Résumé
Objective To lay a foundation for the cultivation of disease-free plantlet of Citrus medica L.var.sarcodactylis Swingle(CMS) by discussion of the factors affecting survival rate for micro-grafted shoot-tip of CMS.Methods With citrus limon seedlings cultured in the dark as the rootstock,shoot-tip of CMS was grafted to the inverse T-shaped cut in the cotyledon of rootstocks.Then an investigation was carried out on the factors which affected the survival rate for micrografted shoot-tip of CMS.The factors included age of rootstocks,scion size,culture medium with different concentrations of phytohormones,and rootstocks with or without cotyledon.Results The survival rate for micro-grafted shoot-tip of CMS was closely related to the factors mentioned above.A higher survival rate was obtained when CMS shoot-tip with 4 pieces of leaf primordia was grafted to the inverse "T" shaped cut in the cotyledon of parental stock(citrus limon seedlings) which was cultured in the dark for 14 days,and when the citrus limon seedlings were cultivated in MT-based medium with ?-naphthylacetic acid 0.1 mg?L-1 added.Conclusion The optimized micro-grafting conditions for CMS shoot-tips improve the propagation of micro-grafted plantlet and the survival rate of micro-grafted shoot-tip of CMS.
Résumé
Objective To provide a new approach of preservation for germplasm resources of Citrus medica var.sarcodactylis(CMS).Methods A micrografting test was made on CMS shoot-tips after vitrified cryopreservation,resulting in living shoot-tip.Results First the CMS shoot-tip is inoculated in medium consisting of MS,5%DMSO,and 5%sucrose for 48 h preincubation.Then the shoot-tips were cut off 3—4 mm long and fore-treated by 60%PVS_2 at room temperature(25℃) for 30 min.After that,they were treated by PVS_2 at 0℃for another 80 min and conserved in liquid nitrogen for 24 h.Next the shoot-tips were defrosted by aqueous bath at 40℃and cleaned twice at room temperature(25℃) by MT minimal medium with additive 1.2 mol/L sucrose,10 min once.Finally data collected recorded a higher survival rate of 81.25%by TTC inspectation and a better regeneration rate of 52.94%if the CMS shoot-tips had been grafted on darkly-cultured parental stock,growing,and differentiating normally afterwards.The plantlets detected by polymerase chain reaction(PCR) showed that the pathogens of Huanglong disease had been removed.Conclusion Therefore a conclusion is made that the approach of vitrified cryopreservation may be applied to the preservation of CMS germplasm resources.