Studies On Tissue Culture And Morphogenetic Cytology Of Elaeagnus Mollis

Elaeagnus mollis, is an endangered perennial arboreal or shrubby plant and a national second-class key protected rare species in China. Its leaves and seeds contain abundant vitamin E, vitaminC, fatty acid, protein and microelement. In addition, its roots are symbiotic with azotobacter, which can increase nitrogen content in soil. However, E. mollis is very difficult to reproduce because its seeds not only germinate difficultly but also have a low livability. The tissue culture in vitro provide a feasible way for its reproduction. Several studies in vitro culture of E.mollis had been achieved but there had many problems need to be solved. In this paper, three plant regeneration types were compared, and the cytology of organogenesis type was studied as well. The main results were as follows:1.Explant surface sterilization methods.The explants were washed using sterile water three times for 1 min, each time 20s, and then 70% ethanol three times for 30s, each time 10s, and sterile water three times, and 0.1% HgCl₂ three times for 9 min, each 3.min,. and sterile water three times in turn. This kind of sterilization method could decrease the explant infection rate to 10%.2.Plant regeneration in organogenesis typeThe effects of explant type, inoculation method, temperature and plant hormone combinentions on callus induction of E. mollis were investigated. The aseptically germinated seedings which had a lowest infection and nigrescence and was easy to obtain, was the best explant resource. On five kind of explant from aseptically germinated seedings, the hypocotyl explants gave a highest callus induction rate of 90.00% in which only 7.50% was browning, and the callus from hypocotyl was green and loose.The explant cutting methods as well as inoculation methods had effects on callus induction. The callus induction rate achieved 75.00% when stem was cut transversely and layed on the medium then inoculate upright 56.25%. The callus induction rate achieved 80.00% when leaf was cut vertically with the nervation then cut parallel 25.00%. The callus induction rate achieved 92.50% when cotyledon was cut transversely and layed with the back airward, then layed with the cut face airward 75.00%.The optimal temperature scope was from 24℃to 27℃for callus induction. The best medium for callus induction was 1/2 MS + 2.0 mg/L 6-BA + 0.2 mg/L 2, 4-D + 0.2 mg/L NAA + 30 g/L glucose + 7.0 g/L agar with the best medium for shoot differentiation being 1/2 MS + 0.5 mg/L 6-BA + 0.02 mg/L 2, 4-D + 30g/L sucrose + 7.0 g/L agar.3 Plant regeneration in organ typeThe best explant resource was the aseptically germinated seedings as well for organ type. Both of the hypocotyl and cotyledon could be used for induction of multiple buds directly. The best medium for buds formation from explant was 1/2 MS + 0.5 mg/L 6-BA + 0.02 mg/L 2, 4-D + 30 g/L sucrose + 7.0g/L agar. The best medium for buds multiplication was 1/2 MS + 2.0 mg/L 6-BA + 0.02 mg/L 2, 4-D. The best medium for strong sprout was 1/2 MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 30 g/L sucrose + 7.0 g/L agar.4. Plant regeneration in leafbud-differentiation typeThe best medium for axillary buds germination from stem explant was 1/2 MS + 2.0 mg/L 6-BA + 0.02 mg/L 2, 4-D and the best medium for buds growth was 1/2 MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 30 g/L sucrose + 7.0 g/L agar. The buds grew faster when inoculated levely, but multiple faster when inoculated upright.5. Root formationThe best medium for buds rooting was 1/2 MS + 0.5 mg/L IBA + 0.1 mg/L AC + 7.0 mg/L agar + 20 mg/L sucrose and a dark culture of 8 days was required.5. Cytology of plant regeneration in organogenesis typeAfte three days inoculated the hypocotyl callus originated from cambium of vascular bundle, phloem parenchyma and cortex parenchyma and the buds were differentiated from callus originated from cortex parenchyma. As well, after 4 days, the cotyledon callus originated from cortex parenchyma and then the buds formed. The bud formation from both cortex parenchyma were externally originated.