Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3-5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5-11 days post-infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.     

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.     

The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana

In planta transformation methods are now commonly used to transform Arabidopsis thaliana by Agrobacterium tumefaciens. The origin of transformants obtained by these methods has been studied by inoculating different floral stages and examining gametophytic expression of an introduced beta-glucuronidase marker gene encoding GUS. We observed that transformation can still occur after treating flowers where embryo sacs have reached the stage of the third division. No GUS expression was observed in embryo sacs or pollen of plants infiltrated with an Agrobacterium strain bearing a GUS gene under the control of a gametophyte-specific promoter. To identify the genetic target we used an insertion mutant in which a gene essential for male gametophytic development has been disrupted by a T-DNA bearing a Basta resistance gene (B(R)). In this mutant the B(R) marker is transferred to the progeny only by the female gametes. This mutant was retransformed with a hygromycin resistance marker and doubly resistant plants were selected. The study of 193 progeny of these transformants revealed 25 plants in which the two resistance markers were linked in coupling and only one plant where they were linked in repulsion. These results point to the chromosome set of the female gametophyte as the main target for the T-DNA.     

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.     

The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Transgenic loci obtained after Agrobacterium tumefaciens -mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6–30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20–36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4–6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24–31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells.     

Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology

Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.     

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.     

High frequency of single-copy T-DNA transformants produced by floral dip in CRE-expressing Arabidopsis plants

For genetic transformation of plants, floral dip with Agrobacterium often results in integration of multiple T-DNA copies at a single locus and frequently in low and unstable transgene expression. To obtain efficient single-copy T-DNA transformants, two CRE/ loxP recombinase-based simplifying strategies for complex T-DNA loci were compared. A T-DNA vector with oppositely oriented loxP sites was transformed into CRE -expressing and wild-type control Arabidopsis thaliana plants. Of the primary CRE -expressing transformants, 55% harboured a single copy of the introduced T-DNA, but only 15% in the wild-type plants. However, 73% of the single-copy transformants in the CRE background showed continuous somatic inversion of the DNA segment between the two loxP sites. To avoid inversion of the loxP -flanked T-DNA segment, two T-DNA vectors harbouring only one loxP site were investigated for their suitability for CRE/ loxP recombinase-mediated resolution upon floral-dip transformation into CRE -expressing plants. On average, 70% of the transformants in the CRE background were single-copy transformants, whereas the single-copy T-DNA frequency was only 11% for both vectors in the wild-type background. Both resolution strategies yielded mostly Cre transformants in which the 35S-driven transgene expression was stable and uniform in the progeny and remarkably, also in Cre transformants with multiple T-DNA copies. Therefore, a role is proposed for the CRE recombinase in preventing inverted T-DNA repeat formation or modifying the locus chromatin structure, resulting in a reduced sensitivity for silencing.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenesmediated transformation of embryogenic calli

Genetically transformed roots and calli were induced from leafsegments of grapevine (Vitis vinifera L. cv. Koshusanjaku) afterco-cultivation with wild-type Agrobacterium rhizogenes strains,but plant regenera tion from them was not achieved. On the otherhand, transgenlc grapevine plants were obtained via somaticembryogenesis after co-cultivation of embryogenic calli withan engineered A. rhizogenes strain including both the neomycinphosphotransferase II (NPT II) and the ß-glucuronidase(GUS) genes, followed by selection of secondary embryos forkanamycin resistance. All these plants showed GUS gene expressionrevealed by histochemical assay. Southern blot analysis revealedthe stable integration of the GUS cording region in their genome.Transformants containing Ri T-DNA exhibited various phenotypes:most of them showed a typical Ri-transformed phenotype suchas wrinkled leaves, while the others looked normal. Key words: Agrobacterium rhizogenes, grapevine, transgenic plants, Vitis vinifera     

根癌农杆菌介导的乙肝表面抗原基因对烟草的转化

构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于农杆菌的转化;通过冻融法将重组质粒pBRSAg转入根癌农杆菌LBA4404中,利用农杆菌介导法转化烟草叶盘,经筛选培养获得烟草植株。抗性植株经GUS染色和PCR检测为阳性,初步表明乙肝表面抗原基因在烟草中得到表达。     

拟南芥根特异表达基因启动子在烟草中的表达

以拟南芥为材料,利用PCR技术分离pyk10启动子序列,构建了该启动子GUS植物表达载体,农杆菌介导转化烟草,分析该基因在烟草中的表达,以明确拟南芥根特异表达基因pyk10启动子在烟草中的表达特性.结果表明:克隆的pyk10启动子与已报道的pyk10启动子一致性为100%,GUS基因在烟草的根部特异表达,表明该启动子为根部特异表达启动子,为揭示植物根的发生、分化和发育机制,以及培育抗根部病虫害和营养高效利用型转基因烟草奠定了基础.     

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%).     

农杆菌介导的玉米原位转化方法改良

原位转化是一种简便的植物转基因方法,在拟南芥中已经应用较为成熟,在玉米上的应用并不多见.本文在玉米开花期间,将含有目标载体pCAMBIA1301的农杆菌菌株LBA4404的细胞悬浮液直接涂抹到事先授粉约8小时后并去除掉苞叶和花丝的幼穗上.转化后种子的幼苗,经过gus活性染色和潮霉素基因(hpt)的PCR扩增,证实有部分的种子T-DNA整合进入了基因组.在潮霉素筛选后,有2.6 %抗性苗存活.其中57.7 %抗性植株表现出GUS阳性,相当于全部检测幼苗的1.5 %.这一结果通过潮霉素基因的PCR扩增,得到进一步的证实.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones

Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon. Using several Arabidopsis: genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes. Transient expression of a T-DNA encoded ss-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten ARABIDOPSIS: genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli). Cultivation of Arabidopsis: tissues on phytohormones for 2-8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2-11-fold and stable T-DNA integration with petiole explants. Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups). The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia. This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones. The importance of pre-cultivation with phytohormones for genetic transformation is discussed.     

Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants

Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells. Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5%. T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells. A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining. When selection for transformation competence is applied, the picture is completely different. Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome. Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors.