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.     

Isolation of tomatine from cultured excised roots and callus tissues of tomato

Tomatine was present in cultured excised tomato roots but in lower concentrations than in seedling radicles of the same age. The alkaloid was not detected in 'spent' root medium. Newly-initiated callus cultures of hypocotyl, radicle and cotyledon origin produced roots, and tomatine was isolated from both roots and callus. Roots contained more tomatine than callus, but neither contained as much as the organ explants from which the cultures were initiated. The number of roots produced decreased with time, as did also the tomatine content of the callus tissues. After 447 days, when no organized structures were produced by callus cultures, tomatine was not detected. An established hypocotyl callus contained small amounts of tomatine when grown on certain nutrient media, but a chlorophyllous sub-isolate of this callus did not produce detectable quantities of the alkaloid. Tomatine was not detected in an established root callus isolate or in suspension cultures initiated from established, tomatine-containing hypocotyl callus.     

Transcriptome analysis of age-related gain of callus-forming capacity in Arabidopsis hypocotyls

Callus-forming capacity is enhanced with hypocotyl maturity in Arabidopsis. However, the genetic regulation of age-related gain in capacity for callus formation is unclear. We used a gene expression microarray assay to characterize the underlying mechanisms during callus formation in young and mature hypocotyl explants of Arabidopsis. As expected, genes involved in photosynthesis and cell wall thickening showed altered expression during hypocotyl maturation. In addition, genes involved in cytokinin perception were enriched in mature hypocotyl tissues. Phytohormone-induced callus formation in hypocotyl explants was accompanied by increased expression of genes mainly related to the cell cycle, histones and epigenetics. The induction level of these genes was higher in mature hypocotyl explants than young explants during callus formation. We identified a number of genes, including those with unknown function, potentially involved in age-related gain in callus formation. Our results provide insight into the effect of hypocotyl age on callus formation. Altered cytokinin signaling components, cell cycle regulation and epigenetics may work in concert to lead to gain of callus-forming capacity in hypocotyls with age.     

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     

Cytodifferentiation and transformation of embryogenic callus lines derived from anther culture of wheat

Three types of callus tissues established from anther culture of eleven doubled haploid (DH) lines of wheat (Triticum aestivum L.) were evaluated for their ability in enhancing friable embryogenic (Type II) culture differentiation and genetic transformation. Differences between types of callus inocula were highly significant (P < 0.001), suggesting that the quality of the initial callus explant is of profound importance in encouraging the proliferation of Type II cultures. Other factors found to be crucial included weekly subculture of friable embryogenic callus tissues on a maintenance medium containing 30 microM dicamba and a predominance of amino-acid nitrogen supplement. Transfer and integration of the beta-glucuronidase gene was also affected by the type of inoculum when suitable embryogenic cell cultures were transformed using silicon carbide whiskers and high velocity microprojectiles. Expression of the hygromycin phosphotransferase selectable marker gene sequence was confirmed in all the stably transformed cell lines maintained on selection media containing lethal levels of hygromycin. Comparatively, there were differences in the frequency of regenerable, transgenic clonal segments between whisker-treated and microprojectile bombarded tissues mainly as a result of the fact that cultures vortexed with whiskers were more capable of post-treatment cell proliferation and embryo differentiation than those bombarded with cDNA-coated microprojectiles. Conditions for obtaining these results are outlined and discussed in relation to the suitability of the two transformation strategies for producing transgenic cell aggregates of wheat.     

影响农杆菌介导白细胞介素-2基因转化番茄的因素研究

利用农杆菌介导法将白细胞介素-2基因（il-2）导入番茄中,对影响其转化的因素进行了分析。结果表明：农杆菌菌种（EHA105和C58C1）、外植体类型（子叶和下胚轴）、带有不同筛选标记（Kan^r、PPT^r、Hyg^r）的载体质粒几个因素对芽诱导分化及转化均有影响。实验共接种转化2018个子叶和下胚轴外植体,获得了47株抗性再生株,对其进行il-2的PCR扩增检测,有44株呈阳性。PCR-Southern杂交证实PCR结果可靠,显示il-2基因已导入到番茄中。     

Induction of embryogenic callus in Cucurbita pepo hypocotyl explants by indole-3-ethanol and its sugar conjugates

Hypocotyl explants of pumpkin ( Cucurbita pepo L.) were inoculated aseptically on solid media containing Murashige and Skoog (MS) macro- and micronutrients, organic supplements, glucose as a carbon source, and indoleacetic acid (IAA) or one of its possible metabolic precursors, i.e. indole-3-ethanol [3-(2-hydroxyethyl)-indole, tryptophol], 2-(indol-3-yl)-ethyl α-L-arabinopyranoside (tryptophol arabinoside), and 2-(indol-3-yl)-ethyl β-D-glucopyranoside (tryptophol glucoside). Embryogenic callus and adventitious roots developed on the explants, the response depending on the source of auxin, its concentration, and endogenous, possibly genetic, factors. Hypocotyl sections did not form embryogenic tissues and did not survive on media which did not contain an auxin or auxin precursors. Fewer explants responded to IAA than to indole-3-ethanol. Its arabinopyranoside was even more effective, while only a few hypocotyl sections proliferated in the presence of the corresponding glucopyranoside. Embryogenic callus induced by any of the four above compounds could be subcultured on media containing the same source of auxin. Selected callus lines have been maintained for more than three years and have continued to form embryoids. Indole-3-ethanol and IAA were suitable for clonal propagation of regenerated plantlets by axillary and apical bud culture.     

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.     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen

A successful in vitro Agrobacterium-mediated transformation protocol was developed for Mimulus aurantiacus, a model species for ecological and evolutionary genetics and a promising ornamental plant. Three binary vectors were tested, each containing the hptII selectable marker gene and one of the reporter genes: gusA, EGFP or ZsGreen, all of them under CaMV 35S promoter. Genetic transformation was achieved through 4 days of co-cultivation of leaf, petiole and hypocotyl explants with Agrobacterium tumefaciens strain LBA 4404. Explants produced transformed callus tissue on solid modified Murashige and Skoog medium supplemented with 1 mg L^?1 6-benzylaminopurine, 0.5 mg L^?1 1-naphthaleneacetic acid, 30 g L^?1 sucrose and 20 or 50 mg L^?1 hygromycin B. All three reporter genes were expressed in callus tissue but the intensity of expression gradually decreased during further plant development. The new reporter gene ZsGreen proved suitable for plant transformation experiments since very intense and bright fluorescence was detected. Out of 1,760 co-cultured explants, 110 plants were regenerated and all of them were found to be PCR positive for the selection and/or reporter genes. Chemiluminescent Southern blot analysis revealed that 91 % of the regenerated plants (100 T₀ plants) contained T-DNA integrated in their genome. Transformation efficiency varied from 1.4 to 23.3 % for hypocotyl and petiole explants, respectively. Integration of some backbone sequences in plant genomes was confirmed in 75.3 % of T₀ plants. Using this protocol, stable transformants expressing selectable marker gene hptII and one of the reporter genes (gusA, ZsGreen or EGFP) were obtained in 4–5 months.     

Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean)

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

In vitro culture of Safflower L. cv. Bhima: initiation,growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake

Summary Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.     

Growth,ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin

Summary Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.Abbreviations PPT Phosphinothricin - PAT Phosphinothricin acetyltransferase     

Stable transformation of peanut callus viaAgrobacterium-mediated DNA transfer

Transformed callus was produced from peanut (Arachis hypogaea L. cv. Okrun) hypocotyl explants after four days of co-cultivation withAgrobacterium tumefaciens strains EHA101, LBA4404 or ASE1 carrying the binary vector pKYLX71GUS on a defined medium followed by selection with kanamycin (200 mg l^–1). Transformed calluses were cultured as independent cell lines potentially derived from a single transformation event. Stable integration and expression of foreign gene(s) in the callus was confirmed by Southern and western blot analyses and enzyme assays. A few cell lines showed a single insert of the foreign gene. Using the above protocol, transformed peanut callus expressing the peanut stripe virus coat protein gene was obtained.     

向日葵悬浮培养再生芽

从54份向日葵(Helianthus annuus L.)材料中筛选出三个再生能力较强的基因型,取其下胚轴诱导愈伤组织,再以愈伤组织制备悬浮细胞系。在 MS 添加 BA(0.5mg/l)和2,4-D(0.001mg/l)液体培养基上确定细胞生长量,悬浮培养细胞在去掉2,4-D 的 MS 培养基上能够诱导芽的再生。     

Agrobacterium tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”

An efficient protocol for genetic transformation of rutabaga (Brassica napus var. napobrassica) cultivar ??American Purple Top Yellow?? was developed by optimizing several factors influencing gene delivery and plant regeneration. A two-step regeneration protocol, adapted from canola, was optimal for rutabaga regeneration using hypocotyl explants. Transient expression studies monitored by histochemical ??-glucuronidase (GUS) assays indicated that several factors, including Agrobacterium tumefaciens strain, cocultivation time, and cocultivation medium, affected gene delivery. For stable transformation, precultured hypocotyl explants were cocultivated with Agrobacterium cells on sterilized filter paper overlaid on callus induction medium containing 100???M acetosyringone for 6?d under a 16-h photoperiod. Selection and regeneration of transformed cells were conducted on media containing 50?mg?l^?1 kanamycin and 250?mg?l^?1 Timentin. Using this protocol, GUS- and PCR-positive transformants were obtained from 3.2 to 4.2?% of hypocotyl explants inoculated with each of the three Agrobacterium strains after 3?C5?mo. Most transformants exhibited a normal phenotype. Southern blot analysis confirmed stable integration of the gusA transgene in T₀ plants.     

Genetic transformation of wheat using mature seed tissues

A protocol for biolistic transformation of bread wheat based on using mature seed tissues as explants has been developed. Embryogenic callus obtained from mature seed tissues was transformed with a psGFP-BAR plasmid containing gfp reporter gene and bar selectable marker gene. The influence of hormone composition of the medium on the efficiency of transformation of mature wheat seed tissues has been demonstrated. The use of auxin 2,4-D resulted in the formation of transgenic plants with a frequency of 0.75%, while the use of Dicamba auxin for the regeneration of plants did not result in transformant development. The transgenic status of the plants obtained in the experiments has been confirmed by PCR and RT-PCR. Stable inheritance of transgenic features in the following generations of wheat (T₁, T₂) has been demonstrated and transgenic plants exhibiting high resistance to herbicides have been obtained. The protocol developed allows for a simplified transformation of wheat in order to obtain transgenic plants with novel features.