The Study On Response Of Cytoplasmic Male Sterility To Nucleus Background In Brassica Oleracea L. Var. Capitata L.

Cabbage (Brassica oleracea L. var. capitata L.) is an important leafy vegetable crop with widely growing area only second to Chinese cabbage in China. Cabbage is typical allogamy plants and has obvious heterosis. Utilization of male sterile lines is becoming the trends in cabbage heterosis breeding. But cytoplasmic male sterility has not yet been found in cabbages. In application, cabbage CMS lines are always obtained by transmitting foreign sterile cytoplasm to cabbage. Studying on the response of cabbage cytoplasmic male sterility to nucleus background is significant to improve the mechanism of cytoplasmic male sterile and make it more convenient to determined the direction of breeding as well as reduce the breeding workload.Plant materials used in the parent study involved many alloplasmic lines which developed by hybridization between introduced CMS lines and different inbred lines, and then successive backcross with those inbred lines as male parents. The CMS types of introduced CMS lines were identified by amplified special cytoplasmic male sterile associated gene, then morphology response of the flowers to nuclear background was observed, after that, anther abortion stages and character were studied by the means of paraffin section method to detect the cytomorphology response of anther to nuclear background in alloplasmic lines developed from CMS lines 78,79,99 backcross with their maintainers. The expression of mitochondrial genes and flower morphogenesis-related genes were analysis using semi-quantitative RT-PCR in different developmental stages of bud. Mitochondrial structure was analyzed by restriction enzyme in and between the three group of CMS lines which with same sources of cytoplasmic and different nuclear background. Isozymes of POD, ATPase, were also analyzed in those materials. The main results are as follows:(1) Cytoplasmic male sterility materials in Brassica oleracea were lake of classification and identification at present. The application of every type of cytoplasmic male sterility material was also not so clear. Fifteen cytoplasmic male sterile materials introduced from home and abroad in Brassica oleracea was identified in this paper. Ogu CMS take up the most part in the materials investigated. Fourteen materials of 15 belong to this class. Only No.78 belongs to Nap CMS. The usage of sterile cytoplasm on radish (Ogu CMS) was overwhelmed.(2) There was an obvious difference between the Nap-type cytoplasmic male sterile and two Ogu cytoplasmic male sterile on floral organs morphology. Nap-type cytoplasmic male sterile petal was horn, and with degraded short stamens, anthers shapes varied greatly, degradation nectary. The first group of Ogu CMS petals wrinkled, and with pistillody stamens, degraded nectarys; the second group of Ogu CMS, their petals were normal, stamens were partly degraded. The response of Nap CMS to different nuclear background showed three types. The first type was similar with original material or show negative response; The second type was positive response; The third type was complicated, varying with floret parts. The Ogu CMS materials either showed a weak positive response to nuclear background or keep unchanged comparing to original material. Morphology response of different source of Ogu CMS to the same nuclear background was varied with unclear background. The inbred-line K1, N4 have similar impact on different cytoplasmic male sterile, but 37, F1 and G7 have different effect on different sources of cytoplasmic male sterile in flower morphology.(3) Cytomorphological analysis indicated that the abortion stages and characteristics of Nap CMS and Ogu CMS were affected by nucleus background. Nap-type cytoplasmic male sterile lines with different nucleus background were divided into two type, some materials were no pollen sacs differentiated, others abortion occurred after pollen sacs differentiated. As far as the later with pollen sac, some materials inhibition of pollen development occurred at tetrad stage, but microspores could be released from tetrads in several other materials, abortion in those CMS lines caused by anther failed to cracking.In the first group of Ogu CMS materials, their anther show a trend of Pistillody, but nucleus background had a deep affection on the performance of abortion. Absolutely, differentiation of anther were affected by nucleus background. In spite of anthers were pistillody in almost all CMS materials in this group, material coded 95 still had relatively normal anther development. In the second group of Ogu CMS materials, their abortion stages were relatively steady in different nucleus background, which were concentrated in tetrad stage. The abnormity of tapetal and anther development show some difference between different nucleus backgrounds in this group.(4) Expression analyze to CMS-associated mitochondrial gene at different development stages reveal that, as far as Nap CMS materials, expression level of orf222, cox1, atp6 always had same trend during bud development in same Nap CMS lines. Expression of the three genes (orf222, cox1, atp6) also affected by nucleus background. The expression tendency of the three genes were varied with nucleus background, which could been classified in two types:expression of these three genes were reduce in middle and large bud compared to that in small bud or show no difference among three stages investigated. This reveals some relationship with corresponding abortion character. Nap-CMS materials which expressed orf222, coxl, atp6 in small bud mostly had no pollen sacs differentiated. Whereas in these CMS lines which had pollen sacs development, orf222, coxl and atp6 express in similar abundance. atpA, atp9 shown no significant difference among three development stages of all the Nap-CMS materials investigated, so the expression of these two genes was not regulated by nucleus background.Performance of these mitochondrial genes in Ogu-CMS materials was different with that in Nap-CMS materials, and they were not identical between two groups of Ogu-CMS lines. In the first grope of Ogu-CMS materials whose cytoplasm sourced from No.79, the expression of orf138 show a little difference in different development stages in part of materials in this group. Expression of coxl, atp6 atpA, atp9 was identical among different development stage and different Ogu-CMS lines in this group.In the second grope of Ogu-CMS materials whose cytoplasm sourced from No.99, there were no obvious differences in such five mitochondrial genes orf138, cox1, atp6 atpA and atp9. Difference was detected neither in three development stages nor in different CMS lines. Expressions of these five genes were hardly affected by the nucleus background.(5) Expression analysis of genes which related to the development of floral organ was carried out by semi-quantitative RT-PCR, the results show that expression of AP3, AG were identical among three development stage of all the CMS materials, whether in Nap-CMS lines or in Ogu-CMS lines. However, the expression of PI show a significant change among these materials with different nucleus background, it suggests that PI may has an important role causing diverse abnormality of floral organ in CMS lines.(6) Mitochondrial DNA was isolated from three groups of CMS materials, restriction enzymes Pstl, Xhol, BamHl and EcoRl were used to digest the mtDNA. It was found that Restriction patterns of Mitochondrial DNA were identical among 13 Nap-CMS lines, However rearrangements of mitochondrial DNA were observed in both two groups of Ogu-CMS lines. In 8 Ogu-CMS lines of first group, one of them was distinct from others in BamHl-digested restriction map. It suggests that structure of mitochondrial genome have changed in this material, which may be result from its nucleus background.In 8 Ogu-CMS lines of second group, we found three CMS lines whose restriction map diverge considerably from those of other five CMS lines. It suggests that the structure of mitochondria genome was affected by the nucleus background in this group. The result also indicate that rearrangement tend to occur in same set, although the impact come from different nucleus background.(7) Isoenzymes of ATPase and POD were analyzed in this paper. The result show that, comparing to male fertile line, activity of ATPase reduced markedly in large-bud of Nap-CMS lines. In some of Nap-CMS lines, several band even have not been detected in large-bud. There was on significant difference in activity of ATPase in Ogu-CMS lines, whether first group or second group, but number of bands were digressed in middle and large bud in some Ogu-CMS lines. The absence of bands was varied with nucleus background.No.93 and No.107 belong to two groups of Ogu-CMS material respectively, but they had same maintainer. There was difference between these two lines in POD isozyme bands. The band Rf=0.787 was found in No.93, but obscene in No.107. In CMS line No.79, the band Rf =0.382 was detected which result from interaction of cytoplasm and nuclear. The results show the influence of cytoplasmic on the composition of POD enzyme.