Thearrestofmegagametophytedevelopmentresultsinthefemalesterilityofadominantgenicmale-sterilelineinBrassicanapus.doc

精品論文the arrest of megagametophyte development results in the female sterility of a dominant genic male-sterile line in brassica napus5han xue, hong dengfeng, yang guangsheng(college of plant science and technology, huazhong agricultural university, 430070)abstract: the phenomenon of female sterility on all male-sterile plants was observed in a genic male-sterile (gms) brassica napus line designated as fm195ab (a represents sterile plants and b refers to its wild type ones).for the purpose of clarifying the causal mechanism of this female10sterility, we contrastively studied the morphological characteristics of reproductive structures and procedures involved in seed formation between fm195a and fm195b. first, scanning electron microscopy (sem) investigations on stigma papilla cells showed no apparent difference between the fertile and sterile plants. second, alien pollen grains can normally germinate and pollen tubes can enter the stigma papilla cells of both fm195a and fm195b; however, pollen tubes in fm195a pistils15appear morphologically abnormal in comparison with fm195b, even if they can normally elongate and reach the ovules. furtther observations of the paraffin-sectioned pistils revealed that earlymagasporogenesis in fm195a is similar to that of fm195b; however, mono-nucleate and thesubsequent developmental stages of the female gametophytes can hardly seen in the ovules of fm195a, suggesting. approximately at the eight-nucleate embryo sac stage in fm195b, five major types of the20abnormal embryo sacs in fm195a can be observed. similarly, we found that microsporogenesis in fm195a is also stopped before the entrance of meiosis. because both of the male and female sterility occur at the same plant, it can be assumped that the male sterility gene, ms, may play an important role in prompting the transition from maintaince of the spore mother cells into meiosis.key words: crop genetics and breeding; brassica napus; genic male sterility; female sterility;25megagsporogenesis; microsporogenesis0introductionfemale sterility can be briefly defined as the developmental defects within the female reproductive organ (pistil) which will result in the failure of a functional seed.in angiosperm, the female sterility can be generally classified into three types according to the architecture of a30pistil in combination with the process of a seed formation. the first type can be called as pollination obstacle. normally, the viable pollen grains can be captured and recognized by the pistil, and then complete invasion into it. this process depends on the interaction between pollen grain and stigma papillae cell (edlund et al., 2004). however, abnormalities in stigma structure or function will potentially repress this interaction process and generate low or even no seed setting.35for example, the antisense suppression of a papillar cell-specific gene (pis63) expression inbrassica napus blocks the pollen germination and causes female steritly (kang and nasrallah,2001). in the second type, the germinative pollen tubes can elongate but can not enter the ovules. a typical case of this kind sterility type is the spontaneous mutant rice reported by li et al. (2006), in which the pollen tubes abnormally proceed and cease growth finally in the style-transmitting40tissue, and therefore the double fertilization is completely hampered. the third type is the one in which the ovule development is arrested. the ovule shows great importance in seed development because it is the precursor of a seed and provides the place where the double fertilization occurs. atypical angiosperm ovule contains one or two integuments that develop into the seed coat, afoundations: doctoral fund of the ministry of education of china (20090146110011)brief author introduction:han xue (1984-), female, master of crop genetics and breeding, genetics and breeding for oilseed rapecorrespondance author: yang guangsheng (1962-), male, professor, genetics and breeding for oilseed rape. e-mail: - 18 -funiculus that connects the ovule to the ovary wall, and a nucellus in which megasporogenesis and45magagametogenesis sequentially occur and generate the female gametophyte (embryo sac). the genetic control and regulation of ovule development have been comprehensively reviewed, and many important genes involved in these complex processes especially the female gametophyte development have been identified (schneizt, 1999; skinner et al., 2004; yadegari and drews,2004). in most cases, the function loss of any one of those genes will likely affect the ovule or50zygotic development, and as a result, will inevitably give rise to the abortion of a seed formation.the characterization of the sporocyteless (spl) gene is a good illustration. in the spl mutation, the archesporial cells fails to differentiate into magesporocytes and no embryo sac is formed even if the integument development is not affected (yang et al., 1999).early research showed it was common for plant species to have loci capable of producing55male-sterile, female-fertile mutations, or both male- and female-sterile mutations (gottschalk and kaul, 1974), but less common for male-fertile, female-sterile or partial female mutations (ilarslan et al, 2003). nevertheless, recent works on sexual reproduction also extensively revealed many mutants with individual female-sterile mutation, especially in model plants, arabidopsis and rice, due to the identification of artificial mutants in a large scale (ji hoon ahn et al, 2007). contrarily,60sterility mutants both in female and male were relatively less reported. this phenomenon may beattributed to the possibility that genes exclusively expressed in one sexual organ may predominate over the ones specifically acting in both reproductive apparatuses. an additional reason may come from the fact that only the recessive sporophytic mutations that affected both male and female gametophyte development can be successfully transmitted to the subsequent generation. in65soybean, several mutants have been discovered displaying the failure of both fertility. further research showed that the synapsis defects were observed in these mutants. therefore, the utilization of female and male sterility mutants will accelerate the discovery of functionally conserved genes of great importance in plant gametogenesis.yi3a is a natural dominant gms mutant in brassica napus (li et al. 1985), and its fertility70restoration was once genetically interpreted as the interaction between a dominant male sterility gene (ms) and its dominant epistatic restoration gene (mf or rf) (li et al., 1985; 1988; 1990). however, recent detailed genetic analyses and molecular validation has demonstrated that this line is inherited in a multiple allelic pattern in which the ms and mf genes are two alleles of a locus (song et al., 2005; 2006; liu et al., 2007). as an important alternative of cms systems, elite75dominant gms lines derived from yi3a has been successfully applied as an efficient pollination control system for the utilization of heterosis during the past decade in china (zhou et al. 2003, shi and dong 2004). however, an intrinsic trouble of this gms system remains to hamper its more extensive application that the gms lines especially the homozygous ones generally exhibit a low seed setting rate. furthermore, a complete female sterility line was unexpectedly bred through80our practical breeding schedule (not published, see details from materials and methods).consequently, it provides us a good material for the thorough investigation of the cytological and even the molecular mechanism of the female sterility of this dominant gms mutation.a prerequisite for an understanding of the mechanism of female sterility is to dissect the possible morphological defects into distinct steps (schneitz et al., 1995). however, it is still85difficult to directly observe plant megagametophyte development, fertilization, and early embryogenesis because these processes occur in the embryo sac embedded deeply in the sporophytic tissues (wei and sun, 2002). to solve the problem, four available techniques are developed such as sectioning (misra 1964), whole-mount clearing of ovules (schneitz et al., 1995), enzymatic isolation of embryo sacs (wei and sun, 2002) and a confocal laser scanning microscope9095100105110115120125130(clsm) (christensen et al., 1997). sectioning can be applied in most cases, and more remarkably, it demands low experimental equipments and cost effectively. inconveniently, this technique alwaysundergoesatime-consuming sample-dealing procedure and can onlyobtain two-dimensional photos. whole-mount clearing is a simple and easy-to-handle technique, but it is not efficient for clear cytological observation, especially for distinguishing abnormal events (wei and sun, 2002). enzymatic isolation is an ideal method for the observation of embryo sac development, but the type, concentration and ph value of enzyme solution may range from different plant species. clsm only demands simple specimen preparation and can be completed in a short time. moreover, it provides thin optical sections with excellent resolution and high contrast, and these sections can be assembled into three-dimensional digital volume sets (christensen et al., 1997; zeng et al., 2009). however, it remains a challenge for the observation of megagametogenesis in brassica napus, due to its thickness and large size of ovules. hence, sectioning is still comparatively feasible in investigation of ovule development in brassica napus.similarly, in most reports of male sterility mutants, the female fertility is normal.soybean and maize (ling et al. 1991; ilarslan et al. 1997, 1999, 2003; pamer et al. 2000; cheng et al. 2002; chen et al., 2005), and cytological investigations revealed that the arrest of ovule development before and after fertilization is the main cause for these female infertility mutants. however in brasscia napus, only a brasscia napus mutant altered in the stigma papillae cell figure and number has been reported to arrest the adhesion of pollen grains and result in female sterility (chen et al., 2005).fm195ab is the first report of a line carrying both male and female sterility in brassica napus. in the present study, we comparatively observed the morphology of stigma papillar cell, alien pollen germination and pollen tube elongation between fm195a and fm195b, to examine whether female sterility results from the abnormality in sporophytic tissues. we further systematically investigated the ovule development in fm195ab, and found that the arrest of magagametogenesis in fm195a is the causal mechanism of the female sterility. in combination with the male sterility phenotype of fm195a, we can conclude that the failure of both female and male gametogenesis occurs in meiosis and is caused by the introduction of ms.1materials and methods1.1preparation of materialsthe dominant gms line rs1046ab is a two-type line derived from yi3a (lu et al., 2004; liu et al., 2007). a rs1046a (msms) plant was randomly selected as the mother line to cross with a double haploid canola line dh195 (mfmf), and the offspring individuals plants carrying the ms allele were successively backcrossed with dh195 for three times aided by molecular marker-assisted foreground and background selection. several individuals having highly similar genetic background with dh195 and carrying the heterozygous alleles in ms locus (msmf) were self-pollinated to generate a bc3f2 population. then for the purpose of obtaining a new homozygous gms line, we hybridized the sterile plants (msms) with the fertile ones (msmf). however, we observed almost no hybrid seed and even open pollination seed can be harvested from all the sterile plants, due to their female sterility. alternatively, the heterozygous fertile plants (msmf) were self-pollinated to maintain this material. in this research, fm195a and fm195b referred to the male-sterile individuals (msms) and heterozygous male-fertile individuals (msmf) from the bc3f4 segregation population, respectively. thus, fm195a and fm195b can be regarded as near-isogenic lines (nils). the open pollination cultivar registered in china,135140145150155160165170175huashuang 5, was used as an alien pollen source. all the plant materials were grown in the experimental farms of huazhong agricultural university.male fertility was scored during flowering time by examination the anthers, because the anthers of fertile plants are yellow and well-developed whereas that of the sterile ones were white and shrunken with no pollen. the seed setting was assessed at the seed ripening stage according to the seed number per silique under open pollination circumstance and artificial pollination.pike off the inflorescences, fix in faa solution (50% ethanol 89ml , formalin 5ml , acetic acid 6ml , glycerol 5%) at room temperature or 4c where the test materials are needed to be stored for a long time. at the experiment time, pistils and stamens were dissected with forceps from the flower buds.1.2molecular markers selection at seeding stagein order to obtain all early stages flower buds at initial, we confirm the male-sterile plants with molecular marker-assister selection at seedling stage. in this study, two scar makers (be10 and scd8-2) which are linked with ms gene, be10 is a co-dominant marker and scd8-2 is a dominant marker.1.3scanning electron microscopysamples for sem observation were prepared according to the method described by wan et al. (2010). pistils from fm195a and fm195b one day before anthesis and at 4 h after artificial pollination were prefixed overnight in 2.5% glutaraldehyde adjusted to ph7.4 with 0.1 m sodium phosphate buffer and postfixed in 2% osmium tetraoxide (oso4) in the same buffer, and subsequently dehydrated with a serious of graded ethanol solutions (30%-70%). samples were then dried to the critical point in liquid co2 and coated with palladiumgold in a sputter coater. samples mounted on specimen stubs using double-sided tape were then examined in a jsm-6390 scanning electron microscope.1.4fluorescence micrographs of pollen tube germination and growthmature flowers one day prior to anthesis were peeled away petals and artificially pollinated with mature pollens from huashuang 5. flowers were collected and their whole gynoecium was dissected at 2, 4, 24, 48h after pollination. for pollen tube staining, we followed the protocol described by wei (wei q et al 2000). sample were fixed in faa solution at least 24h, and then washed them in distilled water, softened with 6 m naoh for 12h at room temperature .washed two or three times in distilled water, and stained for 24h with 0.1% aniline blue in 0.14 m k2hpo4 (ph 8.2) at room temperature. a fluorescence microscopy was used to photograph and record those images that indicated germination or growth of the pollen tubes.1.5paraffin section and light microscopythe preparation of paraffin-sectioned materials was performed following the procedure described as li sc et al. (2007) with minor modification.pistils and stamens were dissected from the flower buds and fixed at room temperature for a minimum of 24 h in ethanol 50% (v/v), formalin 5% (v/v), acetic acid 6% (v/v) and glycerol 5% (v/v). following fixation, the fixed organizations were washed in low concentration ethanol (10%) and stained in hematoxylin original solution for 72 h. after rinsed in distilled water for 6 times, the samples were then dehydrated in a graded series ethanol solution from 20% to 100% (20% steps for 2 h each). subsequently, the dehydrated samples were cleared in graded mixture (dimethylbenzene:180185190195200205210anhydrous alcohol=1:4, 1:2, 2:1, and pure dimethylbenzene, each step for 1.5h). finally, after infiltrated and dipped in paraffin, pistils and stamens were transversely and longitudinally sectioned into 6 um slices. bright-field photographs of the samples were taken using a compound microscope.1.6tabletting experimental of male gametophytecollecting flower buds at appropriate size, fixing in the carnoys solution (70%ethanol:acetic acid 3:1) until the sample are discolored (d rosellini et al 2003). dissecting an anther with forceps from the flower bud, softening it in hydrochloric acid solution at 60c, puting on a glass slide, mashing, staining with fuchsin for few seconds, covering with microscopic glass, and test by microscope.2results2.1scanning electron microscopyas a typical dry stigma, the mature stigmas of brassica npaus have intact surface papillar cells, providing necessary material and identification signal for pollen germination and penetration of pollen tubes. thus, we compared the stigma surface structure between fm195a and fm195b before and after pollination to examine whether it has blocked these processes in fm195a. from the scanning electron micrographs, we observed no obvious difference in the stigma papilla cells between the sterile and fertile plants one day before anthesis (fig.1 a and c). similarly, a