Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated byAgrobacterium following liquid-phase wounding of embryogenic tissues with caborundum

Summary Generation of transgenic papaya (Carica papaya L.) has been hampered by the low rates of transformation achieved by conventionalAgrobacterium infection or microprojectile bombardment. We describe an efficientAgrobacterium-mediated transformation method based on wounding of cultured embryogenic tissues with carborundum in liquid phase. Embryogenic tissues were obtained from cultured immature zygotic embryos collected 75–90 days after pollination. The expressible coat protein (CP) gene of a Taiwan strain of papaya ringspot virus (PRSV) was constructed in a Ti binary vector pBGCP, which contained the NPT-II gene as a selection marker. The embryogenic tissues were vortexed with 600 mesh carborundum in sterile distilled water for 1 min before treating with the disarmedA. tumefaciens containing the pBGCP. Transformed cells were cultured on kanamycin-free medium containing 2,4-D and carbenicillin for 2–3 weeks and then on the kanamycin medium for 3–4 months. The developed somatic embryos were transferred to the medium containing NAA, BA and kanamycin and subsequently regenerated into normal-appearing plants. Presence of the PRSV CP gene in the putative transgenic lines was detected by PCR and the expression of the CP was verified by Western blotting. The transgene was nuclearly inherited as revealed by segregation analysis in the backcrossed R₁ progeny. From five independent experiments, the average successful rate of transformation was 15.9% of the zygotic embryos treated (52 transgenic somatic embryo clusters out of 327 zygotic embryos treated), about 10–100 times higher than the available methods previously reported. Thus, wounding highly regenerable differentiating tissues by carborundum vortexing provides a simple and efficient way for papaya transformation mediated byAgrobacterium.     

Somatic embryo cycling: Evaluation of a novel transformation and assay system for seed-specific gene expression in soybean

Somatic embryo cycling, a modification of soybean somatic embryogenic suspension culture, was developed as an efficient and rapid method of producing tissue suitable for stable transformation of soybean germplasm by biolistic particle bombardment. Instead of using immature seed explants, cotyledon-staged somatic embryo hypocotyls were placed on auxin-containing medium, where they initiated new somatic embryos primarily from single epidermal cells. By bombarding hypocotyls prior to initiation of subsequent embryo formation, we have effectively transformed soybean somatic embryos with the reporter genes neomycin phosphotransferase,gb-glucuronidase, and a mammalian stearyl CoA delta-9 desaturase, controlled by a seed-specific promoter. These embryos contain significantly reduced levels of saturated palmitic and stearic fatty acids, and significant amounts of monounsaturated palmitoleic acid, which is not normally abundant in soybean seeds. This study demonstrates the effectiveness of somatic embryo cycling for soybean transformation, and for testing expression of genes for seed-specific proteins. Abnormal flower development in recovered plants is a limitation for application of the technique to produce transgenic seed at present.     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Somatic embryo cycling: Evaluation of a novel transformation and assay system for seed-specific gene expression in soybean

Somatic embryo cycling, a modification of soybean somatic embryogenic suspension culture, was developed as an efficient and rapid method of producing tissue suitable for stable transformation of soybean germplasm by biolistic particle bombardment. Instead of using immature seed explants, cotyledon-staged somatic embryo hypocotyls were placed on auxin-containing medium, where they initiated new somatic embryos primarily from single epidermal cells. By bombarding hypocotyls prior to initiation of subsequent embryo formation, we have effectively transformed soybean somatic embryos with the reporter genes neomycin phosphotransferase,gb-glucuronidase, and a mammalian stearyl CoA delta-9 desaturase, controlled by a seed-specific promoter. These embryos contain significantly reduced levels of saturated palmitic and stearic fatty acids, and significant amounts of monounsaturated palmitoleic acid, which is not normally abundant in soybean seeds. This study demonstrates the effectiveness of somatic embryo cycling for soybean transformation, and for testing expression of genes for seed-specific proteins. Abnormal flower development in recovered plants is a limitation for application of the technique to produce transgenic seed at present.     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE ZYGOTIC EMBRYOS OF MUSCADINE GRAPE (VITIS ROTUNDIFOLIA) CULTIVARS

Embryogenic cell lines of Vitis rotundifolia were produced from immature zygotic embryo explants obtained by culturing ovules, harvested at 20 d postanthesis, for 8 wk and then dissecting embryos from them. Ovules cultured on Nitsch and Nitsch medium with naphthoxyacetic acid and benzyladenine (BA) produced a brown exudate, necessitating three transfers to fresh medium at 2-wk intervals during the 8-wk culture cycle. Zygotic embryos that were subsequently isolated from cultured ovules and placed on the same medium produced a heterogenous callus from which eventually emerged embryogenic cell lines. A higher percentage of ovules from cultivars ‘Dixie’, ‘Fry’, ‘Nesbitt’, and ‘Welder’ produced zygotic embryos (31%–39%) than did those from ‘Carlos’ (3%). A higher percentage of ‘Fry’ ovules produced embryogenic lines from cultured zygotic embryos (6.3%) than did those of the other four cultivars (1%–1.6%). Embryogenic cell lines were white and composed of variably sized cell clusters, somatic embryos, and embryonic tissue embedded in a watery matrix. These lines were maintained for over 1 yr on modified Murashige and Skoog (MS) medium lacking growth regulators by transfer of selected cell clusters every 6 wk. White, opaque somatic embryos grew directly from cell clusters and passed through recognizable developmental stages. Germination was induced by transfer of somatic embryos to MS medium with BA. Although 80%–100% of embryos germinated, plant recovery was low due to poor shoot development.     

转基因番木瓜研究进展

番木瓜环斑病毒 (PRSV)使热带亚热带的重要水果番木瓜的生产受到严重影响 ,在众多方法防效不佳的情况下 ,利用病原获得抗性防治PRSV给番木瓜的生产带来了光明。综述了近年来转PRSVCP基因番木瓜中影响番木瓜转化因素和转基因番木瓜的抗性因素。转PRSV外壳蛋白 (CP)基因的番木瓜中多以胚性组织为转化材料 ,被转化材料的生理状态和基因型 ,是影响转化效率和转基因植株质量的主要因素。所获得的转基因番木瓜对PRSV的抗性在很大程度上依赖于接种PRSV与所转化PRSVCP基因的序列同源性、转基因拷贝数和所转基因的位置等。     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus. Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000     

Multiple paternal genotypes in embryogenic tissue derived from individual immature loblolly pine seeds

Pine embryogenic tissue derived from immature zygotic embryos may consist of multiple genotypes due to simple polyembryony. To test this hypothesis, megagametophytes with intact zygotic embryos were cultured from immature loblolly pine (Pinus taeda L.) seeds of clone WV42 control pollinated with a 1:1:1 pollen mix of clones WV44, WV47, and WV48. Each pollen parent contained a marker allele at one or more of the following loci: aconitase, malic dehydrogenase, 6-phosphogluconate dehydrogenase, and shikimate dehydrogenase, allowing determination of the paternal parent. After two to four weeks in culture, embryogenic tissue derived from zygotic embryos extruded from megagametophytes was separated into individual embryos and sectors of embryogenic tissue. The paternal genotype of each resulting cell line was determined by starch gel electrophoresis. Three of thirty-six explants produced multiple cell lines with genotypic differences among the cell lines within each explant. Our results unequivocally show that it is possible to initiate embryogenic tissue from more than one zygotic embryo of a loblolly pine seed and that the resulting cell lines may be genetically different.Abbreviations ACO aconitase - MDH malic dehydrogenase - SKDH shikimate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency

Summary Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos. In an effort to improve the transformation protocol, the effects of gold particle size and callus morphology on transformation efficiency were investigated. Reducing gold particle size from 1.0 μm or 0.6 μm resulted in a significant increase in the rate of recovery of bialaphos-resistant clones from Type II callus. The average transformation efficiency of pre-embryogenic, early embryogenic and late embryogenic callus did not vary significantly. Rates of transformation, regeneration and fertility achieved for Type II callus are summarized and compared to those achieved for greenhouse- and field-derived immature zygotic embryos.     

Evaluation of key factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of somatic embryos of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l⁻¹ kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l⁻¹ partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ timentin for 2 months. Then, kanamycin level was increased to 100 mg l⁻¹ for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA₃ for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l⁻¹ agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.     

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.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Somatic embryogenesis from immature zygotic embryos of annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl⁻¹ activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.     

超声波辅助农杆菌介导CP基因转化番木瓜（Carioca papaya L.）的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PT—NP—YKT)插入双元栽体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1／2MS 6％蔗糖 1％葡萄糖,pH5．7)调整至光密度OD600nm=15-0．20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26．3％),而在对照处理64块胚性愈伤中仅获得1个转化系(1．6％);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16．7％),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP基因转化上均表现出相似的效果。试验还表明：120mg／L是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定。     

华北落叶松体细胞胚胎发生及遗传转化实验系统的建立(简报)

华北落叶松(Larix principis-Rupprechtii)是我国北方中高山地区重要的针叶速生用材树种,进行其体细胞胚胎发生和植株再生的研究,在针叶树无性快速繁殖及基因工程育种上有其特殊的用途,既可为针叶树无性系林业提供产业化途径,也可作为目的基因遗传转化实验系统。针叶树的基因转化相对较难,再生更属不易,Lelu等报道过杂种落叶松与欧洲落叶松体细胞胚胎发生方面的研究;而我国尚未见有落叶松体细胞胚胎发生的研究报道。我们