火龙果多倍体诱导体系的优化

【目的】探讨适宜的火龙果多倍体诱导技术体系,以期解决火龙果品种单一、遗传相对狭窄的问题。【方法】以2个主栽的火龙果品种‘大红’和‘白水晶’为材料,通过秋水仙素浸渍法和混培法进行多倍体的诱导,比较不同秋水仙素浓度和处理时间的组合后代变异率和死亡率。【结果】随着秋水仙素浓度升高和处理时间的延长,诱导率和死亡率增加;5 g·L^-1秋水仙素浸渍萌动种子24~48 h和50 mg·L^-1秋水仙素在培养基中与无菌苗共培养3~5 d多倍体诱导效率较高且死亡率适中,混培法30 d表型变异率高于浸渍法,死亡率低于浸渍法。变异株气孔密度和气孔长度分别为对照的81%和115%,变异株染色体数目包括2n=2X=22,2n=4X=44,2n=6X=66等几种不同类型,此外还存在非整倍体。流式细胞仪检测结果发现:5 g·L^-1秋水仙素浸渍萌动种子24 h多倍体和同源四倍体诱导效率最高;50 mg·L^-1秋水仙素在培养基中与无菌苗共培养5 d多倍体和同源四倍体诱导效率最高,混培法多倍体和同源四倍体诱导效率高于浸渍法。【结论】50 mg·L^-1秋水仙素与无菌苗混培养5 d能提高火龙果多倍体诱导效率,火龙果混培法多倍体和同源四倍体诱导效率高于浸渍法,无菌苗与50 mg·L^-1秋水仙素混培5 d是火龙果多倍体诱导的最佳组合,本实验为火龙果多倍体新种质创建奠定了技术基础。

Optimization of polyploidy induction system in dragon fruit

【Objective】Dragon fruit(pitaya,pitahaya)is a new and highly commercial tropical fruit species in China.However,the less variety diversity,self-incompatibility and small fruit size have limited the development of dragon fruit industry.Polyploid breeding,an effective method for fruit crop breeding,has been successfully applied in a lot of plants but little has been done about Dragon fruit.In the experiment,the dragon fruit polyploid induction system was optimized,which would be helpful to enlarging the genetic background and innovating new polyploidy germplasm.【Methods】Two dragon fruit cultivars‘Dahong’(2n=2X=22)and‘Baishuijing’(2n=2X=22)were used as the materials.Seeds and plantlets were soaked in colchicine or co-cultivated with colchicine.Germinated seeds were soaked in different concentrations of colchicines(2.5、5、10 g·L^-1)for different times(12,24,36 h).In addition,aseptic seedlings were co-cultivated with different concentrations of colchicines(25,50,100 mg·L^-1)for different times(3,5,7 d).Then germinated seeds and aseptic seedlings were washed with clean water after treated with colchicine,and the mutation rate and death rate after 7 and 30 days were evaluated and statistically analyzed.Stomatal density,length and width of guard cells,chromosomes counting and Ploidy Analyser were used to identified the ploidy of the variants.【Results】The variation of dragon fruit plants showed the following features that longer and wider leaves and wider hypocotyledonary axis appeared,and the mutation and death rates increased with increase of colchicine concentrations and treatment time.The germinated seeds soaked in 5 g·L^-1 colchicine for 24-48 h had better induction effect,and the mutation and death rates after 7 d were 13.83%-27.75%and 3.53%-29.28%,respectively,while the mutation and death rates after 30 d were 11.87%-15.08%and 37.56%-52.42%,respectively.Lower and higher concentrations of colchicines were less effective.The hypocotyl enlargement rates at lower concentration of colchicine(2.5 g·L^-1)were much lower(3.12%-22.83%),and the mutation rate after 30 d was lower than 5%.The higher concentration of colchicine(10 g·L^-1)had higher hypocotyl enlargement rate,but the death rate was very high,and the mutation rates after 30 d were only 3.16%-7.10%.Aseptic seedling co-cultured with 50 mg·L^-1 colchicine for 3-5 d had better induction effect.The mutation and death rates after 7 d were 26.17%-44.97%and 10.06%-14.15%,respectively,while the mutation and death rates after 30 d were 20.36%-26.32%and 22.51%-45.50%,respectively.The lower or higher concentrations of colchicine were less effective.Lower concentration of colchicine(25 mg·L^-1)had lower hypocotyl enlargement rate(7.73%-34.44%)and mutation rate after 30 d(3.66%-6.59%),and higher concentration of colchicines(100 mg·L^-1)had higher hypocotyl enlargement rate(30.47%-67.55%),but the death rates after 7 d(17.16%-26.56%)and 30 d(31.20%-66.88%)were very high,and the mutation rates after 30 d were very low(8.13%-17.46%).The stomatal density decreased,and the guard cells became larger in variation plants.The stomatal density and size were 81%and 115%of the control group respectively.Ploidy analyser was used to identify the ploidy levels of the variants,which indicated that more than half of the variants were diploid,the polyploid/variation rates of variants induced by seed-soaked method were 0.00%-47.00%,and the Polyploid/Variation rates of variants induced by co-cultured method were 3.85%-37.31%.Results from chromosome counting showed the chromosome numbers of variants were unstable(2n=44,56,66),while that of diploids was 2n=22.The results with Ploidy analyser showed the variants contained tetraploids(2n=44),hexaploid(2n=66)and mixoploid or chimera(2n=22,2n=44).Autotetraploid/polyploidy rates in co-cultured trial(0.00%-28.57%)were higher than seed-soaked trial(0.00%-28.57%).The germinated seeds soaked in 5 g·L^-1 colchicine for 24 h had better induction effect,and the polyploidy and homolog tetraploid induction efficiency were 4.04%-5.51%and 1.15%-1.38%,respectively.Aseptic seedling co-cultured with 50 mg·L^-1 colchicine for 5 d had better induction effect,the polyploidy and homolog tetraploid induction efficiency were 8.25%-8.68%and 1.92%-2.43%,respectively.And the polyploidy and homolog tetraploid induction efficiency with co-culture method were higher than soaked method.【Conclusion】Dragon fruit aseptic seedling co-cultured with 50 mg·L^-1 colchicine for 5 d was the best combination for tetraploid induction,which had higher mutation and tetraploid rates.Co-culture method was more suitable for dragon fruit polyploidy induction than seed-soaked method,but the tissue culture technique was needed.Although the induce efficiency of seed-soaked method was lower,but it may be a better choice for the breeder without tissue culture condition.Ploidy Analyser is a simple and rapid method to identify the ploidy levels of variants,especially to distinguish tetraploid,mixoploid and chimera.This result will be helpful to germplasm innovation and breeding of dragon fruits.