大豆基因型对根癌农杆菌菌株敏感性的研究

以栽培大豆［Glycine max (L.) Mer］吉林30、吉林43、绥农8、黑农35和东农42等的下胚轴为外植体,用EHA105和LBA4404 2个根癌农杆菌菌株（分别含有pGBI121S4ABC和pGBI4A2B质粒）研究大豆基因型对根癌农杆菌的敏感性,以及根癌农杆菌对大豆的侵染能力。结果表明,大豆基因型对根癌农杆菌的敏感性存在显著差异,以吉林43最敏感。根癌农杆菌菌株对大豆下胚轴侵染能力不同,含有pGBI121S4ABC质粒的LBA4404侵染能力较强,但差异未达显著水平。 Abstract:The sensitivity of genotypes in soybean to lines of Agrobacterium tumefaciens and the ability of A.tumefaciens infecting to soybean were investigated with hypocotyls of soybean (Jilin30,Jilin43,Suinong8,Heinong35 and Dongnong42) and lines of A.tumefaciens LBA4404 and EHA105 which including plasmid pGBI121S4ABC and pGBI4A2B respectively.The results showed that the sensitivity of genotypes in soybean to A.tumefaciens was significantly different.Jilin43 was the most sensitive materials to A.tumefaciens.The ability of A.tumefaciens infecting hypocotyls in soybean was different.LBA4404 including plasmid pGBI121S4ABC was easier to infect hypocotyls of soybean.     

A Novel System for Proliferation，Maintenance and Plantlet Germinationfrom Somatic Embryo of Soybean

Somatic embryogenesis was successfully induced with auxin in high concentration from immature cotyledons of 18genotypes of soybean(Glycin emax(L)Merr．)adaptable for planting in Northeast China．The frequency of somatic embryogenesis varied from 0．29％to77．62％among 18genotypes of soybean．The proliferative somatic embryos were obtained from 10genotypes of soybean at rate of 5．2％-22．1％．For the first time．plantlets were obtained even after subcultured and maintained on solid medium for over one year．This system provided a new and efficient receptor for genetic transformation of soybean．     

农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究

以杂交育种中广泛使用的优良玉米自交系340、4112为材料,用带有质粒pGBIL04（Pactin-Bt-Tnos）的根癌农杆菌LBA4404转化其幼胚及其初始愈伤组织,共培养3天后,在含PPT的培养基上连续筛选培养3代,然后分化获得再生植株。PCR检测证明目的基因已整合到再生植株的基因组中。实验结果表明幼胚预培养后形成的新鲜的初始愈伤组织是比较适宜的转化受体,结果还发现将共培养温度降到22℃可以提高农杆菌介导的玉米遗传转化的筛选频率。 Abstract:Excellent inbred-lines of maize,340 and 4112,which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens.The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04.After 3 days of co-cultivation,the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations,then plants were regenerated.It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants.The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22℃.     

Effect of 2,4-dichlorophenoxyacetic acid concentration on somatic embryogenesis and heritable variation in soybean [Glycine max (L) merr.]

Summary The frequency and quality of embryogenic response from cotyledons of immature zygotic soybean embryos varied with 2,4-dichlorophenoxyacetic acid (2,4-D) concentration in the culture medium. The frequency of variants among progeny of regenerated plants decreased with an increase of 2,4-D concentration. Teratogenic effects on embryo morphology and development were greatest at 22.5μM 2,4-D and decreased with increasing 2,4-D. At the lowest 2,4-D concentration tested, 22.5μM, morphologically abnormal, cotyledonary-stage somatic embryos were produced. Ten percent or less of these embryos converted to plants. Over the nine genotypes tested, 40% of the families derived from plants regenerated under a low 2,4-D concentration manifested heritable variation. In contrast, embryogeny was suppressed at the globular stage by the highest 2,4-D concentration tested, 200μM. Eighty to one-hundred percent of the embryos organized under this latter 2,4-D level converted to plants. Only 3% of the families from the progeny of plants regenerated under a high 2,4-D concentration exhibited heritable variation. This is Journal Paper No. J-14217 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2974. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or Iowa State University and does not imply its approval to the exclusion of other products that may be suitable. This work was supported, in part, by American Soybean Association grant no. 400-46-73-15-2763.     

植物激素对体细胞胚胎发生的诱导与调节

以作者自己的工作为背景,结合国内外近几年的有关报道,综述了几种外源和内源激素对植物体细胞胚胎发生的诱导与调节作用。外源生长素和细胞分裂素是诱导离体培养细胞分化与增殖所必需的,2,4-D是诱导胚性愈伤组织的重要激素。在体细胞胚胎发生中内源激素含量和代谢的平衡起着关键的作用,而且外源和内源激素对诱导体细胞胚胎发生起相互调节作用。ABA在提高体细胞胚胎发生频率和质量上具有重要作用,同时,外源与内源ABA对体细胞胚胎发生起相互促进作用。本文还较为深入地讨论了这些激素诱导体细胞胚胎发生的可能作用机制。 Abstract:The paper summarizes the induced and regulatory effects of a few exogenous and endogenous hormones in plant somatic embryogenesis by our studies and related international reports.The exogenous auxin and cytokinin are necessary to induced differentiation and proliferation of cells of culture in vitro.2,4-D is an important hormone of induced embryogenic calluses.The contents and the metabolic balances of endogenous hormones have key effects for somatic embryogenesis.In addition,the exogenous and endogenous hormones have mutual regulatory effects for somatic embryogenesis.ABA has an important effect to improving the frequency and quality of somatic embryogenesis.Meanwhile,the exogenous and endogenous ABA have mutual promoted effects for somatic embryogenesis.The paper discusses possible mechanism of hormones-induced somatic embryogenesis in a deep-going way.     

抗生素对大豆愈伤组织的诱导和生长的影响

用红霉素、头孢唑啉钠、头孢拉定、头孢霉素(国产和进口)等5种抗生素对农杆菌LBA4404进行抑菌试验,以头孢霉素的抑菌效果最好.头孢霉素作为抑菌剂用于大豆遗传转化试验时,在下胚轴浓度以300mg/L,在子叶节以500mg/L为宜.大豆品种对卡那霉素的反应在出愈率上表现相似,在褐化率上表现有些不同.大豆不同外植体对卡那霉素的反应存在较大差异,以真叶反应最敏感,下胚轴反应最迟钝.在以卡那霉素作为抗性选择标记时,选择压力真叶和子叶节以50～100mg,L为好,下胚轴以100～200mg/L为宜。 Ahstract:The experiment of inhibiting Agrobacterium LBA4404 was undertaken with 5 antibiotics (the Erythronycin Base,Cefazolin Sodium,Cefradine,2 kinds of Cefotaximes).Among them,Cefotaxime showed the best effect.When Cefotaxime is used in transformation,the ideal concentration is 300mg/L in hypocotyl and 500mg/L in cotyledon node.The response of soybean varieties to Kanamycin is similar in induction of callus rate and is different in brown rate of callus.Differences of the response of soybean explants to Kanamycin were found.The young leaves are sensitive to Kanamycin,but hypoeotyl is not.The ideal selecting pressure of Kanamycin is 50- l00mg/L in young leaf and cotyledon node,andl00-200mg/L in hypocotyl when Kanamycin is used as selection marker.     

Somatic embryogenesis from chickpea (Cicer arietinum L.) inmature cotyledons: The effect of zeatin, gibberellic acid and indole-3-butyric acid

Studies were conduced to test the effects of various cytokinins on somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons. Zeatin (13.7 μmol) added, to B5 basal medium, supplemented with 1.5 % sucrose and 0.2 μmol indole-3-acetic acid, was the most effective cytokinin. Lobular structures obtained from cotyledons cultures were transferred to B5 basal medium supplemented with gibberellic acid and indole-3-butyric acid at different concentrations. The most effective treatment was B5 medium containing 14.4 μmol gibberellic acid plus 1.0 μmol indole-3-butyric acid in which 42.8 % of lobular structures cultured formed normal somatic embryos. High conversion of embryos into plantlets (61.0–65.2 % embryos regenerated plants) was observed when germinated embryos were placed on plant development medium.     

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]

Summary Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1⁻¹ (45.2 μM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1⁻¹ (90.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1⁻¹ (11.3 μM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.     

大豆体细胞胚增殖保存与萌发植株体系的建立(英文)

以大豆(Glycine max(L.)Merr.)未成熟子叶为外植体,用高浓度生长素诱导东北地区主栽大豆的18个基因型体细胞胚胎发生,诱导率为0.29％～77.62％。在此基础上成功地诱导10个大豆基因型产生可继代增殖的体细胞胚,诱导率在5.2％～22.1％之间。经在固体培养基上多次继代增殖,首次建立了可在固体培养基上继代增殖的大豆体细胞胚萌发再生体系,继代一年以上的体细胞胚仍具有萌发能力和正常育性,得到了结荚植株。此体系的建立为大豆的遗传转化提供了新的、更为有效的受体体系。     

大豆体细胞胚增殖保存与萌发植株体系的建立

以大豆(Glycine max(L.)Merr.)未成熟子叶为外植体,用高浓度生长素诱导东北地区主栽大豆的18个基因型体细胞胚胎发生,诱导率为0.29%～77.62%.在此基础上成功地诱导10个大豆基因型产生可继代增殖的体细胞胚,诱导率在5.2%～22.1%之间.经在固体培养基上多次继代增殖,首次建立了可在固体培养基上继代增殖的大豆体细胞胚萌发再生体系,继代一年以上的体细胞胚仍具有萌发能力和正常育性,得到了结荚植株.此体系的建立为大豆的遗传转化提供了新的、更为有效的受体体系.     

大豆主栽品种体细胞胚胎发生的影响因素及再生植株

Factors on in vitro somatic embryogenesis of soybean (three elite cultivars) were studied using cotyledons of 3.0-6.0 mm immature seed as explants. Not only the kinds, concentrations and combinations of plant growth regulatory substances but also immature embryo length and inoculum density have main effects on the approaches of embryogenesis. The results of two-factors analysis of variance experiments showed that immature embryo length, plant growth substance concentration and basic medium type have very significant effects on the frequency of embryogenic response, furthermore, interactions exist between the former two factors and are just very significant(at 1% level). The best combinations between 2,4-D concentration and cotyledon length are 10 mg/L 2,4-D & 4.0 mm immature embryos, 20-40 mg/L 2,4-D & 5.0 mm immature embryo. Under these combinations, the salt composition of E1 are very significantly better than that of MS. In conclusion, in the regeneration system established by us the frequency of somatic embryogenesis from the soybean immature cotyledons is greater than 50% and the frequency of conversion of normal (not fused) somatic embryos is about 52.9%-62.6%.     

金花茶子叶在离体培养中胚状体的发生和小植株的形成

研究了金花茶(Camellia chrysantha(Hu)Tuyama)子叶在离体培养中体细胞胚状体发生的条件。在MS基本培养基中附加苄基嘌呤(BA)或苄基嘌呤与萘乙酸(NAA)组合,诱导了胚状体发生。组织学观察表明,胚状体起源于子叶的表皮细胞。在增添细胞分裂素和生长素的MS或改良B_5液体培养基里振荡培养,明显地促进了胚状体根的生长和茎的发育。胚状体在继代培养中能保持旺盛的再生能力。已得到两个繁殖率较高的胚状体无性系。在合适的条件下,胚状体能长成正常的小植株。     

华山松胚性愈伤组织诱导与幼胚离体培养

探索华山松(Pinus armandii)体细胞胚胎发生技术对其实施规模化无性繁殖和开展遗传转化具有重要意义。本文以1/2LM为基本培养基, 通过激素调节等措施对华山松的胚性愈伤组织诱导和幼胚的离体培养技术进行了初步研究。研究结果: 胚性愈伤组织诱导率最高可达52.71%, 但愈伤组织继代培养后没有体细胞胚胎的分化; 首次从其子叶期的幼胚中直接诱导出具有根和茎的完整植株, 诱导率达92%以上。文章确认了采集的幼胚发育状态对胚性愈伤组织的诱导有重要影响, 并对诱导的培养条件等进行了探讨。     

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.     

Somatic embryogenesis and plant regeneration in Chinese soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)—impacts of mannitol,abscisic acid,and explant age

From a preliminary experiment on 98 Chinese soybean varieties, 12 varieties with somatic embryogenesis frequency ranging from 0.0% to 85.7% were selected for further study in order to enhance the efficiency of somatic embryogenesis and plant regeneration. The effects of different mannitol concentrations, abscisic acid (ABA) concentrations, and embryo explant ages (sizes) were investigated. Significant differences in somatic embryogenesis were found among the 12 soybean varieties, with initiation frequencies varying from 22.1% to 89.0% under suitable mannitol concentration, and with N25281, N25263, and N06499 having the highest somatic embryogenic capacity. The results showed that all three factors were relevant for raising rates of callus initiation and somatic embryogenesis, but with differential responses among the genotypes. The treatment of 3.0% (w/v) mannitol, 5 mg l⁻¹ ABA, and a 4- to 5-mm-sized explant was found to be optimal for somatic embryogenesis, generating the highest explant-based regeneration rate at 83.0%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. The above results provide a basis for efficient regeneration of soybean and are informative for the development of genetic transformation systems in Chinese soybean germplasm.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Optimization of direct somatic embryogenesis from mature zygotic embryos ofPanax ginseng C. A. Meyer

Culture conditions were optimized for somatic embryogenesis ofPanax ginseng. The highest frequency of embryo formation was obtained when tissues were excised from the middle region of the cotyledon segments of zygotic embryos. Only treatment with light could stimulate the formation of single-type somatic embryos, whereas multiple-type somatic embryos and calli were observed under dark conditions. The highest production of somatic embryos was found with an NH₄ ⁺:NO₃ ratio of 21:39. Among the tested media (MS, B5, and SH), maximum formation of somatic embryos was obtained when cotyledon expiants were cultured on an 1% agar MS medium supplemented with 5% sucrose. Regenerated ginseng plantlets were transferred to an autoclaved soil mixture in the greenhouse. These transformants showed no detectable variations in their morphology or growth characteristics compared with the donor plant.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.