Studies On The Conservation Methods Of 'Hongxiangyu' Taro(Colocasia Esculenta(L.)Schott) In Vitro

Taro?Colocasia esculenia?L.?Schott?is a typical vegetative propagation plant with underground corms as products and propagation organs.The corms of taro are rich in nutrients,with a higher content of starch and other carbohydrates.Taro is not only an important vegetable and food crop,but also has a high medical value.China is one of the original countries of taros with abundant taro germplasm resources and long plantation history.However,the long-term vegetative propagation,diseases and insect pests easily result in degeneration of taro good species,causing a serious loss in the production and quantity of taros.Thus,the conservation of taro fine varieties is especially important for biodiversity conservation,as well as quality and efficient production.Compared with conservation in field,preservation techniques in vitro of germplasm resources have more advantages.In this study,Jianchang 'hongxiangyu',the fine local variety of Jiangsu province,was used as the material.The main researches were as follows;1)the effect of ABA,CCC,mannitol and sorbitol on subculture preservation time of taro seedlings was studied;2)taro shoot-tips were used as the material to study the effect of pre-culture,loading and dehydration,thawing process on the cryopreservation by vitrification;3)effect of sucrose,KNO₃,SA on the formation of taro in vitro corms was studied;4)we also studied the changes of the major carbohydrate content and related enzyme activities during the expanding process of corms in vitro.The main results are as follows:1.Four kinds of chemicals could increase the subculture time to varing degrees:CCC had the best effect on conservation of taro plantlets and the second best is ABA.The survival rates kept over 72%of cases after cultured for 150 days with 0,5-8 mg·L^-1 CCC.When ABA concentration was 2.5 mg·L^-1,52%shoots survived after cultured for 150 days.Mannitol and sorbitol did poorly with mortality increasing continuously with time.After being preserved for 90 days through these methods,taro plantlets were transferred to normal medium and they could recover growth,and had no significant differences with normal plantlets in terms of morphological and physiological indexes?such as the content of chlorophyll and MDA?.2.Taro shoot tips could be cryopreserved by vitrification,the results indicated that taro shoot tips got the highest survival rate of 55.56%,when precultured on MS medium with 0.6 mol·L^-1 sucrose for 3 days;After being loaded by 60%PVS2 for 30 min and dehydrated by PVS2 for 15 min,taro shoot tip had the lowest water content?57.23%?,but the survival rate was the highest?65.08%?;Defrosting shoot tip with 1 min or 2 min has no significant effect on survival rate under the same temperature,and 40? was the suitablethaw temperature than 30?,50? and 60?.After being cryopreserved by vitrification,taro shoot tips had a regeneration rate of 39.68%in normal regeneration culture.And there were no remarkable differences between the regenerated plantlets and the normally cultured plantlets.In conclusion,a suitable system for cryopreservation by shoot tip vitrification of taro was built.The procedure was as follows:shoot tips of taro were firstly preconditioned on MS with 0.6 mol·L^-1 sucrose for three days,then loaded with 60%PVS2 for 30 min and dehydrated with PVS2 for 15 min before immersion into liquid nitrogen,subsequently thawed in 40? for 1 or 2 min after cryopreservation,washed shoot tips by MS liquid medium with 1.2 mol·L^-1 sucrose and finally cultured shoot tips in regeneration culture medium to obtain normal taro plantlets.3.Different chemical agents were added into medium to induce taro microcorms.The results showed that suitable concentrations of sucrose,KNO₃ and SA could induce taro corms in vitro.60 g·L^-1 sucrose treatment was the suitable concentration and the induction rate was 89.3%with a bigger and earlier corms formation.When KNO₃ was 50 mmol·L^-1,the induction reached 94.6%and the fresh weight of corm was 0.80 g on average.The induction frequency was over 80%by 0.1-0.5 mmol·L^-1SA,but the quality of microcorms was weaker than sucrose and KNO₃.4.Taro shoots in vitro began to expand by high glucose(60 g·L^-1)induction after 20 days.Being cultured for 50 days,most plant leaves and petioles withered,and the microcorms formed.During the development of corms,fructose,glucose and soluble sugar content presented a trend of increase firstly and then decrease.The fructose content of corms reached the maximum after 27 days of induction,and the highest content of total soluble sugar and glucose appeared on the 34th day.The sucrose content rose at first,went down latter and then rose again.When cultured for 48 days,sucrose accumulation reached the highest.Total starch content,amylose and amylopectin content increased with the incubation time.The total amount of starch,mainly amylopectin,accounted for about 76%of dry weight.Both of ADPGPase and Q-enzyme activity reached the maximum on the 41st day(1.22 ?mol·g^-1·min^-1 and 2.39 ?mol·g^-1·min^-1,respectively).By correlation analysis between ADPGPase activity and total starch content,as well as Q-activity and amylopectin content,from shoot beginning to expand?20 d?to ADPGPase and Q-enzyme activity reaching the peak?41 d?,the correlation coefficient were 0.819?P<0.01,n=12?and 0.738?P<0.01,n=12?,showing a significant positive correlation.