Construction Of Slow Growth Storage In Vitro Preservation And Droplet-vitrification Cryopreservation System Of Morinda Officinalis How.

Morinda officinalis How.is a perennial vine belonging to the genus Morinda of Rubiaceae Juss.It is one of the “four famous southern medicines” in China.It has high medicinal value and is an important export-earning medicinal material.However,due to the limitations of its cultivated varieties and the continuous depletion of wild resources,it is urgent to preserve its germplasm resources.At present,the research on in vitro preservation of M.officinalis mostly focuses on the in vitro tissue culture and rapid propagation technology.Related research on slow growth in vitro preservation and cryopreservation is rarely reported.Therefore,the conduct of these works can provide some theoretical basis for the preservation of germplasm resources of M.officinalis and similar tropical medicinal plants.In this study,M.officinalis was used as the experimental material to explore the optimal container for the growth of in vitro shoots and the optimal medium for prolonged in vitro preservation.In addition,a droplet vitrification cryopreservation method was developed.The obtained results are as follows:1.The tissue culture bag can maintain the normal growth of M.officinalis in vitro shoots,and the survival rate of plants in the medium-term preservation is not significantly different from those cultured in the two containers of glass bottle and plastic bottle.High sugar concentration and low hormone medium(MS + 2.0 mg/L 6-BA + 0.4 mg/L NAA +50 g/L Sucrose + 7.5 g/L Carrageenan,p H 5.8)can extend the in vitro preservation of M.officinalis to 6 months,and reduce the water content of the plant to a certain extent.2.In this experiment,the cryopreservation procedures for M.officinalis was established.The procedure is as follows:The axillary buds induced from nodal segments by high sugar and low hormone medium for about 21 days were excised.After the overnight incubation in the transition medium,they were transferred to the liquid pre-medium of 0.3 M sucrose for 1 d,and then transferred to the liquid pre-medium of 0.6 M sucrose for another 2 d;Then they were transferred into Loading solution containing 0.6 M sucrose for 30 min loading,before an incubation in plant vitrification solution 3(PVS3)for 60 min.Then shoot tips were transferred onto aluminum foils and plunged into liquid nitrogen for 30 min of storage,prior to an immediate transferring to Unloading solution containing 0.8 M sucrose for 20-30 min for thawing.After an overnight post-thaw culture in the liquid premedium with 0.3 M sucrose,it was finally transferred to the regeneration medium for regrowth,and the regeneration rate was 23.33 %.Through the morphological and histological observation of the shoot tips after cryopreservation,it was found that only the materials with most of the cells in the apical meristem survived had the opportunity to regenerate normal plants.