Study On Somaclonal Variation Of Three Lily Species And Selection Of Heat-resistant Clones Of Lilium Regale

Lily,belonging to the genus Lilium,is becoming one of the most popular cut flowers.Lily likes cold humid climate,when the temperature is higher than 28℃,it will seriously affect the quality of cut flowers and cause the degradation of lily bulbs.So,lily production over summer in southern and northern areas has become one of the problems of producing perfect cut flowers.Given this,breeding heat-resistant lily varieties is the key factor to solve the problem.There has been only 8 cultivars from China among more than 5000 cultivars in International Registry of New Varieties,although our country is blessed with lily resources.Therefore,It is of profound significance to make full use of own resources for breeding heat-resistant cultivars grown well in China.So in this paper,firstly the law of somaclonal variation of Lilium concolor,L.regale and L.henryi were investigated systematically from three ways, including Morphology,cytology and molecular biology.Secondly,breeding heat-resistant cultivars for Lilium regale was carried out.The main results are as follows:(1) In the process of lily tissue culture,the genotype plays a decisive role in somaclonal variation rate:L.concolor is the highest(11.44%),L.regale higher(5.65%) and L.henryi the lowest(1.10%) according to the results of ISSR molecular marker.In the matter of explant selection,the somaclonal variation rate is as follows:leaf>scale>seed.Somaclonal variation rate determined by ISSR molecular marker of clones regenerated from leaf in L.concolor was 46.48%higher than that of clones regenerated from scales(32.74%) and seed(11.44%).In L.regale somaclonal variation rate of clones regenerated from leaf was 29.70%higher than that of clones regenerated from scales(21.20%) and seed(5.65%). Somaclonal variation rate of clones regenerated from leaf in L.henryi was 20.73%,higher than that of clones regenerated from scales(1.10%) and seed(14.11%).Different exogenous hormone had different effects on lilies.Addition of NAA,6-BA and IBA had no effects on somaclonal variation rate. But 2,4-D and TDZ can reduce genetic stability of L.concolor which increased 0.74～1.09 times in somaclonal variation and L.regale which increased 0.28~0.42 times respectively.There were less significant effets on L.henryi.Somaclonal variation rate of regenerated plants from callus of L.concolor, L.regale and L.henryi was 42.10%,11.98%and 8.56%which was higher than that of regenerated plants from adventitious buds.The somaclonal variation rate of L.concolor after 5 generation times(36.71%) as well as L.regale after 8 generation times(15.22%) reaches the peak respectively,while L.henryi was increasing slowly in the 2～8 subcultures and rose sharply to 4.88%after 15 subcultures.Therefore, when generic resources preservation required to maintain exactly generic information is the purpose,it is better to select seed as the explant,NAA,6-BA or IBA as the exogenous hormone,adventitious buds as the regeneration way,and bulbs diameter of Lconcolor and L.regale is more than while L.henryi less than 1 cm as the state of preservation.When mutation breeding required more mutants is the objective,it is better to select leaf as the explant,2,4-D or TDZ as the exogenous hormone,callus as the regeneration way,and bulbs diameter is less than while L.henryi more than 1 cm as the state of preservation.(2) Transplanted seedlings of L.regale were treated under high temperature.The results showed that heat-resistance index and chlorophyll content in leaf were decreased significantly under high temperature stress,while malondialdehyde content increased.SOD activity(5457.86 U/g),free proline content(16.84 mg/g) and soluble protein content(0.61 mg/g) in leaf of seedlings treated for 32 hours under 42℃reached the peak,with increasing 0.66,1.77 and 9.78 times respectively compared the control but 48 hours later,they declined.The results indicated that discontinuous stress under 42℃for 48 h had irreversible heat injury effects on seedlings.(3) The study applied 5 screening methods on clones of L.regale,including high temperature stress (one-step or multiple -step screening),chemical stress(one-step or multiple-step of HYP) and compounded stress(HYP with high temperature).Identification and analysis on seedlings screened by 5 above-mentioned methods were carried out.The survival rate of seedlings screened by high temperature stress was 26%,which was higher than that of HYP stress(17%) and compounded stress(23%).The heat-resistant index of seedlings screened by high temperature stress was 0.17,which was higher than that of HYP stress(0.055) and compounded stress(0.07).During high temperature environment,the SOD activities of screened seedlings increased to a large extent with free proline and soluble protein content higher than the control.All in all,30 mutants were obtained from 5000 in vitro plants.Thus, with the respect of difficulty and efficiency in experiment,it is better to choose adventitious buds as regeneration ways,multiple-step high temperature under 37℃or 40℃as the screening methods,so that more genus mutants of L.regale can be obtained.This paper provides theoretical basis for genetic resources preservation of lilies as well as lays a foundation for mutant breeding.