Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids between Brassica napus and Sinapis alba 1

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1 Journal of Integrative Agriculture Advanced Online Publication: 2013 Doi: /S (13) Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids between Brassica napus and Sinapis alba 1 WANG Juan 1, GAO Ya-nan 1, Kong Yue-qin 1, JIANG Jin-jin 1, LI Ai-min 2, ZHANG Yong-tai 2, and WANG You-ping 1 1 Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou , China 2 Jiangsu Institute of Agricultural Science in the Lixiahe District, Yangzhou , China Abstract Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was fertile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther. Key words: Brassica napus, anther abortion, cytoplasmic male sterility (CMS), semi-thin sections, somatic hybridization 1 *Correspondence WANG You-ping, Tel: , Fax: , wangyp@yzu.edu.cn

2 INTRODUCTION Rapeseed (Brassica napus L.) is the fifth most important crop after rice, wheat, maize, and soybean in China and one of the most important oil crops worldwide (Mei et al. 2011). Heterosis has a key function in increasing rapeseed production because it improves the quality of rapeseed and enhances its resistance (Fu and Yang 1995). Cytoplasmic male sterility (CMS) can be used to effectively promote large-scale hybrid seed production (Fu and Yang 1995; Havey et al. 2004). Pol CMS line is a natural male sterile line identified by Fu (1981) in To date, pol CMS is the most extensively used CMS line for hybrid seed production in China and abroad (Yuan et al. 2003). Sexual crossing is an alternative way to obtain CMS lines. A typical example is the nap CMS line, which was detected in the F 2 generation of the cross between spring rape cv. Bronowski and winter rape cv. RD58 and possesses various restorer lines (Thompson 1972). Another example is the NCa CMS line, which was bred from the descendants of the distant hybridization between B. napus and B. carinata (Wei et al. 2005). Somatic hybridization is also a main source of CMS lines, by which sterile genes can be transferred from donor to receptor parents; new sterile genes can also be produced by recombination and reconstruction of mitochondrial genomes from biparents during protoplast fusion (Liu et al. 2005; Prakash et al. 2009). Orf138, a sterile gene originally discovered by Ogura (1968) in radish, was successfully transferred into B. napus by protoplast fusion (Pelletier et al. 1983; Uyttewaal et al. 2008). Fusion experiments between B. napus and X-ray-treated B. tournefortii protoplasts were performed to develop tour CMS in B. napus. mtdna recombination between B. rapa and B. tournefortii was verified in the CMS line (Stiewe and Roebbelen 1994). Liu et al. (1996) produced cybrids or partial hybrids through somatic hybridization between X-ray-irradiated mesophyl protoplasts of B. tournefortii and iodoacetamide-inactivated hypocotyl protoplasts of B. napus. The nuclear mitochondrial constitution of these plants indicated that the combination of B. tournefortii cytoplasm and B. napus nucleus resulted in CMS. Hu et al. (2004) obtained the Nsa CMS line in the offspring from the somatic hybridization between B. napus and Sinapis arvensis. The putative candidate Rf gene was cloned based on the pentatricopeptide repeat protein encoded by the restorer gene from CMS rapeseed plant (Hao et al. 2011). In China, the wide utilization of the pol CMS system greatly improved rapeseed yield. However, these rapeseed hybrids are at risk for disease epidemics and may decrease the yield because of its single cytoplasm (Liu et al. 2012). Therefore, developing and creating a novel CMS system is very important for the production of rapeseed hybrids in China (Wei et al. 2009). Sinapis alba, a member of the Brassicaceae family, possesses desirable agronomic characteristics such as yellow seed color; drought stress tolerance; reduced pod shattering; and resistance to viral diseases, blackleg disease, black spot, and beet cyst nematodes (Li et al. 2009; Prakash et al. 2009). Somatic hybrids between B. napus and S. alba obtained through electrofusion have been previously described (Wang et al. 2005). These hybrids were subsequently backcrossed with B.

3 napus cv. Yangyou 6 to the BC 3 generation and then self-pollinated to obtain BC 3F 2, which has valuable agronomic characteristics such as introgression lines with yellow seeds and disease resistance (Li et al. 2009; 2012). A stable male sterile line SaNa-1A was selected from the progenies of B. napus + S. alba somatic hybrids (Wang et al. 2013). In the present study, light microscopy was used to carefully investigate the cytological characteristics of the fertile anther (SaNa-1B) from the maintainer line and sterile anther (SaNa-1A) from this novel CMS line to provide insight into the abortive stage and developmental features of the sterile anther. MATERIALS AND METHODS Plant materials The CMS line used in this study, SaNa-1A, was obtained from the BC 3F 2 progenies from the somatic hybridization between S. alba and B. napus (Wang et al. 2012). The SaNa-1B, B. napus cv. Yangyou 6, is the maintainer of SaNa-1A. When SaNa-1A was crossed with the normal fertile SaNa-1B, the sterility characteristic was inherited from the female parent and stably maintained. The plants were grown in the experimental field in Jiangsu Institute of Agricultural Science in the Lixiahe District. Anatomical observation of anther The samples were collected at 8:00 am. The developmental stages of the anther were estimated according to their lengths of buds, i.e. less than 1 mm, 1-2 mm, 2-3 mm, 3-4 mm, 4-5 mm, 5-6 mm, 6-7 mm, 7-8 mm. The length of fifty buds of SaNa-1A and SaNa-1B was measured. The anthers were immediately fixed in 2.5% glutaraldehyde. After 24 h, the fixative solution was replaced with a fresh one. After 1 d, the anther was washed with phosphate buffer (PB; ph 7.2) three times for 15 min each and then post-fixed with 1% osmium tetroxide for 4 h. The post-fixed tissue was again washed with PB three times. The anther was dehydrated through a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) for 15 min each, infiltrated with acetone, and embedded in 812 resin. Semi-thin sections (1 µm) were cut from the polymerized blocks and stained with 1% toluidine blue for 3 min for light microscope observations. RESULTS Phenotype characteristics of SaNa-1A and SaNa-1B floral organs The sterile line SaNa-1A was distinct from the fertile line SaNa-1B with regard to floral organ phenotypes. SaNa-1A showed shortened filaments and thinner anthers with a shriveled appearance compared with SaNa-1B. No obvious pollen grains were attached to the surface of SaNa-1A (Fig.

4 1). Compared with that of the fertile line, adjacent didynamous stamens of the sterile line adhered to one other. The sepal, pistil, and petal of the two lines were similar (Fig. 1). The bud size in different developmental stages was also compared (Table 1). Before the pollen mother cell (PMC) stage, the sterile bud size was similar to that of the fertile bud. However, when the microspore entered the early uninuclear phase, the fertile bud was significantly larger than the sterile bud, and the difference in bud size was amplified as the vacuolated microspore developed to a mature pollen grain (Fig. 2). Anther development of the fertile line SaNa-1B Based on the significant characteristic changes in floral development, seven stages were used as benchmarks for further comparison. The stages according to developmental order are archesporial cell, sporogenous cell, PMC stage, tetrad stage, early uninuclear phase, late uninuclear stage, and mature pollen (Table 1, Fig. 2). Archesporial cell: In early development, the initial oval structure of the anther formed by the stamen primordium was relatively simple, consisting of an outermost epidermal layer and a group of meristem cells inside (Fig. 3a). When the flower buds grew up to 0.40 mm ± 0.10 mm, the meristem cells rapidly divided at four corners to form archesporial cells that are slightly larger than the other cells, which resulted in a quadrangular shaped anther. Subsequent periclinal division of the archesporial cells produced the primary parietal cells (Fig. 3b). Sporogenous cell: Archesporial cells from buds with length of 1.05 mm ± 0.45 mm underwent periclinal division to generate the precursors of PMC, which are sporogenous cells. The primary parietal layer divided into second parietal cells consisting of two layers. The outer layer next to the epidermis formed the middle layer and endothecium, and the inner layer divided and differentiated into the tapetum. In this phase, the anther developed into a butterfly-like shape (Fig. 3b). Given that the two lower locules usually develop more rapidly than those the two upper locules, these two developmental stages are easy to detect in an anther (Fig. 3b). PMC: The division of the secondary parietal cell formed a three-layered mature anther wall composed of an endothecium, a middle layer, and a tapetum (from outside to inside) (Fig. 3c). The endothecium has a greater volume and is located adjacent to the epidermis; the middle layer contains rich nutrients for the meiosis of PMC. However, the middle layer gradually disappeared from the locule wall as microspores were released from the tetrad stage (Fig. 3f). The innermost wall is the tapetal layer, which is directly in contact with the developing pollen grains in the anther locule. The tapetal layer possesses the greatest volume and is uninucleate at early stages (Fig. 3c). However, nuclear division and the absence of new cell wall synthesis resulted in a dicaryotic or polynucleate structure (Fig. 3d). Within the tapetum, RNAs, proteins, and carotenoids serve as nutrients for meiosis and microspore development. Early PMCs are adjacent to one another (Fig. 3c). When the anther developed into the late PMC stage, the intercellular layer between the PCMs

5 disintegrated, and the PMCs slightly separated (Fig. 3d). The shape changed from irregular to round shape (Figs. 3c and 3d). Tetrad stage: The separated PMCs immediately underwent meiotic division. The tapetum continuously secreted nutrients into the sac to form haploid microspores (Fig. 3e). The middle layer became tabular and showed a disintegrating trend when tetrads formed. The primary microspores were enclosed by callose walls and displayed a tetrahedron shape. Another characteristic of this stage is the degeneration of the tapetum with the emergence of small vacuoles (Fig. 3e). The entire process is brief and occasionally observed in buds with size of 2.00 mm ± 0.20 mm. Uninucleate stage: The callase secreted by tapetal cells entered the sacs to dissolve the callose wall for the release of microspores (Fig. 3f). The early microspores were spherical with thin cell walls and nucleus located at the center. The middle layer completely disappeared (Fig. 3f). Upon development, a vacuole occupied most of the microspore, squeezing the central nucleus into the cell periphery (Fig. 3g). A thicker wall consisting of exine and intine formed on the microspore surface, which led to a prismatic shape (Fig. 3g). This developmental phase occurred over a long period, especially the late uninucleate stage, which increased the floral length to 3.40 mm ± 1.10 mm. Nucleate pollen to mature pollen: The vacuolated microspore underwent asymmetric division, which generated a larger vegetative cell and smaller generative cell. The generative cell underwent second symmetric mitosis to form two sperm cells that were surrounded by the vegetative cell. The three-layered cells became spherical again (Fig. 3h). However, these two generative cells were difficult to recognize because of their small size. The endothecium stretched and expanded in a radial direction, forming a ribbon of thick or fibrous layer (Fig. 3h). The epidermal cells located in adjacent pollen sacs appeared as lip-shaped cells and changed into stomium to facilitate the release of pollen grains when the microspores matured. The tapetum was fully degenerated (Fig. 3i). Abortive process of the sterile anther of SaNa-1A Aside from the phenotypic difference between sterile and fertile anthers, cytological observation showed significant differences between the anthers. Before PMC stage: Anther development including floral size was the same in fertile and CMS lines before PMC stage. The anther was quadrangular and divided into sporogenous cells at four corners (data not shown). These sporogenous cells, which contained a condensed cytoplasm, divided and differentiated into PMCs. The locule wall also developed normally with three-layered cells (Fig. 4a). From PMC to tetrad stage: Abnormality appeared at the late PMC stage with small vacuoles located in the tapetal cells, which had a tendency for radial extension (Fig. 4b). By contrast, this

6 phenomenon emerged at the tetrad stage in the fertile line (Fig. 3e). The PMCs of the CMS line also contained several vacuoles with less condensed cytoplasm and continued developing into less-condensed tetrads. The middle layer disappeared earlier at this stage (Figs. 4b and 4c). Uninuclear stage to fully abortive stage: The microspores released from the tetrad of the sterile line resembled those of the fertile line except for the less-condensed cytoplasm (Fig. 4d). A large vacuole with a marginal nucleus began to appear but had no pollen wall (Fig. 4d). Although the tapetum elongated, the microspore maintained a vacuolated and uncoated state. The tapetum radically and persistently elongated and became vacuolated until the entire locule was occupied. The vacuolated microspores failed to separate and adhered to one other with the development of the hypertrophic tapetum, which resulted in a shriveled anther sac (Figs. 4e and 4f). The middle layer gradually disappeared at the tetrad stage (Fig. 4d). However, no fibrous layer was attached to the endothecium, and no stomia were found open for pollen release (Fig. 4g). DISCUSSION Several CMS types of B. napus have been developed such as nap CMS (Thompson 1972), pol CMS (Fu 1981), ogu CMS (Pelletier et al. 1983), and tour CMS (Pahwa et al. 2004; Bang et al. 2011). Only the pol CMS system is extensively used in commercial rapeseed hybrid production in China. The discovery of SaNa-1A provides an alternative choice for producing hybrid seeds. The anther of SaNa-1A initially aborted at the late PMC stage with the tapetum showing prior vacuolization and formation of vacuolated PMCs (Fig. 4b). The microspores released from the less condensed tetrads developed into late uninucleate grains with no visible surrounding anther wall and then ceased to grow into mature pollen grains (Fig. 4d). The tapetal cells radically elongated and became hypertrophic until the entire locule was occupied (Figs. 4e and 4f). The hypertrophic tapetum squeezed the vacuolated microspores, which resulted in the adherence of microspores to one other (Fig. 4f). In pol CMS, anther development is blocked at the archesporial cell stage, and traces of pollen grains are detected at lower or higher temperatures (Fu and Yang 1995). By contrast, the SaNa-1A line is completely sterile and stable at various temperatures. Another well-known CMS line, ogu CMS, aborts at the uninuclear cell stage and its restorer line is difficult to find in rapeseed. The novel CMS line SaNa-1A derived from the somatic hybrids between B. napus and S. alba enriches the source of rapeseed CMS and may be a potential germplasm for hybrid production of rapeseed in the future. Pollen development is often associated with tapetum regulation. The abortion of SaNa-1A is consistent with the finding in Allium, where an abnormal tapetum behavior causing male sterility was characterized by extremely early degeneration and tapetum hypertrophy and autolyzation (Holford et al. 1991). Yi et al. (2010) found that the lack of sporopollenin or exine in pollen wall formation at the tetrad stage coincides with the maximum BnCYP704B1 mrna accumulation in the tapetal cells at the haploid stage in Arabidopsis. This finding demonstrated that BnCYP704B1,

7 a homologous gene of BnMs1 and BnMs2 detected in rapeseed (Yi et al. 2006; Lei et al. 2007), is involved in the lipid metabolism of exine formation and basic tapetal cell development. Arabidopsis male sterility 1 (ms1) mutation is caused by tapetal layer breakdown, which does not occur through the normal process of programmed cell death (PCD). Thus, immature pollens are aborted after the microspores are released from the tetrads (Dawson et al. 1993; Wilson et al. 2001). Vizcay-Barrena and Willson (2006) speculated that MS1 regulated the tapetal gene transcription involved in pollen wall development or tapetal development by directly regulating tapetal PCD. Ogu CMS sterile anthers form because of the premature death of tapetal cells presumably by an autolysis process rather than normal PCD, which impairs pollen development at the vacuolated microspore stage (Gonzalez-Melendi et al., 2008). In the nap CMS system, sterility results from delayed pollen development and indehiscent anthers (Barthowiak-Broda et al. 1977), whereas in the pol CMS system, sterility is due to the failure of microsporangium formation (Wei et al. 2009). In this study, a CMS gene located in the mitochondria disturbs tapetum development. The formation of a hypertrophic tapetal layer contributes to the adherence of uncoated microscopic cells. For future work, mitochondrial sequencing of this CMS line has been performed, and identification of potential sterile gene(orf122)is ongoing. Acknowledgments This work was supported by the NSFC project, Priority Academic Program Development of Jiangsu Higher Education Institutions, the Program of International S & T Cooperation of China (1021) and Jiangsu Province Graduate Innovation Fund. References Bang S, Tsutsui K K, Shim S, Kaneko Y (2011) Production and characterization of the novel CMS line of radish (Raphanus sativus) carrying Brassica maurorum cytoplasm. Plant Breeding, 130, Bartkowiak-Broda I, Rousellle P, Renard M (1977) Investigations of two kinds of cytoplasmic male sterility in rapeseed (Brassica napus L.). Genetics Polish, 20, Dawson J, Wilson ZA, Aarts MGM, Braithwaite AF, Briarty LG, Mulligan BJ (1993) Microspore and pollen development in six male-sterile mutants of Arabidopsis thaliana. Candian Journal of Botany, 71, Fu TD (1981) Production and research of rapeseed in the People s Republic of China. Eucarpia Cruciferae Newsletter, 6, 6 7. Fu TD, Yang GS (1995) Cytoplasmic male sterility in rapeseed. In: Fu TD (ed), Breeding and utilization of rapeseed hybrid, Wuhan, Hubei Science and Technology Press, pp 1-6.

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10 Figure legends: Fig. 1 Comparison of the floral phenotype between sterile and fertile lines. (a) and (c) Novel CMS line derived from Brassica napus + Sinapis alba hybrids (SaNa-1A). (b) and (d) Male fertile maintainer line (SaNa-1B)

11 Fig. 2 Comparison of the anther size at different stages between fertile (SaNa-1B) and sterile (SaNa-1A) lines. Ap, archesporial cell; Sp, sporogenous cell; Td, tetrad stage; PMC, pollen mother cell; early Uni, early uninuclear stage; late Uni, late uninuclear stage; MP, mature pollen

12 Fig. 3 The anther development of fertile line SaNa-1B. (a) Initial anther showing meristem cell; (b) anther of asymmetric development including archesporial cell and sporogenous cell (Sp), primary parietal cell (PPC), and secondary parietal cell (SPC); (c) early PMC; (d), late PMC; (e) tetrad stage; (f) early uninuclear stage; (g) late uninuclear stage, vacuolated microspore, enlarged microspore shown at the lower right corner; (h) mature pollen stage, a mature pollen with normal cell wall was shown at the lower right corner; (i) releasing pollens. Ep, Epidermis; Ap, archesporial cell; En, endothecium; ML, middle layer; T, tapetum; PMC, pollen mother cell; Td, tetrad; Ms, microspore; MP, mature pollen; FL, fibrous layer; S, stomium

13 Fig. 4 The anther development of the male sterile line SaNa-1A showing its abortive process. (a) Early PMC, similar to the fertile line SaNa-1B; (b) late PMC, showing tapetal cells with small vacuoles, which emerges at the Td stage in the fertile line as Fig. 3e; (c) tetrad stage, PMCs containing less condensed cytoplasm and disappeared middle layer; (d) early uninuclear stage I; (e) vacuolated microspores I, an enlarged microspore without normal cell wall indicated at the lower right corner; (f) vacuolated microspores II, showing the hypertrophic and elongated tapetum, leading to the adherence of microspores to each another; (g) fully abortive stage. Ep, Epidermis; En, endothecium; ML, middle layer; T, tapetum; PMC, pollen mother cell; Td, tetrad; Ms, microspore

14 Table 1 Comparison of the anther development between sterile line SaNa-1A and fertile line SaNa-1B Stage Bud length (mm) Anatomical characteristics SaNa-1B SaNa-1A SaNa-1B/fertile SaNa-1A/sterile Archesporial cell 0.40± ±0.10 Epidermal layer and meristem cells form; meristem cells Similar with the fertile line divide to form archesporial cells; primary parietal cells Sporogenous cell 1.05± ±0.35 Periclinal division of archesporial cell; sporogenous cell appears; butterfly-like shape Similar with the fertile line, sporogenous cells with condensed cytoplasm PMC 1.65± ±0.10 Three-layered mature anther wall; tapetal layer with great Slightly-vacuolated and radically-extended volume and uninucleate; PMCs with condensed cytoplasm tapetum; mildly-vacuolated and less-condensed PMCs Tetrad 2.00± ±0.15 Marginally-vacuolated and radically-extended tapetum; Heavily-vacuolated and radically-extended microspores enclosed by callose; normal tetrads form tapetum; less-condensed tetrads Early uninucleate Microspores released; spherical microspores with thin wall Heavily-vacuolated and radically-extended and central nuclear tapetum; less-condensed microspores Late uninucleate 3.40± ±0.95 Prismatic microspores with thick wall and peripheral Heavily-vacuolated and radically-extended nuclear tapetum; vacuolated microspores with uncoated wall Mature pollen ~7.5 ~5.8 Three-nuclear pollen; stretched endothecium and radically expanded; fibrous layer; lip-shaped cells appeared and pollen grains released Vacuolated microspore adhered with hypertrophic and vacuolated tapetum; no fibrous layer; shriveled anther sac