Pollination control technologies for hybrid breeding

Efforts in hybrid breeding have made this technology one of the main factors contributing to the substantial global rise in agricultural output over the last few decades. For hybrid breeding, an efficient pollination control system is necessary to avoid the unwanted self-pollination or sib-pollination of the female parental line. This review will provide a historical overview of pollination control systems and their use in hybrid crop breeding. We outline the prerequisites for commercial hybrid breeding and summarize the most important non-biological and biological technologies. Our main focus is on hybrid systems that are based on genetically engineered plants. We describe their suitability for pollination control, propagation of the male-sterile crossing partner, fertility restoration and mixed planting. Additionally, we report on the latest findings in the development of inducible sterility systems and various technologies that enable pollination control via metabolic engineering. We discuss the pros and cons of the different pollination control strategies.     

A straight-forward seed production technology system for foxtail millet (Setaria italica)

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility(CMS)and environment-sensitive genic male sterility(EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet(Setaria italica). Here, we report on the establishment and application of a seed production technology(SPT)...     

Loss of CMS-specific mitochondrial DNA arrangement in fertile segregants of Petunia hybrids

The progeny of somatic hybrid Petunia plants derived from the fusion of a male-fertile line and a cytoplasmic male-sterile (cms) line were examined. Male-fertile progeny derived from three different male-sterile somatic hybrid plants did not exhibit the mitochondrial DNA (mtDNA) arrangement which has previously been correlated with cms in Petunia. The cms-associated mtDNA arrangement was present in the male-sterile predecessors of these fertile revertants. Thus, it is concluded that the loss of this mtDNA arrangement is associated with reversion to fertility in the progeny of the unstable somatic hybrid petunia plants.     

Development of the “Third-Generation” Hybrid Rice in China

Rice is a major cereal crop for China. The development of the ‘‘three-line" hybrid rice system based on cytoplasmic male sterility in the 1970 s(first-generation) and the ‘‘two-line" hybrid rice system based on photoperiod-and thermo-sensitive genic male-sterile lines(second-generation)in the 1980 s has contributed significantly to rice yield increase and food security in China. Here we describe the development and implementation of the ‘‘third-generation" hybrid rice breeding system that is based on a transgenic approach to propagate and utilize stable recessive nuclear male sterile lines, and as such, the male sterile line and hybrid rice produced using such a system is nontransgenic. Such a system should overcome the intrinsic problems of the ‘‘first-generation" and‘‘second-generation" hybrid rice systems and hold great promise to further boost production of hybrid rice and other crops.     

Strategy and utilization of a herbicide resistance gene in two-line hybrid rice

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.     

Floral biology and pollen viability of parental lines of onion hybrids

Parental lines (inbred and hybrid male-sterile lines and inbred pollinators) of prospective F, hybrids from the NVRS spring-sown bulb onion breeding programme were examined to study their floral biology and aspects of pollination under glasshouse conditions. Inbreeding depression occurred in both pollinator and male-sterile inbreds in terms of reduced flower numbers per umbel and a greater proportion of defective pistils. Male-sterile and male-fertile lines differed in their stigma and style characteristics and pattern of development. At flower opening, the styles of male-sterile lines were longer and the stigmatic knob became receptive relatively sooner. The inbred male-sterile line II/3 ms appeared to have a reduced receptive area on its stigmatic knob and remained receptive for a relatively shorter period. Individual lines differed for time of flowering, nectar content and its sugar concentration, and in their effectiveness as male and female parents as judged from pollen tube growth down the style in specific pairings. Assessment of pollen viability using the fluorescein diacetate test did not correlate with assessments based on pollen grain germination and subsequent growth down the style, perhaps as a result of differential receptivity in the inbred lines tested. These results are discussed in relation to future hybrid seed production.     

Male sterility systems in wheat and opportunities for hybrid wheat development

The common wheat (Triticum aestivum L.) is a poly(hexa)ploid, derived from an amphi-diploidization process involving the donor species—Triticum urartu, Aegilops speltoides, Triticum turgidum, and Aegilops tauschii. The genetic diversity of the autogamous wheat is narrow, which is a major reason for lesser rate of yield gain in wheat, in contrast to rice and maize. It is desirable to encourage hybrid breeding, i.e., combining different lines into genetically divergent heterotic pools. Thus, hybrid plants are a unique combination of desired alleles produced by crossing between genetically different parental lines. Hybrid seed production in a crop requires male-sterile female parents along with a reliable outcrossing system. The male-sterile female parent prevents pollen shedding and self-fertilization, maintaining the purity of hybrid seeds. An outcrossing system enhances hybrid seed production. This article emphasizes the biological relevance of crossbreeding and self-pollination in wheat, and reviews different male sterility systems which could be utilized for the development of hybrid wheat. Several biotechnological approaches and their practical utility in generating cross-compatible male-sterile female parent lines have been discussed.     

Evolutionarily inspired solutions to the crop pollination crisis

The global decline in insect diversity threatens pollination services, potentially impacting crop production and food security. Here, we argue that this looming pollination crisis is generally approached from an ecological standpoint, and that consideration of evolutionary principles offers a novel perspective. First, we outline that wild plant species have overcome ‘pollination crises’ throughout evolutionary history, and show how associated principles can be applied to crop pollination. We then highlight technological advances that can be used to adapt crop flowers for optimal pollination by local wild pollinators, especially by increasing generalization in pollination systems. Thus, synergies among fundamental evolutionary research, genetic engineering, and agro-ecological science provide a promising template for addressing a potential pollination crisis, complementing much-needed strategies focused on pollinator conservation.     

甘蓝型油菜CMS微粉活力研究及其对杂交制种纯度的影响

通过对微粉和正常花粉在不同和同一雌蕊上的结实力比较和结实竞争研究,表明微粉虽能够授粉结实,但活力低、萌发慢、持续时间短、受精结实能力差,与正常花粉的竞争力弱,授微粉后间隔３－７２ｈ,再授恢复系花粉,其结实表现仍可达到或超过单独授恢复系花粉的结实率,最高的还超过了只授恢复系花粉结实率的９．５７％;授微粉后间隔０－４８ｈ再授恢复系花粉仍可获得７０．３５％－９１．００％的异交率,说明微粉对油菜杂交制种过     

A novel hybrid seed system for plants

A two-component hybrid seed system has been developed that is broadly applicable and provides for effective generation and maintenance of the male-sterile parent, hybrid seed production and full restoration of fertility in the hybrid seed. The technology is based on the functional interaction of two loci that are inserted in the same position on two homologous chromosomes, and thus are 'linked in repulsion', and that jointly code for male sterility and herbicide resistance, both traits being expressed in heterozygous plants only. The localization to the same locus on a chromosome is achieved by the genetic transformation of plants with a construct containing both genetic elements (loci), and subsequent derivatization from the primary pro-locus of the two precursor lines using site-specific deletions. The functional interaction of the two loci is achieved through intein-based trans -splicing of two pairs of complementary protein fragments that provide for male sterility and herbicide resistance. Unlike the hybrid seed systems that are currently in use, the technology relies on the genetic modification of just one parent, and is therefore much simpler to develop and use. Arabidopsis has been used for the proof of principle presented here, but the essential elements of the technology are generic and have been shown to work in many crop species.     

利用生物技术创建主要作物雄性不育杂交育种和制种的技术体系

雄性不育技术在作物杂种优势利用和杂交种生产中发挥着重要作用。基于核质互作雄性不育的“三系法”与光温敏核不育的“两系法”已经在水稻等主要作物的杂交制种中获得了广泛应用,但是存在着资源利用效率低、育性不稳定、易受外界环境影响等诸多问题。近三十年来,利用生物技术创建不同类型的植物雄性不育系取得了一系列突破性进展。主要针对玉米、水稻、小麦三大作物的基因工程雄性不育技术的最新进展进行总结,特别详细地描述了本实验室最近研究创制的玉米多控不育技术体系,以期为相关研究和产业化应用提供技术参考。     

Human welfare and its connection to nature: What have we learned from crop pollination studies?

Human welfare depends on the function of natural systems. This idea is paradigmatic to ecologists and has been the theme of a growing branch of applied ecology. I examine the narrative of human dependence on nature by considering the literature on crop pollination by animals and its importance for food production. Making the connections between human welfare and natural systems is seen as a way to better motivate society to make better decisions, but the debate around crop pollination has been surprisingly contentious. There have been confusing messages, disagreements on the facts, an unfortunate focus on dire projections for the future and a lesser focus on solutions. Most of these problems arise not from poor science but instead from poor communication of complex ideas and differences in perspective, such as the deep disciplinary gap between agricultural scientists and ecologists. By understanding these problems, we can improve the way we do our science and communicate our ideas. I argue that ecologists should continue to communicate the principle that human welfare depends on the function of natural systems and discuss how we can do so in a way that is more genuinely connected to society's needs, such as growing food. If we succeed, we will be changing an intellectually interesting conversation into a dialogue that influences how society interacts with nature.     

Buttercup squash provides a marketable alternative to blue hubbard as a trap crop for control of striped cucumber beetles (Coleoptera: Chrysomelidae)

Winter squash is a vital agricultural commodity worldwide. In the Northeastern United States, the primary insect pest is the striped cucumber beetle, Acalymma vittatum F. Using a Blue Hubbard squash (Cucurbita maxima Duchesne) perimeter trap crop system can reduce insecticide use by >90% in butternut squash (C. moschata Poir), the primary winter squash grown in this region. Despite the savings in insecticide costs, growers may be reluctant to give up field space for a perimeter crop of Blue Hubbard squash, which comprises only 5% of the winter squash market in New England as compared with 19% for buttercup squash. Finding a more marketable trap crop would lower the barrier for adoption of this system. We tested eight varieties of three species of cucurbits for attractiveness to beetles relative to Blue Hubbard and butternut squash, and chose buttercup squash as the most promising replacement. We compared the effect of a buttercup border, Blue Hubbard border, or control (no border) on beetle numbers, herbivory, insecticide use, pollination, and pollen limitation in the main crop. We found that buttercup squash performed equally well as Blue Hubbard as a trap crop, with 97% reduction in total insecticide use compared with control fields. Honey bees (Apis mellifera L.) and squash bees (Peponapis pruinosa Say) were the predominant pollinators, and border treatments did not affect visitation. Hand pollination did not increase reproduction or yield, indicating that natural pollination was sufficient for full yield. This study confirms the effectiveness of perimeter trap crop systems and offers growers a more marketable trap crop for managing cucumber beetle damage.     

玉米核雄性不育的分子机制研究与应用分析

玉米细胞核雄性不育突变体是研究花粉发育和减数分裂的理想材料,也是杂种优势利用的重要种质资源。随着分子生物技术的快速发展,部分玉米细胞核雄性不育基因陆续被成功克隆,为其在工程不育化杂交种生产中的应用奠定了基础。综述了近年来对玉米细胞核雄性不育的细胞学鉴定、基因克隆和分子机制的研究进展,并对其应用途径和前景进行了分析。     

Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination

Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T₀-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained.     

Crop management affects pollinator attractiveness and visitation in oilseed rape

Ecological intensification of agriculture implies managing ecological processes to improve performance of agricultural systems. However, impacts on relevant ecological functions such as insect pollination from other crop management factors are poorly explored. Pest insects and crop resources such as water availability can directly affect crop yields, but it is unknown if there are indirect effects through effects on insect pollination. With a factorial experiment, we examined how irrigation and control of pollen beetles affected crop attractiveness and pollinator visitation in an open-pollinated spring oilseed rape cultivar. We studied how irrigation and pest control modified the production of flowers and nectar in oilseed rape, and if this in turn affected the flower-visitation of honey bees and bumble bees. Pest control increased the number of oilseed rape flowers by 69%, and the amount of nectar per flower with 36%, but for the latter only in non-irrigated plots. Furthermore, we found higher pollinator densities in plots with reduced pollen beetle densities. Pest control also reduced the number of non-legitimate flower visits, suggesting higher pollination efficiency in plots with reduced pollen beetle densities. We show that crop management affects the value of mass-flowering crops as a resource for pollinating insects. Development of pest control tools that are harmless to pollinators could increase the value of flowering crops as food resources for pollinating insects.     

On the inconsistency of pollinator species traits for predicting either response to land‐use change or functional contribution

The response and effect trait framework, if supported empirically, would provide for powerful and general predictions about how biodiversity loss leads to loss in ecosystem function. This framework proposes that species traits will explain how different species respond to disturbance (i.e. response traits) as well as their contribution to ecosystem function (i.e. effect traits). However, predictive response and effect traits remain elusive for most systems. Here, we use data on crop pollination services provided by native, wild bees to explore the role of six commonly used species traits in determining both species’ response to land‐use change and the subsequent effect on crop pollination. Analyses were conducted in parallel for three crop systems (watermelon, cranberry, and blueberry) located within the same geographical region (mid‐Atlantic USA). Bee species traits did not strongly predict species’ response to land‐use change, and the few traits that were weakly predictive were not consistent across crops. Similarly, no trait predicted species’ overall functional contribution in any of the three crop systems, although body size was a good predictor of per capita efficiency in two systems. Overall we were unable to make generalizable predictions regarding species responses to land‐use change and its effect on the delivery of crop pollination services. Pollinator traits may be useful for understanding ecological processes in some systems, but thus far the promise of traits‐based ecology has yet to be fulfilled for pollination ecology.     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties

Key message

We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties.

Abstract

In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F₁ seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F₁ seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.