Temperature sensitive diphtheria toxin confers conditional male-sterility in Arabidopsis thaliana

A gene encoding a temperature-sensitive diphtheria toxin A chain (DTA) polypeptide was fused to the Arabidopsis thaliana tapetum-specific A9 promoter. Expression of the chimaeric gene in transgenic A. thaliana lines resulted in plants that were male-sterile, but female-fertile, when grown at 18 degrees C, and fully self fertile at 26 degrees C. No pollen grains were found on the anthers of transgenic plants grown at 18 degrees C, although aggregated pollen grains were found inside the anthers. Electron microscopy revealed discrete alterations in the tapetal cells of the male-sterile transgenic plants. The strength of the phenotype observed in segregants correlated with the level of expression of the gene and the copy number. The low frequency at which fully male-sterile plants were generated suggests that the temperature-sensitive DTA protein is disabled as a cytotoxin, relative to the wild-type protein activity.     

RNAi-mediated male sterility of tobacco by silencing TA29

The superior performance of F₁ hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.     

Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor

Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.     

Gibberellin regulates post-microsporogenesis processes in petunia anthers

Previous studies have suggested that gibberellins (GAs) are produced in petunia anthers and transported to the corolla to induce growth and pigmentation. In this work, we studied the role of GA in the regulation of anther development. When petunia plants were treated with the GA-biosynthesis inhibitor paclobutrazol, anther development was arrested. Microscopic analysis of these anthers revealed that paclobutrazol inhibits post-meiotic developmental processes. The treated anthers contained pollen grains but the connective tissue and tapetum cells were degenerated. A similar phenotype was obtained when the Arabidopsis GA-signal repressor, SPY, was over-expressed in transgenic petunia plants, i.e. anther development was arrested following microsporogenesis. The expression of the GA-induced gene, GIP , can be used in petunia as a molecular marker to study GA responses. GA₃ treatment of young anthers promoted, and paclobutrazol inhibited, GIP expression, suggesting that the hormone controls the natural activation of the gene in the anthers. Analyses of GIP expression during anther development revealed that the gene is induced only after microsporogenesis. This observation further suggests a role for GA in the regulation of post-meiotic processes during petunia anther development.     

Distribution of the glutamine synthetase isozyme GSp1 in maize (Zea mays)

In maize (Zea mays L.), GSp1, the predominant GS isozyme of the developing kernel, is abundant in the pedicel and pericarp, but absent from the endosperm and embryo. Determinations of GSp1 tissue distribution in vegetative tissues have been limited thus far to root and leaves, where the isozyme is absent. However, the promoter from the gene encoding GSp1 has been shown to drive reporter gene expression not only in the maternal seed-associated tissues in transgenic maize plants, but also in the anthers, husks and pollen (Muhitch et al. 2002, Plant Sci 163: 865-872). Here we report chromatographic evidence that GSp1 resides in immature tassels, dehiscing anthers, kernel glumes, ear husks, cobs and stalks of maize plants, but not in mature, shedding pollen grains. RNA blot analysis confirmed these biochemical data. In stalks, GSp1 increased in the later stages of ear development, suggesting that it plays a role in nitrogen remobilization during grain fill.     

Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation

Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. ‘Hillary’ was 55.3% whereas that of cv. ‘Tenorio’ reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l⁻¹ allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.     

Overexpression of The Calcium-Dependent Protein Kinase OsCDPK2 in Transgenic Rice is Repressed by Light in Leaves and Disrupts Seed Development

Independent transgenic rice lines overexpressing the rice CDPK isoform OsCDPK2 were generated by particle bombardment. High levels of OsCDPK2 were detected in leaves removed from etiolated plants, as well as in stems and flowers. However, there was no overexpression in green leaves that had been exposed to light, confirming that OsCDPK2 protein stability was subject to light regulation. The morphological phenotype of transgenic plants producing high levels of recombinant OsCDPK2 was normal until the onset of seed development. Flowers developed normally, producing well-shaped ovaries and stigmas, and mature anthers filled with pollen grains. However, seed formation in these plants was strongly inhibited, with only 3–7% of the flowers producing seeds. Seed development was arrested at an early stage. We discuss these data with respect to the possible requirement for specific CDPK isoforms during rice seed 4.4ptdevelopment.     

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.     

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

Transgenic studies on the involvement of cytokinin and gibberellin in male development

Numerous plant hormones interact during plant growth and development. Elucidating the role of these various hormones on particular tissue types or developmental stages has been difficult with exogenous applications or constitutive expression studies. Therefore, we used tissue-specific promoters expressing CKX1 and gai, genes involved in oxidative cytokinin degradation and gibberellin (GA) signal transduction, respectively, to study the roles of cytokinin and GA in male organ development. Accumulation of CKX1 in reproductive tissues of transgenic maize (Zea mays) resulted in male-sterile plants. The male development of these plants was restored by applications of kinetin and thidiazuron. Similarly, expression of gai specifically in anthers and pollen of tobacco (Nicotiana tabacum) and Arabidopsis resulted in the abortion of these respective tissues. The gai-induced male-sterile phenotype exhibited by the transgenic plants was reversible by exogenous applications of kinetin. Our results provide molecular evidence of the involvement of cytokinin and GA in male development and support the hypothesis that the male development is controlled in concert by multiple hormones. These studies also suggest a potential method for generating maintainable male sterility in plants by using existing agrochemicals that would reduce the expense of seed production for existing hybrid crops and provide a method to produce hybrid varieties of traditionally non-hybrid crops.     

Alloplasmic male-sterile <Emphasis Type="Italic">Brassica juncea</Emphasis> with <Emphasis Type="Italic">Enarthrocarpus lyratus</Emphasis> cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species

A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; E(l)E(l)). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration ( Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and E(l) chromosomes in monosomic addition plants (2n = 18II+1E(l)). The percent pollen fertility of restored F(1) ( lyr CMS x putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS ( lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F(1) hybrids, remains a limitation.     

Overexpression of <Emphasis Type="Italic">Zm401</Emphasis>, an mRNA-like RNA,has distinct effects on pollen development in maize

We previously identified a 0.7 Kb cDNA fragment of Zm401, a novel pollen-specific gene in maize (Zea mays). However, little information is known about the function of Zm401 in pollen development. The full-length of Zm401 cDNA was amplified by 5′ RACE and 3′ RACE and both sequence analysis and in vitro translation of Zm401 showed that it belonged to an mRNA-like non-coding gene. To analyze its possible biological roles in pollen development, the Zm401 cDNA was overexpressed in transgenic maize under the control of a pollen specific promoter Zm13 or a CaMV 35S promoter. RT-PCR and RNA gel blot analysis indicated that the expression level of Zm401 in leaves and anthers of transgenic plants was much higher than that of non-transformants. Compared with the non-transformed maize, transgenic maize showed distinct phenotypes, such as abnormal tassels and degenerate anthers. The histological observation showed that the development of pollen grains and anthers in transgenic plants were abnormal. These abnormalities include delayed degradation of tapetum, asynchronous fusion of pollen sacs, and aborted pollen grain development. Furthermore, the pollen viability in six transgenic plants ranged from 1.24% to 6.63%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. These results suggest that Zm401 is involved in the regulation of pollen development. This article demonstrated Zm401, as a non-coding RNA, plays an essential role in pollen development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of Somatic embryogenesis receptor-like kinase 1 in controlling pollen production of the Gossypium anther

In flowering plants, male gametophytes are generated in anthers from microsporocytes. However, more evidence is needed to reveal the genetic mechanisms which regulate the differentiation and interaction of these highly specialized cells in anthers. Here we report the characterization of a series of male-sterile cotton (Gossypium hirsutum) mutants, including mutants with normal fertility, semi-sterility and complete sterility. These mutants are forms of transgenic cotton containing RNAi vectors with partial cDNA fragments of GhSERK1. The GhSERK1 gene encodes a putative leucine-rich repeat receptor protein kinase (LRR-RLK), and generally has 11 domains. In previous research, we found plants containing GhSERK1 produce an abundance of male reproductive tissue. In this paper, three RNAi constructs were designed separately to analyze its function in anther. After the three RNAi vectors were transformed into the cotton, transgenic plants with the specialized fragment exhibited normal fertility or the pollen energy decreased slightly, as ones with the homologous fragments exhibited various degrees of male sterility with different expression levels of GhSERK1 mRNA. In conclusion, for the transgenic plants with conserved fragments, lower expression levels of GhSERK1 mRNA were in transgenic plants, and a higher degree of male sterility was observed. Taking together, these findings demonstrate the GhSERK1 gene has a role in the development of anthers, especially in the formation of pollen grains. Also, we infer there must be another homolog of GhSERK1 in cotton, and both of GhSERK1 and its homolog function redundantly as important control points in controlling anther pollen production.     

Towards male sterility in Pinus radiata--a stilbene synthase approach to genetically engineer nuclear male sterility

A male cone-specific promoter from Pinus radiata D. Don (radiata pine) was used to express a stilbene synthase gene (STS) in anthers of transgenic Nicotiana tabacum plants, resulting in complete male sterility in 70% of transformed plants. Three plants were 98%-99.9% male sterile, as evidenced by pollen germination. To identify the stage at which transgenic pollen first developed abnormally, tobacco anthers from six different developmental stages were assayed microscopically. Following the release of pollen grains from tetrads, transgenic pollen displayed an increasingly flake-like structure, which gradually rounded up during the maturation process. We further investigated whether STS expression may have resulted in an impaired flavonol or sporopollenin formation. A specific flavonol aglycone stain was used to demonstrate that significant amounts of these substances were produced only in late stages of normal pollen development, therefore excluding a diminished flavonol aglycone production as a reason for pollen ablation. A detailed analysis of the exine layer by transmission electron microscopy revealed minor structural changes in the exine layer of ablated pollen, and pyrolysis-gas chromatography-mass spectroscopy indicated that the biochemistry of sporopollenin production was unaffected. The promoter-STS construct may be useful for the ablation of pollen formation in coniferous gymnosperms and male sterility may potentially be viewed as a prerequisite for the commercial use of transgenic conifers.