种子萌发及其调控的研究进展

大多数有花植物通过有性生殖和产生种子繁衍后代, 种子的成功萌发和正常成苗决定植物物种的繁衍与生存。种子萌发容易受到机械伤害、病害和环境胁迫的影响, 是植物生活周期中最重要和最脆弱的阶段, 对于一年生和二年生植物则更为重要。种子萌发是一个复杂的多步骤过程, 在这个过程中静止的干燥种子迅速恢复代谢活性, 完成胚伸出周围结构的细胞事件, 以及为随后的幼苗生长做准备。本文综述了近年来种子萌发及其调控的研究进展, 主要包括种子萌发过程中的重要生理事件, 与种子萌发有关的蛋白合成、翻译后修饰和蛋白质组, 以及植物激素对种子萌发的调节。此外, 我们还提出了种子萌发的能量刺激假说, 此假说为回答植物学、农学和园艺学中的两个基本问题, 即胚怎样从它的周围结构中伸出完成萌发？胚的伸出怎样被阻断以至于种子被维持在休眠状态？以及减少禾谷类作物种子和粮食生产中发生的穗萌现象提供了新的研究思想。     

Germination characters in wild and cultivated Anemone coronaria L.

Summary Achenes of wild forms of Anemone coronaria growing in Israel differ in their germination requirements from achenes of the cultivated de Caen type. The optimum temperature for dark germination was between 10–15°C in the former and between 15–20°C in the latter. Maximum daily rates of germination were higher, reaching 16% per day, and the minimum lag period between sowing and seedling emergence was shorter in achenes of the cultivated than in those of the wild anemone when the two types were incubated under identical conditions. Wild achenes showed a marked post-harvest maturation requirement, and attained full germination, a minimum pre-emergence lag period after sowing, and a maximum daily germination rate only when dry-stored for several months subsequent to harvesting. In the cultivated plant this requirement was far less pronounced or absent. Most embryos in freshly harvested achenes of both the wild and the cultivated anemone were torpedo-shaped but in the cultivated form embryos were larger. In both types embryos remained unchanged in shape and size during dry storage.     

Shortening the generation cycle in faba bean (Vicia faba) by application of cytokinin and cold stress to assist speed breeding

The aim of this study was to reduce the length of the breeding cycle for faba bean by accelerating seed setting. We examined the mode and time of exogenous 6-benzylaminopurine (BAP) (cytokinin) application, and cold treatments and their combinations in two faba bean genotypes. Acropetal node number of pod and seed set and pollen viability were recorded during the experiments. Application of BAP improved pollen germination. The application of 10^–5 M BAP 4 days after flowering increased seed set at the lower nodes. Cold treatment (8/4°C day/night for 2 days) after the onset of flowering induced the formation of more pods and faster pod set compared to the non-cold treatment. The time to first seed was significantly reduced, and pollen viability was increased in plants exposed to cold treatment. Increased pollen viability also showed a significant positive correlation with seed setting. The combinations of 10^–5 BAP and cold treatment together had similar and independent effects. These results will accelerate plant breeding in faba bean by providing additional tools for reducing generation time.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific hybridization between Cicer arietinum and C. pinnatifidum

An interspecific hybrid between Cicer arietinum cv. GL 769 and a wild species C. pinnatifidum was obtained after emasculation, pollination and application of growth regulators. Ovules were cultured and embryos were later dissected to obtain hybrid plants. These plants were albinos and morphologically resembled C. pinnatifidum. Shrivelled seeds were also obtained in 2% of the crosses, which on germination gave rise to albino plants. These plants did not survive beyond 20 days. The hybrid nature of these plants was confirmed by esterase isozyme studies. Hybrid shoots obtained from germinating embryos were cultured on modified ML-6 medium with BAP 2 mg/1, IAA 0.5 mg/1, where they turned green after 3–4 weeks. Transmission electron microscopy (TEM) studies on leaf sections from green hybrid shoots showed an improvement in the chloroplast structure, with better organized grana.     

Seasonal changes in the incidence of apomixis of diploid,triploid, and tetraploid plants of Paspalum cromyorrhizon

Summary Changes in incidence of apomixis were determined at different intervals of the flowering period in a highly sexual diploid cytotype of Paspalum cromyorrhizon Trin., a natural tetraploid cytotype of the same species, and in the triploid and tetraploid intraspecific hybrids that were produced by pollinating the 2x cytotype with pollen of the tetraploid. Reproductive behaviour was determined from observations of embryo sacs in mature ovules. Gametophytic apomixis in this species in characterized by aposporous embryo sacs which differ from normal sacs by their number, orientation inside the ovule, their dimensions and shape, and the absence of antipodal cells.The potential for apomictic reproduction increased in relation to the ploidy level, suggesting a gene dosage effect in the incidence of apomixis. In the three ploidy levels, the highest incidence of apomixis was observed when the plants reached the maximum number of flowering heads. These findings suggest that the same environmental conditions that favour flowering should also be responsible for increasing the incidence of apomixis. An additional experiment under controlled conditions indicated that the light regime should be one of the factors that govern the amount of apomictic reproduction. Thus, polyploidy and apomixis should be of special significance in the dispersion and evolution of this grass species. The 4x facultative apomictic cytotype is far more frequent in nature than the highly sexual 2x. Moreover, most of the seeds produced in a flowering season should arise asexually. So, apomixis rapidly increases the number of plants bearing the successful maternal genotype, and sexual reproduction becomes important in adverse environmental conditions.     

Microspore culture of white cabbage, Brassica oleracea var. capitata L.: Genetic improvement of non-responsive cultivars and effect of genome doubling agents

For the efficient application of haploid induction procedures in cabbage breeding, a sufficient number of regenerants should be achieved in a broad spectrum of genotypes. However, the majority of genotypes are somewhat recalcitrant. The efficiency of microspore culture was tested by crossing a responsive (28.7 embryos per Petri dish) and a non- responsive (0.1 embryo) cabbage cultivar. The embryo yield of one progeny was intermediate (18.9) while two were superior to the best parent cultivar (52.9 and 64.0 embryos). Thus, genes for haploid embryogenesis, present in responsive lines, can be effectively transmitted to responsive × non-responsive hybrids. Abscisic acid-induced desiccation of embryos was used for the efficient regeneration of plants. High germination percentages (54.7-70.6%) followed by normal plantlet development were achieved. Spontaneous genome doubling measured at the plantlet stage differed markedly in untreated genotypes. The percentage of diploids ranged from 21 to 67%. The effects of two antimitotic drugs applied to freshly isolated microspores were determined in two experiments. In the first experiment, trifluralin (0.5 and 1.0 mg:l) had no effect on embryo induction while oryzalin partly (0.125-0.25mg/l) or completely (0.5.mg/l) inhibited the formation of embryos. In the second experiment, higher concentrations of trifluralin increased the proportion of diploidized plants. Application of anti-mitotic drugs to microspores did generally not improve the overall production of haploid plants, which was higher in an untreated control.     

In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis

Embryo development in vivo has been studied in four Citrus aurantium L. polyembryonic genotypes. Seeds were collected 65, 85, 105, 125 and 220 days after pollination (DAP). None of the immature seeds harvested 65 and 85 DAP contained visible embryos. A single embryo at a more advanced developmental stage was observed in the central position at the micropylar apex of the embryo sac in about 74% of seeds harvested at 105 DAP, while at 125 and 220 DAP the majority of seeds had two or more embryos at the same developmental stage crowded together. Restriction fragment length polymorphism (RFLP) analysis of lowand high-copy-number nuclear DNA was used to distinguish zygotic from nucellar seedlings. Analysis of plantlets derived from in vitro culture of the bigger embryos, located in the central position at the micropylar apex of the embryo sac of seeds harvested at 105 DAP, established that the frequencies of zygotic embryos ranged from 82 to 88%. Media for immature embryo germination in vitro were based on the nutrients and vitamins of Murashige and Skoog (MS) and Murashige and Tucker (MT) media supplemented with various concentrations of sucrose and growth regulators. A total of 76% of globular stage embryos (<0.3mm) germinated on MT medium containing 150 mM sucrose and 14.4 μM gibberellic acid. Heart stage embryos (0.3-0.8 mm) germinated at 95% on MT medium supplemented with 150 mM sucrose and 2.9 μM gibberellic acid. The addition of 500 mg/l malt extract to MS medium increased the germination of early cotyledon stage (0.8-2.0mm) embryos to 98%. The optimum sucrose concentration for embryo rescue was 150 mM for the three embryo developmental stages. The ability to form plants in vitro strongly increased with increasing embryo developmental stage.     

花生体细胞胚诱导及植株再生研究

为了建立花生体细胞胚诱导及植株再生体系,为花生分子育种提供技术支撑,以花生品种‘桂花30’和‘桂花771’为材料,采用预培养3天的种子胚小叶、下胚轴、子叶节为外植体,在添加外源激素的MS培养基中使体细胞脱分化形成体细胞胚,再分化成植株。结果表明,在所设定2,4-D浓度（3,5,10,15,20 mg/L）范围内,胚小叶最容易诱导形成体细胞胚,2,4-D的适宜浓度为10 mg/L,经过约30天培养,可产生大量体细胞胚,‘桂花30’和‘桂花771’的平均诱导率分别为55.37%和36.72%。平均每个外植体产胚量分别为5.68个和4.27个。将诱导形成的体细胞胚转接到6-BA浓度由5 mg/L逐渐降低到1.5 mg/L的MS培养基中,体细胞胚萌发再生成无根小苗,正常植株再生率‘桂花30’为32.6%,‘桂花771’为23.5%。将无根苗转接到生根培养基中可获得完整植株。花生是较难诱导体细胞胚形成的作物之一,筛选合适的基因型、外植体和激素浓度是获得较高体细胞胚发生率和植株再生率的关键技术。     

Plant Regeneration from in vitro Cultured Anthers of Black Mustard (Brassica nigra Koch)

Anthers of Brassica nigra, excised from fresh as well as cold-pretreated (3 days at 3 ± 2°C) buds cultivated on modified B₅ medium (Gamborg et al. 1968) containing sucrose level varying from 2 % to 10 %, along with 1O^?6M BAP (benzylaminopurine) and 9 × 10^?6M 2,4-D (2,4-dichlorophenoxyacetic acid), developed calli and/or embryos. The latter response was observed only in anthers reared on media containing 6 % or higher levels of sucrose. On media containing two or four per cent sucrose, the anthers produced calli, exclusively. The growth of embryos was inhibited or else they started callusing if left on the media containing higher levels of sucrose. However, on transfer to MS medium (Murashige and Skoog 1962), containing 2 % sucrose, embryos started callusing and subsequently a few secondary embryos differentiated. Such embryos were sub-cultured on MS + 5 × 10^?6M BAP + 2 % sucrose, wherein numerous shoots developed from embryos. The shoots were rooted by transferring to a medium containing 5 × 10^?6M NAA (naphthalene acetic acid). Within two months of culture, some of these plants started flowering in vitro.     

Embryo rescue in plants—a review

Summary The plant breeders usually rescue inherently weak, immature or hybrid embryos to prevent degeneration. The successful production of plants from the cultured embryos largely depends upon the maturation stage and the composition of the medium. Abortion of embryos at one or the other stage of development is a characteristic feature of distant hybridization. For the first time successful embryo culture to obtain an interspecific cross between Linum perenne × L. austriacum was demonstrated by Laibach (1925, 1929). Since then several refinements have been made in embryo culture/embryo rescue techniques which have been a popular approach for raising hybrids from a number of incompatible crosses. Currently embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining plants from inherently weak embryos, obtaining haploid plants as well as for shortening the breeding cycle.     

Vernalization of immature embryos of winter wheat genotypes

Summary The effect of direct vernalization of immature embryos on flowering was studied in six winter wheat genotypes. Fourteen-, 17-, and 20-day-old embryos were excised and vernalized for 0–6 weeks on synthetic medium during a conditioning period. Percent germination of embryos was high (overall 96.1%), and free from genotypic effects. Genotypes differed for flowering in response to cold treatment of excised embryos. Embryo vernalization was as effective as or more than conventional vernalization (control, seedling vernalization for 6 weeks). Seventeen-day-old embryos were the most responsive to vernalization. With a 5-week vernalization of 17-day-old embryos, the percentage of plants anthesed was higher than those from 14-and 20-day-old embryos. For 17-day-old embryos vernalized for 5 weeks, the mean number of days from culture to anthesis was less than that of 6 week vernalization, less than that of 14- and 20-day-old embryos, and less than controls.Purdue Univ., Agronomy Dept., W. Lafayette, IN 47907, USA.     

Plant Regeneration from the Hybridization of Brassica juncea and B. napus Through Embryo Culture

Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocals with the aid of embryo culture techniques. A better response of hybrid embryo culture was obtained from two cross combinations of B. juncea × B. napus (Ames 24521 × Huyou 15 and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg l^?1 naphthylacetic acid (NAA) + 1.5 mg l^?1 6‐benzylaminopurine (BAP) and with 0.3 mg l^?1 NAA + 2.0 mg l^?1 BAP. Callus formation and plant regeneration on these two media reached 55.43 and 26.65 %, and 66.98 and 24.61 %, respectively.     

Ovule and embryo culture to obtain hybrids from interspecific incompatible pollinations in chickpea

Many of the wild species of chickpea were not accessible to the improvement of chickpea due to cross incompatibility. In these interspecific incompatible crosses, fertilization takes place but the embryo aborts a few days later. The only way to obtain hybrid plants is by the application of growth regulators to pollinated pistils to prevent initial pod abscission and to save the aborting hybrid embryos by embryo rescue techniques. Although there are a few papers on regeneration from different explants of chickpea, information on embryo rescue techniques is not available. The paper summarises the embryo rescue techniques developed for chickpea, by the use of which hybrid plants between C. arietinum and C. pinnatifidum were produced. The paper also emphasises the effect of genotype to successfully obtain hybrids. The morphology of the hybrid plants resembled the male parent in leaf structure and growth habit. The colour of the flowers produced on the hybrid plant was pale violet, resembling the male parent whose flowers were violet in colour. The flower colour of the female parent was white. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Effectiveness of Vernalization of Immature Embryos of Winter Wheat var. Grana as Related to Age and Exogenous Phytohormones

Starting from the 10th day after pollination, immature embryos of winter wheat var. Grana were isolated and then vernalized for 4 to 8 weeks on Murashige and Skoog nutrients containing BAP (0.5 or 2 mg/dm³), NAA (0.5 or 2 mg/dm³), or GA₃ (5 or 20 mg/dm³). Vernalized seedlings were cultivated in a greenhouse and the number of days to the heading of individual plants as well as the percentage of plants capable of generative development were estimated. The lower limit of size for 50 % survival of embryos strongly depended on the phytohormone used: from 0.9 mm in control, 1.1 mm in nutrient containing BAP, 1.2 mm for NAA, up to 1.7 mm in nutrient with GA₃. Exogenous GA₃ was lethal for embryos younger than 18 days but induced elongation of older embryos. Embryos isolated 2.5 to 4 weeks after pollination showed minimal requirements for the length of vernalization. BAP increased the percentage of heading plants originated from the youngest embryos. GA₃ improved partial vernalization, strongly increasing the percentage of heading plants, but did not change the time from the end of vernalization to heading. It has been postulated that GA₃ increases number of plants capable of overcoming the threshold of induction of generative development but does not accelerate the flowering process. Hormonized plants showed no deformation of generative development. As the effectiveness of vernalization increased, the heading of plants was faster but they were shorter, forming spikes with a smaller number of spikelets and producing fever lateral shoots. The very young embryos probably have in reserve sufficient amounts of auxins and gibberellins and therefore exogenous GA₃ decreases their viability or even exerts a deleterious effect. However, as the embryos' ageing, gibberellin starvation develops. This being especially pronounced during vernalization. The de novo synthesis or activation of gibberellins takes place during the second stage of vernalization. This is why exogenous hormone improves the effectiveness of partial vernalization, though it is not possible to substitute by gibberellins the vernalization requirements of immature embryos.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

Clonal propagation of Triticum durum Desf. from immature embryos and shoot base explants

Summary Immature embryos from five durum wheat cultivars were grown on Murashige and Skoog medium supplemented with two concentrations of kinetin or 6-benzylaminopurine (BAP). The embryos cultured on the medium containing 5 mg/l of BAP proliferated several axillary shoots. Shoot base segments subcultured on the same medium gave more shoot proliferation. The shoots developed into ear-bearing plants. This technique could be used for clonal propagation of wheat.     

Comparison of callus culture with embryo culture at different times of embryo rescue for primary triticale production

Summary Callus culture of immature wheat-rye hybrid embryos was compared with embryo culture in two experiments. Embryos were rescued from field grown mother plants at two day intervals 13–21 days after pollination and plated for 1) callus culture on Murashige and Skoog medium (MS) supplemented with 2 mg/12,4-dichlorophenoxy acetic acid followed by plant regeneration on hormone free MS medium with half strength mineral salts, 2) embryo culture on Taira and Larter medium (TL). Observations were made on embryo size and condition at time of rescue (Experiment 1) and embryo development directly into plants (embryo culture) or through embryogenesis (callus culture). Fewer 19 and 21 day old embryos developed into plants from callus culture than from embryo culture in Experiment 1. Callus culture was more efficient than embryo culture in promoting plant recovery from 17 day old embryos in Experiment 2. The number of plants per embryo was significantly higher from callus culture than from embryo culture. In both experiments callus culture promoted embryogenesis in more embryos than developed in embryo culture. Embryo rescue 15–17 days after pollination was optimal in both experiments.     

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上;不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B5培养基幼胚萌发率为70.9%~88.3%, 平均82.0%;1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%;两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。     

Development and characterization of interspecific hybrids of Trifolium alexandrinum×T. apertum using embryo rescue

This is the first report on the development of interspecific hybrids between Trifolium alexandrinum and T. apertum using embryo rescue and characterization of F₁ plants. T. apertum was used as the male parent and T. alexandrinum as the female parent. Development of interspecific hybrids under natural conditions is not successful and so embryo rescue was attempted. Of the several combinations tried, pollination 2 days after emasculation and embryos rescued 11 days after pollination was found to be the best. For embryo culture, EC3 medium consisting of MS basal supplemented with 2.3 μM kinetin and 3% sucrose was used. Germinated embryos were transferred to LSP3 medium 25 days after inoculation wherein most of the cultures showed multiple shoots that were split and subcultured on RL1 medium for rooting. After hardening, about 75% of hybrids were successfully transferred to the field. The hybrids, in general, showed morphological traits intermediate between the two parents; however, a few hybrids showed better growth than either parent. Some F₁ plants were almost 3 weeks later in flowering than the female parent. Pollen fertility among these plants ranged from 78 to approximately 100%. Chromosomal associations at diakinesis and isozyme banding patterns for acid phosphatase (ACP), superoxide dismutase (SOD) and peroxidase also confirmed the hybrid nature of the plants.