Agrobacterium-mediated Gene Transfer Research Articles

Functional gene‐mining for salt‐tolerance genes with the power of Arabidopsis

Here we report on a functional gene-mining method developed to isolate stress tolerance genes without any prior knowledge of the genome or genetic mapping of the source germplasms. The feasibility of this approach was demonstrated by isolating novel salt stress tolerance genes from salt cress (Thellungiella halophila), an extremophile that is adapted to a harsh saline environment and a close relative of the model plant Arabidopsis thaliana. This gene-mining method is based on the expression of salt cress cDNA libraries in Arabidopsis. A cDNA expression library of the source germplasm, salt cress, was constructed and used to transform Arabidopsis via Agrobacterium-mediated gene transfer. A transgenic seed library consisting of >125,000 independent lines was generated and screened for salt-tolerant lines via a high-throughput genetic screen. A number of salt-tolerant lines were isolated, and the salt cress cDNAs were identified by PCR amplification and sequencing. Among the genes isolated, several novel small protein-encoding genes were discovered. The homologs of these genes in Arabidopsis have not been experimentally analyzed, and their functions remain unknown. The function of two genes isolated by this method, ST6-66 and ST225, and their Arabidopsis homologs, were investigated in Arabidopsis using gain- and loss-of-function analyses, and their importance in salt tolerance was demonstrated. Thus, our functional gene-mining method was validated by these results. Our method should be applicable for the functional mining of stress tolerance genes from various germplasms. Future improvements of the method are also discussed.

A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression.

Agrobacterium-mediated gene transfer, or agroinfiltration, can be a highly efficient method for transforming and inducing transient transgene expression in plant tissue. The technique uses the innate DNA secretion pathway of Agrobacterium tumefaciens to vector a particular plasmid-encoded segment of DNA from the bacteria to plant cells. Vacuum is often applied to plant tissue submerged in a suspension of A. tumefaciens to improve agroinfiltration. However, the effects of vacuum application on agroinfiltration and in planta transient transgene expression have not been well quantified. Here we show that vacuum application and release act to drive A. tumefaciens suspension into the interior of leaf tissue. Moreover, the amount of suspension that enters leaves can be predicted based on the vacuum intensity and duration. Furthermore, we show that transient expression levels of an agroinfiltrated reporter gene vary in response to the amount of A. tumefaciens vacuum infiltrated into leaf tissue, suggesting that vacuum infiltration conditions can be tailored to achieve optimal transient transgene expression levels after agroinfiltration.

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering

The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium-mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (beta-carotene ketolase) and CrtZ (beta-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO2 concentrations of 1500 micromol photons m(-2) s(-1) light intensity.

Mining GOLD and new model organisms in biotechnology

Mining GOLD and new model organisms in biotechnology

Recent Patents on Agrobacterium-Mediated Gene and Protein Transfer,for Research and Biotechnology

Agrobacterium has been widely used, in the last decades, for genetic transformation of a large number of plant species, and the genes and DNA sequences involved in this process have been subject of numerous patents. This review focuses on recent discoveries, which have shown new possibilities for the utilization of this versatile microorganism. For example, the identification of an ever-increasing number of the bacterial and plant factors involved in the Agrobacterium-mediated DNA transfer and integration may lead to new applications in various fields of research and biotechnology. One of the main challenges in the Agrobacterium-mediated gene transfer technology is to achieve a better control of the integration and expression of transferred genes in the host cells and to apply it for targeted integration into the host genome or gene replacement (a technique not yet available in plants). In addition to genetic transformation of plants, under laboratory conditions, the host range of Agrobacterium can be extended to virtually all eukaryotic species, as demonstrated for various fungi, sea urchins, and animal cells. Not only can Agrobacterium transfer DNA to these very diverse hosts, but also its virulence machinery is able to inject proteins into the host cell, independently of the DNA transfer. Thus, Agrobacterium represents a universal gene and protein transfer machine.

Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen

We generated transgenic tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) plants harboring a synthetic Myxococcus xanthus protoporphyrinogen oxidase (MxPPO) gene through Agrobacterium-mediated gene transfer. Successful integration of the transgene into the genome of transgenic plants confirmed by polymerase chain reaction (PCR) and Southern blot analysis, and the functional expression of the MxPPO gene at the mRNA level in transgenic lines was validated by Northern blot analysis. Responses of transgenic and non-transgenic tall fescue plants to diphenyl-ether herbicides such as oxyfluorfen and acifluorfen have been evaluated in respect of various physiological and biochemical parameters. Differential responses were observed in chlorophyll content, in vivo H2O2 deposition and lipid peroxidation in both transgenic and non-transgenic plants exposed to oxyfluorfen or acifluorfen. Isozyme profiles of four antioxidant-enzymes, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), were also investigated in transgenic and non-transgenic plants using native PAGE analysis. Compared to the transgenic lines, higher staining activities of the examined antioxidant-enzymes observed in non-transgenic plants subjected to 100 μM of oxyfluorfen or acifluorfen suggests that non-transgenic plants are unable to prevent the photodynamic induced oxidative stress caused by herbicides. In addition, both transgenic and non-transgenic plants exposed to oxyfluorfen exhibited proportionally increased band-staining patterns in contrast to acifluorfen, which suggests that oxyfluorfen has relatively greater or more rapid effects on leaves than acifluorfen.

T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.

SummaryEctomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium‐mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T‐DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T‐DNA right border (RB)–genomic DNA junctions in Escherichia coli. Fifty‐one Agrobacterium‐transformed fungal strains, picked up at random from a larger collection of T‐DNA tagged strains (about 500), were analysed. Sixty‐nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T‐DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T‐DNA borders indicating non‐homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium‐mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.

Analysis and optimization of DNA delivery into chickpea (Cicer arietinum L.) seedlings by Agrobacterium tumefacience

The main purpose of this study was to develop a non-tissue culture based Agrobacterium mediated transformation method for chickpea. The influences of several factors were investigated on the transfer of
-glucuronidase (GUS) gene into chickpea (Cicer arietinum) seedlings during the early stages of Agrobacterium-mediated gene transfer, including cocultivation period in liquid induction medium (2, 8, 16 and 24 h), strains of Agrobacterium tumefaciens (C58C1, EHA105, KYRT1) containing the plasmid pTJK136, developmental stage (16 h imbibed and 40 h germinated), microwounding, vacuum infiltration (200, 400, 600 mmHg for 20 and 40 min) and genotype (5 different). The number of GUS-expressing foci was counted to evaluate the gene transfer process. The KYRT1/pTJK136 strain of A. tumefaciens was significantly more effective for transformation than the 58C1/pTJK136 and EHA105/pTJK136 strains. The highest transient GUS activity was obtained from 16 h imbibed seedlings of cv.Uzunlu wounded with a needle and co-cultivated in liquid induction medium for 24 h with the KYRT1 strain (226 GUS foci/per explant).

Elimination of selectable marker genes via segregation of uncoupled T-DNAs in populations of doubled haploid barley

Barley represents one of the economicallymost important and most widely distributed crops worldwide, and genetic engineering is expected to play a crucial role in its further improvement. Once transgenic plants are obtained, the selectable marker gene is not any more necessary or even unwanted. Here we present a fast and efficient method to produce selectable marker-free, homozygous transgenic lines. Primary co-transgenic barley plants (T0), containing both the selectable marker gene (hygromycinphosphotransferase, in this case coupled with a green fluorescent protein gene) and a ’gene of interest’ (s-glucuronidase, without selectable marker) were generated via Agrobacterium-mediated gene transfer to immature embryos using separate T-DNAs. The binary vectors were introduced into the Agrobacterium strains AGL1 and LBA4404. The efficiency of the method was optimised by comparing more than ten different protocols to co-transfer independent T-DNAs to immature embryos. Uncoupled TDNAs present at hemizygous state in the primary transformants are randomly and independently distributed during male meiosis to the pollen grains. Homozygous selectable marker-free transgenic regenerants of such segregating androgenetic cultures can be efficiently produced and identified among doubled haploid individuals of the resultant T1. The method presented is not covered by complicated intellectual property rights as is the case in some alternative approaches. Moreover, only comparatively small populations are needed to find selectable marker-free, true-breeding transgenic T1plants, and further segregation analyses are unnecessary.

Efficient generation of transgenic barley: The way forward to modulate plant–microbe interactions

Stable genetic transformation represents the gold standard approach to the detailed elucidation of plant gene functions. This is particularly relevant in barley, an important experimental model widely employed in applied molecular, genetic and cell biological research, and biotechnology. Presented are details of the establishment of a protocol for Agrobacterium-mediated gene transfer to immature embryos, which enables the highly efficient generation of transgenic barley. Advancements were achieved through comparative experiments on the influence of various explant treatments and co-cultivation conditions. The analysis of representative numbers of transgenic lines revealed that the obtained T-DNA copy numbers are typically low, the generative transmission of the recombinant DNA is in accordance with the Mendelian rules and the vast majority of the primary transgenics produce progeny that expresses the respective transgene product. Moreover, the newly established protocol turned out to be useful to transform not only the highly amenable cultivar (cv.) 'Golden Promise' but also other spring and winter barley genotypes, albeit with substantially lower efficiency. As a major result of this study, a very useful tool is now available for future functional gene analyses as well as genetic engineering approaches. With the aim to modify the expression of barley genes putatively involved in plant-fungus interactions, numerous transgenic plants have been generated using diverse expression cassettes. These plants represent an example of how transformation technology may contribute to further our understanding of important biological processes.

An Agrobacterium-mediated system for gene transfer in Pinus patula

A pioneer study investigating the feasibility of using Agrobacterium-mediated gene transfer was undertaken in Pinus patula, the most important softwood species in South African forestry. This was achieved via the introduction of pAHC25 under the control of the ubiquitin promoter. A variety of target tissues were tested including: mature zygotic embryos, mature somatic embryos and embryonal suspensor masses (ESM). Expression of positive histochemical GUS activity in transformed material was confirmed by polymerase chain reaction (PCR) analysis. Results indicated that Pinus patula tissue was amenable to transformation. Use of the ESM tissue type, together with inclusion of l-proline in the medium, provided the most suitable transformation system.

두 가지 항산화유전자를 동시에 발현시킨 형질전환 톨 페스큐 식물체의 환경스트레스에 대한 내성 특성 해명

환경스트레스에 강한 내성을 지닌 신품종 톨페스큐를 개발할 목적으로 산화스트레스에 의해 강하게 유도되는 SWPA2 promoter 하류에 CuZnSOD와 APX 유전자가 엽록체에 동시에 발현하도록 제작한 벡터를 Agrobacterium법을 이용하여 톨 페스큐에 도입하였다. Hygromycin이 첨가된 선발배지에서 내성을 가지며 재분화된 형질전환 식물체를 pot로 이식하여 기내 순화시킨 후, Southern 분석을 실시하여 본 결과, 발현벡터의 T-DNA 영역이 형질전환 식물체의 genome에 성공적으로 도입되었음을 확인하였다. 형질전환 식물체 잎 절편을 산화스트레스와 중금속을 포함하고 있는 용액에 처리하여 엽록체의 손상정도를 조사한 결과, 비형질 전환체에 비해 형질전환체는 강한 내성을 나타내었다. 또한 유식물체 수준에서 MV를 처리하여 내성을 비교한 결과, 비형질전환체에 비해 형질전환체는 손상을 덜 받았다. 이와 같은 연구결과는 CuZnSOD와 APX 유전자를 엽록체에 동시발현시키는 기술이 다양한 환경스트레스에 대해 복합재해내성을 가지는 다양한 작물을 개발하는데 유용하게 이용될 수 있음을 나타낸 결과이다. Environmental stress is the major limiting factor in plant productivity. As an effort to solve the global food and environmental problems using the plant biotechnology, we have developed transgenic tall fescue (Festuca arundinacea Schreb.) plants via Agrobacterium-mediated gene transfer method. To develop transgenic tall fescue plants with enhanced tolerance to the environmental stresses, both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes were incorporated in a pIG121 binary vector and the both of the genes were controlled separately by an oxidative stress-inducible sweet potato peroxidase 2 (SWPA2) premoter expressed in chloroplasts. Leaf discs of transgenic plants showed 10-30% less damage compared to the wild-type when they exposed to a wide range of environmental stresses including methyl viologen (MV), <TEX>$H_2O_2$</TEX> and heavy metals. In addition, when <TEX>$200{\mu}M$</TEX> MV was sprayed onto the whole plants, transgenic plants showed a significant reduction of visible damage compared to wild-type plants that were almost damaged. These results suggest that over expression of CuZnSOD and APX genes in transgenic plants might be a useful strategy to protect the crops against a wide range of environmental stresses.

BPMADS4 - A MADS BOX GENE OF BIRCH INDUCES FLOWERS ON TRANSGENIC APPLE PLANTS IN VITRO

In comparison to herbaceous plants, the breeding of fruit trees, such as apple (Malus x domestica Borkh.), is more time-consuming due to its long generation time. Shortened juvenility and precocious flowering are, therefore, important breeding goals. Flower initiation has been intensively studied in Arabidopsis and orthologues/ homologues of LEAFY (LFY), APETELA1 (AP1), and TERMINAL FLOWER (TFL1) have been cloned from apple. One approach to shortening the juvenile phase of perennial crops is to reduce juvenility/vegetative maintenance factors, such as TFL1, by gene silencing. An alternative approach is to induce flowering by the over-expression of genes involved in the formation of floral meristems. In this context, the bpMADS4 gene of silver birch, which is similar to FRUITFULL of Arabidopsis, was overexpressed in apple. This gene is normally expressed in the apical meristem before the appearance of meristems (Elo et al., 2001). In transgenic apple, the expression of bpMADS4 was controlled by the constitutive CAMV35S promoter. Twenty transgenic lines were obtained after Agrobacterium-mediated gene transfer into apple. The integration and expression of the nptll and bpMADS4 genes were characterized by PCR analysis and RT-PCR. To quantify the level of transgene transcription a Real Time PCR procedure was carried out. Solitary flowers were detected on in vitro shoots of line T1165 thirteen weeks after transformation. Four other lines also produced flowers during in vitro propagation. The initial flowers were solitary, but shoots later developed clusters with about five flowers. Most of these flowers appeared morphologically normal but some were abnormal. In spring 2005, shoots of these lines were rooted and transferred to the greenhouse for further evaluation. To our knowledge, this is the first report of in vitro flower induction in transgenic apple.

Agrobacterium-mediated transformation of indica rice with chitinase gene for enhanced sheath blight resistance

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.

Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants

cDNA of human fibroblast growth factor 8 isoform b (FGF8b) was cloned for the first time into a plant expression vector with or without endoplasmic reticulum retention signal (KDEL) and was transiently expressed as His tagged fusion protein in Nicotiana tabacum leaves through Agrobacterium mediated gene transfer by vacuum infiltration method. Expression of FGF8b was confirmed by ELISA and Western blot using anti-FGF8b antibody and the expression level was measured as 4.1% of total soluble protein of tobacco leaves. The expressed recombinant protein was purified by Ni-NTA affinity chromatography and its molecular weight was determined by MALDI-TOF-MS. Schiff's test, Concanavalin A (Con A) immunoblot and enzymatic deglycosylation indicated that the high molecular mass was due to glycosylation of the FGF8b expressed in plant cells. Measurement of its biological activity in NIH3T3 cells by thymidine incorporation and MTT assay showed induction of cell proliferation. These results indicate that biologically active recombinant FGF8b could be expressed in tobacco plants.

Transient co-expression of post-transcriptional gene silencing suppressors and beta-glucuronidase in harvested lettuce leaf tissue does not improve recombinant protein accumulation in planta.

Agrobacterium-mediated gene transfer was used to co-express three virus-derived post-transcriptional gene silencing (PTGS) suppressors, P19 from tomato bushy stunt virus and two species of helper component proteinase (HcPro) from tobacco etch virus (TEV) and turnip mosaic virus, with beta-glucuronidase (GUS) in harvested lettuce leaf tissue to investigate whether GUS accumulation increases in the presence of PTGS suppressors. Co-expression incubations were 3-5 days at 4 and 22 degrees C. GUS activity and leaf viability were measured after incubation. Co-expression of PTGS suppressors did not elevate GUS expression levels. Under certain incubation conditions, co-expression of TEV HcPro significantly lowered transient GUS expression and was detrimental to leaf viability, suggesting that expression of PTGS silencers may have a negative effect on transient expression levels that outweighs any effects of PTGS suppression in harvested leaf tissues.

Agrobacterium-mediated gene transfer in higher plants

The genetic engineering of plants actually started with the discovery of a pathogenic bacterium, Agrobacterium tumefaciens, causing a disease known as crown gall (tumor). The bacterium transfers a set of genes from its plasmid into infected plant cells, where the transferred genes are expressed and produced gall at the junction of root and stem (i.e., crown region of the plant). This discovery has revolutionized the plant transformation method through which any gene can be introduced into plant genome to change their genetic make-up for commercial benefits, spaning from disease resistance to the production of pharmaceutically important proteins.

In vitro regeneration and transient gene expression in mango cv. ‘Vellaikolumban’

SUMMARYA protocol to regenerate shoots through somatic embryogenesis was developed in the polyembryonic mango cv. ‘Vellaikolumban’, and the feasibility of gene transfer using the βglucuronidase (GUS) reporter gene was demonstrated. Young, 25–30 d-old fruits were found to be ideal for culture initiation. MS and B5 media were equally effective for initiation of nucellar cultures. A higher percentage of somatic embryo induction was obtained from nucellar cultures using B5 or MS medium supplemented with 20% (v/v) coconut water compared to MS medium without coconut water. Maturation of somatic embryos, 1.0 – 1.5 cm in length, was optimised in M3 medium composed of B5 salts, 400 mg l–1 L-glutamine and 1 mg l–1 abscisic acid. Faciation and necrosis of embryogenic cultures could be effectively controlled through the use of 0.1 mg l–1 salicylic acid. For germination of mature somatic embryos, with well-developed roots and shoots, a semi-solid medium was found to be superior to a bilayer medium. Various stages of nucellar culture were transformed with Agrobacterium tumefaciens strain LBA 4404 containing the binary vector, pCambia 2301, harbouring the npt II gene as a selectable marker and GUS as a reporter gene. During kanamycin sensitivity tests, it was found that 200 mg l–1 kanamycin completely inhibited regeneration of fresh new callus in nonco-cultivated embryogenic callus derived from nucellus. Among the various stages of nucellar culture tested, embryogenic callus was found to be amenable to Agrobacterium-mediated gene transfer. Maximum transformation was obtained using 150 µl bacterial culture with 3 d of co-cultivation. The expression of the reporter transgene was confirmed by GUS assay.

Production and analysis of transgenic wheat (Triticum aestivum L.) with improved insect resistance by the introduction of cowpea trypsin inhibitor gene

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T2 lines obtained from T1 PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T1 PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.

Transformation of carnation with genes related to ethylene production and perception: towards generation of potted carnations with a longer display time

Potted carnation (Dianthus caryophyllus L. cv. Lillipot) plants were transformed with cDNAs for carnation 1-aminocyclopropane-1-carboxylate (ACC) synthase (DC-ACS1, s/aACS transgenes) or ACC oxidase (DC-ACO1, s/aACO transgenes) in sense or antisense orientation or mutated carnation ethylene receptor cDNA (DC-ERS2′) by Agrobacterium-mediated gene transfer. The presence of acetosyringone at 100 µM in media for shoot culture prior to leaf explant preparation and preculture of Agrobacterium in addition to the conventional method of addition to media for infection and coculture, and the use of water instead of nutrient media for infection and coculture increased the transformation efficiency to 4.0% compared to the 0.1% obtained by the conventional method. PCR analysis as well as Southern blot analysis confirmed the integration of the ethylene-related transgenes. Leaflet segments of cultured shoots of some lines transformed with s/aACO transgenes had less activity to convert ACC to ethylene than that of the non-transformed control plant, indicating that the integrated s/aACO transgenes reduced the expression of endogenous ACC oxidase gene (DC-ACO1) in the cultured shoots.