A Set Of Primary Trisomics Obtained And Its Characteristics Studies In Chinese Cabbage

In Chinese cabbage,development of trisomics and monosomics provides a useful cytogenetic tool to associate genes and molecular markers with their respective chromosomes.It is very difficult to obtain trisomics and monosomics using triploid,because the crossed of diploid and tetraploid Chinese cabbage could not obtain triploid.In the present study we applied the isolated microspore culture of autotetraploid Chinese cabbage to identify trisomics and monosomics,explore the cytological basis of this approach.A complete set of trisomics and some monosomics were identified,and their gamete transmission rate was observed.The tissue cytological cause differentiated gamete transmission rates among various aneuploids were further discussed.Fluorescene in Situ Hybridization(FISH) technology was used to detect the positions of 25S rDNA and 5S rDNA on chromosome of Chinese cabbage diploid and trisomics.Furthermore,the monosomic,disomic and trisomic of satellite chromosome were used to examine the effect of addition and reduction of chromosome on photosynthesis.The research results are listed as follows:1 Plant Regeneration from Isolated Microspore Culture of Autotetraploid Chinese Cabbage(Brassica campestris ssp.pekinensis)Autotetraploid Chinese cabbage was used to study microspores culture response and plant regeneration.The results showed that the late uninucleate stage was the best period to induce embryos.Embryogenesis was related to different genotypes,of which 9405 line was the highest yield with 81 embryos per bud.Medium NLN supplemented with 15%sucrose,BA 0.05-0.2mg/L and NAA 0-0.5mg/L induced high frequency embryogenesis,which is suggested a basic medium for microspore culture.And addition of 0.05-0.1g/L activated charcoal to microspore suspension had significant effect upon the yield and developmental uniform of embryos and percentage of cotyledary embryos.Furthermore,the optimal response was obtained at 33℃for 24h at initial culture period,which the frequency of embryogenesis was the highest. Cotyledary embryos had a higher rate of plant regeneration,reaching 62%-65%when embryos germination was conducted on B₅ or MS medium with 1.2%agar.2 Cytological Research on Trisomics and Monosomies Obtained by Microspore Culture in Autotetraploid Chinese CabbageThe observation of male gametophyte formation indicated that the chromosome pairing in PMC forms at diakinesis wereâ… ,â…¡,â…¢,â…£.The irregular separation ofâ… ,â…¢,â…£, straggling chromosomes at metaphaseâ… ,resulted in the chromosome number of PMC at anaphaseâ…¡were varied from 15-24,the type with 20 chromosomes was the most,and then they were 19 and 21 chromosomes.The plant regeneration rate of microspore(2x+1) with 21 chromosomes was similar to that of the normal microspore(2x) with 20 chromosomes. The irregular behaviour of chromosome and developmental rate of trisomics were different among the strains.Seventy nine aneuploid plants with one extra chromosome(2x+1),7 with two extra chromosome(2x+2) and 9 with loss one chromosome(2x-1) were obtained from the isolated microspore cultured plantlets of the tetraploid Chinese cabbage line 9405. Through karyotype analysis of the root tip metaphase chromosome,meiosis observation and plant characters investigation,a complete set of primary trisomics(Tri-1,Tri-2,Tri-3, Tri-4,Tri-5,Tri-6,Tri-7,Tri-8,Tri-9 and Tri-10),two double trisomics(Double tri-3,5 and Double tri-3,6) and four monosomics(Mono-3,Mono-5,Mono-6,Mono-10) were identified, each of which had some unique characters on plant morphological traits.Thus,the isolated microspore culture of tetraploid is a convenient and effective way to select trisomics in Chinese cabbage.3 Gamete Formation and Transmission Rate of a Set of Primary Trisomics and Several Monosomies in Chinese CabbageA set of primary trisomics and several monosomes in Chinese cabbage was used as parents to make reciprocal crosses with diploid,the seeding set and the male and female gamete transmission rate were investigated,and chromosome behaviours of each trisomic and monosomic during meiotic divisions,gamete development structure,pollination, fertilization and embryo development were systematically studied.These results indicated that the seeding set of four monosomes were zero without transmissibility,n+1 gametes of the Chinese cabbage trisomics transmitted through female and male parents,except for Tri-10,with different transmission rate among various trisomics.The main influencing factors of n+1 or n-1 male gamete transmission rate were the trivalent formation and abnormal chromosome segregation at anaphaseâ… andâ…¡during meiotic divisions of each trisomics and monosome.The observation of male gamete development tissue anatomy showed:male gamete development of Tri-2,Tri-5,Tri-7 and Tri-9 was similar to diploid, whereas abnormal tapetum cells were commonly found in Mono-3,Mono-5,Mono-10, Tri-4 and Tri-10 with delayed degeneration of the tapetum cells.It was the main factor influenced the transmission rate of these aneuploid,thereafter microspores were serious abortion generally,and finally produced vast abortive pollen.Additionally abnormal vascular tissue of the anther and the anther chambers unified also caused pollen abortion. Embryological observation results showed that the embryo sac abortion was main factor that led to the Tri-10 seeding set with zero and female gametes without transmission rate. However,the low transmission rate of Tri-6 through n+1 female gametes was due to the low fertilization rate of the ovule,and the main factor of the low transmission rate of Tri-3 and Tri-9 through n+1 female gametes was young embryo abortion.The experimental results described above were a valuable reference for the rarity material propagation and preservation and for carrying out gene orientation and molecular and cytogenetical study using aneuploid of Chinese cabbage.4 FISH Mapping of 25S rDNA and 5S rDNA in Chromosome of Chinese Cabbage Diploid and TrisomicsIn order to clarify the number and location of rDNA sites in Chinese cabbage genome, and identify special molecular markers related to different chromosomes,fluorescene in situ hybridization(FISH) technique system was established.25S rDNA and 5S rDNA were further mapped in the mitotic metaphase chromosomes of Chinese cabbage diploid and different trisomics based on this method.The results were showed that:(1) With the study of correlative parameters during FISH,the technique system on Chinese cabbage was established,including chromosome preparation,chromosome pre-treatment,probe hybridization,washing and signal surveying.(2) The hybridization signals of 25S rDNA were observed in metaphase chromosomes of Chinese cabbage diploid and trisomics (except for Tri-3),which were located on the long arms of chromosomes1-4 and the satellite of chromosome 10.But the hybridization signal position was different in the same chromosome of each trisomics,and the hybridization signal intension was various in different chromosomes,the weakest signal appeared on chromosome 1 and the strongest signal appeared on the satellite of chromosome 10.(3) The hybridization signals of 5S rDNA were observed in the metaphase chromosomes of Chinese cabbage diploid and three trisomics,in which they were located on the long arms of chromosome 2 and the short arms of chromosome 9 and 10.The number of hybridization signals was 6 for diploid,7 for Tri-2, 6 for Tri-3,and 7 for Tri-9 respectively.This study provides accurate identified marks for chromosome 1-4,9 and 10 of Chinese cabbage by two rDNA-FISH location.5 Photosynthetic characteristics of satellite chromosome monosome,trisome and diploid in Chinese cabbageIn order to research the effect of addition or reduction of satellite chromosome on the photosynthetic characteristics of Chinese cabbage,the satellite chromosome monosome, diploid and trisome of Chinese cabbage were used to examine the photosynthetic rate(Pn), diurnal variation and chlorophyll content,Hill reaction activity,Rubisco activity so on.The result showed that the addition or reduction of sat-chromosome had a negative effect on the photosynthetic rate(Pn),when PAR was over 900μmol·m^-2.s^-1.The curves of diurnal change of Pn of trisome and diploid were quite similar,displaying typical two peaks and conspicuous "noon sleep".The addition or reduction of sat-chromosome had a positive effect on chlorophyll content and a negative effect on chla/b and boosted the degree of thylakoid stacking.The decline of Hill reaction activity and Rubisco activity were direct factors resulting in low photosynthetic rate of sat-monosome and sat-trisome.These results could offer valuable information to realize the function of sat-chromosome of Chinese cabbage and high-photosynthetic efficiency breeding using the chromosome engineering.