Induction And Proliferation Of Embryonic Callus Of Pinus Massoniana's

Masson pine(Pinus massoniana) is one of the dominant afforestation species in south China and it could be used for timber, resin tapping, papermaking, and etc. However, the elite cultivars of masson pine are not be able to satisfy the market demand due to limited supplies of seed sources and out of date breeding techniques. In this study, we selected immature seeds as ex-plant, investigated the effect of important factors, including collection time, mother tree, induction media, plant regulators, and sugar concentration, on embryonic callus induction. Morphology and cytology of embryo callus on sequential culture period had been observed and discussed. The goals are to establish the better and effective protocol for embryogenesis of P. massoniana and to provide reference for other pine tree embryogenesis. The major findings are as the following:1. The developmental process of zygotic embryo of P. massoniana in Hunan was similar as that of other pine trees. During late May to late June, it was difficult to separate seed coat and endosperm and zygotic embryo was not visible or existed in liquid form. In July, when zygotic embryo was forming, embryo head began to inflate and the suspensor started to degraded. Hypocotyl elongation was in the early cotyledon stage. Seed coat and endosperm were able to be separated easily. In August, zygotic embryo tended to mature. The hypocotyl elongated and suspensor degraded. The head of embryos (meristematic tissues) was clearly visible and endosperms had formed. Seed coat and endosperm separated easily.2. The better seed collection time was on July25, when the zygotic embryos in the early cotyledon stage. The callus induction rate reached up to15%. During late May to late June, most of callus inducted from the explants were non embryonic callus and browning frequently showed up.. The highest browning rate was85%. In July, the explants were easily to be induced into embryonic callus and had much lower browning percentage. The induction rate was up to15%. In August, the explants were induced into a small amount of embryonic callus, bigger quantity of non-embryonic callus, and majority of explants did not do anything at all. Regression analysis showed that collection time had significantly influence on the embryonic callus and somatic callus induction rate. 3. No significant difference was found among genotypes (mother trees) on embryonic callus induction rates. For the three genotype explants collected on July25, embryonic callus induction rates for Tree A, B, and C were15%,6.7%, and3.3%, respectively. However, regression analysis concluded that genotypes had significant influence on the non embryonic callus induction.4The better culture medium is LP for embryonic callus induction. Embryonic callus induction rates for LP, DCR, and LM were10%,13.3%, and1.7%, respectively. LP and DCR medium had no significant difference on embryonic callus induction percentage. But the embryonic callus produced in DCR medium was seriously hydrated and died after the2nd subculture during proliferation process. Under LP medium, callus proliferation could be sub-cultured multiple times and the callus physiological characteristics were stable. LM medium induced a little embryonic callus and died during the proliferation. The results indicated that culture media had significant effect on embryonic callus induction and the LP medium should be used.5. For embryonic callus induction, the better hormone combination was1.5mg/L2,4-D+1.0mg/L6-BA. Under this hormone treatment, embryonic callus induction rate was up to8.33%. Sucrose's impact was not significant in embryonic callus induction. But the embryonic callus produced much higher capacity under the high concentration of sucrose than that of under lower concentrations. Embryonic callus produced under different hormone levels had significant impact on proliferation frequency and quality. Under the same hormone concentration, hormone types had no significant effect on times of sequential subculture and quality of embryonic callus.6. Morphology and cytology were investigated for both embryonic and non-embryonic callus during the sequential subculture. The results demonstrated that surface of embryonic callus had many early embryos. The cells were regularly arranged and the cell nuclear was bigger. Also the cell had significant polarity. Non-embryonic callus had no early embryos. The cells were almost circle and irregularly arranged. The cell had no clear polarity. During the proliferation, the morphological and cytological features were different under different subculture period.