High-frequency transformation ofArabidopsis thaliana byAgrobacterium tumefaciens

Incorporation of 5 mg/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density of the root explants and duration of bacteria-plant cocultivation period, were also found to influence the efficiency of recovery of transformants.     

Extracellular enzymes ofErwinia carotovora eliminate the need for azacytidine treatment for high frequency transformation ofArabidopsis thaliana

Summary This paper reports a part of our studies on large-scale T-DNA-mediated gene tagging inArabidopsis thaliana. To enhance the chance of tagging specific stress-responsive genes of this species by monitoring the preferential insertion of the T-DNA into the actively transcribed loci, we exposed the root explants to low temperature (LT), abscisic acid (ABA), and extracellular enzymes (EXE) of the plant pathogenErwinia carotovora prior to transformation byAgrobacterium tumefaciens. Both LT and ABA reduced the frequency of transformation; with these treatments, the average transformation frequencies were 8.1% and 2.6%, respectively. However, in explants pretreated with EXE the transformation frequency was 89.0%, similar to that obtained in control materials (92.6%). Transgenic calli developed from these explants did not require any treatment with azacytidine (azaC) for efficient shoot regeneration. Furthermore, this treatment enhanced multiple insertion of the T-DNA into the plant genome; within a population of EXE-treated transgenic plants, the number of lines harboring at least three copies of the integrated T-DNA was much higher (61%) than that observed in an untreated population (34%).     

Germ-line transformation of Arabidopsis lasiocarpa

In planta transformation methods have opened up the possibility of transforming plant species for which no regeneration protocols currently exist. In this study, the suitability of the germ-line transformation method developed for Arabidopsis thaliana was examined for four taxa in the Brassicaceae that have not been previously transformed: Arabidopsis griffithiana, Arabidopsis lasiocarpa, Arabidopsis petraea and Capsella bursa-pastoris. Numerous transformants were obtained for A. lasiocarpa. Transformation of A. lasiocarpa was confirmed at the phenotypic and molecular levels for stably transformed lines and for backcrossed lines segregating the T-DNA insert. Parameters affecting transformation efficiency of A. lasiocarpa were also explored. As with A. thaliana, sucrose and surfactant in the inoculation medium are required for high levels of transformation, although the suitable concentrations of these are different for A. lasiocarpa. Other components present in earlier versions of the inoculation medium had little effect on transformation efficiency. Vacuum infiltration (rather than simple floral dipping) led to higher rates of transformation and did not seriously affect seed production in A. lasiocarpa. Identification of species susceptible to germ-line transformation will aid in determining the factors important for applying this technology to more recalcitrant species.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the genome-sequenced poplar clone,nisqually-1 (<Emphasis Type="Italic">Populus trichocarpa</Emphasis>)

The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood (Populus trichocarpa). To improve its utility for functional genomics research, having an efficient means for transformation and regeneration is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants with a strain ofAgrobacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was incorporated in the selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during the early stages of shoot induction. Histochemical assays revealed expression of theGUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol, transgenic plants were recovered within 6 mo at an efficiency of 6%, adequate to produce a large number of transgenic events with modest effort.     

Phosphomannose isomerase: A versatile selectable marker forArabidopsis thaliana germ-line transformation

A new selection system using mannose has been evaluated for germ-line transformation ofArabidopsis thaliana. Although mannose itself has no adverse effects on plant cells, it leads to an accumulation of mannose-6-phosphate, which depletes intracellular stores of inorganic phosphate. This results in an inhibition of plant cell growth. The selection system uses theEscherichia coli pmi gene that encodes phosphomannose isomerase (PMI). Transgenic plants carrying thepmi gene can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the PMI activity. Germ-line transformation ofA. thaliana followed by sterile selection on 2–5 mM of mannose resulted in the isolation of mannose-6-phosphate-resistant progeny in about 2.5% of the treated seed, consistent with transformation rates using other selection schemes. Integrative transformation was confirmed by Southern hybridization. Analysis of PMI enzyme activity demonstrated a 5-fold range of activity levels, although these differences had little effect on the ability to select transformed plants or on the growth of transformed plants on mannose. Finally, mannose selection using thepmi gene could be accomplished in sterile plates and in soil, making this an extremely versatile tool forA. thaliana transformation.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

Infiltration with <Emphasis Type="Italic">Agrobacterium</Emphasis> — the method for stable transformation avoiding tissue culture

A number of in planta transformation protocols that avoid long culture under sterile conditions were developed for Arabidopsis thaliana. The most widely used methods are based on vacuum infiltration and floral dip. These methods were adapted for transformation of other species as well. Successful in planta transformations of alfalfa, radish, pakchoi and petunia were reported recently. In this short review we present several modified procedures originally developed for Arabidopsis thaliana and in some cases adapted to other species. We emphasize the crucial parameters involved in in planta transformation. We also describe here the studies attempting to shed light on the mechanisms and estimating the cellular target of transformation, which may help in transforming new plant species.     

Non-destructive transformation ofArabidopsis

Culture conditions are reported in which roots ofArabidopsis thaliana grow abundantly along the surface of, rather than down into, a solid matrix. Such root segments can be explanted without killing of the plant, which can then complete its life cycle. Non-destructive explanting permits transformation of heterozygous plants without preventing subsequent segregation and of plants that can only be obtained from a population segregating for a marker expressed late in growth. Moreover, the speed and abundance of surface root growth provides a rapid non-destructive phenotypic assay of response to selective conditions.     

Ectopic expression of Atleafy in Brassica juncea cv. Geeta for early flowering

High temperature stress during pod filling severely affects the yield of Brassica juncea. Early flowering can evade the terminal heat stress and result in early maturity of the crop. In this study, a regeneration and transformation protocol has been standardized for B. juncea cv. Geeta. Hypocotyl from 5-day-old seedlings were used as explants. Of the various combinations of auxins and cytokinins tried along with Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium, MS + IAA (0.2 mg/l) + BA (3 mg/l) proved best for shoot regeneration with 89.9 % regeneration efficiency. To induce early flowering Leafy gene from Arabidopsis thaliana was transformed using Agrobacterium mediated transformation method. After 12 weeks transgenic plants showed flowering in vitro whereas their untransformed counterpart did not flower even after 16 weeks. The maximum transformation frequency was 4 %.     

Agrobacterium tumefaciens-mediated transformation of recalcitrant crops

The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general guidelines for the development of a transformation protocol are discussed.     

Somatic embryogenesis and in vitro flowering inBrassica nigra

Summary This report describes a protocol for regeneration ofBrassica nigra in vitro from unorganized callus to a highly differentiated stage of flowering. Callus is initiated from seedling hypocotyl, and root explants and plantlets are obtained via somatic embryogenesis. Shoot cultures can be established from these plantlets. These shoots can either be induced to flower in vitro or rooted to produce plants which flower ex vitro. Each stage of development is marked with a specific growth regulator requirement. This has potential as a model system to understand the cellular and molecular mechanisms involved in morphogenesis, and it can be used to understand the mechanism of change of phase from vegetative to reproductive. An advantage of this system is that in vitro flowering can be obtained repeatedly in the shoots raised from the axillary buds of the flowering shoots. The protocol can also be used to procureB. nigra gametes under aseptic condition.     

Morphological, physiological and molecular genetic characterization ofArabidopsis himalaica, with reference toA. thaliana

Arabidopsis himalaica (Edgeworth) O.E. Schulz, a poorly characterized species typical of HimalayanArabidopsis, was analyzed in terms of its morphology, physiology, chromosome number and molecular genetics, in comparison withA. thaliana which is the standard species in the genusArabidopsis. From view point of developmental genetics, several features which are specific toA. himalaica seem not to be derived by single-gene mutations inA. thaliana. Phylogenetic analyses based onrbcL sequences suggested that genusArabidopsis is not monophyletic. The detailed characterization ofA. himalaica should provide clues to understand the trait of evolution of particular features of Himalayan species ofArabidopsis and their genetic basis.     

Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianBrassica juncea lines

In recent years,Brassica species have acquired an important position in the oilseed industry. Even thoughBrassica transformation protocols are well established,there is still a need for the development of new transformation protocols for elite AustralianB. juncea lines,because regeneration inB. juncea is highly genotype-dependent and in addition, their hypocotyl explants are susceptible to necrosis.Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianB. juncea lines is described here. To overcome necrosis, we have adopted 2 strategies: extension of precultivation time of hypocotyl explants, and use of a 2-stage hygromycin selection process.The frequency of recovery of transformants from AustralianB. juncea andBrassica napus lines was 1.7% and 0.9%, respectively. Polymerase chain reaction tests confirmed that allBrassica plants that survived through stringent screening procedures were positive for the inserted hygromycin resistance gene,hph. Progeny from 6Brassica lines tested segregated for thehph gene, and χ² analysis suggested a 3:1 segregation ratio.This is in line with a tDNA integration into a single locus, which is an important feature of a transformation protocol for subsequent breeding purposes. Although the scientific content of this article has been reviewed,the full-text Web publication has not been edited in detail.     

Relative regeneration proficiency ofArabidopsis thaliana ecotypes

We have compared regeneration proficiency for cultured explants from different tissues of different ecotypes ofArabidopsis thaliana. Proficiency varies widely with both tissue and ecotype, and is highest when the flux of light during regeneration is low. Analysis of F1 hybrids suggests that high proficiency is dominant to low proficiency.     

Differences in in vitro plant regeneration ability among four Arabidopsis thaliana ecotypes

Summary The Arabidopsis ecotypes Columbia (Col), Landsberg erecta (Ler), Cape Verde Island (Cvi) and Wassilewskija (WS) have been tested for their regeneration response in vitro. A characteristic morphology of leaf-derived calluses has been found for each ecotype. Differences in regeneration ability have been detected depending on the plant strain. the explant source and on the culture medium composition. In CIR/SIR media, which contain 0.5 mg l⁻¹ (2.26 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and glucose, root explants from the four ecotypes are able to reach a considerable regeneration level, while leaf explants do not regenerate beyond a basal level (5% approximately). In CIH/SIH media, which contain 2.2 mg l⁻¹ (9.95 μM) of 2,4-D and suerose, leaf explants from all the ecotypes, with the exception of Col, are able to regenerate, but they do it at variable levels (Ler 5.75%, WS 75.09%, and Cvi 27.53% as regeneration rates). With these media all root explants are able to regenerate, but again the four ecotypes show different rates (Col 27.7%, Ler 57.25%, WS 98.54%, and Cvi 42.25%). The variation of the different medium components affects differentially the regeneration ability of the four ecotypes depending also on the kind of explant. Thus, when the 2,4-D concentration is raised WS duplicates its regeneration rate in both leaf and root explants. Changing glucose for sucrose in CIR/SIR media diminishes to the basal level the regeneration of Cvi root explants, while the CIH/SIH salts and vitamin concentration permit the regeneration of leaf explants from all the ecotypes except Col. The genes responsible for those observed differences in regeneration ability could be identified and mapped by analyzing the in vitro regeneration behavior of the recombinant inbred lines (RILs) obtained by crossing these ecotypes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated genetic transformation of summer squash (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L. cv. Australian green) with <Emphasis Type="Italic">cbf-1</Emphasis> using a two vector system

Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore, experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation. 6-benzylaminopurine at 0.05 mg l^−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets in indole-3-aceticacid 0.5 mg l^−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent.     

Shoot regeneration of dwarf dogwood (Cornus canadensis L.) and morphological characterization of the regenerated plants

Dwarf dogwoods (or the bunchberries) are the only suffrutex in Cornaceae. They are attractive ground cover ornamentals with clusters of small flowers surrounded by petaloid bracts. Little has been reported on plant regeneration of dogwoods. As a step toward unraveling the molecular basis of inflorescence evolution in Cornus, we report an efficient regeneration system for a dwarf dogwood species C. canadensis through organogenesis from rejuvenated leaves, and characterize the development of the plantlets. We used the nodal stem segments of vegetative branches as explants. Micropropogated shoots were quickly induced from axillary buds of nodes on an induction medium consisting of basal MS medium supplemented with 4.44 μM BAP and 0.54 μM NAA. The new leaves of adventitious shoots were used as explants to induce calli on the same induction medium. Nearly 65% of leaf explants produced calli, 80% of which formed adventitious buds. Gibberellic acid (1.45 μM) added to the same induction medium efficiently promoted quick elongation of most adventitious buds, and 0.49 μM IBA added to the basal MS medium promoted root formation from nearly 50% of the elongated shoots. The growth of plantlets in pot soil was characterized by the development of functional woody rhizomes, which continuously developed new aboveground vegetative branches, but not flowering branches, within the past 12 months. Potential reasons causing the delay of flowering of the regenerated plants are discussed. The establishment of this regeneration system facilitates developing a genetic transformation system to test candidate genes involved in the developmental divergence of inflorescences in Cornus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High frequency stable transformation ofArabidopsis thaliana by particle bombardment

Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl⁻¹ hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots.     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.)

In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.