Transformation Of Tomato Research Articles

Expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic tomato (Solanum lycopersicum L.) fruit

We report a feasibility study for expressing the LTB protein (Escherichia coli heat-labile enterotoxin B subunit) via Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.). We produced five regenerated plants obtained on the selection medium supplemented with an antibiotic. Stable integrations of the LTB gene into the genome of these plants were confirmed by Southern blot hybridization. Western blot analysis showed that only two of the five T<sub>0 </sub>transgenic tomato plants expressed the pentameric LTB protein in the fruits. An enzyme-linked immunosorbent assay indicated that these two plants synthesized the LTB protein bound specifically to GM1 ganglioside, suggesting that the LTB subunits formed active pentamers. The LTB protein produced in tomatoes can be a potential candidate for inexpensive, safe, and effective plant-based vaccines.

Use of proximal hypocotyl segment for high-through put transgenic development of tomato

Achieving high-through put and efficient regeneration are the top priorities of any Agrobacterium mediated plant transformation experiments to develop large numbers of in vitro transformants. The type of explant plays a critical role in shoot regeneration efficiency. In the present investigation, an attempt was made to study the effect of various segments of hypocotyl and abaxial/adaxial orientation of cotyledon explants on regeneration efficiency in tomato. A plant transformation vector, pGRNAi-harboringds RNA expressing construct stargeted to two genes of Helicover paarmigera, serine protease and chymotrypsin independently were used to transform tomato. Of the three segments of hypocotylsobtained from 12 day old seedlings, the proximal (closest to shoot apex) segment had yielded highest regeneration (28.65%)compared to the middle (11.86%)and the distal segments (11.20%). In cotyledon explants, those incubated with their abaxial surface in contact with media exhibited highest regeneration (20.83%) compared to adaxially placed cotyledon explants. However, of the two-explant types, hypocotyls had higher regeneration compared to cotyledons. The molecular characterization of putative transformants through PCR and Southern blot analysis revealed the presence of the transgene. Thus, these results will aid in obtaining high-throughput regeneration in transformation of tomato in particular and other crops in general.

Generation of boron-deficiency-tolerant tomato by overexpressing an Arabidopsis thaliana borate transporter AtBOR1.

Nutrient deficiency in soil poses a widespread agricultural problem. Boron (B) is an essential micronutrient in plants, and its deficiency causes defects in both vegetative and reproductive growth in various crops in the field. In Arabidopsis thaliana, increased expression of a major borate transporter gene AtBOR1 or boric acid channel gene AtNIP5;1 improves plant growth under B-deficient conditions. In this study, we examined whether high expression of a borate transporter gene increases B accumulation in shoots and improves the growth of tomato plant, a model of fruit-bearing crops, under B-deficient conditions. We established three independent transgenic tomato plants lines expressing AtBOR1 using Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L. cv. Micro-Tom). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that two lines (Line 1 and Line 2) more strongly expressed AtBOR1 than Line 3. Wild-type plants and the transgenic plants were grown hydroponically under B-sufficient and B-deficient conditions. Wild-type and Line 3 (weakly expressing transgenic line) showed a defect in shoot growth under B-deficient conditions, especially in the development of new leaves. However, seedlings of Line 1 and Line 2, the transgenic lines showing strong AtBOR1 expression, did not show the B-deficiency phenotype in newly developing leaves. In agreement with this phenotype, shoot biomass under low-B conditions was higher in the strongly expressing AtBOR1 line. B concentrations in leaves or fruits were also higher in Line 2 and Line 1. The present study demonstrates that strong expression of AtBOR1 improved growth in tomato under B-deficient conditions.

Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids. In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYb, EC:1.14.-.-) is a key regulatory enzyme in the catalysis of lycopene to β-carotene, an important dietary precursor of vitamin A for human nutrition. Two closely related lycopene β-cyclase cDNAs, designated CsLCYb1 and CsLCYb2, were isolated from the pulp of orange fruits (Citrus sinensis). The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopene-accumulating genotype Cara Cara than that in common genotype Washington, and this might be correlated with lycopene accumulation in Cara Cara fruit. The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system, but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene. In tomato transformation studies, expression of CsLCYb1 under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene, and the ripe fruits displayed a bright orange colour. However, the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation. In fruits of the CsLCYb1 transgenic plants, most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g−1 DW. Unexpectedly, most transgenic tomatoes showed a reduction in total carotenoid accumulation, and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits. Collectively, these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency, and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

Utilization of the plant methionine sulfoxide reductase B genes as selectable markers in Arabidopsis and tomato transformation

Plant transformation is an important tool for basic research and agricultural biotechnology. In most cases, selection of putative transformants is based on antibiotic or herbicide resistance. Overexpression of plant genes that provide protection from abiotic or biotic stresses can result in a conferred phenotype that can be used as a means for selection. We have demonstrated herein that specific methionine sulfoxide reductase B (MsrB) genes that are overexpressed in transgenic plants may constitute a new selectable marker with concomitantly increased tolerance to methyl viologen (MV) treatment. Arabidopsis transformants overexpressing cytosolic MsrB7, MsrB8 or MsrB9 are viable and survive after MV selection. To establish whether these native plant origin genes serve as new non-antibiotic markers that can be applied to crop transformation, tomato cotyledons were used as transformation materials. MsrB7 transgenic tomato plants were successfully obtained by Agrobacterium-mediated transformation and selection on medium supplemented with MV. We suggest that specific MsrB genes that are overexpressed in transgenic plants may constitute a new selectable marker with increased tolerance to oxidative stress concomitant with MV treatment.

Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200mM) acetosyringone and minimal concentrations of (0-20mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28%. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5mg L(-1) thidiazuron, 1.5mg L(-1) indole-3-butyric acid, 30mg L(-1) kanamycin, and 0-1.5mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90%. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T1 seedlings of tomato exposed to metal ions

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T0). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T1) produced the highest GUS activity when treated with 150μM Cu2+ compared to the control (without Cu2+). However, Zn2+ and Fe2+ treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T1 seedlings of tomato when subjected to Cu2+ ions.

Plastid transformation of tomato (Solanum lycopersicum L.) with HIV-1 p24 gene by biolistic particle delivery system

Plastid transformation in tomato (Solanum Lycopersicum L.) is carried out using vector carrying HIV-1 p24 gene and a selection of aadA gene, spectinomycin for resistance to antibiotics, and streptomycin. We used the hepta adaptor to replace the rupture disk retaining cap and microcarrier launch assembly. Three days after bombardment, the leaves and cotyledons were cut into small pieces each of 3 × 3 mm and placed on medium MS salts and B5 vitamins containing 2.0 mg/l Zeatin, 0.2 mg/l IAA, 50 mg/l spectinomycin for leaves, and medium MS salts and B5 vitamins containing 2.0 mg/l BAP, 1.0 mg/l IAA, 0.5mg/l Kinetin, 50 mg/l spectinomycin for cotyledons. Selected further time on the medium containing spectinomycin was up to 300 mg/l. PCR analysis of aadA and HIV-1 p24 genes of the tomato plastid transformation lines showed the presence of DNA amplification with the corresponding 250bp and 700bp. Southern blot analysis confirmed that the foreign genes were inserted into the tomato plastid genome. The result indicated the presence of HIV-1 p24 in the tomato plastid genome with the presence of DNA band of 5.8 kb. However, it is heteroplasmic transformant because it has a DNA band of 3.5 kb of wide type plant.

Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog’s (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l−1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l−1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.

Tomato plants transformed with the inhibitor‐of‐virus‐replication gene are partially resistant to several pathogenic fungi

AbstractThe inhibitor‐of‐virus replication (IVR) gene associated with the local lesion response to Tobacco mosaic virus in tobacco codes for a putative protein with a molecular mass of 22 kDa. Earlier work revealed that when tomato (Solanum lycopersicum) cv. VF36 plants are transformed with a cDNA clone encoding this gene, they become partially resistant to Botrytis cinerea infection. The transformation of tomatoes with the IVR gene induced partial resistance to in vitro seedling infection by Alternaria alternata, Pythium aphanidermatum and Rhizoctonia solani and faster seedling growth. Resistance to damping‐off was observed in transgenic seedlings planted in soil infested with R. solani and P. aphanidermatum. Early blight (Alternaria solani) and powdery mildew (Oidium neolycopersici) were also partially controlled in mature transgenic tomato plants.

PL1 fusion gene: a novel visual selectable marker gene that confers tolerance to multiple abiotic stresses in transgenic tomato

Visual selectable markers, including the purple color caused by the accumulation of anthocyanins, have been proposed for use as antibiotic-free alternatives. However, the excessive accumulation of anthocyanins seriously inhibits the growth and development of transgenic plants. In our study, the AtDWF4 promoter from Arabidopsis and the tomato LeANT1 gene, encoding a MYB transcription factor, were used to construct the PL1 fusion gene to test whether it could be used as a visual selectable marker gene for tomato transformation. All the PL1 transgenic shoots exhibited intense purple color on shoot induction medium. In the transgenic tomato plants, PL1 was highly expressed in the cotyledons, but expressed only slightly in the true leaves and other organs. The expression of PL1 had no significantly adverse effects on the growth or development of the transgenic tomato plants, and conferred tolerance to multiple abiotic stresses in them. With the “cut off green shoots” method, multiple independent 35S::GFP transgenic tomato lines were successfully obtained using PL1 as the selectable marker gene. These results suggest that PL1 has potential application of visual selectable marker gene for tomato transformation.

Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes

Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L(16) (4(5)) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin. We also examined the effect of these parameters on contamination rate, necrosis rate, mortality, cut-surface browning rate, and undamaged explant rate. Both the bacterial and carbenicillin concentrations had a significant influence on the rate of infected explants. The time of co-cultivation also had a significant influence on the transformation parameters. The optimal transformation protocol consisted of an Agrobacterium suspension of 0.5 × 10(8) cells/mL (OD(600) = 0.5) and an infection time of 5 min, one day of co-cultivation and 500 mg/L carbenicillin. Under these conditions, the transformation efficiency of the shoots reached 5.1%; the mean transformation frequency was 3.9% (N = 838).

Construction and Genetic Transformation of Tomato ARF4 RNA Interference Expression Vector with Fruit Specific Promoter

SlARF4 RNA interference vector expressing only in tomato fruits was constructed to investigate its effect on fruit characteristics of tomato.The full-length cDNA sequence of SlARF4 gene was amplified by RT-PCR and a plant RNAi expression vector harboring inversely repeated sequence of ARF4 driven by a fruit-specific promoter TFM7 was constructed,and transferred into tomato plants through a reproducible Agrobacterium-mediated transform method.Double digest assay confirmed the expression vector pBI121-TFM7-A4Ri was successfully constructed.The transgenic plants were obtained through PCR assay.The analysis of semi-quantitative RT-PCR showed significant reduction of ARF4 expression levels in transgenic tomatoes.The content of chlorophyll in green mature fruit,the weight per fruit and pericarp thickness of the transgenic lines were higher than those of the control plants.These results suggested that fruit-specific expression of SlARF4 interference in tomato could improve tomato fruit quality.

Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses

Genetically engineered tomato ( Lycopersicon esculentum) with the ability to synthesize glycinebetaine was generated by introducing the codA gene encoding choline oxidase from Arthrobacter globiformis. Integration of the codA gene in transgenic tomato plants was verified by PCR analysis and DNA blot hybridization. Transgenic expression of gene was verified by RT-PCR analysis and RNA blot hybridization. The codA-transgenic plants showed higher tolerance to salt stress during seed germination, and subsequent growth of young seedlings than wild-type plants. The codA transgene enhanced the salt tolerance of whole plants and leaves. Mature leaves of codA-transgenic plants revealed higher levels of relative water content, chlorophyll content, and proline content than those of wild-type plants under salt and water stresses. Results from the current study suggest that the expression of the codA gene in transgenic tomato plants induces the synthesis of glycinebetaine and improves the tolerance of plants to salt and water stresses.

Development of a simple and effective protocol for Agrobacterium tumefaciens mediated leaf disc transformation of commercial tomato cultivars

The transformation of tomato (Solanum lycopersicum) through Agrobacterium tumefaciens is still far from being routine, particularly when it comes to commercial varieties. In the present paper, we present an efficient and simple protocol for leaf disc transformation of three Vietnamese tomato cultivars (DM8, MTS, FM372C) by comparing shoot regeneration media for expanding leaves and examining different parameters of inoculation, co-culture and selection conditions. The present transformation method requires neither feeder layers of cell suspension cultures nor pre-culture. The data clearly show that appropriate cytokinin- and auxin combinations and concentrations provide competent tissues for transformation. Supplementing of 8µM trans-zeatin and 5 µM indoleacetic acid (IAA) into pre-treatment, inoculation and co-culture media resulted in higher frequency of transformation and stronger GUS-expression than that of media supplemented with 4 µM trans-zeatin and 2 µM IAA. The experiments also exhibited that tomato leaf tissues were more sensitive to glufosinate after inoculation with Agrobacteria compared to the untreated controls, so a more sophisticated scheme for the glufosinate selection had to be established.

토마토 재분화 효율 향상 및 엽록체 형질전환 조건

국립원예특작과학원에서 분양받은 토마토 18계통을 공시하여 재분화가 잘되는 적정 품종을 탐색한 결과, 계통번호 2001-58에서의 재분화율이 93%로 양호하였다. 또한 식물체로의 재분화 과정에서 보여 지는 갈변현상과 phenolic compound에 의한 식물조직의 괴사현상을 막기 위하여 항산화제인 ascorbic acid와 cystein을 단용 또는 혼용으로 첨가한 후 토마토 재분화에 미치는 영향을 살펴 본 결과, ascorbic acid <TEX>$200{\sim}300\;{\mu}M/L$</TEX> 처리구에서 줄기형성율 및 생체중이 증가되는 현상을 관찰할 수 있었다. 토마토 엽록체 형질전환체 선발을 위해 spectinomycin의 적정 농도를 살펴본 결과, 재분화배지에 spectinomycin 20~25 mg/L 농도가 첨가되어진 처리구에서 재분화가 거의 이루어지지 않았다. 토마토 엽록체 형질전환을 위해 토마토 엽록체 게놈 일부를 분리하여 염기서열을 분석하여 담배와 비교 분석한 결과, homology가 매우 높음을 알 수 있었다. Homologous recombination에 의한 엽록체 형질전환이 되기 위해서 분리한 토마토 엽록체 게놈 일부를 border sequence로 이용하였고, transient assay를 위해 GFP 유전자가 포함된 토마토 엽록체 형질전환용 운반체 pKRT22-AG를 제작하였다. Bombardment을 한 후 원형질체를 나출하여 공초점 현미경하에서 관찰한 결과 엽록체 내에서만 GFP가 발현됨을 알 수 있었으며, DNA 농도 <TEX>$1\;{\mu}g$</TEX>, <TEX>$0.6\;{\mu}m$</TEX> gold particle 1 mg, target distance 9 cm 조건이 가장 좋았다. Eighteen cultivars of tomato were tested for their regeneration response. Lycopersicon esculentum cv. 2001-58 showed a very high frequency of regeneration (93%). We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cystein). The use of ascorbic acid (<TEX>$200\;-\;300\;{\mu}M/L$</TEX>) has a positive effect on shoot regeneration. To develope a system for plastid transformation in tomato via homologous recombination, we constructed the tomato plastid expression vector (pKRT22-AG) harboring 2.2 kb flanking sequences cloned from intact plastid genome and gfp gene. To investigate the factors affecting the delivery system of the pKRT22-AG into chloroplast using bombardment, We assessed the optimal DNA concentration, gold particle volume and target distance. Expression of the GFP protein was observed within chloroplast on protoplast of cotyledon explant by confocal laser scanning microscopy, which indicates that the protocol developed in this study be useful for the production of plastid transgenic plants in tomato.

English

&nbsp; &nbsp; As part of our efforts to improve tomato tolerance to abiotic stress, we have undertaken this study to introduce two candidate genes encoding: a sodium antiporter and a vacuolar pyrophosphatase, previously shown to enhance drought and salt tolerance in transgenic&nbsp;Arabidopsis&nbsp;plants.&nbsp;First, we evaluated the potential of primary leaves from three to four week-old in&nbsp;vitro-grown tomato seedlings as alternative explants to cotyledons for tomato transformation. Our results demonstrated that primary leaves are three times more efficient then cotyledons in terms of regeneration percentage, productivity, and transformation frequencies independently of the medium and genetic construct used. Second, primary leaves were used to introduce the genes of interest using&nbsp;Agrobacterium-mediated transformation. Many transgenic tomato plants were easily recovered. The presence of the transgenes and their expression were confirmed by PCR and RT-PCR analysis. The transformation frequencies for primary leaf explants ranged from 4 to 10% depending on the genetic construct used. The time required from inoculation of primary leaves with&nbsp;Agrobacterium&nbsp;cells to transfer of transgenic tomato plants to soil was only 2 months compared to 3 to 4 months using standard tomato transformation protocols. The transgenic tomato plants obtained in the current study were more tolerant to salinity and drought stress than their wild-type counterparts. &nbsp; &nbsp; &nbsp; &nbsp; Key words:&nbsp;Agrobacterium-mediated transformation, cotyledon, primary leaf, Regeneration, tomato.

A simple and efficient Agrobacterium-mediated procedure for transformation of tomato

We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and beta-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

Production of transgenic tomato plants with the Fe-dependent superoxide dismutase gene

Agrobacterial transformation of tomatoes by an expression vector containing the Fe-SOD gene is carried out. The transgenic status of the transformants obtained is confirmed by PCR analysis. Differences are noted in the cell cycle and ultrastructure of transgenic and nontransgenic plants under the effect of salt stress.

Influence of bacterial density during preculture on Agrobacterium-mediated transformation of tomato

An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and resuspended in a liquid cocultivation medium-I, adjusted to OD600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency is stable and highly efficient.