In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis

A procedure is described for the isolation and culture of protoplasts from embryogenic callus (gel-like callus — GLC) and embryogenic suspension cultures (ESC) of Cucumis sativus c.v. Borszczagowski. Maximal protoplast yields from GLC and ESC were 5×10⁶ and 1×10⁷ protoplasts/g tissue respectively. They were obtained following 14–16 h digestion with 1.2% Cellulase Onozuka R-10, 1.2% Macerozyme R-10 and 0.3% Driselase. At a plating density of 2×10⁵ / ml, first divisions occurred in 4–5 days and 7–8 days in ESC-and GLC-derived protoplasts respectively. The highest percentage of direct embryogenesis (over 80%) was observed with ESC. It was possible to obtain approximately 5000 embryo structures / g tissue. Some embryos converted into plants after 6 weeks, but most of them after 2 months of culture. ESC-derived plants, when transferred into the glasshouse, bloomed normally, and set seeds.Abbreviations CMS Murashige & Skoog (1962) medium for cucumber - GLC gel-like callus - ESC established embryogenic suspension culture - 2,4-d 2,4-dichlorophenoxyacetic acid     

Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants

Summary Transgenic cucumber (Cucumis sativus L.) plants were successfully obtained from hypocotyl explants inoculated with Agrobacterium tumefaciens, which harbored a binary vector plasmid with NOS-nptII, CaMV 35S-I-gus and CaMV 35S-hph genes. Acetosyringone enhanced the efficiency of transformation at the cut surface cells of hypocotyl explants during five days of co-cultivation. Transformed cells were more effectively selected using 20–30 mg/l hygromycin B than using 50–100 mg/l kanamycin. Shoot regeneration occurred within 4–6 wks, and 12 of 21 regenerated plantlets displayed strong GUS expression in the very young leaves. All of 8 GUS-positive R0 plants examined showed single or a few positive bands by Southern blot analysis. The expression of the CaMV 35S-I-gus gene was observed in various tissues and organs of R0 and R1 transgenic cucumber plants.     

Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants

Cotyledons of cucumber seedlings (Cucumis sativus L. cv. Poinsett 76) were co-cultivated with disarmed Agrobacterium strain C58Z707. The Agrobacterium strain contained the Agrobacterium-derived binary vector plasmid pGA482, its T-DNA region contains a plant expressible bacterial derived neomycin phosphotransferase II (NPT II) gene which upon transfer, genome integration, and expression in plant tissues confers resistance to the antibiotic kanamycin. After growth of inoculated cotyledon sections on selective medium containing 100 mg/l kanamycin, transformed embryogenic calli were obtained followed by the development of embryos and plant regeneration. Transformed R₀ and R₁ cucumber plants appeared normal and tested positive for NPT II enzyme activity. Genomic DNAs isolated from the NPT II positive plants all showed hybridization to the characteristic 2.0 kb (BamHI to HindIII) NPT II gene-containing fragment. These results show that the Agrobscterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for the transfer of genetic material into plant species belonging to the family Cucurbitaceae.Abbreviation Cb carbenicillin - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - KN kinetin - MS Murashige and Skoog - NAA naphthaleneacetic acid - NPT II neomycin phosphotransferase II     

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo₃ in the infection medium and adding AgNo₃ in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×10⁹ c.f.u. ml^–1 for bacterium inoculation; 100 mg l^–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T₀ plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T₁ plants were analyzed and transmission of transgenes to the T₁ generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.     

Transfection and transformation of Agrobacterium tumefaciens.

Summary The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10^-7 transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum frequency of 3.5 10^-7 transformants per total recipient population was obtained. Agrobacterium Ti-plasmids were introduced in the strain GV3100 with a maximal efficiency of 4.5 10^-8. These experiments provide further evidence that the Ti-plasmid is responsible for the oncogenic properties of A tumefaciens and for its capacity to induce opine synthesis in Crown-gall plant cells.     

Daily temperature gradients and processes of organogenesis in apical meristem of Cucumis sativus L

We studied the influence of daily temperature gradients on organogenesis in apical and axil shoot meristems at different developmental stages in Cucumis sativus L. The level of organogenic activity of meristems was determined according to the number of leaf primordia on the main and lateral shoots, number of 2nd order shoots, and rudiments of flowers of different levels of development. At the studied ontogenetic stages (mesotrophic seedling or juvenile state), plants were grown under the controlled conditions: photoperiod 12 h, light intensity 100 Wt/m2, range of mean daily temperatures 20 ... 30 degrees C, and daily temperature gradients -20 ... +20 degrees C. After the temperature treatment, some plants were returned to the optimal, for growth and development, conditions for two weeks (aftereffect). Three types of organogenic activity of meristems in response to the influence of variable daily temperatures were described: stimulation, inhibition, or absence of effect. The phenomenon of stimulation includes two subtypes: optimization, when a maximum effect, observed at other constant temperatures, was attained under the influence of variable temperatures and maximization, when maximum values markedly exceeded those at constant temperatures. The patterns described are preserved on the whole in the aftereffect of daily temperatures.     

黄瓜子叶和下胚轴的离体培养

1　植物名称　黄瓜 (Ｃｕｃｕｍｉｓｓａｔｉｖｕｓ)品种“津绿 4号” ,由天津农业科学院黄瓜研究所育成。。2　材料类别　种子萌发的无菌苗子叶和下胚轴切段。3　培养条件　 (1 )种子萌发培养基 :1 /2ＭＳ0 ;(2 )诱导分化培养基 :ＭＳ 6 ＢＡ 1 0ｍｇ·Ｌ- 1 (单位下同 ) ＩＡＡ 0 2～ 0 4;(3 )生根培养基 :ＭＳ ＩＡＡ 0 5。上述培养基内琼脂浓度均为 0 8% ,蔗糖为 3 % ,ｐＨ5 8～ 6 0。培养温度 (2 5± 2 )℃ ,光照时间 1 0ｈ·ｄ- 1 ,光照度 2 0 0 0ｌｘ。4　生长与分化情况4.1　无菌苗的培养　将黄瓜种子去壳后 ,以 75 %乙醇…     

Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes

Summary Transgenic cucumber plants (Cucumis sativus L., cv. Straight Eight) were regenerated from roots induced by inoculation of inverted hypocotyl sections with Agrobacterium rhizogenes containing the vector pARC8 in addition to the resident Ri-plasmid. The DNA transferred to the plant from the vector (T-DNA) included a gene which encoded the enzyme neomycin phosphotransferase II, and thus conferred on the plant cells resistance to kanamycin. The transgenic plants looked normal and were positive for the neomycin phosphotransferase II. Southern blot analysis of the transgenic plants revealed that all plants contained vector DNA, but only some of them contained DNA from the Ri plasmid.     

A rapid and efficient method for in vitro shoot organogenesis and production of transgenic Bacopa monnieri L. mediated by Agrobacterium tumefaciens

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Bacopa monnieri L. (Scrophulariaceae), a plant well known for its medicinal properties. Leaf explants were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), and in combination with either indole-3-acetic acid (IAA) or napthalene-3-acetic acid. A combination of BAP (17.80 μM) and IAA (2.28 μM) maximized shoot initiation (85.2 ± 2.43) with greatest shoot length (2.8 ± 0.22), and was obtained directly from leaf explants without an intervening callus phase. Leaf segments from in vitro grown plants were co-cultivated with Agrobacterium tumefaciens LBA4404 harboring pCAMBIA1301 with ?-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes. The co-cultivated explants were transferred to selective shoot induction and elongation medium. The elongated hygromycin-resistant shoots were subsequently rooted on MS medium supplemented with 4.9 μM indole-3-butyric acid and 25 mg/l hygromycin (SSRM). Successful transformation was confirmed by monitoring histochemical GUS activity during shoot elongation and PCR analyses using uidA- and hpt-specific primers. Integration of hpt into the genome of transgenic plants was also verified by Southern blot analysis. The highest transformation efficiency achieved was 70.6%, with an average of 10.4 ± 0.15 transgenic plantlets per explant using the present transformation system. Therefore, these highly efficient and rapid regeneration and transformation systems create significant potential for engineering of B. monnieri with a view to detailed biomolecular analyses or for further enhancement of its medicinal properties.     

根癌农杆菌介导灭蚊卵菌贵阳腐霉的遗传转化

Pythium guiyangense is a mosquito pathogen, and has been proved to be a promising agent for biological control of mosquitoes. In order to develop the strains adaptable to different ecological environment having stable virulence to mosquito larvae, and being able to prolong the shelf life, an effort was made on transforming the fungus by using homologous or heterologous virulence genes. In this paper, a genetic transformation experiment of P. guiyangense mediated by Agrobacterium tumefaciens is reported. As a result, an A. tumefaciens mediated genetic transformation system was established successfully.     

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.     

In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts

A method for leaf mesophyll protoplast isolation and plant regeneration of cucumber (Cucumis sativus L.) is described. Using an isolation solution complemented with 0.1 M glycine, 8.2·10⁶ viable protoplasts were isolated from 1 g of fresh leaves. The effect of the growth substances indole-3-acetic acid, naphthalene acetic acid, 2,4,-dichlorophenoxy acetic acid, 6-benzylaminopurine, 2-isopentenyladenine and kinetin at concentrations from 0.5 to 5 mg·1^–1 was studied using the multi-hanging drop technique. The optimal growth substance combination, namely 5 mg·1^–1 naphthalene acetic acid and 3 mg·1^–1 2-isopentenyladenine, together with agarose medium in a so-called bead culture resulted in a plating efficiency of 21%. Some of the colonies obtained regenerated to plantlets which developed to plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog - NAA naphthalene acetic acid     

Flower Formation In vitro by Hypocotyl Explants of Cucumber (Cucumis sativus L.)

Hypocotyl explants of cucumber (Cucumis sativus L.) producedcallus when grown in Murashige and Skoog medium with 0.5 or1.0 µM benzyladenine and 1.5 or 5.0 µm 2, 4-D. Somaticembryos and adventitious buds were formed when callus was transferredto medium without growth regulators. Flowers that were formedin vitro were either staminate or pistillate. Cucumis sativus L, cucumber, embryogenesis, organogenesis, flowering in vitro     

Agrobacterium tumefaciens-mediated genetic transformation of Digitalis purpurea L.

Genetic transformation is a tool of special interest for developing new biotechnological strategies for the production of bio-active compounds such as cardenolides, which are exclusively obtained from plants. To date, Digitalis plants are the main economically viable source of cardenolides for the pharmaceutical industry. This study describes the development of efficient plant regeneration and Agrobacterium-mediated genetic transformation protocols for Digitalis purpurea L. First, a plant regeneration procedure starting from leaf segments of in vitro-cultivated plants was established and the minimal inhibitory concentration of G-418 (geneticin) for callus induction was determined. Both leaf segments and callus tissue were sensitive to G-418 70 mg l^?1. Afterwards, two Agrobacterium strains were used to test their T-DNA transfer ability on D. purpurea leaf tissues, EHA105 and C58C1Rif^R (pMP90), both harboring the binary vector pTJK136. Strain C58C1Rif^R (pMP90) yielded a higher number of transformed plants than EHA105. Successful transformation was confirmed by histochemical β-glucuronidase (GUS) assays of the putative transgenic tissues and PCR analyses using β-glucuronidase (uidA)- and neomycin phosphotransferase II (nptII)-specific primers. Southern blot hybridization confirmed the stable integration of the nptII gene in the transgenic plants. In total, 518 independent transgenic lines were regenerated with an average of 6.91 transgenic lines per initial leaf segment infected with A. tumefaciens strain C58C1Rif^R (pMP90). To date, only a few studies have been published on the genetic transformation of Digitalis species. The protocols for plant regeneration and genetic transformation described in this paper will contribute to functional studies for a better understanding of cardenolide biosynthetic pathways and the metabolic engineering of cardenolides to develop high-yielding improved genotypes.     

根癌农杆菌介导库拉索芦荟遗传转化因素优化

以美国库拉索芦荟的横切薄片(transversethincelllayer,tTCL)作为转化外植体,初步研究了以根癌 农杆菌介导的多种因子对芦荟遗传转化的影响。结果表明:菌株EHA105比LBA4404及AGL1转化率高; 除了乙酰丁香酮(acetosyringone)外,菌液的预处理和重悬液的pH值也是影响转化的主要因子;菌液的预处 理和适合的蔗糖浓度对转化也有促进作用;感染时间为12～18min,共培养的温度和时间分别以25℃及5d 为佳。     

根癌农杆菌介导的金边狗牙根遗传转化条件的优化

为建立根癌农杆菌(Agrobacterium tumefaciens,菌(L.)Pers.]最佳遗传转化体系,以葡萄糖醛酸糖苷酶(GUS)基因的瞬间表达率为指标,从愈伤组织继代时间、根癌农杆菌侵染时间、负压条件及光照时间等方面进行了筛选.结果表明,根癌农杆菌介导的金边狗牙根最佳的转化体系是:以继代培养2周的愈伤组织为起始材料,根癌农杆菌介导感染10 min,经负压处理(抽拉20次)并在全光条件下共培养转化.     

根癌农杆菌介导Bt基因转化水稻的研究

为了培育出无筛选标记基因的转基因水稻,试验将loxp-hpt-loxp基因与成基因连锁在-起转化水稻方法,得到loxp-hpt—loxp—Bt转基因水稻植株,再与同质的带有ere基因的水稻杂交,以定向删除潮霉素抗性筛选标记。试验表明以水稻品种“皖粳97”为供试材料,将成熟胚来源的愈伤组织用根癌农杆菌EHA105／pCAMBIA1305．1感染后,筛选出抗性愈伤组织并获得再生植株。经PCR验证,得到20棵转基因水稻植株。     

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii,C. melo,and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F₁ hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 M), NAA/Z (5.0/5.0 M) or 2,4-D/BA (5.0/5.0 M). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F₁ hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 M. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F₁ hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 M) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 M), but plantlets were recovered only in C. melo.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indoleacetic acid - K kinetin - MS Murashige & Skoog's medium - NAA naphthaleneacetic acid - Z zeatindihydroside