Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

Improving micropropagation of Mentha × piperita L. using a liquid culture system

In the current study, in vitro shoot proliferation and plant regeneration of Mentha × piperita L. (peppermint) cultivar ‘Black Mitcham’ was compared in semi-solid and liquid culture systems. Shoot tips from field-grown plants were used as explants to study shoot proliferation response on either Murashige and Skoog (MS) or Chee and Pool (C2D) medium containing varying levels of 6-benzylaminopurine (BAP), kinetin, and 6-γ,γ-dimethylallyl aminopurine (2iP). Differences in leaf ultrastructure and antioxidant capacity of greenhouse-grown and micropropagation-derived plants were studied to identify potential changes occurring during in vitro culture. Among the various media treatments tested, the maximum number of shoots was produced on the C2D medium with 4.0 μM BAP (40.7) followed by the MS medium with 4.0 μM BAP (32.2). Among the rooting treatments, shoots on the MS medium with 1.0 μM indole-3-butyric acid (IBA) produced the maximum number of roots (14.4). The number of shoots produced in Liquid Lab Rocker® (LLR) vessels containing liquid C2D medium with BAP (103.4) was significantly higher than that produced on semi-solid medium (40.7). No differences were observed in the leaf ultrastructure and antioxidant capacity of leaf extracts obtained from greenhouse-grown and micropropagation-derived plants. The study indicates that the liquid culture system under the described conditions can enhance peppermint micropropagation, with plant material being potentially valuable for use in herbal supplements and essential oil production.

     

Bar-expressing peppermint (Mentha × Piperita L. var. Black Mitcham) plants are highly resistant to the glufosinate herbicide Liberty

Weed control is a substantial economic input for production of mint oils, the most commercially important of which are obtained from peppermint. The objective of this research is to obtain peppermint plants resistant to the broad-spectrum herbicide glufosinate, which can be used for development of economically efficacious weed control strategies and, perhaps, serve as a paradigm in perennial crops. The bar gene, which encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate-ammonium or phosphinothricin (PPT), was constructed into Agrobacterium tumefaciens binary vectors under the nopaline synthase (NOS) or a chimeric promoter containing a trimer of the OCS-upstream-activating sequence (UAS) to a MAS promoter/activator region[(OCS) ₃ MAS]. A total of 142 independent transgenic peppermint (cv. Black Mitcham) plants were obtained (107 and 35 were obtained with pGPTV (and pCAS1) and pATC940 vectors, respectively) and evaluated for herbicide resistance in the greenhouse after foliar application of glufosinate herbicide Liberty as the commercial product. All transgenic plants exhibited substantially less herbicide symptom development than non-transgenic Black Mitcham or untransformed tissue cultured-derived plants, albeit variation for herbicide resistance occurred amongst the transformed lines. Plants from 35 of the 142 lines were selected at random and all were PCR-positive for the presence of bar. Five lines, that were least affected, exhibited no injury symptoms to Liberty concentrations that are 4 times the standard level for control of weeds in peppermint fields. The most resistant transgenic plants had the greatest steady-state PAT mRNA levels and PAT activities. No experimental difference in herbicide resistance was evident between plant populations obtained with pGPTV (pCAS1)-bar or pATC940-bar vector. However, 4 of 35 lines transformed with (ocs) ₃ MAS-bar exhibited maximal resistance while only 1 of 107 NOS-bar lines has comparable resistance. These herbicide resistant peppermint plants will facilitate development of post-emergent herbicide control strategies that use newer generation herbicides, like glufosinate, which have reduced environmental and product residual because of metabolism by microbes and the transgenic plants.     

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus × P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.     

Correction to: Improving micropropagation of Mentha × piperita L. using a liquid culture system

In Vitro Cellular & Developmental Biology - Plant - This correction reflects Sadanand A. Dhekney’s updated e-mail address and affiliation.     

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     

基于RAPD标记的薄荷属(Mentha L.)植物亲缘关系分析

应用RAPD标记方法分析了薄荷属(Mentha L. )7个种38个种源间的遗传多样性,并采用UPGMA聚类分析方法探讨了38个种源间的亲缘关系.结果表明,用20条随机引物从38个种源的总DNA中共扩增出111条带,其中多态性条带91条,多态性条带百分率达81.98%,表明薄荷属植物种间和种内存在丰富的遗传多样性.聚类分析结果表明,在遗传相似系数0.43 处,供试的38个种源被分为2大类,其中第1大类包含日本薄荷(M. arvensis L. )、灰薄荷(M. vagans Boriss. )、留兰香(M. spicata L. )、皱叶留兰香(M. crispata Schrad. ex Willd. )、椒样薄荷(M.×piperita L. )和薄荷(M. haplocalyx Briq. )的37个种源,第2大类仅包含唇萼薄荷(M. pulegium L. )1个种源.在遗传相似系数0.74处,38个种源被分为6组:A组仅包含日本薄荷1个种源;B组包含灰薄荷的4个种源;C组包含留兰香的2个种源和皱叶留兰香的6个种源;D组包含椒样薄荷的5个种源和留兰香的2个种源;E组包含薄荷的17个种源;F组仅包含唇萼薄荷1个种源.在遗传相似系数0.83处,B组、C组、D组和E组可各自进一步划分为不同的亚组.研究结果显示,基于RAPD标记分析的聚类分析结果与传统形态学分类结果基本相吻合;同一种类来源相同或相近的种源聚在一起,说明薄荷属植物种内的遗传关系与地理分布和环境差异有一定的相关性.     

Inheritance of Organelle DNA in Rye (Secale cereale L.) with Triticale (×Triticale Thch.) Hybrids

The mode of inheritance of chloroplast and mitochondrial DNA (mtDNA) in rye × triticale intergeneric hybrids has been studied with the use of specific PCR markers for loci 18S/5S and 3rbcL in organelle DNA. In rye × triticale BC₁, mtDNA copies of two types, paternal and maternal, have been found; in BC₂ plants, only paternal mtDNA and chloroplast DNA (cpDNA) have been detected. Mechanisms determining the inheritance and/or differential amplification of organelles of a specific type are discussed.     

Production,morphology, and cytogenetics of Triticum aestivum (L.) Thell × Elymus scabrus (R. Br.) Love intergeneric hybrids obtained by in ovulo embryo culture

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD), and an apomictic Triticeae species, Elymus scabrus (syn. Agropyron scabrum) (2n=6x=42, HHSSSS), the first successful report of this cross. Nine tiny, underdeveloped, and structureless embryos were obtained in vitro only by in ovulo embryo culture at 4 days after pollination, which gave rise to five mature hybrid plants. All the hybrid plants were vigorous and possessed a phenotype intermediate to the two parents. There were 2n=6x=42 (ABDHSS) somatic chromosomes in the hybrids. There was little or no homology between the parental genomes, as shown by an overall meiotic chromosome association of 32.83 I + 4.08 rod II + 0.21 ring II + 0.18 III + 0.02 IV. The hybrids were completely sterile and so far backcrosses to wheat parent have not been successful. Alternate approaches to induce gene transfer(s) from E. scabrus to wheat are being attempted.Agriculture Canada Contribution No. 398.     

Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation

Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD₆₆₀ ≅ 0.6, diluted to a density of 10⁹ cells ml⁻¹, followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml⁻¹). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T _L -DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T _R -DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.     

Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens

Vectors for transformation of higher plants mediated by Agrobacterium tumefaciens were modified so that one, two or three additional copies of the left border (LB) sequences were inserted close to the original LB of the T-DNA. A gene for -glucuronidase (gusA) was placed outside the T-DNA to monitor the transfer to plants of 'vector backbone' sequences. The expression of GUS in immature embryos of rice that had been co-cultivated with A. tumefaciens carrying these constructs was around one tenth of that with A. tumefaciens carrying an unmodified control vector. Between 88 and 127 of independent transformants were regenerated from rice tissues infected with A. tumefaciens carrying each of these vectors. The GUS expressors among the rice transformed with the modified vectors were much less frequent than ones among the control transformants, and rate of reduction in the ratio of transgenic plants that expressed GUS was higher than 93%. Detection of a fragment across the LB region by the polymerase chain reaction and the gusA gene by Southern hybridization correlated well with GUS expression. These results indicate that transfer of the 'vector backbone' from the control vectors resulted mainly from inefficient termination of formation of the transfer intermediate of the T-DNA and additional LB sequences effectively suppressed such transfer. This approach is simpler than the strategy to place a 'lethal gene' outside the T-DNA and will likely help produce 'clean' transformants efficiently.     

水稻(Oryza sativa L.)×狼尾草(Pennisetum sp.)胚胎发育的细胞学观察

1.狼尾草花粉可以在水稻柱头上萌发并能长入胚囊,但受精过程缓慢而且不正常,单受精现象经常出现。2.胚胎发育滞缓,长时间停留在球状胚阶段,很难进一步分化,而且不时败育,只在传粉后16—24天的一些胚囊中看到有简单分化的胚胎。3.胚乳发育异常,由游离核形成细胞的时间推迟到传粉后第八天。整个胚乳组织由形状及功能上均有很大差异的细胞团块组成。其中一部分细胞缺乏合成淀粉的能力,胚乳在发育过程中不时出现解体现象。4.在反足细胞附近的一些珠心细胞中出现多量淀粉积累,反映因杂交而出现的胚囊代谢上的某些变化。讨论了胚和胚乳发育困难的原因和得到杂种种子的可能性。     

Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’

Efficient callus formation was achieved from cotyledon, stem, and leaf expiants of the domestic safflower cultivar Centennial on MS salts medium containing 1 mg/L BAP and 1 mg/L NAA. Shoot buds were regenerated from 26% of leaf-derived calli on callus induction medium, although attempts to root regenerated shoots were not successful. Centennial expiants inoculated with Agrobacterium tumefaciens containing NPT II and GUS genes produced kanamycin-resistant calli from which buds were regenerated. Transformation and stable integration of transgenes was confirmed by GUS assay and DNA hybridization in kanamycin-resistant calli, and GUS assay in regenerated shoots.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - GUS -glucuronidase - IAA indole-3-acetic acid - NPT II neomycin phosphotransferase II     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Micropropagation of fig (Ficus carica L.) ‘Roxo de Valinhos’ plants

Summary An in vitro protocol for Ficus carica cv. ‘Roxo de Valinhos’ was optimized. Nodal explants containing two buds were excised from field-grown mature plants, and transferred to different proliferation media consisting of combinations of distinct concentrations of activated charcoal with benzyladenine (BA), kinetin with gibberellic acid (GA₃), and WPM (woody plant medium) with kinetin. The regular strength of WPM in combination with 0.5 mgl⁻¹ kinetin was the best condition for shoot proliferation of Ficus carica ‘Roxo de Valinhos’ plants. The addition of activated charcoal in the medium completely inhibited shoot proliferation. The inclusion of BA in the medium induced excessive callus formation as well as small and vitrified shoots, while GA₃ induced excessive elongation associated with vitrification, chlorosis, and tip-burned shoots.     

水稻(Oryza sativa L.)×狼尾草(Pennisetum sp.)杂种F_1的细胞学观察

1.本工作对于邓炎棠所获得的水稻北陆12-狼尾草杂种 F_1不孕植株进行了细胞学观察,附带对于胚囊的形成及雌配子体的分化作了简单的描述。2.在孕穗期固定的材料中观察到多数大孢子在形成过程中即退化而不能发育成胚囊。一些已形成的胚囊中没有细胞而也呈退化现象。雌配子体大多数不能正常分化为卵器、极核与反足细胞,或能分化而细胞的数目不正常,有时卵器中助细胞与卵细胞不能区分。在有一些情况下子房膨大而胚囊中充满了液体。仅在少数情况下观察到雌配子体的正常发育。3.母本水稻的染色体为 n=12,父本狼尾草的染色体为 n=9,而杂种 F_1的体细胞染色体变化在12—42之间,而在花粉母细胞中的染色体则变化在12—20之间,但也有多至30余者。4.在减数分裂中期Ⅰ及Ⅱ和后期Ⅰ及Ⅱ都经常看到有大量落后的染色体。在后期Ⅰ及Ⅱ偶然也遇到染色体桥。在末期Ⅰ及Ⅱ或甚至在形成四分孢子时仍经常观察到遗留在核外的染色体。但并不形成小核。5.在减数分裂的两次分裂中纺锺体的形成都是正常的,没有发现细胞的不等分裂,多极纺锺体或多纺锺体等现象。四分体的形成同样也是正常的,没有发现二分体或五分体等的形成。但是,小孢子形成后,其核大多数不能分裂,因而产生了败育的花粉粒。仅在极少数花的花药中见到正常的花粉粒。6.正常发育的雌雄配子非常少,因而导致了杂种 F_1植株的高度不孕性。     

Developing phosphinothricin-resistant transgenic sunflower (Helianthus?annuus L.) plants

Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants. Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta^? at 3 l/ha.