Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

抗生素对大豆愈伤组织的诱导和生长的影响

用红霉素、头孢唑唑钠、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大于豆遗传转化试验时,在下胚轴浓度以300mg/L,在子叶节以500mg/L。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。     

Somatic embryogenesis in Eucalyptus globulus

Somatic embryos were obtained from 1% of cotyledon pieces and hypocotyls of mature embryos of Eucalyptus globulus Labill. cultured on media containing a high concentration of picloram or IBA. 2,4-D and other synthetic auxins did not yield somatic embryos or embryogenic callus. Somatic embryos arose indirectly via callus, being visible after four months, and directly, where little callus or adventitious root initiation occurred. Somatic embryos, formed directly from explants, were visible within five weeks. Various structural abnormalities of somatic embryos were observed, especially after induction on media containing picloram. Only two out of fifteen somatic embryos showed hypocotyl and radical elongation, but plantlets did not develop further.     

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.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

Effects of hygromycin on cotton cultures and its application in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated cotton transformation

We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l⁻¹ completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not grow on media supplemented with 10 mg l⁻¹ hygromycin and were killed at 15 mg l⁻¹. In seed germination assay, the presence of 20 mg l⁻¹ hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin selection protocol for Agrobacterium-mediated cotton transformation and regeneration.     

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.     

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses??caulogenesis, direct somatic embryogenesis, and callusing??were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08???M TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46???M indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.     

Glufosinate as an efficient inhibitor of callus proliferation in coffee tissue

Summary To improve selection of transgenic Coffea spp. tissue after transformation treatments, the effects of the selective agents chlorsulfuron, glufosinate, glyphosate, hygromycin, and kanamycin were studied on callus development from leaf explants (from greenhouse-grown plants and somaplants) and in embryogenic suspension cultures. Studied genotypes were from C. arabica, C. canephora, and the interspecific hybrids Arabusta and Congusta. A culture system based on “direct” somatic embryogenesis from C. canephora leaf explants proved to be more sensitive to selective agents than high frequency somatic embryogenesis from C. arabica or Arabusta leaf explants. With respect to the selective effect, chlorsulfuron and hygromycin provoked strong inhibition and severe necrosis, whereas glyphosate and kanamycin showed variable inhibition. Glufosinate appeared to efficiently inhibit growth of both leaf callus and callus suspensions of all genotypes tested without inducing necrosis. These properties may make the use of glufosinate advantageous in a selective growth system for detection of transformed coffee tissues.     

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l^–1 or on geneticin within 12.5–50 mg l^–1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l^–1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l^–1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l^–1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l^–1) to avoid the adverse effects of kanamycin on embryogenesis.     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

Tissue position,explant orientation and naphthaleneacetic acid (NAA) affect initiation of somatic embryos and callus proliferation in Norway spruce (Picea abies)

Sections from mature zygotic embryos of Norway spruce exhibited different capacities for somatic embryo initiation. The upper hypocotyl part (Zone 2) was the most embryogenic, followed by the lower hypocotyl (Zone 3) and the apical zone (Zone 1); the root part (Zone 4) never initiated embryonal-suspensor masses (ESM). The embryogenic capacity of mature zygotic embryo is narrowly located in the vicinity of Zone 2. The frequency of embryos differentiating simultaneously ESM on Zones 1 and 3 is very low (0.6%) compared to those initiating ESM on Zones 1 and 2 (7%) or Zones 2 and 3 (16%). Elevated concentrations of naphthalene acetic acid (40 and 80 M) reduced ESM initiation and callus proliferation on all sections but Zone 1. Highest initiation rate was obtained when explants were cultured with an apical-end-up orientation. Placing the explant basal-end-up partially inhibited the expression of embryogenic capacity, as well as decreasing the callus proliferation on Zone 3. A weak positive correlation (r=0.19, p < 0.001) was found between embryogenic capacity of the explant and proliferation rate of the derived callus.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

长白落叶松体胚发生再生体系优化

以长白落叶松(Larix olgensis)未成熟合子胚为外植体诱导胚性愈伤组织, 通过调节影响体胚发生的营养物质和植物生长调节剂配比, 进行愈伤组织的胚性恢复与保持以及体胚发生再生体系的优化。结果表明: 不同无性系之间胚性愈伤组织诱导率差异显著, 胚性愈伤组织在S+0.2 mg·L ^-1NAA+0.5 mg·L ^-1BA+0.5 mg·L ^-1KT+0.5 g·L ^-1谷氨酰胺+0.5 g·L ^-1水解酪蛋白+30 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶培养条件下, 可以恢复胚性并长久保持。在S+20 mg·L ^-1ABA+60 g·L ^-1PEG₄₀₀₀+60 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶条件下分化培养6周, 体胚发生率可达100%。将正常发育的体胚先在WPM+ 6 mg·L ^-1间苯三酚+1.0 g·L ^-1活性炭+3.0 mg·L ^-1VB₁+20 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶条件下培养2周, 再转接至B₅+ 0.4 mg·L ^-1NAA+1.0 mg·L ^-1IBA+0.5 mg·L ^-1GA₃+2.0 mg·L ^-1VB₁+1.0 g·L ^-1活性炭+20 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶条件下培养2周, 可见子叶舒展、下胚轴伸长且根系正常的体胚苗。该研究建立了长白落叶松胚性愈伤组织胚性恢复与保持方法, 并进一步优化了体胚发生的植株再生体系, 为林木资源快速繁育和遗传改良奠定了基础。     

Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Analysis of cell wall polysaccharide composition of embryogenic and non-embryogenic calli obtained from hypocotyl and petiole explants from Medicago arborea L. revealed significant differences. For calli induced from both hypocotyls and petioles, levels of total sugars, pectins, and hemicelluloses were higher in embryogenic than in non-embryogenic calli. Whereas in the residual cellulose fraction, the highest levels of sugar were detected in non-embryogenic calli. When comparing the two donor sources of callus explants, the highest total sugar levels were detected in embryogenic calli induced from petioles, mainly in the pectin fraction and to a lesser extent in the hemicellulose fraction. Moreover, analysis of uronic acids revealed higher levels in embryogenic calli, primarily in the pectin fraction. Analysis of those sugars associated with cell walls of calli suggested that these polysaccharides consisted of pectic polysaccharides and glucans, and that their levels were higher in embryogenic than non-embryogenic calli.     

亚麻遗传转化体系的建立及几丁质酶基因导入的研究

报道了亚麻遗传转化体系的建立和几丁质酶基因对亚麻遗传转化的研究。亚麻下胚轴切段培养在不同激素浓度的ＭＳ培养基上,诱导分化出不定芽。最佳的激素组合是ＭＳ＋ＢＡ１ｍｇ／Ｌ＋ＩＡＡ０．５ｍｇ／Ｌ,分化频率可达９７％。亚麻的下胚轴经带有几丁质 根癌农杆菌感染后,在含有１００ｍｇ／Ｌ卡那霉素的选择分化培养基上,１４￣２１ｄ就能产生抗生小芽,小芽进一步伸长后可在１００ｍｇ／Ｌ卡那霉素的ＭＳ选择生根培养基（ＭＳ     

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.