植物实验生殖生物学与生殖细胞工程:现在与未来

植物实验生殖生物学是植物实验胚胎学发展的新阶段,其主要特征为操作技术水平的提高与多学科综合性研究的加强。花粉原生质体、生殖细胞、精子,胚囊、卵细胞的操作、雌雄配子体外融合、配子-体细胞杂交等,代表了当前的主要研究趋势。实验生殖生物学与基因工程相结合,开辟了植物生殖工程新技术领域的前景。对生殖工程的意义与内容提出了轮廓设想。     

植物实验生殖生物学与生殖工程：现在与未来

植物实验生殖生物学是植物实验胚胎学发展的新阶段,其主要特征为操作技术水平的提高与多学科综合性研究的加强。花粉原生质体、生殖细胞、精子、胚囊、卵细胞的操作、雌雄配子体外融合、配子-体细胞杂交等,代表了当前的主要研究趋势。实验生殖生物学与基因工程相结合,开辟了植柏物生殖工程新技术领域的前景。对生殖工程的意义与内容提出了轮廓设想。     

花粉原生质体,精子与生殖细胞的实验操作

当前,一个可以称为“植物实验生殖生物学”的分支学科正在形成之中。其主要特点是:由以往实验胚胎学的器官、组织水平的操作发展为细胞、原生质体水平的操作;由传统的比较单一的研究发展为多学科的综合性研究;因而无论在研究的手段与内容方面均进入更高的层次。例如,就雄性系统而言,已由花药培养发展到花粉粒培养,最近又向花粉原生质体、精子和生殖细胞的操作深化。就雌性系统而言,亦由子房、胚珠培养向胚     

Experimental Manipulation of Pollen Protoplasts Sperms and Generative Cells

This review deals with the up-to-date advances in manipulation of male sexual cells, i.e. pollen protoplasts, sperms, and generative cells of flowering plants, including the isolation, purification, culture and/or fusion, as well as cell-biological studies of these haploid protoplasts or cells. These experimental approaches show fascinating prospects for both genetic engineering and fundamental researches on plant reproductive cells.     

植物原生质体在分子细胞生物学研究中的应用

植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。     

植物受精工程

植物受精工程是正在发展之中的高技术领域。近年来,雌雄配子的人工分离取得了较大进展,两者的体外模拟受精在玉米中已首次成功。本文对该领域的理论体系形成和实验系统创建作了评述,对目前存在的问题和今后的发展趋势进行了讨论。     

Protoplasts in organelle research: Transfer and transformation of plastids

Protoplasts, because they lack the wall of a typical higher plant cell, offer unique opportunities for experimental manipulation of their organellar constituents. Here, we report on modification of the organellar content of Nicotiana tabacum protoplasts by microfusion-induced transfer of defined numbers of chloroplasts into albino recipient cells. A single chloroplast is found to be sufficient for establishing a new plastid population in the progeny of the recipient cell. The frequency of green or variegated regenerants is shown to be genotype dependent. It can be drastically increased by using selection pressure for the transferred organelle. We also report on transient expression of plastid specific reporter gene constructs in plastids after PEG-mediated direct gene transfer into Nicotiana plumbaginifolia protoplasts. The expression is shown to be localized in the plastids by determining gene expression in isolated chloroplasts under conditins which completely remove cytoplasmic enzyme activity derived from a nuclear reporter gene construct. These data demonstrate for the first time that functional DNA, introduced into the cytoplasm by direct gene transfer, enters the organellar compartment and is expressed.     

THE QUANTITATIVE GENETICS AND COEVOLUTION OF MALE AND FEMALE REPRODUCTIVE TRAITS

Studies of experimental sexual selection have tested the effect of variation in the intensity of sexual selection on male investment in reproduction, particularly sperm. However, in several species, including Drosophila pseudoobscura, no sperm response to experimental evolution has occurred. Here, we take a quantitative genetics approach to examine whether genetic constraints explain the limited evolutionary response. We quantified direct and indirect genetic variation, and genetic correlations within and between the sexes, in experimental populations of D. pseudoobscura. We found that sperm number may be limited by low heritability and evolvability whereas sperm quality (length) has moderate V_A and CV_A but does not evolve. Likewise, the female reproductive tract, suggested to drive the evolution of sperm, did not respond to experimental sexual selection even though there was sufficient genetic variation. The lack of genetic correlations between the sexes supports the opportunity for sexual conflict over investment in sperm by males and their storage by females. Our results suggest no absolute constraint arising from a lack of direct or indirect genetic variation or patterns of genetic covariation. These patterns show why responses to experimental evolution are hard to predict, and why research on genetic variation underlying interacting reproductive traits is needed.     

Developmental biology meets with reproductive engineering; interdisciplinary science area as a breakthrough.

It has been postulated many times that different scientific topics and strategies often encounter each other, which create cutting edge research field resulting in further significant progresses of science. This was also a lesson bestowed by Prof. Raymond Wegmann where he created innovative research field for biology, molecular biology and biochemistry-biophysics. Progresses of developmental biology were boosted by molecular biology and reproductive engineering where ES cells and embryonic manipulation are necessary. There are no questions about the utility of their technologies. Reviews on their contributions with respect to the condition of genome manipulation are addressed.     

Individual selection,culture and manipulation of higher plant cells

Summary Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts — mainly performed with cells of the crop plant Brassica napus L. — are presented.This article is dedicated to the memory of H.-G. Schweiger, initiator and mentor of the experimental single cell approach reviewed herein     

VARIATION IN MALE REPRODUCTIVE INVESTMENT AND MALE REPRODUCTIVE SUCCESS IN WHITE SPRUCE

An experimental population composed of ramets of white spruce clones was studied to determine the influence of different levels of clonal male reproductive investment on clonal male reproductive success, where the male reproductive success of different clones in the population was defined as the proportion of the seed crop sired by each clone. We present a multilocus estimation procedure which provides unbiased estimates of the proportion of seed sired by each parental genotypic class from pollen gamete frequency data, whenever gametic segregation frequencies are known. Significant linear and quadratic components were found for the relationship between the number of male cones produced and the estimated proportion of the seed crop sired. A clone that produced an exceptionally large number of male cones, however, did not have the greatest male reproductive success. In general, the results support an assumption made in theoretical models of selection on mating system modifiers regarding the relationship between male investment and male reproductive success. They also support the hypothesis that male reproductive success in wind-pollinated species continues to increase substantially with increasing male reproductive investment.     

COMPONENTS OF REPRODUCTIVE EFFORT AND YIELD IN GOLDENRODS

Four components of reproductive yield (the weight of reproductive tissue) were examined in relation to their effect on reproductive effort and their relative contributions to reproductive yield in five species of goldenrods (Solidago, Compositae). The yield components were number of flowing stems per plant, number of flowering branches per stem, number of flowering heads per branch, and number of seeds per seed head. Individuals within populations increase their reproductive effort by increasing their reproductive weight, not by decreasing their vegetative weight. Each species shows a different pattern of positive correlations of yield components with reproductive yield and reproductive effort, indicating that each species has its own mechanisms for regulating reproduction. The yield components were not significantly intercorrelated.     

Infrared picosecond laser for perforation of single plant cells

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. In this report, we demonstrate the use of an infrared (1,064 nm) picosecond laser for the perforation of tobacco cell protoplasts. A single pulse was sufficient to perforate the plasma membrane enabling the uptake of dye from the surrounding medium into the cytosol. Moreover, the procedure was shown to be suitable for the efficient delivery of DNA expression constructs to the nucleus, as demonstrated by the subsequent expression and correct targeting of a recombinant fluorescent protein. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.     

空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。     

Towards molecular breeding of reproductive traits in cereal crops

The transition from vegetative to reproductive phase, flowering per se , floral organ development, panicle structure and morphology, meiosis, pollination and fertilization, cytoplasmic male sterility (CMS) and fertility restoration, and grain development are the main reproductive traits. Unlocking their genetic insights will enable plant breeders to manipulate these traits in cereal germplasm enhancement. Multiple genes or quantitative trait loci (QTLs) affecting flowering (phase transition, photoperiod and vernalization, flowering per se ), panicle morphology and grain development have been cloned, and gene expression research has provided new information about the nature of complex genetic networks involved in the expression of these traits. Molecular biology is also facilitating the identification of diverse CMS sources in hybrid breeding. Few Rf (fertility restorer) genes have been cloned in maize, rice and sorghum. DNA markers are now used to assess the genetic purity of hybrids and their parental lines, and to pyramid Rf or tms (thermosensitive male sterility) genes in rice. Transgene(s) can be used to create de novo CMS trait in cereals. The understanding of reproductive biology facilitated by functional genomics will allow a better manipulation of genes by crop breeders and their potential use across species through genetic transformation.     

REPRODUCTIVE BIOLOGY OF WEEDY AND CULTIVATED MIRABILIS (NYCTAGINACEAE)

The reproductive biology of Mirabilis nyctaginea (Michx.) MacM. contributes to the plant's success as a weed and invader of disturbed habitats whereas that of M. Jalapa L. reflects its success as a cultivar. Plants of M. nyctaginea flowering in early summer produce chasmogamous flowers whereas plants flowering in late summer produce cleistogamous flowers. Plants of both species are self-compatible and flower behavior contributes to self-pollination. The flowers open late in the afternoon and close late the following morning. In M. nyctaginea flowers are effectively pollinated by bees both in the afternoon and morning, and during the night moths are effective pollinators. Mirabilis Jalapa is self-pollinating. Anther closure during rain showers, a result of water droplets falling into open anthers, minimizes pollen loss and is highly adaptive. The reproductive biology of M. nyctaginea and M. Jalapa is contrasted with that of the obligate outcrossing species M. multiflora (Torr.) Gray and M. froebelii (Behr) Greene.     

SHIFT IN EGG‐LAYING STRATEGY TO AVOID PLANT DEFENSE LEADS TO REPRODUCTIVE ISOLATION IN MUTUALISTIC AND CHEATING YUCCA MOTHS

Through the process of ecological speciation, insect populations that adapt to new host plant species or to different plant tissues could speciate if such adaptations cause reproductive isolation. One of the key issues in this process is identifying the mechanisms by which adaptation in ecological traits leads directly to reproductive isolation. Here I show that within a radiation of specialist moths that pollinate and feed on yuccas, shifts in egg placement resulted in changes in female moth egg‐laying structures that led to concomitant changes in male reproductive morphology. As pollinator moths evolved to circumvent the ability of yuccas to selectively abscise flowers that contain pollinator eggs, ovipositor length became shorter. Because mating occurs through the ovipositor, shortening of the ovipositor also led to significantly shorter and wider male intromittent organs. In instances where two pollinator moth species occur in sympatry and on the same host plant species, there is one short and one long ovipositor species that are reproductively isolated. Given that many plant‐feeding insects lay eggs into plant tissues, changes in ovipositor morphology that lead to correlated changes in reproductive morphology may be a mechanism that maintains reproductive isolation among closely related species using the same host plant species.     

Plant protoplasts: status and biotechnological perspectives

Plant protoplasts ("naked" cells) provide a unique single cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Several parameters, particularly the source tissue, culture medium, and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Importantly, novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. Nevertheless, isolated protoplasts are unique to a range of experimental procedures. In the context of plant genetic manipulation, somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of pharmaceutical products, and toxicological assessments. This review focuses upon the most recent developments in protoplast-based technologies.     

Fungus propagules in plastids: the mycosome hypothesis

A stress-induced "mycosome" phase of Aureobasidium pullulans consisting of minute reproductive propagules that may revert directly to walled yeast cells is described. Mycosomes detected by light- and electron-microscopy reproduce within senescent plant plastids, and display three developmental pathways: wall-less cells (protoplasts), yeast cells, or membrane-bounded spherules that harbor plastids. Widespread in plant and algal cells, mycosomes are produced by both ascomycete and basidiomycete fungi. Electronic Publication