水稻游离小孢子培养最新研究进展

水稻小孢子培养技术不但可用来生产单倍体植株和加倍单倍体植株,而且可望成为转基因的理想受体系统。目前转基因技术、分子杂交技术与小孢子培育技术的有机结合已成为加快水稻育种速度的有效途径。本文从水稻小孢子分离纯化、影响愈伤组织或胚状体发生的因素以及植株再生三个方面综述了水稻游离小孢子培养的最新研究进展。最后就水稻小孢子培育技术急需解决的问题进行了讨论,并对小孢子培育技术发展前景进行了展望。     

秋水仙碱对大麦离体培养小孢子存活与成苗的影响

以4份大田生长的优良大麦品种/品系为材料,采用超速旋切法分离小孢子进行培养.提取液和预处理液中添加适当浓度的秋水仙碱可明显提高大麦小孢子的存活率、胚状体的成苗潜力.     

Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern and Coss)

Summary The influence of donor plant growth environment, microspore development stage, culture media and incubation conditions on microspore embryogenesis was studied in three Indian B. juncea varieties. The donor plants were grown under varying environments: field conditions, controlled conditions, or a combination of the two. The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same does not hold true for the field-grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive with genotypic variability ranging from 10 to 35 embryos per Petri dish, irrespective of the other factors. NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis The fortification of this medium with activated charcoal, polyvinylpyrrolidone, colchicine, or growth regulators (6-benzylaminopurine and 1-naphthaleneacetic acid) was observed to be antagonistic for microspore embryogenesis, while silver nitrate (10 μM) had a significant synergistic effect. A post-culture high-temperature incubation of microspores at 32.5±1°C for 10–15 d was found most suitable for high-frequency production of microspore embryos. The highest frequency of microspore embryogenesis (78 embryos per Petri dish) was observed from the late uninucleate microspores (contained in bud sizes 3.1–3.5 nm irrespective of genotype) cultured on NLN medium containing 13% sucrose and silver nitrate (10 μM), and incubated at 32.5°C for 10–15 d.     

In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays)

Haploid induction has potential application for maize breeding. This paper reports that maize haploid plants have been induced by in vitro culture of pollinated ovaries. From a total of 26,400 cultured ovaries, 24 haploid plants were obtained and two of them were doubled after colchicine treatment. The maximum frequency of gynogenesis was 0.17% at 19.5 h post-pollination (HPP). The results showed that HPP was an important factor affecting plant induction from ovaries. Regenerated diploid R₀ plants were then subjected to genetic analysis using SSR molecular markers. One R₀ plant, whose progeny revealed a high level of homogeneity for several agro-morphological traits, was homozygous at 20 loci tested, with 11 showing paternal and 9 maternal banding pattern. This demonstrates that it is feasible to induce maize haploid plants by in vitro culture of pollinated ovaries.     

Effects of Copper on Root Growth, Cell Division, and Nucleolus of Zea Mays

The effects of different concentrations (10^–5 – 10^–2 M) of copper sulfate on root growth, cell division and nucleoli in root tip cells of Zea mays L. were investigated. 10^–5 M Cu stimulated root growth, but at higher concentrations (10^–4 – 10^–2 M) inhibited it. Cu had toxic effects on chromosomal morphology: c-mitosis, anaphase bridges, and chromosome stickiness were induced. Some nuclei had irregular shape and particles extruded from nucleoli to nuclei and finally from the nuclei into the cytoplasm.     

Induction of Haploid Callus from Isolated Microspores of Peony in vitro

The in vitro culture of isolated microspores of two cultivarsof Paeonia, P. lactiflora cv. "Kumoinotsuru" (2n=10) and cv."Toyonoakari" (2n = 10), was attempted. With both cultivars,some cultured microspores (3–6%) began to divide afterfive to seven days of culture. Those of "Kumoinotsuru" grewinto calli, whereas the others degenerated. Cytological examinationof the callus developed from a microspore revealed haploids,diploids or a mixture of both. No direct embryo formation frommicrospores was observed in this experiment. (Received December 1, 1980; Accepted January 16, 1981)     

Auxin induces exocytosis of acid phosphatase in coleoptiles from Zea Mays

Auxin-stimulated elongation growth of maize coleoptiles has been suggested to be associated with enhanced exocytotic activity. However, the problem in plants is one of finding a soluble parameter, which can be used as a direct measure of exocytosis (H. D. Blackbourn and N. H. Battey [1993]. Physiol. Plant. 89: 27–32). In yeast, acid phosphatase (EC 3.1.3.2) is used as a marker for secretory activity (E. Harsay and A. Bretscher [1995]. J. Cell Biol. 131: 297–310). Therefore, extracellular acid phosphatase activities in maize tissues were investigated. Coleoptile (7.36 nkat mg^-1) and mesocotyl (8.9) showed higher specific extracellular acid phosphatase activities than primary leaf (6.0), root (4.9) and root tip (2.7). In coleoptiles extracellular acid phosphatase activity was 6.7% of total homogenate activity (mesocotyls 10.6%). Auxin (30 μM IAA) increased the extracellular acid phosphatase activity of coleoptiles (146% of control). This effect was tissue-specific; extracellular acid phosphatase activity of mesocotyls was not enhanced by IAA. The stimulating effect of auxin on extracellular acid phosphatase activity in coleoptiles was reversed by the protonophore nigericin (0.3 μM). Furthermore, localization of an acid phosphatase activity in Golgi vesicles was shown by co-migration of the Golgi marker latent IDPase (EC 3.6.1.6) and acid phosphatase activity (65% of total microsomal activity) on isopycnic continuous sucrose density gradients. Tonoplast-enriched membrane frctions (24% of microsomal acid phosphatase) and plasma membrane-enriched fractions (11%) contained lower amounts of acid phosphatase. The data presented suggest that acid phosphatase activity is a useful marker for hormone-induced secretory activity in plant cells.     

一类依赖于NAD的玉米胞质型苹果酸脱氢酶基因的克隆及其序列分析

苹果酸脱氢酶普遍存在于各种生物中,它负责催化草酰乙酸和苹果酸之间的相互转换.根据其辅酶的特异性和在细胞内的分布及其生理功能的不同,苹果酸脱氢酶在高等植物中可以区分出不同的类型,依赖于NAD的细胞质型苹果酸脱氢酶是其中研究较少的一类.根据已发表的其他高等植物的依赖于NAD的胞质型苹果酸脱氢酶基因的保守序列,运用SMART RACE RT-PCR技术,从玉米叶片中分离了cyMDH 的1 264 bp全长cDNA序列,通过生物信息学分析发现,该序列含有一个999 bp的完整的开放阅读框,其共编码332个氨基酸(GenBank登陆号 EU625276).序列联配与树状分析结果表明,该玉米cyMDH 序列与多个物种的cyMDH 基因具有高度的同源性.组织特异性表达分析显示MDH基因在玉米叶片中表达量最高,在茎、根中亦有低水平表达.本研究将为更深入的研究玉米cyMDH 基因的分子调控机理奠定基础.     

Nuclear DNA amounts in the egg and zygote of maize (Zea Mays L.)

The nuclear DNA content of isolated eggs and zygotes of maize was estimated using 4,6-diamidino-2-phenylindole (DAPI) staining and microspectrofluorometry. The data indicate that egg nuclei contain the 1C level of DNA (basic haploid amount) at the time of karyogamy, and that, by inference, the sperm nuclei are also at 1C. Fertilization occurred in most ovules by 24–28 h post-pollination (hpp), and DNA synthesis was well underway by 27–31 hpp. By 30–34 hpp, 80% of the zygotes were at the 3C DNA level or above, and many were undergoing mitosis. This study provides information that is pertinent to experiments on the microinjection of exogenous DNA into isolated zygotes of maize, and it will serve as a comparative base for future determinations of the DNA content of zygotes produced and cultured in vitro.Abbreviations DAPI 4,6-diamidino-2-phenylindole - hpp hours post-pollination We would like to thank R. Blanc, D. Aldon, and C. Digonnet for their expert technical assistance and advice during the course of this study. Partial support for this study by the Organized Research Fund, Northern Arizona University, is gratefully acknowledged. The bulk of this study was carried out while H.L.M. was visiting research professor at the Ecole Normale Supérieure de Lyon.     

The Effect of D-Glucose and Other Sugars on the Trans-root Potential of Zea Mays

The addition of D-glucose and certain other sugars to the bathingsolutions of young excised maize roots (Zea mays L., var. Pioneer)gives rise to rapid changes in the electrical potential differencebetween the bathing solution and the xylem fluid. The resultssuggest the presence of a fairly non-specific sugar transportsystem in the plasma membranes of the root epidermal cells inwhich the hydroxyl group in the I-carbon position of n-glucoseis cntically involved. D-Mannose, 2-deoxy-D-glucose, and 2-deoxy-D-galactose have adelayed but more profound effect on this potential, reducingit to a much smaller value. The subsequent addition of adequateamounts of D-glucose restores this potential to about its formervalue, suggesting that these three compounds interfere withthe supply of endogenous glucose or glucose-derived productswithin the root.     

Two male-sterile mutants of Zea Mays (Poaceae) with an extra cell division in the anther wall

Two recessive male-sterile mutants of maize with similar patterns of pollen abortion were studied. Genetic studies showed that one of the two mutations was allelic with a previously identified male-sterility locus (ms23) and the other mutation was in a newly identified male-sterility locus (ms32). Cytological characterization of homozygous mutants and fertile heterozygous control siblings was performed using brightfield, fluorescence, and electron microscopy. During normal anther development, the final anther wall periclinal division divides the secondary parietal anther wall layer into the middle layer and tapetum, forming an anther with four wall layers. This is followed by differentiation of the tapetal cells into protoplastic binucleate, secretory tissue. In both the ms23 and ms32 mutants, the prospective tapetal layer divided into two layers, termed t1 and t2, forming an anther with five wall layers. Neither the t1 nor the t2 layers differentiated normally into tapetal layers, as determined by examination of cell walls, nucleus number, and cytoplasmic organization. Pollen mother cells aborted after the onset of prophase I of meiosis, suggesting that an early developmental coordination may exist between tapetum and pollen mother cells.     

Effect of exogenous ammonium gluconate on growth,ion flux and antioxidant enzymes of maize (Zea Mays L.) seedlings under NaCl stress

• Ammonium gluconate (AG) provides both an organic carbon source and a nitrogen source, which can positively improve soil fertility and delay soil degradation.
• We investigated the underlying mechanisms of both NH₄⁺‐ and C₆H₁₁O₇^?‐mediated resistance to high salt concentrations in maize (Zea mays L.), and how they relate to antioxidant cellular machinery, root system architecture, root activity and lignin content in roots.
• Seedlings treated with AG maintained lower Na⁺ content, higher chlorophyll content, higher CAT and POD activity, compared with those without AG and ammonium carbonate (AC). The total size of the root system, primary root length and number of lateral roots detected on the primary root treated with AG decreased compared with those not treated with AG at the same NaCl concentration. However, average root diameter and root activity when treated with AG were significantly higher than roots without AG at the same NaCl concentration. Furthermore, total size of the root system, primary root length and number of lateral roots detected on primary rootsof seedlings treated with AG were higher than those treated with AC at the same NaCl concentration.
• These results suggested that AG may be a good organic fertiliser under salt stress by decreasing Na⁺ content and increasing chlorophyll content, activity of antioxidant enzymes, root diameter and root activity in maize seedlings.

     

Effects of Water Stress and High-Temperature Stress on the Structure and Activity of Photosynthetic Apparatus of Zea Mays and Helianthus Annuus

Effects of high-temperature stress (HTS) and PEG-induced water stress (WS), applied separately or in combination, on the functional activity and ultrastructure of the photosynthetic apparatus (PSA) of maize (Zea mays L.) and sunflower (Helianthus annuus L.) plants were investigated. In maize plant tissues WS provoked the decrease in RWC by 10.9 %, HTS by 7.0 %, and after simultaneous application of the both treatments the decrease was 32.7 % in comparison with control plants. Similar but more expressed changes were observed in sunflower plants. Sunflower was more sensitive to these stresses. Net photosynthetic rate decreased significantly after all treatments, more in sunflower. In mesophyll chloroplasts after separately applied WS and HTS the number of grana and thylakoids was reduced and electron-transparent spaces appeared. At combined stress (WS+HTS) granal and stromal thylakoids were considerably affected and chloroplast envelope in many of them was partially disrupted. This revised version was published online in August 2006 with corrections to the Cover Date.     

An overview of preliminary studies on the development of doubled haploid protocols for nutraceutical species

There is very little activity underway to improve the genetics of herbs, spices, and nutraceutical crops. Much of the industry relies on the harvest of “wild” plants; therefore, the potential for variability in performance and active ingredients is high. This presents significant challenges for an industry that is striving to achieve market credibility and meet current regulatory standards. Uniform varieties would also be beneficial for use in clinical trials. The development of plants performing consistently in cultivation under various environmental conditions and producing a stable quality and quantity of desired active ingredients cannot solely rely on traditional plant breeding, but must be supported by the development of tissue culture methods targeted to the species of interest. We have screened over 80 herb, spice, and nutraceutical species for microspore culture response using the Brassica napus microspore culture protocol. The majority of the species did not respond. Swelling and initial divisions of the microspores were observed in some species. Embryogenesis, however, was observed in the Apiaceae and the Caryophyllaceae. Species within these families were selected for further optimization. Improvements in embryogenic frequency were observed in both families. Haploid and doubled haploid plants have been regenerated in anise (Pimpinella anisum), carrot (Daucus carota), caraway (Carum carvi), dill (Anethum graveolens), fennel (Foeniculum vulgare), lovage (Levisticum officinale), laceflower (Amni majus), parsnip (Petroselinum crispum), and cow cockle (Saponaria vaccaria).     

Androgenic response to preculture stress in microspore cultures of barley

Summary. Various stresses such as starvation and cold or heat shocks have been identified as triggers in the induction of the microspore embryogenesis. This study attempts to quantify the effects of different pretreatment conditions for successful microspore culture of malting barley (cv. Scarlett). While the sporophytic microspore development could be induced from treated and nontreated microspores, abiotic stress was essential for embryo formation and plant regeneration. The type of stress treatment applied affected the numbers and the ratios of albino and green plants regenerated, as well as their fertility. The highest number of green plants was obtained after the treatment of anthers in 0.3 M mannitol at 32 °C for 24 h before microspore culture. Correspondence and reprints: Department of Plant Biotechnology and Cytogenetics, Institute of Plant Breeding and Acclimatization, Radzików, 05-870 Blonie, Poland.     

Improvement of Longevity and Viability of Sperm Cells Isolated from Pollen of Zea mays L

Our previous studies showed that the common maize (Zea mays L.) sperm isolation medium (Brewbaker and Kwack salts in 0.44 m sucrose without buffering) caused cell lysis in vitro. In an attempt to remedy this situation, 6 sugars, 10 buffers, 5 pH values, and 3 membrane protective agents were screened to improve longevity and viability of isolated Zea mays sperm cells as estimated by hemacytometry and flow cytometry. Use of 0.55 m galactose in the isolation solution increased sperm yield by 2.5-fold compared with sucrose, and suspension of isolated sperm cells in the galactose solution gave the best longevity among the six sugars. Buffering the galactose solution with 2 mm 2-(N-morpholino)ethanesulfonic acid significantly improved longevity, whereas other buffers had no effect or decreased the longevity and/or viability. Among the five pH values tested (5.0, 6.0, 6.7, 7.0, and 8.0), pH 6.7 appeared to be optimal for maintenance of both longevity and viability. Screening of membrane protectants showed that cysteine caused a rapid decrease in cell viability and increased lysis, whereas dithiothreitol increased the cell numbers but lowered their viability. Addition of 0.1% bovine serum albumin increased cell numbers and viability, and about 70% of the cells remained viable after 72 h of suspension. Cell longevity and viability were also improved in 0.44 m sucrose when the solution was conditioned with 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin. Use of 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin inthe isolation and suspension medium significantly improved the viability and longevity of sperm cells isolated from Zea mays pollen.     

Leaf area establishment of a maize (Zea Mays L.) field crop under potassium deficiency

The effect of K deficiency on leaf area index (LAI) establishment of a maize field crop (Zea Mays L.) was studied. The experimental work was carried out in 2000 and 2001 on a long-term K fertilization trial. Three K fertilization regimes (K0, K1 and K4) have been applied since 1995, thus leading to contrasted levels of available K in soils (14, 23 and 44 µg exchangeable K per g of dry soil for the three fertilization regimes, respectively). The rate of leaf appearance, the leaf elongation rate (LER), the leaf elongation duration (LED), their final length and width and the number of senescent leaves were investigated. K concentrations in shoot tissue water were lower in K0 plants, whereas concentrations of Ca and Mg were higher. The LAI was reduced in the K0 treatment, mainly because of a slower rate of leaf appearance and a reduced final size of individual leaves. The reduced final length of individual leaves was almost entirely accounted for by a reduced LER during the quasi linear elongation phase. The LED was only slightly affected. A rough parallelism was observed between the relative reduction of leaf length and the relative reduction of plant water content during leaf elongation. Conversely, there was no evidence that leaf elongation was limited by carbohydrate availability in leaf growing zones. This suggests that K deficiency reduced LER probably because of altered plant-water relationships. On the whole, these results strengthen the idea that leaf growth is a key variable for analyzing, and later on modeling, crop growth under K deficiency.