Research On Spore Germination Culture And Cryopreservation Of Several Liverworts

Liverworts, an important component of biodiversity, are the early of land plants. Due to the high dependency of habitats, changes and deterioration of environment can pose a serious threat to the survival of most liverworts. In order to protect the biodiversity of liverworts effectively, germplasm preservation studies mainly focusing on gametophyte stem apex, gemmae and protonemata of liverworts have been actively carried out based on the tissue culture worldwide, but spore preservation of liverworts has not been known. Domestic researches of germplasm resources cryopreservation of bryophytes are just in an early-stage, except for the gemma cryopreservation of Marchantia polymorpha and the spore preservation on several mosses. There is no liquid nitrogen preservation study on spores, protonema and gametophytes of liverworts at present. This work conducted spore germination culture and groped the sterilization conditions of spores and optimum preservation of spores and gametophytes of four species of liverworts. A study was also proceeded on drought resistance of five species of liverworts for suitable materials of cryopreservation. The results are as follows:1. Capsules (unruptured) from four species of liverworts(Acrolejeunea sandvicensis (Gottsche) Steph., Haplomitrium mnioides (Lindb.) R. M. Schust., Dumortiera hirsuta (Sw.) Nees, Solenostoma truncatum (Nees) R. M. Schust.) were sterilized by 75% alcohol (2 s) and 75% sodium hypochlorite solution (1 min) and then were cultivated in modified Knop Medium. Three aseptic liverworts were obtained within 50 days, except H. mnioides, which cost 356 days; Using the dosage of 1.5 mL 0.05% NaCIO solution to sterilize the spores was the best scheme on liverwort capsules (ruptured) and this scheme was successfully applied in A. sandvicensis spore disinfected.2. Before cryopreservation, the spores of A. sandvicensis, D. hirsuta and S. truncatum were treated in different drying time (0 h and 2 h,4 h,8 h) and temperature (15℃,-4℃ and -20℃). Apply the optimal processing conditions for A. sandvicensis, S. truncatum in the different time (1 d,50 d,100 d,150 d) of cryopreservation. Results were that:(1) The best pretreatmeat for D. hirsuta is:dry spores for 4 h, keep them in -20℃ condition for 1 day. The germination rate is 53.23%; The best pretreatmeat for A. sandvicensis is:dry spores for 4 h, keep them in 4℃ condition for 1 day. The germination rate is 62.36%; The best pretreatmeat for S. truncatum is:dry spores for 2 h, let them stay in -20℃ condition for 1 day. The germination rate is as high as 74.09%; (2) when kept in liquid nitrogen for 1 d, the spore germination of A. sandvicensis and S. truncatum reached the maximum rate, at 62.24% and 74.90% respectively. As the preservation time was prolonged to 50 d,100 d and 150 d, germination rate fell down. No significant difference existed (P< 0.05) among the rates of the three time points. Before and after liquid nitrogen preservation, no significant differences were found in morphology.3. The cryopreservation of gametophyte stem apex on H. mnioides and D. hirsuta was tested by encapsulation-vitrification from dehydration time (0 h,0.5 h,1 h and 2 h) and thaw temperature (4℃ and 40℃ and 42℃). The results showed that: The optimum treatment of the beads of two species is:dehydrate the beads for 1 h, and after 1 day’s liquid nitrogen preservation, thaw them at the temperature of 40℃ under rapid water bath. The bead regeneration rate is the highest, at 32.03% and 29.03 % respectively. For the time of differentiating mature gametophytes, the liquid nitrogen preservation of embedding beads is obviously more slowly than control group (preserved in room). The differentiating time rose by 71 d and 58 d respectively. The morphology of the regeneration gametophyte and the normal liverwort was almost the same.4. To explore the drought resistance of liverworts, three leafy liverworts (Plican--thus hirtellus (F.Weber) R.M.Schust., Plagiochila elegans Mitt. and H. mnioides) and two simple thalloid liverworts (Asterella cruciata (Steph.) Horik. and Pallavicinia ambigua (Mitt.) Steph.) collected in field were chosen and treated by 15%PEG-10000 simulating drought stress environment. The measurement of superoxide dismutase (SOD), chlorophyll content, peroxidase (POD), proline (Pro), soluble protein and malondialdehyde (MDA) was determined every 10 hours (0h,10 h 20 h,30 h 40 h, 50h). According to the results, the total chlorophyll content showed a trend of declining, the content of MDA and Pro showed the increased trend. Except the P. ambigua, whose SOD activity presented dropping trend, the figure for SOD activity in the other species showed an increase at first and then a remarkable decrease. POD activity appeared a rising trend in three leafy species, while it declined in two thalloid liverworts. According to the combined analysis of the habitat they live, morphology and the physiological changes under dry stress, leafy liverworts body of liverwort show better ability than the two thalloid liverworts. P. elegans has the strongest tolerance to drought, while H. mnioides was the weakest. A. cruciata seemed to possess better dry resistance than P. ambigua. Compared with thalloid liverworts, leafy liverworts may need to extend dry processing time before they are cryopreservated in the future.