石刁柏胚乳愈伤组织内胚胎发生的组织细胞学观察

石刁柏(Asparagus officinalis L.)的幼嫩胚乳接种于MS+NAA 5ppm+6-BA 1ppm和MS+NAA 1ppm+6-BA 0.5ppm的培养基上诱导产生愈伤组织。将愈伤组织转移到MS+6-BA 1ppm+NAA 0.5ppm的分化培养基上,培养30天后即可形成胚状体,组织细胞学观察表明:胚状体起源于愈伤组织内或近表层的单个胚性细胞。在胚状体发生的早期阶段,观察到与柳叶菜型和藜型胚胎发育大致相似的细胞分裂方式,从而出现了T型或线型的四个细胞的原胚。在多细胞原胚及球形胚期具有单列或不规则排列的多细胞胚柄。石刁柏胚乳愈伤组织中的胚胎发生是不同步的,在同一块愈伤组织的切片中可以观察到不同发育阶段的胚状体。在外形上还可以观察到一个子叶、两个子叶或四个子叶等多种不同形态的胚状体。一部份胚状体能发育成完整小植株。     

Establishment and characterization of a maize Hi-II endosperm culture

An in vitro continuous endosperm callus culture derived from developing endosperm of transformation-amenable maize Hi-II genotype was obtained. The endosperm callus was composed of cells that differentiated into aleurone-like and starchy endosperm-like cell types. This callus has been maintained for 4?yr. Endosperm callus cells transcribe and produce zein proteins at a level similar to developing endosperm tissue. Starchy endosperm cells of the endosperm callus displayed active starch biosynthetic activity. The dual cell physiology of this culture limited the utility of the cell line for promoter analysis and transient assays of gene expression in the current culture conditions. However, because such cell line can be readily initiated and easily maintained for a long period of time, it provides an alternative tool for analysis of transgene expression in endosperm callus derived from transgenic maize lines in Hi-II background.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

Investigations of Laticifer Differentiation in Tissue Cultures Derived from Asclepias syriaca L.

Callus cultures of Asclepias syriaca were established from stemexplants and grown in tissue culture. The culture medium onwhich the callus was grown was modified to produce either planfletsof superficial origin on the callus or embryoids which wereanalyzed to determine whether laticifers differentiated in thesestructures. Mature zygotic embryos and adult plants of A. syriacanormally possess a well-developed network of intrusively-growingnon-articulated branched laticifers that arise only once duringplant develop ment from initials differentiated in the youngheart stage embryo. Embryoids were derived from two differentculture media. These embryoids were observed to lack laticifers,although they were similar in their morphology in other respectsto zygotic embryos. Plantlets of superficial origin were formedon each of the media employed in this study. These plantletswere observed to possess laticifers that resemble those in normalshoots. Embryoids and induced shoots represent experimentalsystems in which it may be possible to control for the firsttime the differentiation of the laticifer as a cell type instructures similar to those present in the normal plant.     

Growth and tissue formation from single geranium cells from virus-infected geraniums

Summary Single geranium (Pelargonium hortorum, Bailey) cells from callus isolated originally from stem tips of virus-infected plants were grown in microculture chambers in liquid Murashige and Skoog medium supplemented with 0.1 mg per liter of α-naphthalane acetic acid and 10.0 mg per liter of kinetin. Four of 1,000 of these single cells divided in the microculture chamber and produced colonies of 15 to 20 cells in 9 to 16 days. In all of the cases the plane of the first few cell division was at right angles to the long axis of the cells. Subsequently, one of the masses of cells obtained from a single cell, when transferred to solid Murashige and Skoog medium, established itself as a clone of callus tissue. Although the yield was low, the results were encouraging for the ultimate production of plants. These results suggested that, just as virus-free tobacco plants have been induced from single cell clones from certain tobacco species, virus-free single geranium cells may be useful to establish single cell clones from which pathogen-free geranium plants may be induced to differentiate. This work was supported by funds from USDA Hatch Project 981 and the NASA Institutional Grant. Published with the approval of the Director, Wisconsin Agricultural Experiment Station. The authors are indebted to Mr. E. H. Herrling and Mr. Steve Vicen for preparation of illustrations.     

The Preliminary Studies on Culture of Unfertilized Ovary of Rice in Vitro

The present paper reports the results of the culture of unfertilized ovaries of rice in vitro. The inducting medium was N6 supplemented with 2 mg/L 2,4-D, 500 mg/L casien hydrolysate and sucrose was 4%. The differentiated medium was N6 supplemented with 2 mg/L Kinetin, 500 mg/L casein hydrolysate and the concentration of the sucrose was 3%. The 4 cultivars and 2 crossed combinations were used as the experimental materials. The experiments were shown the differentiation of the callus occurred amony various cultivars. The induced frequency in the crossed combinations was higher than that in the cultivars. Now 12 green plants and 3 albino plantlets have been obtained. The chromasomes of 11 green plantlets have been examined. Among them, 6 plantlets were haploid (n =12 ) and 5 plantlets were diploid. The embryoids were located in the micropylar end. Some of them possessed the suspensor, similar as zygote embryos. The callus was found from different origin. One of them was originated from haploid tissue derived from the nuclear in the embryo sac. Another was originated from the diploid tissue in the integument or ovary wall. The origin of the callus from the unfertilized ovary was discussed.     

Cytology and Histology in Somatic Embryogenesis of Indica Rice

The somatic embryogenesis was established from mature dehulled seeds. The histological research showed that embryogenic calli were initiated first from absorbed cells of scutellum of mature seed. And then the embryoids derived from the surface of embryogenic callus. Having been the same structure like a zygotic embryo of rice, the embryoids possessed the major parts of scutellum, coleoptile and coleorhiza. In an embryoid, several developmental stages of pro-embryoid, including single embryogenic cells, two, four and multiple cell stage pro-embryeids and some abnormal embryoids were observed. It could be concluded from this experiment that the embryoid from somatic cell culture in Indica rice possessed an original form of a plant in structure like a zygotic did and derived from a single cell.     

Morphogenic studies in tissue cultures of the parasiteSantalum album L.

Whole seeds, excised embryos, and excised endosperm ofSantalum album were aseptically cultured with a view to studying seed germination in isolation from the host species, and to establishing callus cultures from both embryo and endosperm for comparative studies et their morphogenesis. Seed germination and seedling formation occurred normally only on modified White's medium supplemented with casein hydrolysate or coconut milk, or with both substances. Neither the excised embryo nor the endosperm grew on any of the culture media tested. However in about 17 per cent seed cultures on White's medium supplemented with 2,4-D, kinetin, and yeast extract, the endosperm degenerated, whereas the embryo callused and subsequently differentiated into innumerable embryoids; eventually the embryoids developed into normal plantlets. Callusing of the endosperm occurred also in seed cultures on four media supplemented variously with 2,4-D, kinetin, and yeast extract. Although the endosperm tissue grew through several passages no organ fornation was observed.     

Induction of Endosperm Calluses and Regeneration of Endosperm Plantlets of Asparagus officinalis

The endosperm callus has been induced from the young endosperm of Asparagus officinalis L. on the MS supplemented with auxin. The induction frequency of callus amounts to 65.9%–83.1%. When the callus was transferred to the medium supplemented with lower concentration of NAA 0.1 ppm or containing BA 1 ppm and NAA 0.5 ppm, the differentiation of shoots, roots and a few embryoids began to occur. A few calluses and embryoids can develop into plantlets. The chromosome number in the cells from the same root tip and shoot apex of endosperm plantlet is very unstable. They can be euploids (n=10, 2n=20, 3n=30, 4n=40). or aneupl0ids (n=6, 7, 17, 25, 53).     

The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice

Summary Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.Dedicated to the memory of the late Dr. J. P. Nitsch.     

Cytological and Embryological Studies on Haploid Plant Production from Cultured Unpollinated Ovaries of Nicotiana tabacum L.

We obtained mature haploid (n = 24) ovary plants from in vitro cultured unpollinated young ovaries. These ovaries were induced to form embryoids which then developed into plants. The results obtained are summarized as follows: 1. The origin of development of the ovary haploid plants has been followed by light microscopy. Embryological abservations revealed that there are two ways of plantlet production: (1) Ovary haploid plant was derived from the macrospore without an intervening callus phase. (2) Ovary haploid plant was derived directly from the egg cell of mature embryo sac. In addition, Callus derived haploid plant was also obtained from the base and the tip of a bud of the above mentioned haploid plantlet. In same medium embryoids was derived from callus. Finally, plantlet was developed. 2. The exogenous hormones are necessary for high induction frequency of embryoid from unpollinated isolated young ovary, but these are not definitely necessary for induction of embryonic callus to form embryoids which then developed into plant. 3. The induction frequency of embryoid from in vitro cultured ovary and embryonic callus significantly increased when the concentration of thiamine, pyridoxine, ascorbic acid, nicotinic acid, inositol and folic acid was raised.     

Initiation, growth and cryopreservation of plant cell suspension cultures

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Enigma of apogamety

Summary Apogamety, the occurrence of which has been either denied or criticized by some authors, presents several problems that should be carefully considered in argumentation. Gynogenesis, which comprises zygotic, parthenogenetic, and apogametic embryos, an actively developing branch of tissue culture, is of no help here being itself subject to similar criticism. The discussion on apogamety awaits methodological progress which would yield answers to the following questions: are all cells of embryo sac potentially gametic; in what way are the mechanisms of cell differentiation and specialization of cells within embryo sac correlated with their totipotency; what are the limitations of plant cell totipotency in embryo sacs. The above problems of apogamety are not only an enigma for embryology and reproduction of angiosperms but they belong also to the crucial problems of the general biology which might be solved by studies of corresponding molecular mechanisms and experiments in tissue culture.     

茶（Camellia sinensis Kuntze）离体培养体细胞胚胎发生的组织细胞学研究

以茶树叶片为外植体．以ＭＳ培养基附加４ｍｇ·Ｌ－１６－ＢＡ,２ｍｇ·Ｌ－１ＩＡＡ．３ｍｇ·Ｌ－１ＧＡ３和０．２—０．３％活性碳诱导出愈伤组织和胚状体,进一步形成小植株．切片观察表明．茶叶愈伤组织胚状体的发生,起源于愈伤组织表层及其内部的单个细胞和细胞团,胚状体发育顺序与合子胚大致相似．经球形胚、心形胚、鱼雷胚和子叶胚阶段．但发育过程中常有畸形胚出现．     

Mutants with aberrant numbers of gametic cells shed new light on old questions

In contrast to animals, plant gametes form in distinct haploid generations, termed gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg and central cell, which are flanked by accessory cells. The gametic cells differ with respect to morphology, molecular attributes and, importantly, their fate: whereas the egg cell, upon fertilisation, gives rise to the embryo, the central cell forms the endosperm. To ensure correct endosperm formation, not only the egg cell but also the central cell has to fuse with a sperm cell. The respective sperm cell pair is delivered by a single pollen tube. In some plant species, the two male gametes appear to express a different bias towards the female gametes. Such a preference consequently determines their respective contribution to either embryo or endosperm development. In Arabidopsis and many other species the sperm cells are indistinguishable and it has been discussed whether they possess an inherent preference for either of the female gametes. The recent isolation of mutants that form an aberrant number of either male or female gametes stimulates discussion, albeit with different results. Furthermore, some data indicate that the central cell is competent to initiate endosperm formation without a paternal contribution. These data support the theory that the endosperm is of gametophytic rather than sporophytic origin.     

Transmission of male cytoplasm during fertilization in Nicotiana tabacum

Serially sectioned embryo sacs of Nicotiana tabacum were examined during fertilization events using transmission electron microscopy. After pollen tube discharge, the outer membrane of the sperm pair is removed, the two sperm cells are deposited in the degenerate synergid and the sperm cells migrate to the chalazal edge of the synergid where gametic fusion occurs. During fertilization, the male cytoplasm, including heritable organelles, is transmitted into the female reproductive cells as shown by: (1) the cytoplasmic confluence of one sperm and the central cell during cellular fusion, (2) the occurrence of sperm mitochondria (distinguished by ultrastructural differences) in the zygote cytoplasm and adjacent to the sperm nucleus, (3) the presence of darkly stained aggregates which are found exclusively in mature sperm cells within the cytoplasm of both female cells soon after cell fusion, and (4) the absence of any large enucleated cytoplasmic bodies containing recognizable organelles outside the zygote or endosperm cells. The infrequent occurrence of plastids in the sperm and the transmission of sperm cytoplasm into the egg during double fertilization provide the cytological basis for occasional biparental plastid inheritance as reported previously in tobacco. Although sperm mitochondria are transmitted into the egg/zygote, their inheritance has not been detected genetically. In one abnormal embryo sac, a pair of sperm cells was released into the cytoplasm of the presumptive zygote. Although pollen tube discharge usually removes the inner pollen-tube plasma membrane containing the two sperm cells, this did not occur in this case. When sperm cells are deposited in a degenerating synergid or outside of a cell, this outer membrane is removed, as it apparently is for fertilization.