Evaluating extraction and storage of a recombinant protein produced in agroinfiltrated lettuce

Extraction and storage of a recombinant protein produced by transient expression following agroinfiltration of lettuce were investigated. Lettuce leaves expressing beta-glucuronidase (GUS) were extracted by homogenization in several buffer combinations, and the yield and stability were assessed. The reducing agent dithiothreitol (DTT) was found to be the most important (significant) component in the extraction buffer. An extraction buffer consisting of 50 mM sodium phosphate at pH 7.0 with 10 mM DTT produced a good yield and stabilized GUS. Leaching of GUS through intact agroinfiltrated lettuce leaves was determined to be infeasible, with a maximum flux of 10 microg GUS/h/m2 and recovery of 1.7% of the GUS content in 24 h. Freeze-drying was evaluated as a method to extend the shelf life of the perishable leaf material containing GUS. First- and second-order kinetic models and the Weibull distribution were compared to describe inactivation of GUS in the freeze-dried leaves and leaf extracts. The first-order model best fit the inactivation data. An Arrhenius model fit the first-order inactivation data with respect to temperature with R2 = 1.00. Freeze-drying the lettuce leaves extended the estimated half-life of GUS to 69 days at 21 degrees C versus 11 days at 4 degrees C for fresh lettuce.     

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.     

Agrobacterium tumefaciens mediated transient expression of plant cell wall‐degrading enzymes in detached sunflower leaves

For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S‐driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA‐Based Overexpression Vector) systems. For 6‐day leaf incubations, codon‐optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two‐phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6‐fold. Production also increased up to 4.2‐fold when the leaf incubation temperature was elevated above the typical temperature, 20°C, to 30°C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co‐expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall‐degrading enzymes in sunflower. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:905–915, 2014     

Substrate Infiltration and Histological Fixatives Affect the In Situ Localisation of β‐Glucuronidase Activity in Transgenic Tissues

The β‐glucuronidase (GUS) gene is a widely used reporter gene in transgenic research. This study shows that although histochemical localisation of GUS activity may be very specific, differences in incubation conditions and tissue status can lead to artificial localisations that are independent of gene activity. The objective of the current studies was to evaluate the factors that affect the in‐situ localisation of β‐glucuronidase using transgenic tobacco plants as model tissues. The aspects considered include tissue size as well as and the addition of surfactants, vacuum infiltration and chemical fixatives. Transgenic tobacco plants exhibited variable staining patterns dependent on the size of tissue assayed and the treatments that affected the infiltration of substrate. A gradient of blue staining was observed in larger tissue pieces (10 mm²), where staining in central areas was light blue in contrast to edges, which stained deep indigo. More intense staining was associated with peripheral cell layers and regions adjacent to leaf veins. Thinner tissue strips incubated under similar conditions exhibited intense and even X‐Gluc staining. Addition of Triton X‐100 (1%) surfactant and vacuum infiltration (2 min) produced considerably quicker and more uniform staining (intense and consistent indigo blue colour) of the examined tissue after a 4 to 6‐h incubation. Chemical fixation of tissues before GUS assay resulted in quantitative and histochemical differences in enzyme activity that were dependent on the fixative type and duration. Quantitative measurements using the MUG fluorometric assay showed that Histochoice? provided the highest retention of GUS activity, maintaining more than 80 and 50% of the activity after fixation for 15 and 30 min, respectively. Activity in decreasing order was obtained with paraformaldehyde, glutaraldehyde, ethanol and FAA. GUS activity was affected not only by the type of fixative, but also by the duration of fixation with longer fixation producing lower GUS activity. From the experiments performed it can be concluded that those treatments that enhance substrate penetration, i.e., the addition of surfactant and vacuum infiltration, improve the consistency and speed of X‐Gluc staining.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.     

Effect of leaf incubation temperature profiles on agrobacterium tumefaciens‐mediated transient expression

Agrobacterium tumefaciens‐mediated transient expression is known to be highly dependent on incubation temperature. Compared with early studies that were conducted at constant temperature, we examined the effect of variable leaf incubation temperature on transient expression. As a model system, synthetic endoglucanase (E1) and endoxylanase (Xyn10A) genes were transiently expressed in detached whole sunflower leaves via vacuum infiltration for biofuel applications. We found that the kinetics of transient expression strongly depended on timing of the temperature change as well as leaf incubation temperature. Surprisingly, we found that high incubation temperature (27–30 °C) which is suboptimal for T‐DNA transfer, significantly enhanced transient expression if the high temperature was applied during the late phase (Day 3–6) of leaf incubation whereas incubation temperature in a range of 20–25 °C for an early phase (Day 0–2) resulted in higher production. On the basis of these results, we propose that transient expression is governed by both T‐DNA transfer and protein synthesis in plant cells that have different temperature dependent kinetics. Because the phases were separated in time and had different optimal temperatures, we were then able to develop a novel two phase optimization strategy for leaf incubation temperature. Applying the time‐varying temperature profile, we were able to increase the protein accumulation by fivefold compared with the control at a constant temperature of 20 °C. From our knowledge, this is the first report illustrating the effect of variable temperature profiling for improved transient expression. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:783–790, 2015     

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

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

Xcc-facilitated agroinfiltration of citrus leaves: a tool for rapid functional analysis of transgenes in citrus leaves

Key message

Xanthomonas citri subsp. citri pretreatment before agroinfiltration could significantly promote transient expression in citrus leaves which were previously recalcitrant to agroinfiltration.

Abstract

Transient expression via agroinfiltration is widely used in biotechnology but remains problematic in many economically important plants. Xanthomonas citri subsp. citri (Xcc)-facilitated agroinfiltration was employed to promote transient protein expression in Valencia sweet orange leaves, which are recalcitrant to agroinfiltration. However, it is unclear whether Xcc-facilitated agroinfiltration has broad application, i.e., whether Xcc-facilitated agroinfiltration could be used on other citrus varieties. In addition, we intended to investigate whether Xcc-facilitated agroinfiltration could be used to hasten transgene function assays, e.g., Cre/lox system and Cas9/sgRNA system. In this report, Xcc-facilitated agroinfiltration was further exploited to enhance β-glucuronidase (GUS) expression in five citrus varieties. Xcc-facilitated agroinfiltration also significantly increased GFP expression in six citrus varieties tested. Both GUS and GFP assays indicated that Xcc-facilitated agroinfiltration had the best performance in grapefruit. After Xcc-facilitated agroinfiltration was carried out in grapefruit, protoplast analysis of the transformed cells indicated that there were more than 20 % leaf cells expressing GFP. In grapefruit, usefulness of Xcc-facilitated agroinfiltration was assayed in three case studies: (1) fast functional analysis of Cre/lox system, (2) the heat shock regulation of HSP70B promoter derived from Arabidopsis, and (3) Cas9/sgRNA-mediated genome modification.     

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis

The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.     

Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions

Transient expression of recombinant proteins in plant tissues following Agrobacterium‐mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub‐optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10⁸, 10⁹, and 10¹⁰ CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break‐Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10⁸, 10⁹, and 10¹⁰ CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1137–1144, 2014     

Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler

The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk. Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed by the GUS assay and PCR analysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ethylene production and respiration in aging leaf segments and in disks of fruit tissue of normal and mutant tomatoes

Leaf segments of tomato plants (Lycopersicon esculentum Mill.) of a normal strain and of two nonripening mutants rin and nor were aged in darkness. Respiration in leaf segments of all strains followed a climacteric-like pattern which was accompanied by a similar pattern of ethylene production. l-Methionine-U-(14)C vacuum-infiltrated into leaf segments at the beginning of the climacteric-like rise in respiration was metabolized to ethylene and CO(2) during the subsequent 48 hours to about the same extent in all strains. Pericarp disks of immature fruits of all strains also metabolized l-methionine-U-(14)C to ethylene and CO(2) to about the same extent during the first 48 hours following cutting and vacuum infiltration. Conversion of methionine to ethylene in disks was much more efficient than in aging leaf segments. The apparent capacity for increased production of ethylene in aging leaf segments and in response to wounding in pericap disks of rin and nor is contrasted with the absence of a respiratory climacteric and an associated large increase in ethylene production during natural aging of intact fruits of these two strains.     

抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响

大豆(Glycine max)下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hpt II(Hygromycin phosphotransferase II)筛选基因的农杆菌(Agrobacterium tumefaciens) LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for β-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.     

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

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

Arabidopsis 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.     

Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco

We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a -glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.