Addition Of Acetosyringone Research Articles

An Attempt to Establish an Agrobacterium-mediated Transient Expression in Euphorbia tirucalli (L.) an Important Medicinal Plant

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Euphorbia tirucalli evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Optimized parameters resulted in high transformation efficiency 3 fold increase at transient GUS expression .The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (100 µM) and cocultivation temperature of 25°C. Methods: Nodal explants were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56, 0.8 and 1.2 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of NAA 2 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400, 600 and 800 µM. Histochemical analysis of gus gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 72% when the infection process was carried out with acetosyringone in co-cultivation medium (100 µM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Optimization of Agrobacterium tumefaciens-mediated Transformation in Oxalis corniculata (L.)

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Oxalis corniculata evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Five fold transformation efficiency is achieved in transient gus gene expression after optimizing various parameters. The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (400 μM) and cocultivation temperature of 22°C. Methods: Oxalis corniculata (L.) were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56,0.80 and 1.34 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of BAP 2 mg L-1 and NAA 0.5 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400,600 and 800 μM. Histochemical analysis of gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 64% when the infection process was carried out with acetosyringone in co-cultivation medium (400 μM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Technology and Application of Hairy Root Culture in Monocotyledons

Background: Hairy root culture has been widely used in the production of metabolites in dicotyledons, and a large number of food crops and medicinal plants in monocotyledons need to be developed. However, there are many difficulties in the induction of hairy roots in monocotyledons. The purpose of this paper is to introduce the inducing methods, influencing factors, and application of hairy roots in monocotyledons, and to promote the development of hairy root system in monocotyledons. Methods: The mechanism of action of Agrobacterium rhizogenes and the current situation of hairy root induction, induction methods and influencing factors of monocotyledons were summarized to provide convenience for efficient acquisition of hairy root of monocotyledons. Results: Monocotyledons are not easy to produce phenols. Cells are prone to lignification, adverse differentiation and selective response to Agrobacterium rhizogenes strains. It is proposed that before induction, plant varieties and explants should be selected, and different infection strains should be screened. In the process of hairy root induction, exogenous inducers such as acetosyringone can be added. Although these factors can provide some help for the induction of hairy roots in monocotyledons, we still need to pay attention to the disadvantages of monocotyledons from dicotyledons at the cellular level. Conclusion: A large number of food crops and medicinal plants are monocotyledons. Hairy root culture can be used to help the breeding and production of medicinal substances. Therefore, it is necessary to pay attention to the selection of varieties and explants, the selection of Agrobacterium rhizogenes and the addition of acetosyringone in the process of hairy root induction to improve the production efficiency and facilitate the development and utilization of monocotyledons.

Optimization of Agrobacterium Mediated Genetic Transformation in Paspalum scrobiculatum L. (Kodo Millet)

An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM-EHA 105, 250 µM-LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.

Stable and Efficient Agrobacterium-Mediated Genetic Transformation of Larch Using Embryogenic Callus.

Larix olgensis or larch is an economically important coniferous tree species with rapid growth in the early stages, strong adaptability, and a short time to harvest. The genetic improvement of larch has garnered considerable attention in recent years for reclaiming timber forests. However, traditional breeding methods are largely ineffective for achieving rapid genetic improvement of L. olgensis. Studies show that the efficiency of plant regeneration can be improved by optimizing somatic embryogenesis. On this basis, we devised a stable, fast and efficient Agrobacterium-mediated genetic transformation method using suspended embryogenic calluses as explants and β-glucuronidase as the reporter. We evaluated the effects of the Agrobacterium load, co-culture period, and addition of acetosyringone and transformant screening antibiotic on the transformation efficiency. In addition, we tested the pCAMBIA 1300-PtHCA 2-1 promoter-GUS binary expression vector, which contains the GUS gene ORF under the control of Populus trichocarpa high cambial activity PtHCA 2-1 promoter, and observed the tissue-specific expression of the GUS gene in the somatic embryos of transgenic larch. This novel technique can not only accelerate the generation of superior transgenic strains of L. olgensis but also aid in future gene functional studies.

Optimization of Factors Influencing Agrobacterium mediated Genetic Transformation in Eclipta alba (L.) Hassk.

An efficient and reproducible protocol for in vitro plant regeneration and Agrobacterium-mediated genetic transformation was developed for Eclipta alba (L.) Hassk. using nodal explants. Several parameters affecting Agrobacteriummediated gene delivery were investigated and optimized. These include antibiotic concentration, preculture period, infection time, use of acetosyringone, co-cultivation period and temperature and increased level of copper in induction, co-cultivation and regeneration medium. Incorporation of elevated level of CuSO4 led to significant improvements in plant regeneration and recovery of transformed plants. The highest frequency of transformation was observed when explants were infected and co-cultivated at 23oC. Higher transformation frequency was attained by augmenting the medium with 1.0 ?M CuSO4 which was ten times the normal MS level. Addition of acetosyringone in the infection and co-cultivation media was very beneficial and resulted in greater transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected explants and leaves of regenerated transformants, respectively. Molecular analysis of transformed plants with nptII and gus A specific primers revealed the amplification of nptII and gus gene thereby demonstrated the efficacy of optimized protocol for A. tumefaciens-mediated genetic transformation in Eclipta alba for the first time.Plant Tissue Cult. & Biotech. 25(2): 155-164, 2015 (December)

Salinity and drought tolerance conferred by in planta transformation of SNAC1 transcription factor into a high-yielding rice variety of Bangladesh

Abiotic stresses such as drought and high salinity unfavorably affect the growth and productivity of crop plants. Therefore, the development of stress-tolerant crops is essential for the affected cultivable areas. It has been shown in the current study that the overexpression of stress-responsive NAC1 (SNAC1) transcription factor (TF) significantly increases salinity and drought tolerance in a farmer-popular high-yielding, transgenic rice. The indica rice variety BRRIdhan 55, which was poorly responsive to tissue culture was transformed with the SNAC1 TF from the rice landrace Pokkali by the in planta method. Addition of acetosyringone in the Agrobacterium suspension and co-culture media increased previously reported transformation efficiencies by four-folds. Integration of foreign genes into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SNAC1) was also quantified by real-time PCR. Genetic segregation ratio for T1 progenies was calculated and found to follow the law of Mendelian inheritance. Phenotypic screening was conducted at T2 and T3 seedling stages where the transgenic lines exhibited much better tolerance compared to their control non-transgenic plants at 120 mM (NaCl) salt as well as drought stress implemented by withholding water for 20 days.

Optimization of Agrobacterium-Mediated Genetic Transformation and Regeneration of Transgenic Plants in Indian Cultivar of Barley (Hordeum vulgare L. cv. BL 2)

An efficient Agrobacterium-mediated genetic transformation protocol has been developed for Indian winter barley. Several parameters affecting Agrobacterium-mediated gene delivery were investigated and optimized. These include use of acetosyringone, surfactants in the infection medium, antinecrotic treatment of explants and heat and centrifugation pre-treatment. Addition of acetosyringone in the infection and co-cultivation media was advantageous and resulted in increase in transformation efficiency from 3.7 to 11.1 %. Addition of surfactants (Tween-20/Pluronic acid F 68) at 0.1 % (v/v) had also resulted in improved gene delivery. Pre-treatment of immature embryos with antinecrotic mixture had significant positive effect on percent callus survival and transformation efficiency. Temperature pre-treatment of immature embryos prior to infection resulted in an increase in the number of GUS positive calli with optimum temperature of 40 °C for 3 min. However, centrifugation of immature embryos had a negative effect on barley transformation. Regenerated transgenic plants obtained after 8 to 10 weeks of selection were further rooted for 2 weeks. The plantlets established in green house condition showed normal growth. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of regenerated transformants, respectively. Molecular analysis of transgenic plants containing II gene (nptII) demonstrated the efficacy of optimised protocol for Agrobacterium-mediated genetic transformation in Indian cultivar of barley for the first time.

Agrobacterium 공동배양을 이용한 포도 재분화율 향상과 GUS 유전자의 발현

Abstract Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. In this study, methods for Agrobacterium tumefaciens -mediated trans-formation and plant regeneration of grapevine ( Vitis vinifera ) were evaluated. Tamnara, Heukgoosul, Heukbosek, Rizamat were co-cultivated with Agrobacterium strains, LBA4404 containing the vector pBI121 carrying with CaMV 35S promoter, GUS gene as reporter gene and resistance to kanamycin as selective agent. Seven percent of the maximum regeneration frequency was obtained from co-cultivated with explants from Rizamat with LBA4404 strain on selection medium with kanamycin. The addition of acetosyringone, 200 μm in virulence induction step was a key factor for successful GUS reporter gene expression in grapevine transformation. Transgenic plants showed resistance to kanamycin and the GUS positive response in leaf (T 0 ) stem (T 0 ) and petiole (T 0

An Easy Method for Agrobacterium tumefaciens-Mediated Gene Transfer to Nicotiana tabacum cv. TAPM26

Research in Agrobacterium tumefaciens mediated gene transfer has advanced and opened new avenue for plant improvement via introduction of various beneficial genes. Current study investigates transformation parameters during co-cultivation to improve T-DNA delivery into Nicotiana tabacum cv. TAPM 26 by monitoring GUS expression level. The techniques involved basic plant tissue culture and establishment of plant transformation systems. Conditions assessed were bacterial inoculum concentration, infection period, wounding and pre-culture of explants, acetosyringone (AS) concentration and co-cultivation temperature. Optimized conditions resulted in high transformation efficiency at transient level were as follows; Agrobacterium tumefaciens growth phase of A600nm 0.8, infection period of 30 min, pre-culture of wounded explants prior to infection, addition of acetosyringone (AS) in bacterial growth culture (100 µM) and in co-cultivation medium (200 µM) and co-cultivation temperature of 22°C. Although higher density bacterial culture used for the infection process gave higher transformation rate, however, it compromised the viability of the explants. On the other hand, dilution of bacterial suspension reduced necrosis in explants and improved transformation events greatly. The transformation efficiency was increased 9 fold when the infection process was carried out at low temperature of 22°C. Current study has proven among the parameters assessed, temperature was the critical factor during co-cultivation process in Agrobacterium tumefaciens mediated gene transfer.

Developing an Agrobacterium tumefaciens-mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus

Agrobacteriumtumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l−1 and thus all putative transgenic plants were obtained on induction medium containing 50 mg l−1 kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes responsible for Se-accumulation in A. racemosus.

Agrobacterium-mediated transformation of Dioscorea zingiberensis Wright, an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.

Agrobacterium tumefaciens-mediated transformation of Narcissus tazzeta var. chinensis

Phytoene synthase (PSY), as a key regulatory enzyme for carotene biosynthesis, plays an important role in regulating color formation in many species. In the present study, a protocol was developed for the transformation of Narcissus tazzeta var chinensis using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1301 plasmid which contained an antisense phytoene synthase gene, a reporter beta-glucuronidase gene and a selectable marker hygromycin phosphotransferase gene. Effects of some factors on efficiency of transformation and regeneration were examined. Preculture of the explants for 6 days before inoculation enhanced the transient GUS expression. The addition of acetosyringone (AS) at 100 micromol l(-1) for inoculation and a period of 3 days co-cultivation yielded efficient transient GUS expression. Transformants were obtained through selection on MS medium containing 5 mg l(-1) 6-benzylaminopurine (BA), 0.1 mg l(-1)alpha-naphthalene acetic acid (NAA) and 40 mg l(-1) hygromycin. The transformation frequency was 1.24% based on PCR analysis of gus gene. One or two copies of transgene were demonstrated in different transformations by Southern blotting analyses. Northern blotting results confirmed that the transcription of the endogenous psy gene in transgenic plants was inhibited or silenced. The method reported here provides new opportunities for improvement of quality traits of Narcissus tazzeta via genetic transformation.

Agrobacterium-mediated transformation of indica rice under Acetosyringone-free conditions

The possibility of developing transgenic indica rices through Agrobacterium-mediated transformation in the absence of acetosyringone at bacterial preinduction or co-cultivation or both stages was assessed. Four-week-old, scutellum derived calluses of indica rice (Oryza sativa L. cv. ‘Pusa Basmati1’) were co-cultivated with A. tumefaciens strain LBA4404 (pSB1), harboring the binary vector pCAMBIA 1301 with the β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in the T-DNA region. Addition of acetosyringone (AS) to both preinduction medium (PIM) and co-cultivation medium (CCM) induced higher levels of transient GUS expression than that obtained with the addition of AS to either of the stages. Addition of only sucrose to both preinduction and co-cultivation media yielded transient expression levels similar to those obtained by the addition of AS. The resultant fertile plants were stable transformants as revealed by GUS histochemical assay and PCR analysis for the GUS and HPT genes. Thus, phenolics like AS may not be essential for induction of vir genes, and development of transgenic indica rice is feasible under AS-free conditions.

Salt-tolerant transgenic perennial ryegrass ( Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na +/H + antiporter gene

The objective of this study was to obtain a salt-tolerant perennial ryegrass ( Lolium perenne L.) by transforming it with a rice vacuolar membrane Na +/H + antiporter gene via the Agrobacterium-mediated method. To optimize the transformation conditions, two Agrobacterium tumefaciens strains, LBA4404 and EHA105, carrying plasmid pCAMBIA3301, were used to transform embryogenic calli of perennial ryegrass; two factors affecting transformation efficiency, acetosyringone and mannitol, were then determined. Addition of acetosyringone during the incubation of bacteria and the co-cultivation process increased the transformation rate. Transformation efficiency could be further increased by osmotic treatment with 0.4 mol/l mannitol before infection. Using this optimized transformation system, the rice vacuolar Na +/H + antiporter gene, OsNHX1, was transferred into perennial ryegrass by Agrobacterium-mediated method. The transformants were confirmed by both PCR and Southern blot analysis. The resultant transgenic ryegrass had better salt-tolerance. After stress treatment for 10 weeks with 350 mmol/l NaCl, transgenic plants survived, while wild-type plants did not. The leaves of transgenic plants accumulated higher concentrations of Na +, K + and proline than those of the control plants. These results demonstrate the OsNHX1 gene was successfully transferred into perennial ryegrass by Agrobacterium-mediated method, and the salt-tolerance of perennial ryegrass was improved by overexpression of the OsNHX1 gene.

Agrobacterium -induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium -mediated DNA transfer

High necrosis and poor survival rate of target plant tissues are some of the major factors that affect the efficiency of Agrobacterium -mediated T-DNA transfer into plant cells. These factors may be the result of, or linked to, hypersensitive defense reaction in plants to Agrobacterium infection, which may involve the recognition of specific signals from the Agrobacterium that triggers the burst of reactive oxygen species at the infection site. Evidences of Agrobacterium -induced necrosis in target plant tissues and its link to reactive oxygen species are presented. Application of antioxidants, addition of acetosyringone and optimization of pre-culture conditions suppress the Agrobacterium -induced hypersensitive necrotic response in target plant tissues, thereby enhancing stable transformation. Key words: Agrobacterium ; hypersensitive reaction; necrosis; signal transduction; oxidative burst; transformation. African Journal of Biotechnology Vol. 4 (8), pp. 752-757

Agrobacterium -induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium -mediated DNA transfer

High necrosis and poor survival rate of target plant tissues are some of the major factors that affect the efficiency of Agrobacterium -mediated T-DNA transfer into plant cells. These factors may be the result of, or linked to, hypersensitive defense reaction in plants to Agrobacterium infection, which may involve the recognition of specific signals from the Agrobacterium that triggers the burst of reactive oxygen species at the infection site. Evidences of Agrobacterium -induced necrosis in target plant tissues and its link to reactive oxygen species are presented. Application of antioxidants, addition of acetosyringone and optimization of pre-culture conditions suppress the Agrobacterium -induced hypersensitive necrotic response in target plant tissues, thereby enhancing stable transformation. Key words: Agrobacterium ; hypersensitive reaction; necrosis; signal transduction; oxidative burst; transformation. African Journal of Biotechnology Vol. 4 (8), pp. 752-757

Agrobacterium tumefaciens-mediated transformation of highly regenerable cell suspension cultures in Dianthus acicularis

SummaryA successful transformation procedure using Agrobacterium-mediated gene transfer was established for the bacterial wilt-resistant Dianthus species, D. acicularis. Highly regenerable cell suspension cultures derived from calli, which originated from leaf segments, were infected with A. tumefaciens strain EHA101 (pIG121Hm) and co-cultivated for 2 d. After selection on medium with 30 mg l–1 hygromycin, approximately five hygromycin-resistant plants were obtained from 1 g fresh weight of cell culture. For the production of hygromycin-resistant calli, addition of acetosyringone to the co-cultivation medium was indispensable, with an optimum concentration of 200 µM. Hygromycin-resistant calli and plantlets showed stable and uniform GUS expression, and integration of the GUS gene into the plant genome was also confirmed by Southern blot analysis.These transgenic plants had a normal morphology, and no somaclonal variation was observed in their level of ploidy.

Preliminary examination of factors affecting Agrobacterium tumefaciens-mediated transformation of marula, Sclerocarya birrea subsp. caffra (Anacardiacease)

In planta Agrobacterium tumefaciens-mediated transformation of marula, Sclerocarya birreasubsp. caffra(Anacardiaceae) resulted in chimeric transgenic in vitro shoots at a rate of 0.8--1.5%. Average transgene expression rates of 33.1 and 1.3% GUS-positive explants were observed on days 3 and 6 after agroinfection, respectively. One to 4 GUS-positive zones were observed per GUS-positive explant section. Addition of acetosyringone (100 μM) during co-cultivation significantly improved transient transformation efficiency as determined by the percentage of GUS-positive explants on day 3 (p< 0.05) whereas wounding did not show a significant effect. However, wounding and acetosyringone acted antagonistically reducing transient transformation rates.

Agrobacterium을 이용한 고추의 Transient Expression 시스템

Agrobacterium을 이용한 GUS 유전자를 효과적으로 발현시키기 위하여 수행되어진 실험 결과를 요약하면 다음과 같다. 박테이라 농도별 실험을 수행한 결과 균의 전처리 배양 농도 O <TEX>$D_{600nm}$</TEX> 0.3일때 원심분리한 후 얻어진 균을 희석한 후의 최종 농도는 O <TEX>$D_{600nm}$</TEX> 0.8로 맞춘 실험 처리구에서 GUS 유전자 발현율이55%로 가장 높게 나타났다. 병원성 유도 배지 내에 Acetosyringone (AS)이 첨가되지 않은 경우 GUS 유전자가 발현된 고추 잎을 얻을 수 없었으나, 200<TEX>$\mu$</TEX>M을 첨가했을 때 90%의 가장 높은 GUS 유전자 발현율을 나타내어 많은 수의 GUS spots을 관찰할 수 있었다. Agrobacterium에 의한 고추 잎의 감염 정도를 조사한 바 Agrobacterium으로 감염시킨지 3일째부터는 박테리아 에한 감염 정도가 심해져서 GUS 유전자 발현 정도가 약해지므로 Agrobacterium으로 감염시킨지 2일째 되었을 때 GUS 유전자 발현이 가장 강하게 나타난 것을 확인하였다. 이 같은 결과는 박테리아에 의한 감염이 일어난지 3일째 부터는 식물체의 감염부위 고사를 일으키는 것과 관련된 것으로 보인다. We established a transient gene expression system in chili pepper leaves based on Agrobacterium-mediated transformation of GUS gene. For the best GUS transient expression, two step culture system was adopted. When the Agrobacterium tumefaciens cell density of pre-culture was <TEX>$A_{600nm}$</TEX> 0.3, the cells were harvested and diluted to <TEX>$A_{600nm}$</TEX> 0.8 with virulence induction medium after cell harvested. The addition of acetosyringone (200 <TEX>$\mu$</TEX>M) in virulence induction step was a key factor for successful transient expression. Additionally, Younger leaves showed more effective transient expression than older leaves. Temporally, the strongest intensity of GUS expression was detected at 2 days after infiltration. These results demonstrate that Agrobacterium-mediated transient expression can be used for a simple in vivo assays of plant promoters, transcription factors and furthermore provide efficient protocol for chili pepper transformation.