Characterization of a male sterile mutant from progeny of a transgenic plant containing a leaf senescence-inhibition gene in wheat
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- Xi, Y., Ma, X., Zhong, H. et al. Euphytica (2011) 177: 241. doi:10.1007/s10681-010-0254-4
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A male sterile plant of wheat (Triticum aestivum L.) segregated from progenies of a transgenic family containing the leaf senescence-inhibition gene PSAG12-IPT in the genetic background of ‘Xinong 1376’, a well adapted winter wheat cultivar. The male sterile plant (named TR1376A) showed no phenotypic changes, except for florets and male organs, compared to its male fertile sibling plants (named TR1376B). The glumes and florets of male sterile TR1376A plants widely opened whereas those of the fertile counterpart TR1376B were closed or opened only briefly at flowing. Anthers of TR1376A were slender and indehiscent, and failed to release pollen. Compared to TR1376B, TR1376A anthers contained greatly reduced amounts of pollen, which was inviable or weakly viable. Ultra-structure studies indicated that cells in the endothecium and middle layers of the anther wall were dissolved or poorly developed in the sterile anthers of TR1376A. Molecular studies showed that the male sterility of TR1376A was caused by a sequence deletion or mutation that occurred in the promoter region of the transgene. F1 hybrids of TR1376A and TR1376B gave 1:1 segregation of male fertility to sterility, indicating that the male sterility of TR1376A was heritable and controlled by a single dominant gene (named Ms1376). To date, only a few dominant nuclear male sterility genes have been characterized and one of them (Ms2) has been successfully used to improve wheat cultivars through recurrent breeding strategies. The discovery of the Ms1376 gene provides another dominant male sterile source for establishing recurrent breeding systems in wheat.
KeywordsGenetic transformationLeaf senescenceMale sterilityMs geneTriticum aestivum
Wheat (Triticum aestivum L.), one of the most important food crops worldwide, has been improved for high yield and quality through various breeding strategies. A great effort has been made to develop male sterile lines for inbred cultivar or hybrid breeding. Several male sterile systems have been developed for various wheat breeding activities, including cytoplasmic male sterility, nuclear (genic) male sterility and ecological (i.e. thermo-photo-sensitive) male sterility (Edwards 2001; Rajaram 2001). While cytoplasmic and ecological male sterilities have been used in hybrid breeding, nuclear male sterility has been successfully applied to improving breeding populations through recurrent selection (Liu et al. 2002; Yang et al. 2009).
Significant breeding gains were obtained through recurrent selection by using nuclear male sterile lines in breeding programs (Yuan and Zhang 2000; Liu et al. 2002; Yang et al. 2009; Li et al. 2007). Thus, there is a growing interest in utilizing male sterile lines to accelerate the development of inbred cultivars. Specifically, the ‘Taigu’ nuclear male sterile system, which has been studied for almost 40 years since its discovery in 1972 (Gao 1987), has been widely used as a major germplasm platform in wheat breeding in China (Yuan and Zhang 2000; Liu et al. 2002; Yang et al. 2009; Li et al. 2007). One well adapted cultivar, ‘Jinghe 5’, was developed through recurrent selection using the ‘Taigu’ nuclear male sterile system (Yuan and Zhang 2000); the cultivar showed lodging resistance, leaf rust resistance, good spike density, good grain ripening, high 1000-grain weight, and high quality and yield. More importantly, a male sterile-dwarf system was established by selecting recombinants that tightly linked the ‘Taigu’ nuclear male sterile gene Ms2 and the dwarf gene Rht10 (new nomenclature Rht-D1c) on chromosome arm 4DS (Liu et al. 2002). Using male sterile-dwarf phenotype-facilitated recurrent selection, a well known winter wheat cultivar, ‘RS987’, was produced and quickly became one of the dominant cultivars in China due to its high yield stability and good quality (Yang et al. 2009). Recently, the nuclear male sterile-dwarf germplasm was also successfully used for breeding a multi-spikelet cultivar (Li et al. 2007).
With the advancement of plant biotechnology, genetic engineering plays increasing roles in plant germplasm improvement. While target gene expressions are expected, many unexpected phenotypic variations, such as male sterility, can occur during genetic transformation. For example, the over-expression of peptidyl prolyl cis-trans-isomerase wFKBP77 in wheat caused major morphological abnormalities, specifically relating to height, leaf shape, spike morphology and fertility (Kurek et al. 2002). The creation of plant male sterility by genetic engineering was pioneered by Mariani et al. (1990), who induced male sterility in transgenic tobacco (Nicotiana tabacum L.) and Brassica napus plants by expression of a ribonuclease gene (barnase) from Bacillus amyloliquefaciens in tapetal tissues of anthers using a tapetum-specific promoter, TA29. Genetic transformation with chimeric barnase genes driven by various pollen tapetum-specific promoters, such as the tobacco TA29 or the rice (Oryza sativa L.) OsgB6, became a routine approach of inducing male sterility in many crop species, including Brassica, tobacco, tomato (Solanum esculentum L.), wheat, rice and alfalfa (Medicago sativa L.) (De Block and Debrouwer 1993; De Block et al. 1997; Rosellini et al. 2001; Wang et al. 2008). Male sterile plants were obtained by genetic transformation of the chimeric construct TA29:Barnase in B. napus (De Block and Debrouwer 1993). The same gene construct was also successfully transformed into alfalfa (Rosellini et al. 2001). The T0 male-sterile plants were crossed with unrelated non-transgenic male-fertile plants and the progenies showed Mendelian segregation. The barnase gene was also placed into tobacco under the control of a different promoter, resulting in almost complete male sterility (Wang et al. 2008). Similarly, in wheat nuclear male sterility was obtained transgenically by introducing the barnase gene under control of tapetum-specific promoters derived from corn and rice (De Block et al. 1997).
It seems sterility could be induced by many transgenic activities other than using the barnase gene or pollen tapetum-specific promoters. For example, Agrobacterium tumefaciens-mediated gene transformation caused a highly sterile phenotype in Arabidopsis because the anthers failed or delayed to dehisce during the flowering stage, resulting in reduced or disordered endothecial secondary wall thickening of the anthers (Ge et al. 2008). The male sterile phenotype was heritable and was independent of the genes and construct types used for transformation. The transformation of a lectin receptor-like kinase gene mutant, At3g53810, also induced complete male sterility in homozygous progenies of Arabidopsis (Wan et al. 2008). Constitutive expression of a human gene affected the morphologies of transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis (Cheon et al. 2004). Tapetum specific expression of mitochondrial gene orfH522 in tobacco made one-third of the transgenics completely male sterile (Nizampatnam et al. 2009). Cone-specific expression of a stilbene synthase gene (STS) in anthers of transgenic tobacco plants resulted in complete nuclear male sterility in 70% of transformed plants (Höfig et al. 2006). In wheat, male sterile phenomena were observed after introducing alien genes through various approaches (Ji et al. 2003; Yang et al. 2004; Wang et al. 2005).
PSAG12-IPT is a chimeric gene construct that was used specifically to auto-regulate the leaf senescence program (Gan and Amasino 1995, 1997). IPT is an isopentenyl transferase gene, which catalyzes the rate-limiting step in cytokinin (CTK) biosynthesis, cloned from the T-DNA of A. tumefaciens; and PSAG12 is a senescence-specific promoter from Arabidopsis (Gan and Amasino 1995, 1997). The promoter PSAG12 is activated to generate CTK only when leaves start undergoing senescence; however, the activity of the PSAG12 promoter is attenuated once sufficient CTK is produced to retard senescence. Therefore, transgenic plants expressing PSAG12-IPT did not exhibit the developmental abnormalities usually associated with IPT expression because the system was autoregulatory (Gan and Amasino 1995, 1997). In order to suppress leaf senescence, the PSAG12-IPT gene was transformed into many plant species, such as tobacco, rice, lettuce (Lactuca sativa L.) and alfalfa (Gan and Amasino 1995; Fu et al. 1998; McCabe et al. 2001; Calderini et al. 2007). Retarded leaf senescence was observed in all studies.
The PSAG12-IPT gene was also transformed into a commercial wheat variety, ‘Xinong 1376’, through the pollen tube pathway and the expected phenotype of delayed senescence was observed in the transformants (Xi et al. 2004). In addition, a male sterile line was found in progenies of the transgenic plants. Since male sterility has played significant roles in both inbred and hybrid wheat breeding, the sterile line provides a potential novel resource of male sterility for wheat improvement. In the present study, the novel sterile line was molecularly and genetically characterized.
Materials and methods
The germplasm used for gene transformation was ‘Xinong 1376’, a well adapted high yielding and early maturing winter wheat variety. Other lines used for test crosses were ‘Xinong 534’, ‘Xinong 9624’, ‘Yuanfeng 99’, ‘Xinong 9872’, ‘Xiaoyan 605’, ‘Xinong 979’ and ‘Xinong 2208’, which are all commercial varieties grown in the Huang-Huai Wheat Zone, the largest winter wheat growing area in China.
The chimeric gene used for transformation
The original plasmid containing the chimeric gene PSAG12-IPT was obtained from the University of Wisconsin-Madison, USA (Gan and Amasino 1995). The chimeric gene construct was then modified by replacing the kanamycin resistance gene with a hygromycin resistance gene in order to facilitate the efficiency of gene transformation in rice (Cao 2000). The modified PSAG12-IPT gene was built into the plasmid pCMLA35-1, which can be transformed into Escherichia coli strain DH5α for amplification. The plasmid DNA containing the modified PSAG12-IPT (Cao 2000) was used to transform wheat through the pollen tube pathway (Xi et al. 2004).
Polymerase chain reaction (PCR) and PCR-Southern hybridization
The original transgenic plants, as well as the progenies derived from self-pollination and backcrosses in each generation, were identified or confirmed by polymerase chain reaction (PCR) screens, followed by PCR-Southern hybridization. Primers amplifying both SAG12 and IPT sequences were used. The primer sequences used for amplifying SAG12 were 5′-GCAAAGAGACGAGGAAGAAA-3′ (forward) and 5′-TGGCTGAAGTGATAACCGTC-3′ (reverse); and the primers for amplifying the IPT gene sequence were 5′-GGACCTGCATCTAATTTTCGGTCC-3′ (forward) and 5′-TTTCAGAATGGGCCTCTTAACTC-3′ (reverse). PCR was initiated at 94°C for 3 min followed by 30 cycles (94°C for 1 min, 56°C for 1 min, and 72°C for 1.5 min) of amplification with a final extension at 72°C for 7 min.
For PCR-Southern hybridization, SAG12 and IPT sequences that were PCR amplified from the plasmid gene construct were used as probes. Gel blotting, probe labeling and Southern hybridization were carried out following standard protocols (Sambrook et al. 1989).
Discovery and maintenance of the male sterile line
All transgenic plants (T0 and later generations) that were confirmed having the chimeric gene PSAG12-IPT were maintained through self-pollination by bagging spikes. A plant was defined as male sterile if (almost) no seeds were set in bagged spikes, but seeds were well set when the plant was cross-pollinated. One plant in a T2 family was found to be male sterile (named TR1376A) and it was immediately pollinated with pollen from male fertile sibling plants (named TR1376B) within the same family. In each of the following generations, the male sterile lines were maintained in the same way by the cross “TR1376A × TR1376B”.
Test crosses and inheritance analyses of the male sterile line
The male sterile line, TR1376A, was crossed with TR1376B and also various non-transgenic inbred lines, including ‘Xinong 1376’, ‘Xinong 534’, ‘Xinong 9624’, ‘Yuanfeng 99’, ‘Xinong 9872’, ‘Xiaoyan 605’, ‘Xinong 979’ and ‘Xinong 2208’. The spikes of all F1 individuals were bagged. Since the male sterile TR1376A line was heterozygous, the F1s of the above crosses segregated for male fertility and sterility. Percentages of seed set were documented for all progenies. The ratio of male sterile to fertile plants from each cross was recorded and tested for a Mendelian segregation ratio of 1:1 using the χ2 test.
Estimation of pollen viability
The viability of pollen was investigated by 2,3,5-triphenyltetrazolium chloride (TTC, Xi’an Chemical Reagent Plant, Xi’an, PR China) staining (Norton 1966; Huang et al. 2004). Briefly, the freshly released mature anthers were collected on glass slides, a drop of distilled water was added to each slide, the anthers were broken carefully with small forceps to release the pollen grains, and one drop of TTC solution (1%, pH 7.0 ± 0.5) was applied. The slides were then covered with cover slips and incubated at 35°C for 15 min for colour development. After staining, the slides were checked under a light microscope. The viability of pollen correlates with the degree of colour staining; red colour (or darkly stained in black-white images) signifies normal viability whereas light red or colourless indicates weak viability or no viability.
The viability of pollen was also investigated by staining with IKI (Eti 1991; Huang et al. 2004), which was a mixture of 0.5% (w/v) I2 and 1% (w/v) KI (Xi’an Chemical Reagent Plant, Xi’an, PR China). Similarly, pollen grains were stained with one drop of IKI solution. The pollen grains stained dark blue (or darkly stained in black-white images) with IKI indicated normal viability, whereas lightly stained pollen indicated weak or no viability.
Ultra structures of the sterile anthers
The ultra structures of anthers were investigated by transmission electron microscope. Mature yellow anthers were collected before dehiscence and fixed in a 25% (v/v) glutaraldehyde (Xi’an Chemical Reagent Plant, Xi’an, PR China) solution at 0–4°C for 6 h, followed by five washes with 0.1 mol/l PBS (Phosphate Buffered Saline, 140 mM of NaCl, 2.7 mM of KCl, 8 mM of Na2HPO4:7H2O, 1.5 mM of KH2PO4, pH 7.4) for 5, 10, 15, 20 and 30 min, respectively. The anthers were then fixed with 1% (w/v) osmium tetroxide (osmic acid anhydride) (Xi’an Chemical Reagent Plant, Xi’an, PR China) at 4°C for 2 h, followed by 10 min of PBS wash, and then dehydrated using 30, 50, 70, 80, 90 and 100% acetone (Xi’an Chemical Reagent Plant, Xi’an, PR China) for 15, 15, 15, 20, 20 and 30 min, respectively. The last dehydration step (100% acetone) was repeated three times. The dehydrated anthers were finally embedded with Epon 812 (Xi’an Wolsen Bio-technology Co., Xi’an). Cross sections of the anthers were viewed for anther wall structure investigations with a transmission electron microscope.
The discovery of the male sterility
The male sterile TR1376A line was crossed with pollen collected from sibling plants (TR1376B) in the same family. F1 progenies of the cross were planted at Yangling, Shaanxi, in September 2004 (i.e. normal winter planting); 23 F1 individuals were obtained. During heading in April 2005, all spikes of the plants were bagged for self-pollination; 13 plants were fully fertile and 10 were sterile. The 10 male sterile plants were pollinated by the maintainer line, TR1376B, and about half of those progeny were again self sterile, indicating that the male sterility was controlled by a single dominant gene. All male sterile plants showed normal pistil development and abortive male organ development like the original TR1376A plant, demonstrating that the male sterility was stably inherited.
Transgene detection of the male sterile TR1376A line
Inheritance of the male sterility
Male fertility and sterility segregation in the F1 progenies of the test crosses involving TR1376A
No. male fertile plants
No. male sterile plants
TR1376A × Xinong 1376
TR1376A × Xinong 534
TR1376A × Xinong 9872
TR1376A × Xinong 9624
TR1376A × Yuanfeng 99
TR1376A × TR1376B
TR1376A × Xinong 1376
TR1376A × Xinong 534
TR1376A × Xinong 9872
TR1376A × Xinong 2208
TR1376A × Xiaoyan 605
TR1376A × Xinong 979
Pollen viability of the male sterile TR1376A line
The difference in TTC staining between male sterile and fertile plants was very clear (Fig. 3a, b). While almost all pollen grains of male fertile plants were darkly stained (Fig. 3a), pollen grains of the male sterile plants were not well stained by TTC, indicating no or very weak viability (Fig. 3b). The few lightly stained pollen grains may explain the one or two seeds sometimes occurring on bagged spikes of TR1376A plants. Similar results were also seen when pollen was stained with IKI (Fig. 3c, d). The data indicated that although a small amount of pollen was seen in the anthers of the male sterile plants, it was rarely viable.
Anther wall ultra structure
Whereas the florets of normal fertile plants were closed or only opened briefly during anthesis, the florets of the male sterile plants opened widely at the flowering stage, thus the anthers and pistils of the male sterile plants were exposed. However, the anthers of the male sterile plants were indehiscent (Fig. 1e). This feature plus rare availability of viable pollen made TR1376A plants (almost) completely male sterile.
Transgenes can induce unexpected phenotypic variation. Male sterility induced by genetic transformation or alien DNA introduction has been reported in many studies (Ji et al. 2003; Cheon et al. 2004; Yang et al. 2004; Wang et al. 2005; Höfig et al. 2006; Ge et al. 2008; Wan et al. 2008; Nizampatnam et al. 2009). However, most of the male sterile lines were not genetically characterized and it is not known if the male sterility of the lines was stably inherited or if the lines had potential value for crop improvement. One male sterile line induced by exogenous DNA in wheat was characterized, resulting in the development of a novel D-type cytoplasmic male sterility system (Yang et al. 2004).
The male sterile line, TR1376A, reported in this study involves nuclear (genic) male sterility, and is controlled by a dominant gene, Ms1376. Test crosses with multiple non-transgenic materials indicated that Ms1376 is stably inherited. Since the TR1376A plant initially segregated from a T2 population, derived from T0 and T1 plants with normal fertility, the occurrence of male sterility should be independent of the expected function of the transgene PSAG12-IPT. Molecular studies showed that there was a mutation in the transgene PSAG12-IPT sequence, in which there was either a sequence loss or another mutation involving the primer binding site(s) of the transgenic promoter.
It is unclear whether the sequence-losing event or mutation was induced by the transgene, and if so, how it related to male sterility. Further studies are needed to understand the mechanisms of the male sterility and molecular basis of Ms1376. Since the male sterile line TR1376A has been maintained by backcrossing with its sibling line in each generation, the TR1376A and TR1376B are highly isogenic and therefore ideal materials for studying the mechanisms of nuclear male sterility.
Multi-layer structures, especially the endothecium, of anther walls are critical for anther dehiscence (Dawson et al. 1993, 1999; Yang et al. 2007). Cellulosic secondary thickening in the anther wall endothecium provides an inwardly directed force, causing the weakened stomium to rupture. Then, desiccation of the endothecium causes differential shrinkage of thickened and unthickened parts of the cell wall, resulting in an outwardly bending force, leading to retraction of the anther wall and full opening of the stomium (Keijzer 1987; Bonner and Dickinson 1989; Yang et al. 2007). Sterile mutants of Arabidopsis demonstrating reduced or disordered endothecial wall thickenings were indehiscent (Sanders et al. 2005; Dawson et al. 1999; Mitsuda et al. 2005; Yang et al. 2007; Ge et al. 2008). In the present study, TR1376A anthers showed disordered endothecial and middle layers, thus failing to dehisce.
Nuclear male sterility occurs in many plant species and is usually controlled by single recessive genes. In wheat, nuclear male sterile mutations either occur spontaneously or are induced by mutagenic treatments. To date, many mutations have been reported, but only a few have been assigned to chromosomes; these include the recessive genes ms1 on chromosome arm 4BS and ms5 on 3AL (Klindworth et al. 2002), and the dominant genes Ms2 on 4DS (Liu and Deng 1986), Ms3 on 5AS (Maan et al. 1987) and Ms4 on the distal portion of 4DS (Maan and Kianian 2001). It is still not clear if Ms1376 is a novel gene or an allelic variant of an existing Ms gene. Since TR1376A arose as either a sequence loss or another type of mutation in the chimeric gene sequence, it is reasonable to speculate that Ms1376 is novel.
Dominant nuclear male sterility cannot be used for hybrid production in agriculture, but it has been used to establish recurrent selection populations for wheat improvement. The only dominant nuclear sterile gene that has been broadly used for wheat breeding is the Ms2-controlled ‘Taigu’ male sterility (Liu and Deng 1986). A dwarf male sterile wheat (Liu 1987) was developed by linking the dominant male sterile gene Ms2 and dominant dwarfing gene Rht10 (Rht-D1c) on chromosome 4D (Börner et al. 1997). A breeding scheme (Liu et al. 2002; Yang et al. 2009) was developed using dwarf-male-sterile-facilitated recurrent selection to improve wheat breeding pools, and resulted in the release of a number of cultivars, such as ‘RS981’, ‘RS987’, ‘RS201’ and ‘RS209’ in the last a few years (Yang et al. 2009).
TR1376A is in the genetic background of ‘Xinong 1376’, a well adapted winter wheat variety with valuable economic traits such as high yield, early maturity, and multiple disease and lodging resistances. In the late 1990s ‘Xinong 1376’ was one of the most widely grown wheat varieties in the Huang-Huai Wheat Zone, the largest wheat growing area of China, and it is still grown in several provinces. In addition, ‘Xinong 1376’ has very good general combining ability, reflected by its many important derivatives, such as ‘Xinong 979’, ‘Xinong 9718’, ‘Xinong 9872’ and ‘Zhengmai 9023’. Therefore, TR1376A has a high potential to facilitate wheat variety improvement and has already been used as a female parent to develop germplasm and varieties.
The authors sincerely thank Kathleen Ross, USDA-ARS and University of Missouri-Columbia, U.S.A. and Dr. Robert McIntosh, Plant Breeding Institute, University of Sydney, N.S.W., Australia for critical reading of the manuscript. This research was supported by the National Transgene Project, China (JY03-B-23-02), the Key Project of Transgene Study and Variety Development, China (2008ZX08002-003), the Academic Cadreman Project of the Northwest A and F University, China (01140306), and the Basic Study Project of the Northwest A and F University, China (CX200903).