Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

一种快速有效的741杨离体叶片再生芽方法

在对杂交品种741杨(Populus alba(P.davidiana×P.simonii)×P.tomentosa)进行农杆菌介导法转基因的试验中发现了一种快速有效的叶片再生芽的方法.首先叶片外植体在培养基Ⅰ(MS培养基添加0.5 mg/L BA和1.0 mg/L 2,4-D)上培养2～3 d,再转移到培养基SH(MSmedium containing 2.0 mg/L of BA and 0.1 mg/L of NAA)上培养10 d,然后再转移到培养基Ⅱ(MSmedium with 0.5 mg/L of BA)上,培养大约5 d之后86.7%的叶片外植体产生的芽,每片叶片外植体(1 cm×1 cm)可产生40～50个芽.但是,如果叶片外植体在培养基Ⅰ上培养的时间长于5 d,再依次转移到培养基SH和Ⅱ上,则叶片会产生大量根.     

Efficient shoot regeneration from internodal explants of Populus angustifolia, Populus balsamifera and Populus deltoids

In the present study, interactions between the duration of treatment with auxin and different cytokinins and their effect on shoot regeneration were evaluated with the aim to establish a rapid and efficient in vitro regeneration method applicable to a variety of Populus species. Three different species, Populus angustifolia, P. balsamifera, and P. deltoids, were chosen for that purpose. We were successful in regenerating plantlets from stem and petiole explants from all three chosen species using a four-step simple procedure. The first step was callus induction when the explants were exposed to an auxin-rich medium for 0-20 days. During the second step, they were transferred onto a cytokinin-rich medium for shoot bud induction. In the third step, the shoots regenerated were transferred onto a medium with reduced levels of cytokinins to promote shoot proliferation and elongation; finally, in the fourth step, the shoots were rooted and acclimated. A short period (6-10 days) of time of exposure to auxin was sufficient for shoot regeneration. A culture time longer than ten days in callus induction medium drastically reduced the efficiency of shoot regeneration. Besides, cytokinin type and concentration also affected the frequency of shoot induction. A 0.2 mg/l concentration of 2,4-D for callus induction followed by 0.02 mg/l of Thidiazuron for shoot formation proved to be the best treatment for adventitious shoot bud multiplication, generating a maximum of 10-13 shoots of P. balsamifera and P. angustifolia in ten weeks. In contrast, for P. deltoids, a combination of 1.1mg/l 2,4-D, 1.0mg/l NAA, 0.1mg/l zeatin for callus induction followed by a combination of 1mg/l zeatin plus 1.0mg/l BA for shoot bud induction was found to be the most effective, generating on average 15 shoots over a period of ten weeks.     

Genetic Transformation of Leaf Explants of Populus tomentosa

The leaf disc method developed by Horsch et al. (1985) has been used for transformation of Populus tomentosa. The strain of Agrobacterium tumefaciens used harbored a reconstructed Ti plasmid which contained gene 4 of T–DNA and the chimeric CAT(chloramphenicol acetyltransferase) gene. Leaf explants from shoot cultures of Populus tomentosa were co-culfivated with the bacterium. On the hormone free medium, teratoma-like shoots developed from the edge of the leaf explants. When the abnormal shoots were excised from the explants and transferred onto rooting medium, a mass of callus formed at the base of shoots, with new shoots developing, but without root formation. The measurement of'endogenous cytokinin showed that the transformed shoots produced 14 times as much iso-pentenyl adenosine as untransformed shoots did. All teratoma-like shoots-tested showed the presence of nopaline, and were able to grow well. on the medium containing 60-100μg/ml chloromycetin, while normal shoots turned white after 40 days. Pretreatment of A. tumefaciens with phenolic compound, salicylic acid, would increase the frequency of transformation significantly.     

Insect-resistant transgenic cabbage plants and their progenies

An insecticidal crystal protein gene of Bacillus thuringiensis was transferred into cabbage genome with the method of Agrobacterium infection. Cotyledons with petioles as explants were cocultivated with Agrobacterial suspension. Calli generated at the basis of petiole were subjected to selection on the MS medium containing 15-30 mg/L kanamycin (Km). About 5% explants produced calli growing continuously on the selective medium. Green shoots appeared on these calli when they were transplanted onto medium with Km and 6-BA for plant differentiation. The shoots were separated and cultivated on medium with kanamycin. About 80% shoots were rooted. Non-transformed control calli could not give normal shoots and roots and brownized and died gradually. Larvae of Pieris rapae showed poisonous symptoms: growth inhibition and mortality when fed with the leaf of the transgenic plants. About 80% of regenerated plants showed positive hybridization bands when their DNA were probed with crystal protein sequence of Bacillu     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

Survival and escape of Agrobacterium tumefaciens in triploid hybrid lines of Chinese white poplar transformed with two insect-resistant genes

Two partly modified insect-resistant genes (BtCryI Ac gene [Bt gene toxin against Lepidopterean insects] and API gene [arrowhead proteinase inhibitor]) were transferred to the triploid hybrid of Chinese white poplar ((Populus tomentosa Carr. × Populus bolleana Louche) × Populus tomentosa Carr.) mediated by A. tumefaciens. The survival of Agrobacterium in transgenic plants was examined during the processes of transplanting and subculturing on the nutrient medium. The results suggested that 80% of the plants, which were obtained by repeated selection on media added with 50 mg/L kanamycin and 300 mg/l carbenicillin, showed positive reactions after examination using molecular methods. The ELISA test indicated that the Bt toxoprotein was expressed in seven of the transgenic sub-clones. Leaves, stems, and roots of all the 28 transgenic plants were cultured on the YEB medium added with 50 mg/L kanamycin, and it was found that Agrobacterium survived in three sub-clones (33, 37, 5) and could have existed for 24 months in the bottle. These three transgenic sub-clones were transplanted and cultivated for one month in the room, and then the target Agrobacterium was found in rhizosphere of the sub-clone 33.     

Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability

Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable.The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics.     

Transformation of Brassica napus Using Agrobacterium tumefaciens and Regeneration of Transgenic Plants

Seeds of Brassica napus L. cv. "Yunbei 2" were surface-sterilized and germinated on hormone-free MS medium. After 4—5 days the cotyledons were excised in such a way that each has a 1—2 mm petiole was remained at its base. These cotyledons were used as the explants for tissue culture and genetic transformation. This paper first deals with the improvement of the medium for shoot regeneration. Of the elements tested, AgNO3 and carbenicillin enhanced shoot regeneration. The highest frequency (52 %) was obtained on MS medium containing 4.5 mg/L BAP, 20 μmol/L AgNOa and 500 mg/L earbenicillin. An efficient gene transfer system based on the regeneration procedure was established. After 2 days of cocultivation with Agrobacterium tumefaciens strain A208SE (pTi T37-SE, pROA93), the explants were transferred onto selection medium containing 25 mg/L kanamycin. After 1.5 months shoots emerged from 27% of the explants inoculated. They were excised and transferred onto rooting medium containing 25 mg/L kanamycin and 200 mg/L cefotaxime which is better than carbenicillin for root induction. Whole plants were transplanted into pots, and grew well in the phytotron. Transformation was confirmed by β-glueuronidase assay and Southern blotting analysis.     

Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea

Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.     

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.     

Genetic transformation ofRhododendron byAgrobacterium tumefaciens

Summary TransgenicRhododendron plants were obtained byAgrobacterium tumefaciens-mediated gene transfer.A. tumefaciens harboring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and (3-glucuronidase (GUS) genes was co-cultivated with stem and leaf segments fromRhododendron tissues culturedin vitro. Adventitious buds were fonned and shoots were regenerated on kanamycin selection medium 3-4 months after inoculation. Integration of the NPTII and the GUS genes was confirmed by polymerase chain reaction (PCR) and by Southern hybridization analyses. Histochemical GUS assay showed that the inserted gene was expressed in all tissues with the cauliflower mosaic virus (CaMV) 35S promoter. This transformation procedure has the potential to expand the range of genetic variation inRhododendron.     

High-frequency transformation of<Emphasis Type="Italic"> Lobelia erinus</Emphasis> L. by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated gene transfer

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a -glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3–4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high—45% per inoculated disc.Abbreviations BA: 6-Benzyladenine - 2,4-D: 2,4-Dichlorophenoxyacetic acid - GUS: -Glucuronidase - IAA: Indole-3-acetic acidCommunicated by G.C. Phillips     

Transformation of Recalcitrant Melon (Cucumis melo L.) Cultivars is Facilitated by Wounding with Carborundum

Transformation of the recalcitrant melon (Cucumis melo L.) cultivars Kιrka?aç 637 and Noi Yarok was accomplished by wounding cotyledon explants by vortexing with carborundum prior to inoculation with Agrobacterium tumefaciens. The addition of silver nitrate to the regeneration‐selection medium reduced the transformation efficiency, as the percentage of the explants forming putative transgenic calli and bud‐like protuberances was decreased and no transgenic shoots were produced. Chimeric transgenic plants were obtained after the regeneration of putatively transformed callus, bud‐like protuberances, buds and shoots on selective medium with kanamycin. The treatments producing the most buds or shoots from explants after 30–40 days of cultivation were the most successful for the production of transgenic plants. Only treatments where explants were vortexed with carborundum produced transgenic melon shoots of either cultivar. Subculture every 18–20 days on fresh regeneration‐selection medium containing 50 mg/L kanamycin after either a relatively high (100 mg/L) or low level (50 mg/L) of kanamycin in the first regeneration‐selection medium was necessary for the successful transformation of cultivar Kιrka?aç 637. These techniques are now being used in breeding programs for the production of melon lines bearing resistances to zucchini yellow mosaic virus and cucumber mosaic virus, important viruses limiting agricultural production.     

Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus

Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of ``escape' shoots produced. Although the use of a wild-type ``shooty' Agrobacterium strain allowed relatively high frequencies of β-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root. Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus. Received: 8 October 1996 / Revision received: 1 April 1997 / Accepted: 18 April 1997     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.