Karyotype Analysis Of Xinhai 33 Malformed Plantlets Of Transgenic Island Cotton

The occurrence of malformed seedlings is a common physiological abnormal phenomenon in plant tissue culture,which severely restricts the development of plant tissue culture technology.In the somatic embryo system ofsea island cotton,long-term cell culture often causes chromosome aberrations,which affects the acquisition of subsequent normal plants,especially transgenic plants.Whether the malformation seedling is a result of gene mutation or chromosomal aberration is one of the key issues that determine the direction of our follow-up research.In order to better detect whether the chromosomes are distorted,we have optimized the karyotype analysis system of chromosomes ofsea island cotton from the aspects of material selection,pretreatment conditions,hydrolysis conditions,and dyeing time.Based on this analysis,the karyotypes of wild-type and the malformated regenerated cotton Xinhai 33 were analyzed.The results showed that there were indeed some differences in chromosome morphology between the two type cotton.Flow cytometry results further confirmed the difference genome between the regenerated cotton Xinhai 33and the wild-type.And the results of SSR molecular markers showed that the ploidy and sequence composition of the regenerated cotton Xinhai 33 genome are different from that of the wild type plants.This study elucidates the molecular mechanism of malformed seedlings,which is helpful for the screening and breeding of normal plant in the somatic embryo system.The main results are as follows:The production effects of root tips,embryonic callus and young leaves were different under the same method.The apical meristem is the most vigorous mitosis,and the cell wall is the thinnest.Therefore,the metaphase division phase is the most,the most clear,the best preparation effect,and it is the best material to observe chromosome and count.Embryonic cell volume of a body is small,so to be able to see under the condition of the oil mirror image,the tender leaves because of cells connected than follow up more closely,not equal hydrolysis under the conditions of processing for single cell,the status of the nucleus of chromosomes.Therefore,apical cells are the optimal material for subsequent nucleotype analysis.2.Select 0.02%colchicine solution,p-dichlorobenzene saturated solution,0.002 mol/L,8 hydroxy quinoline solution under 4 ℃,the pretreatment,the time needed is 4 h.Among them,0.02%colchicine solution is the best pretreatment agent,and the chromosome morphology after treatment is the best,which is conducive to further analysis.Therefore,0.02%colchicine was selected as the best pretreatment for subsequent nuclear type analysis.3.With preheating 60℃ respectively 1 mol/L HCL treatment for 10 min,15 s,snail enzyme treated with concentrated hydrochloric acid,mixed enzyme(1%pectinase+1%cellulase)was conducted at room temperature for 3 h.Preheat 60 ℃ 1 mol/L hydrochloric acid hydrolysis efficiency highest,tissue is mud after dissociation,easy access to unicellular,metaphase chromosomes can be observed.Therefore,we select preheating 60℃ 1 mol/L HCL as subsequent hydrolysis conditions of karyotype analysis.4.The phenol fuchsia solution was stained for 1min,5min,10min and 15min respectively.When the staining time is 10 min,the cytoplasm is pale or even colorless,and the nucleus and chromosome are deeply colored,so the metaphase division of chromosomes can be clearly seen.Therefore,10 min was selected as the dyeing condition for subsequent karyotype analysis.5.On the basis of optimizing the karyotype analysis system of island cotton chromosome,the karyotype of island cotton wild type and transgenic abnormal seedling cells were analyzed respectively.The chromosomes of wild type and transgenic type island cottonXinhai 33 were tetraploid,with a base of 13,and the nuclear type were all type 4B.Both the wild-type chromosome and the transgenic chromosome are neutral chromosome,and the centromere chromosome is the main one,and there is no telomere chromosome.The nuclear type formula is 2n=4x=52=20m+20sm+12st and 2n=4x=52=32m+16sm+4st(1 SAT)respectively.The components are 4L+8M+1S and 3L+8M+2S respectively,all of which belong to the evolutionary type.6.In order to further testing of wild type and transgenic type of sea island cotton cells whether genomic DNA changes,we use flow cytometry instrument technology respectively to the wild type and transgenic type of sea island cotton plants both fresh and young leaf cells are analyzed.DNA content in wild type sea island cotton cells mostly concentrated in the 100 orders of magnitude,and genetically modified DNA content of sea island cotton cells mostly focused on the 120 orders of magnitude,slightly greater than 100,the two cellular DNA content there is a difference.The results of flow cytometry further confirmed the difference between transgenic abnormal seedlings and wild-type genomic duplication.7.In order to verify the abnormal seedlings on the genome sequence information of whether there is a change in sequence,the extraction of wild type and somatic embryogenesis system of sea island cotton plants from genomic DNA of SSR analysis.The extracted DNA was diluted to 10-100ng/hairy L for subsequent PCR amplification,so as to facilitate the subsequent test to proceed smoothly.Each SSR primer was repeated 3 times by sample PCR,and the results were consistent,indicating that the experimental results were credible.Compared with wild type,somatic embryogenesis system of the new DNA bands of Xinhai 33 and the molecular weight size also exists certain difference,the number of somatic embryogenesis system new sea genotypes of Xinhai 33 changed.The results of SSR molecular marker identification showed that the sequence composition of regenerated island cottonXinhai 33 changed compared with that of wild type plants.The results of this study elucidate the mollecular mechanism of abnormal seedlings,which is helpful for the selection and cultivation of plants with normal somatic embryo system.