Karyotype Analysis And Cultivation Evaluation Of Aneuploid Purple Coneflower(echinacea Purpurea L.)

Purple coneflower(Echinacea purpurea L.)is a perennial root herb belonging to the Asteraceae family and isa kind of medicinal plant native to North America.Being a plant,E.Purpureahas important medicinal and ornamental value,however,currently commercial scale cultivationof E.purpurea is still by the traditional way.In order to increase the yield and guarantee the quality of the products,the following experiments were carried out and some positive results have been obtained.1.The establishment of E.Purpurea Giemsa C-staining technique.Due to the high similarity in the morphology of some chromosomes in E.purpurea,it is difficult to distinguish the 11 pairs of chromosomes of E.purpurea on the basis of the common root tip cells squash method.In fact,the extreme similarity of these chromosomes in shape has become a major obstacle to aneuploid breeding and cytogenetics study of aneuploid for this plant species.In this study,the seedling of diploid E.purpurea was used as the starting material,the Giemsa C-banding technique was established through optimization of the experimental procedures.Based on the original chromosomes classification of E.purpurea,the chromosomes can be divided into 3 types by the application of this technology,which are centromeric bands,intercalary bands and telomeric bands.Centromeric bands andintercalary bands aremost distributed on both long and short arms,whereas intercalary bands are mainly distributed on the long arm of chromosomes.According to the karyotype of E.purpurea and the distribution characteristics of Giemsa C-banding,the individual characteristics of the chromosomes of E.purpurea were determined,and each chromosome could be distinguished.The establishment of this technology provides possibility for the identification of specific chromosomes in aneuploid E.purpurea.2.Aneuploid screening and karyotype determination.The ploidy of hybrid seeds obtained through cross between diploid and tetraploid was identified by conventional chromosome counting method,and five aneuploid lines with chromosome number of 31,32,34,less or more than triploid(2n = 33),were randomly selected for Giemsa-C banding and karyotypic analysis.By the above mentioned Giemsa C-staining technique,the karyotypes of these five lines were determined as: D12-1,2n = 32 = 11 m + 9sm + 12st;T2-2,2n = 32 = 10 m + 7sm + 15st;D12-14,2n = 31 = 11 m + 7sm + 13st;D12-13,2n = 31 = 9m +9sm+13st and D12-8,2n = 34 = 12 m + 11 sm + 11 st.According to karyotypic analysis and C-banding distribution,several abnormal chromosome structures were also identified in these 5 lines.3.Observation of chromosome behaviors in meiosis of cells with different ploidy states.Based on the comparison of chromosome behaviors in meiosis of diploid,triploid and three aneuploid lines with different chromosome constitutions,it was found that there was a high proportion of abnormal chromosome behaviors in meiosis for cells of triploid and aneuploid.Lagging chromosomes,fragment,chromosome bridges,multi-split and micronuclei were most frequently observed as meiotic abnormalities.Pollen viability test showed that,the dyeability rate of triploid D12-12(64%)was much higher than the three aneuploids(all less than 30%).Surprisingly,the pollen vitality of diploid was extremely low in this study;the dyeability rate was just 9%.This might reflect the low fertility genotype in the diploid population.In addition,triploid D12-12 with high pollen fertility could be used as an ideal donor line in anther culture.4.Anther culture of triploid and aneuploid.Aneuploidy microspores were usually formed in meiosis of triploid and aneuploid.In vitro anther culture of these microspores would be able to obtain aneuploid plants recovered by the development of these microspores.In this part,triploid D12-12 was used as the main experimental material.The optimal medium composition for callus induction was determined after several testing as 1/2MS + 0.5 mg/L BA + 0.5 mg/L TDZ + 0.2 mg/L NAA + 0.3 mg/L Ag+ + 100 mg/L inositol + 50 g/L sucrose + 4.5 g/L agar,and the optimal medium composition for callus regeneration as MS + 0.5 mg/L BA + 0.5 mg/L ZT + 0.05 mg/L NAA + 100 mg/L inositol + 30 g/L sucrose + 4.5 g/L agar.Karyotypic analysis and Giemsa C-banding of chromosomes of regenerated plants were performed.The karyotype of aneuploid D12-12 a was 2n = 34 = 12 m + 10 sm + 12 st.This karyotype suggested that the aneuploid D12-12a(2n = 34)line was either produced by spontaneous chromosome doubling of an aneuploid gamete with the karyotype of n = 17 = 6m + 5sm + 6st,or developed by a microspore derived from abnormal chromosome segregation caused by nuclear fusion and abnormal spindle filament during meiosis.The karyotype of another regenerated aneuploid D12-8a(2n = 35)line was 2n = 35 =12m + 10 sm + 13 st,it might be speculated as that this line was originated from the nuclear fusion of a microspore during meiosis.5.Observation of the mature plant phenotypes with different ploidy states.Field cultivation of diploid,triploid,tetraploid and a few aneuploid lines found that aneuploids with chromosome numbers close to hexaploidy had generally delayed phonological period,stunted plant growth,and poor adaptability to the cultivated environments,and some of these lines were difficult to survive.It was found that significant differences in vegetative growth period and florescence of different ploidy plants were evidently affected by karyotype.Aneuploid D12-1 and D12-8 had high ornamental value because they possessed ligulate flower in bright color,larger petals,short and strong plant type and long florescence.Histological observation showed that the numbers of stomata per unit leaf area decreased with the increase of ploidy,whereas the stomatal size and the numbers of chloroplast in guard cells increased with the increase of ploidy;the thickness of leaf,palisade tissue and spongy tissue,and the content of cytochrome in different ploidy leaves increased with the increase of ploidy.These results suggested that both genotype and karyotype had certain effect on cytochrome content.6.The determination and evaluation of the content of medicinal components in different ploidy plants.The contents of five kinds of medicinal components were determined,it was found that the content of cichoricry acid was the highest among these five kinds of medicinal components in all plants,and the contents of chlorogenic acid,1,3-dicaffeoylquinic acid,echinacoside and caffeic acid decreased successively,the content of echinacoside and caffeic acid were not detected for some lines.The karyotype,ploidy,genotype,the length of planting time,harvest time,and organ for determination had close relation to the contents of these five medicinal components.Based on the content of medicinal components in whole plant,two lines,D12-12 and T2-1 were screened.