Studies On The Morphological Anatomy And The Physiological And Biochemical Changes During The Formation Of Microtubers In Dioscorea Opposita Cv. Tiegun

Dioscorea opposita a member of the family Dioscoreaceace, is one of the famous "Four HuaiqingChinese medicines". D. opposita cv.Tiegun is a top grade traditional medicine Long-term vegetativepropagation of D. opposita cv. Tiegun has resulted in the decline of production and germplasmdegeneration in. Therefore, studies on the in vitro rapid propagation and related technologies wereperformed to solve the problem. The microtubers are superior to shoots for mass propagation because theyare easily acclimated, stored and transported. In previous work, we have studied the conditions forinduction of microtubers in D. opposita cv.Tiegun and developed a one step oscillation method formicrotubers production.In this study, the morphological anatomy and the physiological and biochemicalchanges were characterized during microtuber formation. The results were as follows:1. The morphological change during microtuber formation. In the oscillation cultivation under thedarkness, the whole period for microtuber formation are42days. It was divided into7stages: Explantsstage (0d), budding stage (7d), bud stretching stage (14d), bud proliferation stage (21d), microtuberformation stage (28d), microtuber enlargement stage (35d), and microtuber mature stage (42d). As acontrol, the nodal segments maintained in stationary condition under the darkness had bud growing but notuber formation.2. The anatomical change during microtuber formation. The microtubers are formed in leaf axil.Periclinal and antiolinal divisions were appeared in the infracutaneous cells under the leaf axil, resulted inthe formation of a group of bud primordium with meristem. When the milky white spherical microtubersare visible, the apical bud had differentiated in its terminal part. The most external part of the youngmicrotuber was a layer of epidermis. Several layers of parenchyma cells under the epidermis were arrangedrather closely. The parenchyma cell in the middle part of microtuber was rich in starch and protein granules.The most outer part of the mature microtuber was phellogen epidermis and its internal part was theperiderm. The amount of the internal parenchyma cell increased and then the cell enlarged to form certain shape and volume of a mature microtuber.3. The changes of the main organic matter and related enzyme activity during microtuber formation.The content of soluble sugar, glucose and fructose maximized during the microtuber formation stage. Afterthat, the sucrose and starch content rised gradually, which maximized at microtuber mature stage. Thesoluble protein content was higher in the bud proliferation stage. The degradation of sucrose synthase（SS）was consistent with the change of sucrose phosphate synthase (SPS) activity, which both maximized in themicrotuber expanding stage. Synthetic activities maximized on day42. It also had significant positivecorrelation with sucrose accumulation. AGPase had the highest activity in the microtuber expanding stage.Soluble starch synthase (SSS) had the highest activity in the microtuber formation stage, and both hadsignificant correlation with starch accumulation at the time of highest activity. These results indicated thatthe accumulation of materials such as sucrose, starch and SSS, SPS activity changes played an importantrole in microtuber formation.4. The changes of endogenous hormone during microtuber formation. The endogenous indole-3-acetic(IAA)、abscisic acid (ABA)、trans-zeatin-riboside (ZR) and trans-zeatin (Z) were measured duringmicrotuber formation by LC-ESI-MS/MS. The results showed that the contents of IAA, ZR, and Z allmaximized at day28and have very obvious rising trend from day21. However, the content of IAA was ina condition of relatively stable before day21. The content of ZR was firstly increased and then decreasingbefore day21. The content of Z showed a tendency of slowly increasing during day0and21. The contentof IAA and ZR all declined after day28and then reached a steady condition after day35. But the contentof Z was continuous declined after day28. The content of ABA began to increase at day0, and reached thehighest level on day28. The content of JA bengan to decline at day0, but it was slightly increased betweenday14and day21, and then got the minimum at day28. After that the content of JA began to increase andgot the maximum at day42. We conclude that IAA, ABA, ZR and Z all showed positively correlation withmicrotuber formation. Our studies about the rules of the change of hormone will laid a foundation for theinduction of the microtubers more efficiently, through the regulation hormone levels.