棉花体细胞胚发生机理的研究进展

棉花是一种较难通过体细胞胚发生完成植株再生的作物,而体细胞胚发生是限制棉花基因工程和细胞工程得以广泛应用的主要因素.研究者从不同角度探讨了棉花体细胞胚发生机理并取得了很大的进展,为棉花的遗传转化提供了重要的技术支持,同时成功建立了一些棉花植株再生体系.本文分别从基因型限制、胚胎学、生理生化变化、分子机理等方面阐述了近年来棉花体细胞发生机制的研究进展,并讨论了本研究领域目前存在的主要问题及相应对策.     

一种新型的棉花体细胞胚胎发生的快速诱导法

用异常苗的茎段和叶片进行培养,可快速高效诱导获得棉花体细胞胚胎发生,激素组膈及其浓度配比影响异常苗的直接胚胎发生,在附加有０．１ｍｇ／Ｌ ＩＡＡ和０．１ｍｇ／ＬＺＴ的改良ＭＳ培养基上,异常苗不仅体细胞胚胎发生率高,而且形成的体细胞胚数目多,用异常苗作外植体获得胚性愈伤组织仅需要时间１０ｄ,获得成熟胚需要２０－３０ｄ,获得再生植株需要６０ｄ,大大短于常规方法获得胚性愈伤组织、体细胞胚和再生植株的时间     

棉花体细胞增殖和胚胎发生中的细胞程序性死亡

棉花组织培养中愈伤组织褐化可能与细胞程序性死亡(PCD)有关.对棉花组培中不同时期的愈伤组织DNA进行琼脂糖电泳,观察到:仅在第一次愈伤组织继代后10 d左右和愈伤组织第一次继代到分化培养基中培养10 d左右,愈伤组织的DNA产生了180 bp左右的片断或其整数倍片断大小的DNA带,呈DNA梯(DNA ladder)状电泳,说明在这两个时期PCD达到了高峰.在这两次PCD高峰后1周均出现愈伤组织大规模的褐化死亡.显微镜观察到第一次PCD发生高峰的愈伤组织存在许多管状、内含物少的细胞,这些细胞分布在愈伤组织的边缘和内部;第二次PCD发生高峰观察到体细胞胚胎分化、PCD细胞的木栓化和水渍化.对体细胞胚胎分化时期的原生质体进行荧光染色(DAPI),荧光显微镜下观察到发生细胞程序性死亡的细胞核浓缩、染色质凝聚、结块、边缘化和形成PCD小体.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

棉花体细胞胚胎发生的研究进展

经过30多年的发展,已经在多个不同的棉花品种中获得了体细胞胚,并再生成苗。但由于体细胞胚胎发生往往受多种因素影响,如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展,分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述,并对研究中存在的问题进行了讨论。     

Oligosaccharide Inhibits Ethylene Synthesis and Stimulates Somatic Embryogenesis in a Cotton Cell Culture

The effect of a three-component oligosaccharide fragment of xyloglucan FucGalXyl (XG3) on callus-tissue growth and somatic embryogenesis was investigated in a cotton (Gossypium hirsutumL.) cell suspension culture. The oligosaccharide introduced into an induction medium at 10^–8and 10^–7M concentrations did not affect the frequency of callus formation from hypocotyl segments; however, it enhanced the monthly increment of callus-tissue weight 1.5- and 3-fold, respectively. Induction and culturing of the callus on an XG3-containing medium adversely affected its morphogenetic potential. Addition of XG3 to the culture medium during the cell suspension preparation stimulated cell division resulting, after 40 days, in a 3.4-fold (at 10^–8M XG3) and a 1.7-fold (at 10^–7M XG3) increase in the cell number as compared to the control. Exclusion of 2,4-D, kinetin, and oligosaccharide from the culture medium caused, after two weeks, a 3.8-fold increase in the number of embryos in the 10^–7M XG3-treated suspension culture as compared to the control. The stimulation of somatic embryogenesis by the oligosaccharide was accompanied by a 12-fold decrease in ethylene emission. The morphogenetic effect of oligosaccharide is suggested to result from its anti-auxin action, which, in particular, inhibited the auxin-dependent ethylene synthesis.     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO₃, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA₃) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO₃. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA₃. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.     

Somatic embryogenesis in cotton (Gossypium) I. Effects of source of explant and hormone regime

Optimal media for induction of somatic embryogenesis from mature and immature tissues ofG. hirsutum L. cv Coker 312 were determined. Explants of three-day-old seedlings form somatic embryos in 100% of cultures when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 0.5 mg/1 kinetin. Mature tissues are more recalcitrant than immature tissues and formed somatic embryos on a limited number of hormone treatments. Stem tissue is most readily induced to form somatic embryos by 2 mg/1 napthaleneacetic acid plus 0.1 mg/1 kinetin, whereas leaf tissue formed embryos best when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 1.0 mg/1 (2-isopentyl)-adenine, or 1.0 mg/1 napthaleneacetic acid plus 0.5 mg/1 (2-isopentyl)-adenine.     

Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Summary Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog’s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog’s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg · liter^−1), and BAP (1 mg · liter^−1) plus NAA (0.1 mg · liter^−1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.     

Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.)

Summary Embryogenic callus was formed from several cultivars of cotton (Gossypium hirsutum L.) when sections of hypocotyl and cotyledon were cultured on medium supplemented with 5 mg/liter 6-(γ, γ-dimethylallyl-amino)-purine (2iP) and 0.1 mg/liter α-naphthaleneacetic acid (NAA) for callus initiation and proliferation, and subcultured on medium supplemented with 5 mg/liter NAA and 0.1 to 1 mg/liter 2iP for embryogenic callus induction. It seems that a high 2iP:auxin ratio is preferred for callus initiation and proliferation, but should be exchanged with a higher NAA:cytokinin ratio before differentiation will occur. Embryogenic calluses were recovered at a frequency of 2 to 85% depending on the cultivar used. Coker cultivars produced embryogenic callus faster and at higher frequencies than other cultivars. Embryogenic callus produced somatic embryos on phytohormone-free medium. This medium was used to maintain and proliferate embryogenic callus for a perid of 18 to 24 mo. Somatic embryos were converted to plants on a lower ionic strength medium supplemented with 0.1 mg/liter gibberellic acid (GA₃) and 0.01 mg/liter NAA. Glucose was the only carbohydrate used through all phases of tissue culture and was much better than sucrose, on which phenolic production was very high. High temperature (30° C) and low light intensity (9 μE · m⁻² · s⁻¹) were optimal conditions for callus initiation, embryogenic callus induction, and maintenance, whereas lower temperature (25° C) and high light intensity (90 μE · m⁻² s⁻¹) were the optimal conditions for somatic embryo maturation, germination, and plantlet development. Plants could be regenerated within 10 to 12 wk in Cokers or 7 to 8 mo. in others.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

Frequency and Diversity of Fungi Colonizing Tissues of Upland Cotton

A study of the mycoflora of upland cotton in Alabama was conducted throughout the 2000 and 2001 growing seasons. Plants were sampled at seedling, first bloom, full bloom, and maturity stages of development. Thirty-seven genera representing 58 species of fungi were isolated, including 9 species of Fusarium. Fusarium oxysporum, F. solani, and F. equiseti were the most common members of this genus occurring at all four sampling stages in both years. Eight species accounted for 67% of the total fungi isolated during the two-year study. Alternaria alternata was the most common fungus encountered, accounting for 19 and 10% of the total fungi isolated in 2000 and 2001, respectively. Twenty species of fungi are reported for the first time colonizing upland cotton tissues.     

Somatic embryogenesis in Vicia faba L.

The paper describes a method of somatic embryo induction in callus and suspension cultures of Vicia faba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichlorophenoxyacetic acid. The medium with 2.5 M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.     

Studies on Somatic Embryogenesis and Histological Observation in Gossypium hirsutum L. cv. Coker 312

A reproducible 3-step procedure of somatic embryogenesis of Gossypium hirsutum L. cv. Coker 312 has been developed. Calli were initiated on LSC medium containing 0.1mg/L 2,4-D plus 0.1 mg/L KT from cotyledon tissue of 5-day-old-seedlings, and subcultured on the same medium with 4 mg/L NAA and 1 mg/L KT. Embryogenic calli and few globular embryos developed at a frequency of 67.5% after 55 days’ culture in the latter medium. When the embryogenic calli were transferred to growth regulator-free medium, embryogenesis occured and all stages of normal zygotic embryos, globular-, heart- and arrowhead- or torpedo-shaped embryo, ,were developed. Cyto-histological study showed that embryogenic calli were very easily distinguished from non-embryogenic calli. Embryoids were mainly initiated from the cells in the peripheral area of embryogenic calli. At the early stage the development of embryoid was limited in a boundary of thicken cell wail. There were 2 peaks of starch accumulation in the process of embryogenesis, one was at the early globular stage, and the other at the later torpedo-shaped stage.     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Effects of brassinosteroid on cotton regeneration via somatic embryogenesis

Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B₅ vitamins supplemented with 1 mg/L 6-benzylaminopurine + 0.5 mg/L kinetin for callus induction. After one month proliferating calli pieces were collected and cultured on MS basal medium containing various concentrations of BR (0.1, 0.5, 1.0 μM) with their controls. BR treatments were negatively effective on the fresh weight of calli when compared to control. Differential somatic embryogenesis maturation rates due to BR treatment were observed. Somatic embryogenesis was stimulated especially for transition to cotyledonary phase at 0.5 mg/L BR. Histological preparations from embryogenic calli and somatic embryos at different stages of development revealed the spontaneous polyploidisation during early somatic embryogenesis on BR-treated calli. Present results suggest that BR negatively effected calli growth, however, had a stimulating role in maturation of somatic embryos.     

Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis

Summary Despite high commercial interest, the success of biotechnological applications in cotton (Gossypium hirsutum) has been limited due to difficulties in genetic transformation. Major problems have been genotype dependence and low frequency of somatic embryogenesis, making it difficult to regenerate plants from transgenic tissue. This study reports an increase in somatic embryogenesis efficiency and the induction of developmental synchrony in embryogenic callus cultures of cotton by a single cycle of myo-inositol depletion in liquid culture. Calluses were initiated on hypocotyl or cotyledon explants of cultivar Coker 312 by culturing these explants on callus-inducing solid medium [Murashige and Skoog salts plus vitamins of Gamborg's B₅ medium, 30 g l⁻¹ glucose, 100 mg l⁻¹ myo-inositol, 2.2 μM 2,4-dichlorophenoxyacetic acid, and 0.88 μM 6-benzyladenine]. The calluses were transferred to an identical liquid basal medium devoid of plant growth regulators. This induced the development of embryogenic cells. Friable clumps of cells formed after 20 d in the medium were selectively collected over filter mesh 40 subjected to one cycle of myo-inositol starvation. This induced a highly synchronized embryogenesis in the culture. The optimized protocol gave 100% embryos at the globular stage, out of which more than 80% developed into bipolar torpedo-stage embryos. About 68% of these were converted to plantlets by subculturing onto a simplified solid medium, and finally grown into healthy, fertile plants.     

Somatic Embryogenesis in Leguminous Plants

Abstract: This review examines recent advances in the induction and development of somatic embryos in leguminous plants. Emphasis has been given to identify the current trends and successful strategies for the establishment of somatic embryogenic systems, particularly in the economically important species. It appears that, in legumes, somatic embryogenesis can be realized relatively easily especially in young meristematic tissues such as immature embryos and developing leaves. In the majority of the species examined, chlorophenoxyacetic acids remained the most active inductive compounds; however, the new generation growth regulators such as thidiazuron are emerging as successful alternatives for high-frequency direct regeneration of somatic embryos, even from well differentiated explant tissues. Low-frequency embryo production, poor germination and conversion of somatic embryos into plantlets and somaclonal variation are the major impediments limiting the utility of somatic embryogenesis for biotechnological applications in legumes. These limitations, however, may be considerably reduced in the near future, as more newly developed growth regulators with specific morphogenic targets become available for experimentation. From the published data, it is apparent that more effort should be given to develop repetitive embryogenic systems with high frequency of germination and regeneration, since such systems will find immediate application in mass propagation and other crop improvement programmes. As our understanding of various morphogenic processes, including growth and differentiation of zygotic embryos, is fast expanding, it is conceivable that development of highly efficient somatic embryogenic systems with practical application can be anticipated, at least for the important leguminous crops, in the foreseeable future.