Studies On In Vitro Conservation Of Chrysanthemum By Slow-Growth Method At Low Temperature

Chrysanthemum,native to China,is one of Chinese top ten flowers and one of the four cut flowers in the world.It has a variety of values such as viewing,eating,drinking,and medicinal use.The chrysanthemum germplasm resource was mainly preserved in the field,which need a lot of manpower and money.The method is susceptible to waterlogging,pests and diseases.The conventional tissue culture technique requires frequent subculture,easily leads to the dwarfing of the plantlets and the somaclonal variations.Therefore,it is trend to use cryogenic storages to preserve chrysanthemum germplasm in vitro.The purpose of this study is to explore the technique of chrysanthemum slow growth in vitro conservation at low temperature.In this study,170 chrysanthemum cultivars were randomly selected to explore the growth differences among cultivars under low temperature firstly.Then,4 different growth types of plantlets,‘Monalisa Yellow','Anastasia Orange','Xiao Yangju' and 'Nannong Chengpingpang' were selected as materials.By adding sucrose and mannitol in the medium,the most suitable preservation scheme for different chrysanthemum varieties were explored.The 'Monalisa Yellow' was selected as material to explore the effect of mannitol together with sucrose.During conservation,the growth and survival rate of plantlets were observed and counted,and after conservation,the recovery growth ability and genetic stability of the plantlets were detected.The main results were as follows:1.Analysis the difference of varieties at low temperature in vitro:170 chrysanthemum cultivars were conservated in MS for 6 months at(7±2)?.The height,number of leaves,number of green leaves and number of withered leaves were measured after 6 months.The number of green leaves and withered leaves number variation maglitude reached more than 50%,while the plant height and total leaves number variation were 27.6%,25.7%;The distribution of plant height and the number of withered leaves showed a non central partial distribution,and the number of leaves and green leaves were positively distributed.There was a significant positive correlation between plant height and the number of leaves,the number of leaves and green leaves,the number of leaves and withered leaves.In the cluster analysis of the leaves rate of plantlets,170 varieties can be grouped into 3 groups,the aging rate of first group was slow,including 6 varieties eg.'Nannong Chengpingpang',the aging rate of second group is moderate,including 101 varieties eg·'Nannong Shuangjiao',the aging rate of third group is fast,including 63 varieties eg· 'Monalisa Yellow'.2.Effects of mannitol and sucrose on the preservation of chrysanthemum at low temperature in vitro:Four chrysanthemum cultivars with different growth potential were conserved in vitro at(7±2)? in medium supplemented with(30,45,60,75 and 90 g·L-1)sucrose,and(0,5,10,15 and 20 g·L-1)mannitol.The survival rate,number of leaves and height were observed every 3 months.After 12 months of preservation,the tissue structure of the leaves and stems of the tube seedlings was observed,and the recovery growth and genetic stability of the plants were also detected.'Nannong Chengpingpang' and 'Xiao Yangju' could be conserved for 12 months in the MS medium supplemented with 15 g·L-1 mannitol,and the survival rates were 86.67%and 93.33%,respectively.But the highest survival rate of 80%and 93.33%after 12 months in vitro conservation was observed for both‘Monalisa Yellow' and 'Anastasia Orange' in the MS medium supplemented with 20 g·L-1 mannitol.It did not work well to supplement with high concentrations(45-90 g·L-1)of sucrose in MS medium for‘Monalisa Yellow' and‘Anastasia Orange';The survival rate of 'Nannong Cheng pingpang' and‘Xiao Yangju' was observed larger than 70%,but with less green leaves and in a poor growth state compared with mannitol treatments.After 12 months in vitro conservation,the cell density increased for the investigated four cultivars,after 12 months of preservation,the plantlets recovered well and grew well.The morphological indexes of plant height,leaf number,stem diameter,internode length and SSR markers were not different from control,which means maintaining genetic stability.3.Effects of mannitol combined with sucrose on the preservation of chrysanthemum at low temperature in vitro:Chrysanthemum 'Monalisa Yellow' was selected as material conserved at(7+2)? in vitro in medium supplemented with a combination of(0,15,30,45 and 60 g·L-1)sucrose and(0,5,10,15 and 20 g·L-1)mannitol.The survival rate,number of leaves and height were observed every 3 months.After 12 months of preservation,the tissue structure of the leaves and stems of the plantlets was observed and the recovery growth and genetic stability of the plants were detected.Results showed that a highest survival rate of 93.33%was observed for the combined treatment of 15 g·L-1 sucrose and 15 g.L-1 mannitol after 12 months in vitroy with a relatively low withered leaves rate of 17.6%;and the survival rate was 86.67%for the treatments with only with 5 g·L-1 mannitol.Under 30 g·L-1 sucrose,the survival rate increased with the increase of mannitol concentration,and a highest survival rate of 80%was observed in 20 g·L-1 mannitol after 12 months,with the withered leaves rate of 35.2%.When the sucrose concentration increased to more than 45 g·L-1,the in vitro chrysanthemum cannot be conserved well.After 12 months in vitro conservation,the cell density increased for the investigated four cultivars,but regenerated plantlets grew normally in terms of plant height,leaf number,stem diameter,and internode length,and SSR analysis revealed a similar fragment pattern.