus could be seen by naked eyes, the plate rate reached0.011%; the fundamental medium of KM8p, the combination of chromosome of NAA 0.5mg/L +2,4-D 0.5mg/L + 6-BA 1.5mg/L, and Ca²⁺ in media of 300±200mg/L were the best; otherwise, in theproportion of NH₄⁺/NO₃^-, with the decreasing of NH₄⁺ and increasing of NO₃^-, the dissociatefrequency and plate rate had the tendency of increasing continuously, added some organic nitrogenin the medium, the tendency increasing could be more obvious.5. To identifying the protoplast regenerate plantlet by cytology, compared with enatic plant, theresults were showed that the chromosome of regenerated plantlet exhibited no obvious variation inlength, arm rate, and the index of centromere, which its karyotype going in accordance with2n=4x=32=29m+3sm(SAT). In the identification by physiology and biochemistry, under the samecondition of low-temperature intimidation, contrasted with enatic plant, various isoenzymes' activityof regenerate plantlet declined, and some resistant-active-substances' content such as MDA,dissoluble proteins and the main substances in the photosynthesis such as chlorophyll a/b alsoreduced.