Floral spray transformation can efficiently generate Arabidopsis transgenic plants

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

喷雾:一种简便的拟南芥转化方法

目的：建立一种改良的且操作更为简便可行的拟南芥转化方法。方法：用含有OD600等于0．8的农杆菌,5%蔗糖和0. 2 ml/L表面活性剂Silwet L-77的MS液体培养基喷洒正在发育的花器官即可实现基因转化。植物生长到高约4cm时进行转化,此时植物具有大量的花和极少数的果荚,可以得到比较高的转化效率。喷洒溶液后将植物用保鲜膜等材料包裹起来以保持湿度,可有利于转化。结果：得到了365棵转化植株,转化效率和花器官浸泡法的基本一致。结论：这种方法可适用于多种生态型的拟南芥植物转化,并且方便大规模的拟南芥植物的转化,便于人们快速获得带有T-DNA标记的植株或基因遗传互补工作的顺利开展。     

An intensive understanding of vacuum infiltration transformation of pakchoi (Brassica rapa ssp. chinensis)

Pakchoi (Brassica rapa L. ssp. chinensis), a kind of Chinese cabbage, is an important vegetable in Asian countries. Agrobacterium mediated in planta vacuum infiltration transformation has been performed in pakchoi since 1998, but a detailed study on this technique was lacking. Pakchoi plants 40-50 days old with inflorescences were vacuum infiltrated with Agrobacterium tumefaciens strain C58C1 harboring the binary vector pBBBast-gus-intron. The transformation frequency in the harvested seeds mainly varied from 1 x 10(-4) to 3 x 10(-4) over several years, and it was lower than the frequency in Arabidopsis thaliana. Transformants were obtained from both the upper and the lower parts of the infiltrated plants with or without an elongated inflorescence. Stained ovules and pollen grains were found in the unopened flower 13 days post-infiltration, which was about 0.5-1 mm in diameter at infiltration time with an open ovary as revealed by paraffin sections. Histochemical assays revealed that Agrobacteria were more abundant in the flower tissue than in stem and leaf tissues at all times after infiltration despite the sharp decrease of live Agrobacteria in plant 14 days post infiltration as revealed by the colony forming units on the Agrobacteria culture medium. The results of vacuum infiltration transformation of pakchoi and Arabidopsis thaliana were compared and a strategy to optimize the transformation conditions to increase the transformation frequency in pakchoi was discussed.     

Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation

Germ-line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens. We have recently identified several Arabidopsis ecotypes and T-DNA-tagged mutants that are recalcitrant to Agrobacterium-mediated transformation of cut root segments. Some of these ecotypes and mutants are deficient in their ability to bind bacteria. Some are deficient in T-DNA integration. We report here that using a germ-line transformation protocol we transformed these ecotypes and mutants, including attachment- and integration-defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild-type plants. However, we could not transform otherwise highly susceptible Arabidopsis plants by germ-line or root transformation using several vir and attachment-deficient Agrobacterium mutants. These results indicate that certain plant factors important for transformation may exist in germ-line tissue but may be lacking in some somatic cells.     

Generation of genetically stable transformants by <Emphasis Type="Italic">Agrobacterium</Emphasis> using tomato floral buds

Tomato (Solanum lycopersicum) is a model crop plant for the study of fruit ripening and disease resistance. Here we present a systemic study on in planta transformation of tomato with Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA1303 binary vector bearing HPTII as a plant selectable marker and mGFP/GUS fusion as the reporter gene. We attempted the transformation of tomato at different developmental stages viz. during seed germination, seedling growth, and floral bud development. The imbibition of seeds with Agrobacterium suspension led to seed mortality. The vacuum infiltration of seedlings with Agrobacterium suspension led to sterility in surviving plants. Successful transformation could be achieved either by dipping of developing floral buds in the Agrobacterium suspension or by injecting Agrobacterium into the floral buds. Most floral buds subjected to dip as well as to injection either aborted or had arrested development. The pollination of surviving floral buds with pollen from wild-type plants yielded fruits bearing seeds. A transformation efficiency of 0.25–0.50% was obtained on floral dips/floral injections. Transgenic plants were selected by screening seedlings for hygromycin resistance. The presence of the transgene in genomic DNA was confirmed by Southern blot analysis and expression of the reporter gene up to the T₄ generation. The amenability of tomato for in planta transformation simplifies the generation of transgenic tomato plants obviating intervening tissue culture.     

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method

Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01-0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20-30 plants per pot) within 2-3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.     

Floral Spray Transformation Can Efficiently Generate Arabidopsis

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

Flower bud dipping or vacuum infiltration—two methods of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transformation

The purpose of this work was to evaluate two methods (floral dip and vacuum infiltration) of in planta transformation of Arabidopsis thaliana. The key issue of this work is the identification of the developmental stages of A. thaliana flower buds subjected to agroinfection, optimal for the successful transformation. Histological tests performed after agroinfection made it possible to establish the patterns of a GUSPlus reporter gene expression in the examined plants and thus precisely define the range of flower developmental stages most appropriate for efficient transformation. Two plasmids, CAMBIA 1305.1 and CAMBIA 2301, were used. Verification of the transgenic nature of plants was carried out by detection of CaMV::gusA and CaMV::GUSPlus transgenes and their expression in transgenic plants by appropriate molecular and histochemical methods. For the flower dip transformation, three concentrations of Silwet L-77 surfactant and two inoculation times were tested. The most efficient treatment appeared to be 2-min-long flower bud inoculation and 400 μl/l surfactant (pCAMBIA 1305.1 − 1.73%; pCAMBIA 2301 − 2.01%). In the case of vacuum infiltration method, the highest efficiency of the transformation occurred when the inoculation time was 4 min (pCAMBIA 2301 − 1.55%; pCAMBIA 1305.1 − 1.37%). Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 619–628. This text was submitted by the authors in English.     

In solium Selection for Arabidopsis Transformants Resistant to Kanamycin

The kanamycin resistance encoded by the neomycin phosphotransferase II gene (nptII) of transposon Tn5 is widely used in higher plant genetic transformation. The general process of plant transformation using nptII as a selectable marker gene, however, requires selecting kanamycin-resistant plants or tissues in culture. Even with the recently developed vacuum infiltration method for Arabidopsis transformation, the plant culture steps are not completely eliminated in selection for kanamycin-resistant transformants. The herbicide resistance genes, such as bar, which provides resistance to bialaphos, allow Arabidopsis transformation to become a true non-culture procedure. In this report, we assessed the feasibility of applying kanamycin as a spray in selecting for kanamycin-resistant Arabidopsis transformants grown in soil. We find that kanamycin-resistant transformants were effectively selected by spraying soil-grown Arabidopsis seedlings.     

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.     

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.     

An efficient Agrobacterium-mediated transient transformation of Arabidopsis

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.     

An effective and reproducible transformation protocol for the model resurrection plant Craterostigma plantagineum Hochst.

Procedures previously established for plant regeneration and Agrobacterium tumefaciens-mediated genetic transformation of the desiccation-tolerant plant, Craterostigma plantagineum, have been further developed. A highly effective tissue culture system was established based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. The undesirable hyperhydricity of Craterostigma tissue in tissue culture was also circumvented by these modifications, which serve as an alternative to the previously described procedures. The high efficiency of plant regeneration from the callus phase provided the basis for optimising genetic transformation in Craterostigma. For gene delivery, both a standard (method A) and a modified protocol (method B), the latter having previously resulted in successful Agrobacterium-mediated transformation of monocot cereals, were applied. Physical and biochemical key variables in transformation were evaluated, such as gene gun-mediated microwounding of plant explants, infiltration of Agrobacterium suspension cultures into target tissues and the influence of in vitro pre-induction of vir genes. While the physical enhancement of Agrobacterium infection (microwounding, infiltration) had no positive effect, the in vitro pre-induction of vir genes (biochemical enhancement) resulted in a twofold increase in the transformation frequency as compared to the conventional protocol (method A).     

HAL1 mediate salt adaptation in Arabidopsis thaliana

INTRODUCTIONSalinity is a major environmental stress that isa substantial constraint to crop production both fordry land and irrigated agriculture. The detrimental impact of this stress is perpetuated and exacerbated by management practices used to facilitatehigh-output crop production. To overcome theselimitations and improve production efficiency in theface of a burgeoning world population, more salt tolerant crops must be developed. In contrast with traditional breeding, the direct ill…     

Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.     

Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation

When Agrobacterium was used to transform Nicotiana plumbaginifolia protoplasts and Arabidopsis thaliana roots and seedlings, a large number of plants were found in which not only the T-region defined by the border repeat sequences but the entire binary vector was integrated, as determined by both PCR and Southern analysis techniques. N. plumbaginifolia protoplast co-cultivation experiments yielded 3 out of 5 transformants with collinear sequence past the left border. In Arabidopsis root transformation experiments, 33% (6/18) of the transformants had T-DNA which exceeded the left border repeat. Vacuum infiltration of Arabidopsis seedlings produced even a greater percentage of transformants with sequences outside the left border repeat (62%, 39/63). The long transfer DNA cosegregated with the T-region encoded hygromycin resistance in the T2 progeny eliminating the possibility that long transfer DNA was of extrachromosomal or Agrobacterium origin. The high frequency of long transfer after vacuum infiltration of A. thaliana needs to be considered when analyzing T-DNA tagged mutants.     

Genetic transformation of Coffea canephora by vacuum infiltration

Agrobacterium-mediated plant transformation protocol was evaluated as a fast method to obtain genetically modified Coffea canephora plantlets. Leaf explants were used as source material for Agrobacterium tumefaciens-mediated transformation involving a vacuum infiltration protocol, followed by a step of somatic embryogenesis induction and a final selection of the transformed plants. A. tumefaciens strain C58CI containing the binary vector pER10W-35SRed was used. PCR amplification of DsRFP gene and visual detection of the red fluorescent protein demonstrated 33% transformed embryos. The protocol presented here produces reliable transgenic coffee embryos in two months.     

Transient β-glucuronidase activity after infiltration of Arabidopsis thaliana by Agrobacterium tumefaciens

Transient expression of the β-glucuronidase (GUS) gene introduced into Arabidopsis thaliana intact plants by T-DNA after vacuum infiltration of Agrobacterium tumefaciens was followed. The first incidence of GUS activity was found 2 - 3 d after treatment and a peak of activity one week after treatment in both A. thaliana races, Columbia and C24. GUS activity was sharply increased by cultivation of Arabidopsis plants at elevated temperature (29 °C) compared to cultivation at 25 °C. The density of inocula also influenced the GUS activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of agrobacterium transformation

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17. We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17. Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants. Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.