Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross‐pollinating crops

We have developed a novel hybridization platform that utilizes nuclear male sterility to produce hybrids in maize and other cross‐pollinating crops. A key component of this platform is a process termed Seed Production Technology (SPT). This process incorporates a transgenic SPT maintainer line capable of propagating nontransgenic nuclear male‐sterile lines for use as female parents in hybrid production. The maize SPT maintainer line is a homozygous recessive male sterile transformed with a SPT construct containing (i) a complementary wild‐type male fertility gene to restore fertility, (ii) an α‐amylase gene to disrupt pollination and (iii) a seed colour marker gene. The sporophytic wild‐type allele complements the recessive mutation, enabling the development of pollen grains, all of which carry the recessive allele but with only half carrying the SPT transgenes. Pollen grains with the SPT transgenes exhibit starch depletion resulting from expression of α‐amylase and are unable to germinate. Pollen grains that do not carry the SPT transgenes are nontransgenic and are able to fertilize homozygous mutant plants, resulting in nontransgenic male‐sterile progeny for use as female parents. Because transgenic SPT maintainer seeds express a red fluorescent protein, they can be detected and efficiently separated from seeds that do not contain the SPT transgenes by mechanical colour sorting. The SPT process has the potential to replace current approaches to pollen control in commercial maize hybrid seed production. It also has important applications for other cross‐pollinating crops where it can unlock the potential for greater hybrid productivity through expanding the parental germplasm pool.     

以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

Pollen dispersal in sugar beet production fields

Pollen-mediated gene flow has important implications for biodiversity conservation and for breeders and farmers’ activities. In sugar beet production fields, a few sugar beet bolters can produce pollen as well as be fertilized by wild and weed beet. Since the crop, the wild beets, and the weed beets are the same species and intercross freely, the question of pollen flow is an important issue to determine the potential dispersal of transgenes from field to field and to wild habitats. We report here an experiment to describe pollen dispersal from a small herbicide-resistant sugar beet source towards male sterile target plants located along radiating lines up to 1,200 m away. Individual dispersal functions were inferred from statistical analyses and compared. Pollen limitation, as expected in root-production fields, was confirmed at all the distances from the pollen source. The number of resistant seeds produced by bait plants best fitted a fat-tailed probability distribution curve of pollen grains (power–law) dependent on the distance from the pollen source. A literature survey confirmed that power–law function could fit in most cases. The b coefficient was lower than 2. The number of fertilized flowers by background (herbicide-susceptible) pollen grains was uniform across the whole field. Airborne pollen had a fertilization impact equivalent to that of one adjacent bolter. The individual dispersal function from different pollen sources can be integrated to provide the pollen cloud composition for a given target plant, thus allowing modeling of gene flow in a field, inter-fields in a small region, and also in seed-production area. Long-distance pollen flow is not negligible and could play an important role in rapid transgene dispersal from crop to wild and weed beets in the landscape. The removing of any bolting, herbicide-resistant sugar beet should be compulsory to prevent the occurrence of herbicide-resistant weed beet, thus preventing gene flow to wild populations and preserving the sustainable utility of the resistant varieties. Whether such a goal is attainable remains an open question and certainly would be worth a large scale experimental study.     

Intein‐mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes

Engineering traits by the assembly of non‐functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N‐ and C‐terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp. gene DnaB and delivered into the wheat genome via biolistic particle bombardment. Both barnase fragments were expressed under the control of a tapetum‐specific promoter. High efficiency of the split barnase system was achieved by introducing GGGGS linkers between the fusion domains of the assembled protein. Depending on the vector version that was transformed, up to 51% of primary transformed plants produced sterile pollen. In the F₁ progeny, the male‐sterile phenotype segregated with both barnase gene fragments. Expression of the cytotoxic barnase in the tapetum did not apparently affect the vegetative phenotype and remained stable under increased temperatures. In addition, the reconstitution of sulphonylurea resistance was achieved by DnaE intein‐mediated assembly of a mutated acetolactate synthase (ALS) protein from rice. The impacts of the technical advances revealed in this study on the concepts for trait control, transgene containment and hybrid breeding are discussed.     

Ultrastructural study of Saffron pollen

Abstract

Pollen of Saffron (Crocus sativus L.), a sterile autotriploid plant was studied using scanning and transmission electron microscopy. The pollen of mature anthers had grains not containing pores or furrows, a discontinuous thin exine and a very thick intine crossed by tubules containing granular electron-dense material. Electron transparent material and lipid bodies were abundant in the cytoplasm which also contained a large number of vesicles. A high percentage of pollen grains showed anomalies.     

Pollen morphology and germination inNarcissus

Pollen grains of several species and varieties ofNarcissus were examined with a light microscope and a scanning electron microscope. Normal pollen grains were kidney- or spindle-shaped with a germination furrow and a reticulate structure similar to that of the pollen grains ofAmaryllis. Pollen grains germinated within 2 to 3 hr. Percentage of germination was dependent upon temperature and treatment. Pollen tubes grew in length up to 1,000 μm and branched occasionally or behaved in strange fashion. Fresh pollen grains germinated more in distilled water at lower temperature than in sucrose-aqueous medium. Both in the presence and in the absence of stigmatic exudate calcium increased the percentage of germination. Gibberellic acid, abscisic acid and coumarin inhibited the pollen germination. Plasmoptysis occurred in all species and in all media tested except in a medium containing coumarin without stigmatic exudates. Plasmoptysis did not seem to be induced by hypotonic medium alone. Pollen of high germination capacity showed a high percentage of plasmoptysis. Based on the results obtained, evolution and sterility of theNarcissus plant was discussed.     

Production of engineered long-life and male sterile Pelargonium plants

ABSTRACT: BACKGROUND: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. RESULTS: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3--4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. CONCLUSION: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.     

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.     

Ectopic expression of <Emphasis Type="Italic">MNX</Emphasis> gene from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> involved in auxin biosynthesis confers male sterility in transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) plants

A transgenic male sterile line of upland cotton was generated by the ectopic expression of the monooxygenase (MNX) gene from Arabidopsis thaliana via Agrobacterium-mediated transformation. The bacterium harbored a plasmid pBinplus carrying a 1.25-kb MNX coding sequence together with a GUS reporter gene; the former was driven by the MS2 promoter of a male sterility gene in Arabidopsis, and the latter was under the control of CaMV 35S promoter. Twenty-seven putative transgenic plants (T₀) were obtained, all of which showed GUS activity and positive signals of NPTII and MNX genes by PCR analysis, and also showed male sterility to some extent. It was further confirmed by Southern blotting that one copy of the NPTII and MNX gene was integrated in the genome of the plants which expressed male sterility to a higher degree. Northern blotting assay also demonstrated that the transgenes stably transcribed in the genome of the transgenic plants in F₄ generation. The male sterile plants usually display lower plant height, shortened internodes, shrunken anthers without pollen grains or with some abortive pollen grains, and unusual leaves with deeper multi-lobes. Microscope observations on the meiosis processes of pollen mother cells (PMCs) showed that the abortion of pollen grains mainly resulted from abnormalities of meiosis such as direct degeneration of PMCs, degenerations of dyad and tetrads, amitosis, lagging chromosomes, and the multi-polar segregations of chromosomes and so on. This study indicates a method of developing novel cotton male sterile materials for potential application in agriculture and for engineering of male sterility in other important crops.     

A study on the allergenicity and ontogeny of Acacia farnesiana pollen grains in guinea pigs

Pollen grains as the angiosperm male gametophytes transfer male genetic material during sexual reproduction. Pollen grains are among the important plant allergens, such that almost 80–90 % of plant allergens are of pollen origin. Acacia farnesiana is a plant with economical values, and due to its resistance to dry climates, it has had a widespread distribution in Southern Iran. This study was aimed at investigating the allergenicity and the ontogeny of pollen grains of A. farnesiana. Pollen grains were collected from the blossoms and flowers of A. farnesiana in the suburbs of Ahvaz-Iran. Pollen extracts (15 %) were prepared in PBS (pH 7.2). 4–6-week-old male guinea pigs (Hartly) were used for allergenicity tests. Skin tests showed a significant increase in flare diameter. Clinical tests also showed a significant change in the levels of eosinophils, neutrophils, and IgE. Histotechnical analysis was performed on male gametophytes, and photographs were taken using a camera-equipped light microscope. Pollen characteristics were identified using both light and electron microscopy. Sporoderm ultrastructure and pollen morphology were studied using Scanning Electron Microscopy. PAGE analysis of the total protein content of mature pollen grains showed seven clear bands of 10–83 kDa.     

The effects of natural variation in pollinator visitation on rates of pollen removal in wild radish,Raphanus raphanistrum (Brassicaceae)

Pollen removal from flowers is an important component of male fitness, but the effects of natural variation in visitation rates on pollen removal are poorly understood. We measured pollen removal over 2 yr in experimental field populations of wild radish, Raphanus raphanistrum. Pollen removal and pollinator visitation over 1-hr periods were measured on previously unvisited flowers. The effects of pollen production and visitation by different insect taxa on pollen removal were determined using multiple regression. Pollen removal rates were extremely high; a median of 84% of pollen produced was removed in 1 hr. Pollen production was far more important than visitation in determining the number of pollen grains removed. Pollen removal increased with increasing numbers of visits by honey bees and small native bees, but increased numbers of syrphid fly visits had no effect. Average visit duration had no effect on pollen removal in 1991, and a marginally negative effect in 1992.     

Anatomical differences on development of fertile and sterile pollen grains of Prunus salicina Lindl.

Anatomical changes occurring during the microsporogenic development of P. salicina Lindl. were studied in male fertile and male sterile genotypes. Male fertile pollen grains showed three well determined pore regions, without ektexine. Intine was thick and surrounded the vegetative cell. Vegetative cells enclosed the generative cells; their cytoplasm was rich in plastids, abundant RER and active mitochondria. Development of sterile pollen was different from the meiosis step. Microspores did not show germination pores and ektexine was continuous around the whole grain. Pollen grains showed an atypical shape. The tapetum persisted after the tetrad stage and showed hypertrophy and vacuole development, resulting in abnormal microspore development. Only a few pollen grains and rudiments of collapsed microspores close to the anther wall were formed at anthesis.     

The Arabidopsis U–box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells,and its mutation leads to conditional male sterility

Pollen formation is a complex developmental process that has been extensively investigated to unravel underlying fundamental developmental mechanisms and for genetic manipulation of the male‐sterility trait for hybrid crop production. Here we describe identification of AtPUB4, a U–box/ARM repeat‐containing E3 ubiquitin ligase, as a novel player in male fertility in Arabidopsis. Loss of AtPUB4 function causes hypertrophic growth of the tapetum layer. The Atpub4 mutation also leads to incomplete degeneration of the tapetal cells and strikingly abnormal exine structures of pollen grains. As a result, although the Atpub4 mutant produces viable pollen, the pollen grains adhere to each other and to the remnants of incompletely degenerated tapetal cells, and do not properly disperse from dehisced anthers for successful pollination. We found that the male‐sterility phenotype caused by the Atpub4 mutation is temperature‐dependent: the mutant plants are sterile when grown at 22°C but are partially fertile at 16°C. Our study also indicates that the AtPUB4‐mediated pathway acts in parallel with the brassinosteroid pathway in controlling developmental fates of the tapetal cells to ensure male fertility.     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis

Very long chain lipids are important components of the plant cuticle that establishes the boundary surface of aerial organs. In addition, these lipids were detected in the extracellular pollen coat (tryphine), where they play a crucial role in appropriate pollen‐stigma communication. As such they are involved in the early interaction of pollen with the stigma. A substantial reduction in tryphine lipids was shown to compromise pollen germination and, consequently, resulted in male sterility. We investigated the role of two long‐chain acyl‐CoA synthetases (LACSs) in Arabidopsis with respect to their contribution to the production of tryphine lipids. LACS was shown to provide CoA‐activated very long chain fatty acids (VLCFA‐CoAs) to the pathways of wax biosynthesis. The allocation of sufficient quantities of VLCFA‐CoA precursors should therefore be relevant to the generation of tryphine lipids. Here, we report on the identification of lacs1 lacs4 double knock‐out mutant lines that were conditionally sterile and showed significant reductions in pollen coat lipids. Whereas the contributions of both LACS proteins to surface wax levels were roughly additive, their co‐operation in tryphine lipid biosynthesis was clearly more complex. The inactivation of LACS4 resulted in increased levels of tryphine lipids accompanied by morphological anomalies of the pollen grains. The additional inactivation of LACS1 neutralized the morphological defects, decreased the tryphine lipids far below wild‐type levels and resulted in conditionally sterile pollen.     

Inducing male sterility of tomato using two component system

Production of hybrid seeds and pursuing heterosis breeding of many crops have been accomplished using male sterile lines. However, not all crops have valuable male sterile lines due to instability of male sterility and absence of a restorer system. In this study, male sterile lines have been induced using a two-component system. The extracellular ribonuclease Barnase was cleaves into two inactive yet complementary fragments, designated as ??Bn-5?? and ??Bn-3??. Both components were controlled by a TA29 promoter. They were transferred into the tomato inbred line ??Yellow tomato?? by Agrobacterium method. Southern blotting identified that 11 transgenic Bn-5 plants (T₀) and 10 transgenic Bn-3 plants (T₀) were obtained. The vegetative phenotypes of all T₀ plants were similar to wild-type, and they were capable of producing viable pollen grains and normal fruit with seeds, indicating that Barnase had lost its function after it being split two partial fragments. After self-pollination, homozygous progenies (T₁) of transgenic Bn-5 and Bn-3 plants were chosen to cross each other, Barnase could be reconstituted and co-expressed in the same cell, which caused the hybrid plants to produce collapsed pollen grains with no viability and thus100?% male sterile plants were obtained. Stamens of male sterile plants were shorter than those of the wild type plants. PCR detection demonstrated that all male sterile plants contained Barnase, but male fertile plants did not. The male sterile plants were crossed with the male fertile inbred lines, and the result showed that hybrid (F₁) plants were capable of producing normal fruit with seeds, and their pollen grain fertility was restored. The co-segregation ratio of Bn-5 and Bn-3 fragments showed 1:1 among hybrid plants. In conclusion, the results verified that the male sterility could be generated by two component system and be used in hybrid seed production. The F₁ between the male sterile plant and the inbred line showed heterotic comparing to both parents. This system needs not breed restoration line.     

Specific features of meiosis in the Siberian Fir (<Emphasis Type="Italic">Abies sibirica</Emphasis>) in the forest Arboretum of the V. N. Sukachev Institute,Russia

Investigating the tolerance of plant reproductive systems to environmental changes has become a research priority under current climate change scenarios. Successful plant conservation requires knowledge of plant reproductive biology, particularly the meiotic characteristics of planted species. Meiosis, as part of microsporogenesis, is a critical plant developmental stage controlling future pollen quality. Meiosis in a Siberian fir (Abies sibirica) plantation, established in the Forest Arboretum of the Sukachev Institute, Russia, was studied from 2002 to 2004. The microsporogenesis pattern found for the Siberian fir appeared to be largely similar to that exhibited by other conifer species. Meiosis in the Siberian fir has the following characteristics: asynchrony, rapid progression of telophases I and II, and parallel and linear spindle arrangements at different meiosis II stages. General and specific meiosis irregularities were recorded at each stage. Some specific features of meiosis and the specific development of some irregularities were revealed. Pollen development analysis showed that irregular pollen grains made up less than 1% of all grains. The specific features of meiosis identified in fir trees growing in the Arboretum indicated low resistance of male reproductive structures to climatic changes and might account for high fir pollen sterility in this new environment.     

水稻非花粉型细胞质雄性不育系及其保持系花药发育过程中Ca2+的分布变化

钙在高等植物中被称为第二信使,与植物的有性生殖有关。为了研究水稻（Oryza sativa L.）花药中钙的定位与花粉败育的关系,利用焦锑酸钾沉淀法研究了非花粉型细胞质雄性不育系G37A及其保持系G37B花药的发育过程及其细胞中Ca^2＋ 的分布变化。研究发现,在2个材料间花药中钙的分布存在大量差异。G37B的可育花药在花粉母细胞时期及二分体时期,很少看到有Ca^2＋的沉积;而在单核花粉时期,Ca^2＋沉积急速地增加,主要定位在绒毡层细胞、花粉外壁外层及乌氏体的表面;随后花药壁上沉积的Ca^2＋减少而花粉的外壁外层仍然有很多Ca^2＋沉积物。相反,G37A的不育花药在花粉母细胞时期和二分体时期有大量的Ca^2＋沉积在小孢子母细胞和花药壁,中间层和绒毡层特别多。在二分体时期之后,不育花药的Ca^2＋沉积减少,特别是绒毡层内切向质膜附近的Ca^2＋几乎消失。但是同时期的可育花药中,有大量的Ca^2＋沉积在绒毡层。不育花药的Ca^2＋沉积在开花几天后消失。根据研究结果推测在不育花药发育早期中更多的钙离子与花粉败育有一定的关系。