Screening for transgenic plant cells that highly express a target gene from genetically mixed cells

Biochemical Engineering Journal 10 (2002) 175–182

Screening for transgenic plant cells that highly express a target gene from genetically mixed cells Hideo Akashi a , Hiroyuki Kurata b,∗ , Minoru Seki a , Kazunari Taira a,c , Shintaro Furusaki d a b

Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan Department of Biochemical Engineering and Science, Kyushu Institute of Technology, 980-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan c Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan d Department of Applied Life Science, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan Received 14 July 2001; accepted after revision 14 November 2001

Abstract To establish a strategy for stably and highly expressing a target gene in transformed plant cell suspensions, we developed the co-selection method that linked the hygromycin phosphotransferase gene (hph) to the ␤-glucuronidase (uidA, GUS) gene in the opposite direction under the same transcriptional regulation of the cauliflower mosaic virus (CaMV) 35S promoter. The linked genes were transferred into a tobacco BY-2 cell suspension, which was cultivated under various levels of the antibiotic pressure. Under the high pressure of hygromycin, GUS expression was increased and maintained over 1.5 years. We presented a successful example for selecting the plant cell suspension that highly expressed the target gene out of genetically heterogeneous cells. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Enzyme production; Genetic stability; Plant cell culture; Recombinant DNA; Tobacco BY-2; Co-amplification

1. Introduction The production of secondary metabolites, foreign proteins including enzymes, antibodies and antibody fragments, or vaccines in transgenic plant cell suspensions offers broad opportunities for the development of useful products [1,2]. Gene manipulation technology is a powerful means for enhancing the production of biologically active compounds, which are generally synthesized in plant cells through multiple catalytic steps requiring enzymes encoded by a set of genes. However, there have been few commercially successful examples of useful material production by transgenic plant cell suspension culture. One of the major problems is that it is quite difficult to significantly enhance the expression level of a target gene and stabilize its expression during long-term cultivation. Genetically homogeneous plant cell line with a foreign gene has been employed to obtain highly expressing cell line [3,4], but it has been often difficult to maintain a genetically unique transformed line of a plant cell suspension because the expression of the transferred gene can be unstable during long-term cultivation [5–11]. In other words, the plant cell ∗ Corresponding author. Tel.: +81-948-29-7828; fax: +81-948-29-7828. E-mail address: [email protected] (H. Kurata).

suspension transformed with a foreign gene is presumed to change into a mixture of genetically heterogeneous cells with time. From a technological standpoint, obtaining the cell line that achieves high expression of a foreign gene can be accomplished either by giving selection pressure to the heterogenous population, or by clonal selection of the highly expressing cell line. The gene amplification technique, which amplifies mRNA transcription and protein production by increasing the copy number of an endogenous gene with inter- or intra-molecular homologous recombination, is one of the most successful methods to overexpress specific endogenous genes in mammalian, Drosophila or yeast cells [12,13]. In plant cells, the gene amplification method was also demonstrated to increase the expression of a target such as glutamine synthetase [14], 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) [15–23] and acetohydroxyacid synthase [24–27]. In mammalian cells several thousands of gene copy number have been observed in gene amplification experiments. In contrast, the increase in the copy number was limited below a few dozen copies in plant cells [15,16,24]. The cell lines established by the gene amplification method were relatively stable and the observed half lives of the expression level of amplified genes were as long as several years even in the absence of selective agent [15–17].

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Regenerated plantlets from the cell suspension containing the amplified EPSPS genes were maintained for years [18]. However, this method was less effective for amplification of a specific gene of interest in a plant cell suspension. To obtain the cells that highly expressed foreign genes, the co-amplification system has been developed and studied extensively in mammalian cells [28]. By linking a target gene to a drug resistance gene, the transformed cells that highly expressed the target gene were expected to show high resistance to the antibiotic, because antibiotic pressure suppressed the growth of the cells whose expression level of the antibiotic resistance gene was low. However, there have been few reports that the co-amplification system in a plant cell suspension remarkably enhanced or maintained foreign gene expression [29]. Strong correlation between the expressions of the linked genes is required for effective selection by the co-amplification method; however, the variability in the expression level of linked transgenes is commonly observed in plant transformation experiments. Even two genes positioned in the same T-DNA region in a binary vector did not express co-ordinately [30–32]. No correlation was observed between the activities of linked neomycin phosphotransferase and chloramphenicol acetyltransferase activities when two genes were tandemly linked under the identical transcriptional control of nopaline synthase (NOS) promoters, where the NOS promoters were about 4 kb distant from each other [30]. In contrast, the expressions of two genes, which were closely placed to each other (0.1 or 1.3 kb) and were divergently transcribed, were well co-ordinated [33,34]. The distance between two linked genes and the orientation of transcription is suggested to be key factors for co-ordinated expression. In this study, we demonstrated that the cell suspension that highly expressed a target gene could be obtained from a mixture of genetically heterogeneous cells. We linked the hygromycin phosphotransferase gene (hph) and the ␤-glucuronidase (uidA, GUS) gene in the opposite direction

under the same transcriptional regulation of the cauliflower mosaic virus (CaMV) 35S promoter and the NOS terminator, transferring the linked genes into a tobacco BY-2 cell suspension, and cultivated the transformed BY-2 under various levels of the antibiotic pressure. The distance between the sites for initiation of transcription of the two 35S promoters was adjusted to be 1.2 kb, which was expected to be well co-ordinated expression. Under the high concentration of hygromycin, the expression of uidA gene was several-fold enhanced during the cultivation period of 1.5 years.

2. Materials and methods 2.1. Plant cells and culture conditions Suspensions of the tobacco Nicotiana tabacum L. cv Bright Yellow 2 (BY-2) cells were grown in the modified MS medium [35] containing 4.3 g/l MS salt, 0.2 mg/l 2,4-dichlorophenoxyacetic acid, 0.2 g/l KH2 PO4 , 0.1 g/l myo-inositol, 1 mg/l thiamine-HCl and 30 g/l sucrose, which was designated MSD medium. The cells were cultured at 27 ◦ C in a reciprocating shaker at 100 spm in darkness. The tobacco BY-2 suspension was subcultured for 7 days by transferring 1 ml of the cell suspension into 50 ml of fresh MSD medium. 2.2. Construction of plasmids Two binary vectors used for establishment of the stable transformants of BY-2 cells were constructed as follows. The unique EcoR I site of CaMV35S-sGFP(S65T)-nos3 (kindly provided by Dr. Y. Niwa, University of Shizuoka) was converted into a Hind III site by inserting a Hind III linker. The resultant Hind III fragment was inserted into the Hind III site of pBI121 [36], which was a binary plasmid vector for plant transformation, to produce pBI121GFP (Fig. 1a). An hph gene from pHTS14 (kindly provided by Dr. H. Uchimiya,

Fig. 1. Schematic representation of the binary plasmid vectors for plant transformation: (a) pBI121GFP was used to determine quantitative correlationship between the expression of the linked uidA gene and GFP gene; (b) pBI121hph harboring the uidA gene and hph gene was used for the co-selection experiments. 35S, cauliflower mosaic virus 35S promoter; NOS-ter, nopaline synthase terminator; NPT II, neomycin phosphotransferase gene that confers the kanamycin resistance in plant cells; RB and LB, right- and left-border of the Agrobacterium Ti plasmid.

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The University of Tokyo) was inserted into the Hind III site of pBI121 to produce pBI121hph (Fig. 1b). The plasmid vector pBI121hph carried the hph gene that conferred antibiotic hygromycin B resistance and the uidA gene as a reporter gene. 2.3. Transformation Agrobacterium tumefaciens strain C58 (pGV2260) was cultured in medium 523 [37] containing 25 ␮g/ml of rifampicin. Transformation of BY-2 cell suspensions were carried out as described by An [38]. The binary vector, pBI121hph or pBI121GFP, was transferred into A. tumefaciens by electroporation. Four milliliters of 4-day cultured BY-2 cells were mixed with 100 ␮l of a fresh overnight culture of A. tumefaciens C58 containing the binary vector. After 48 h incubation at 27 ◦ C in darkness, the bacterial cells were washed off with sterile liquid MSD, and the transformed calli were selected on MSD agar containing appropriate antibiotics. Hygromycin B of 50 ␮g/ml was used for selecting transformants with pBI121hph, kanamycin of 100 ␮g/ml with pBI121GFP. Cefotaxime of 300 ␮g/ml was also added to the MSD agar medium to kill the remaining A. tumefaciens. After 3-week incubation, more than 100 independent antibiotic-resistant calli were visible on a plate. 2.4. GUS fluorometric assay Approximately 100 mg of BY-2 cell suspension was harvested by centrifugation for a few seconds in an Eppendorf tube and stored at −70 ◦ C until further analysis. GUS activity was measured as described elsewhere [3,4,36]. The cells were resuspended in 100 ␮l of ice-cold GUS extraction buffer (50 mM NaPO4 , 10 mM ␤-mercaptoethanol, 10 mM Na2 EDTA, 0.1% sodium N-lauroylsarcosine, 0.1% Triton X-100). Crude extract was prepared by homogenizing with a pellet mixer for 90 s and by centrifugation at 14,000 rpm for 10 min. Protein concentration was measured using the 20 ␮l of extract according to the Bradford method (Protein Assay Kit; Bio-Rad, Hercules, CA, USA). The cell extract of 50 ␮l was incubated in a reaction volume of 500 ␮l GUS extraction buffer containing 1 mM 4-methyl-umberlliferyl-␤-d-glucuronide (4-MUG) for 30 min at 37 ◦ C. To stop the reaction, the 100 ␮l reaction mixture was added to 2 ml of 0.2 M Na2 CO3 . The fluorescence was measured using a spectrofluorometer (FP-777; JASCO, Tokyo) at 448 nm with excitation at 370 nm, and calculated into GUS specific activity by using 4-methyl-umbelliferone (4-MU) as standard. Triplicate assays were carried out in most of the experiments. 2.5. GFP fluorometric assay BY-2 extracts from the cells transformed with pBI121GFP (Fig. 1a) were used for GFP assay. Protein concentration in the extracts was determined by the Bradford method. The

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GUS extraction buffer was added to the BY-2 cell extract containing 100 ␮g of total protein to yield the final volume of 250 ␮l. The GFP fluorescence was measured in a 5 mm × 5 mm cuvette using a spectrofluorometer at 511 nm with excitation at 488 nm [39]. The fluorescence was converted into the amount of GFP per 100 ␮g of total protein. The calibration curve of GFP was prepared by measuring the fluorescence of recombinant GFP protein (CLONTECH, Palo Alto, CA, USA) in nontransformed BY-2 extract containing 100 ␮g of total protein. The calibration curve for the fluorescence and the amount of GFP protein was linearly related over the range from 1 to 1000 ng, showing the R2 value was 0.995 (data not shown). At least two extracts were assayed for each data. 2.6. Biological assay of hygromycin B To estimate the hygromycin concentration in the medium, biological assay was carried out, measuring the quantitative relationships between the growth of bacteria and the hygromycin concentration. In detail, approximately 5 × 105 cells of Escherichia coli strain DH5␣ were prepared by measuring an optical density at 550 nm. The bacterial cells were incubated in the mixture of 2 ml of LB medium, and 2 ml of MSD medium or suspension culture containing hygromycin. After incubation, the optical density was measured at 550 nm to estimate the concentration of bacterial cells.

3. Results 3.1. Quantitative correlation of linked genes We expected that the expressions of the linked genes would be correlated. This was confirmed by the quantitative relationship between the expression of the linked two reporter genes, the uidA gene and GFP gene, in the binary plasmid vector pBI121GFP (Fig. 1a). The plasmid pBI121GFP was transferred into BY-2 cells through A. tumefaciens to obtain stable transformants. Following transformation, 47 of independent antibiotic-resistant calli were picked up and transferred to the MSD agar medium containing 100 ␮g/ml of kanamycin and 300 ␮g/ml of cefotaxime. After the calli were sufficiently grown on the plate, the transformed BY-2 calli were transferred into 50 ml of MSD liquid medium without antibiotics and cultured for at least 10 days. Then, approximately 100 mg of the plant cells were sampled for assaying the GUS and GFP activities. To eliminate false positives, out of the 47 independent transformants, seventeen whose GUS specific activity was under 30 pmol/min mg protein were excluded, because the GUS activity of nontransformed BY-2 was no more than 30 pmol/min mg protein (data not shown). Indeed, GFP fluorescence could not be detected in 15 false positives, though unexpectedly two exhibited very strong fluorescence corresponding to 168 and 308 ng GFP per 100 ␮g total protein

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Fig. 2. The relationship between the expressions of linked genes in BY-2 cells. Tobacco BY-2 cells were transformed with pBI121GFP (Fig. 1a) through Agrobacterium-mediated transformation method. At least duplicate experiments were carried out to show each data point and the average of the GUS and GFP activity was plotted.

Fig. 3. Schematic representation of the co-selection experiment. All the transformed BY-2 calli harboring pBI121hph in a plate were suspended in MSD liquid medium in a single flask. Then the cell suspension was divided into three lines, each of which was subcultured independently with stepwise increased hygromycin B. The number after the letters “Hm” indicates the final concentration of hygromycin in ␮g/ml in the culture medium.

(data not shown), probably because a complex homologous recombination occurred. As shown in Fig. 2, the GUS and GFP activities of 30 transformed calli showed a linear correlation, indicating that the expression level of the linked genes was quantitatively co-ordinated.

concentration of hygromycin was required because the growth of transformants were completely stopped by a high dose at the initial phase (data not shown), as observed by others [16,24]. In these initial stages, longer growth periods of 7–15 days and the large inoculum size of 5 ml were required to grow the cells for subculture, probably because the proportion of highly resistant cells in population was low.

3.2. Establishment of transgenic cell suspensions

3.3. Amplification of uidA gene expression

To examine the quantitative effect of co-selection in our system, we constructed the binary plasmid vector pBI121hph, in which the hygromycin resistance gene and the GUS gene were linked (Fig. 1b). Tobacco BY-2 cells were transformed through A. tumefaciens containing pBI121hph. More than 100 independent hygromycin-resistant transformants appeared as aggregates on MSD agar. All the transformed calli with pBI121hph in a plate were mixed and cultured in a 50 ml MSD liquid medium containing 50 ␮g/ml of hygromycin and 300 ␮g/ml of cefotaxime (Fig. 3). This genetically heterogeneous cell suspension was divided into three cell lines, which were designated as Hm0, Hm100 and Hm500, respectively. In Hm0, the cells were cultivated without hygromycin. In Hm100 and Hm500, the concentration of hygromycin B was increased stepwise to obtain higher antibiotic-resistant cells as follows. In Hm100, the hygromycin concentration was gradually increased from 50 to 100 ␮g/ml over the initial four batch cultures. In Hm500, the hygromycin concentration was stepwise increased from 50 to 500 ␮g/ml over the initial six batch cultures and then maintained at 500 ␮g/ml. Stepwise increase in the

Fig. 4 shows the time courses of the GUS specific activity of Hm0, Hm100 and Hm500, which were measured over 1.5 years. The GUS specific activity of cells without hygromycin, Hm0, was gradually decreased after the seventh batch culture, reducing to that of nontransformed cells at the 13th batch culture. Hm100 obtained the resistance to the 100 ␮g/ml of hygromycin at the fourth batch culture and the growth rate was almost the same as that of Hm0 after the sixth batch culture. The GUS specific activity of Hm100 increased initially, but it decreased after the 13th batch culture and dropped to that of nontransformed cells at the 20th batch. Hm500 acquired the resistance to 500 ␮g/ml of hygromycin at the sixth batch culture and grew well after the 14th batch, although the growth was reduced by 20% compared with that of Hm0. The GUS specific activity of Hm500 gradually increased and was three-fold enhanced at the 40th batch culture. Expression of the uidA gene in Hm500 was maintained at a high level during the entire cultivation period for 1.5 years. To evaluate the effect of hygromycin pressure on cell viability, trypan blue (0.01%) was added to the cells and

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Fig. 4. Time courses of the GUS specific activity during a long-term culture. The BY-2 cell suspensions transformed with pBI121hph were cultured with various amounts of hygromycin concentration. Each cell line was initiated from the same culture and adapted to stepwise increased hygromycin over the first six subcultures. After the sixth batch culture, the concentration of Hm0, Hm100, and Hm500 was maintained at 0, 100, and 500 ␮g/ml, respectively. Triplicate GUS assay experiments were carried out for each data point and the average of the GUS activity was plotted.

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Fig. 5. Inability to restore the lost GUS activity by the high concentration of hygromycin. The first batch culture of Hm100 and Hm0 were subcultured from the 22nd batch culture of Hm100 and Hm0 in Fig. 4, respectively. The concentration of hygromycin in each culture was increased stepwise to the final concentration of 500 ␮g/ml. Triplicate GUS assay experiments were carried out for each data point and the average of the GUS activity was plotted.

3.4. Hygromycin concentration during a batch culture

the blue-stained cells were counted by a microscope. For analysis, we used the cells from the 14th batch culture of Hm0 and Hm500, whose GUS specific activity were 0.2 and 3.0 nmol/min mg protein, respectively. No trypan blue stained cell was observed in Hm0, whereas 10% of Hm500 cells were stained (data not shown). Thus, the antibiotic pressure with 500 ␮g/ml of hygromycin appeared to work well. To measure the population of uidA-expressing cells, GUS histochemical staining assay was performed with X-gluc as a substrate, using the cells from the 14th batch culture of Hm0 and Hm500. The uidA gene expression was detected in approximately 50% of Hm500 cells, but only 1% of Hm0 cells (data not shown). The high GUS activity of Hm500 was found to be caused at least by the large population of highly expressing cells. After the 22nd batch culture, the GUS specific activities of Hm0 and Hm100 were reduced to that of nontransformed cells. In order to investigate whether the GUS specific activity of Hm0 or Hm100 was restored, Hm0 and Hm100 at the 22nd batch culture were transferred to the medium, where the concentrations of hygromycin were stepwise increased to 500 ␮g/ml over the additional five batch cultures (Fig. 5). Neither Hm0 nor Hm100 restored GUS activity within additional 17 batch cultures, although both grew well with 500 ␮g/ml of hygromycin B.

In Hm100, the cell suspensions with high GUS specific activity were not selected. We supposed that the hygromycin activity decreased in medium during a batch culture as the reason for lack of selection. To clarify if hygromycin was effective within the entire period of a batch culture, biological assay was performed using E. coli strain DH5␣. The equal volume of LB medium was added to the test medium, because no growth of bacteria was observed in the MSD medium. The growth of DH5␣ in the mixed medium was strongly related to the concentration of hygromycin B (Fig. 6). Although the growth curve as shown in Fig. 6 depended on the experiments, the cell growth was reproducibly decreased with the increase in the concentration of hygromycin. This biological assay was sensitive enough to detect the difference of 10 ␮g/ml of hygromycin in medium by measuring the cell growth after appropriate period of incubation (Fig. 6 and data not shown). The relative growth of E. coli in the culture medium of Hm100 or Hm500 was compared with that in the medium containing various concentrations of hygromycin. Table 1 indicates the time course of the concentrations of hygromycin estimated. The hygromycin concentration of Hm100 did not drop below 50 ␮g/ml in 10-day incubation. The initial concentration of 100 ␮g/ml would be effective enough to suppress cell growth during each batch culture, because 50 ␮g/ml of hygromycin completely inhibited the growth of nontransformed BY-2 (data not shown).

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were under the identical transcription machinery (Fig. 1). Two 35S promoters, one of which derived from pBI221 and the other pHTS14 (see Section 2), were used to place a spacer region of approximately 500 bp between the palindromic sequences of 35S promoters to be amplified in E. coli [50–53]. 4.2. Correlationship of the linked genes

Fig. 6. Growth curve of E. coli DH5␣ with various concentrations of hygromycin. Growth suppression of DH5␣ was strongly related to hygromycin concentration in the culture medium.

Table 1 The time course of the concentration of hygromycin B during a batch culture Day

Hm100 (␮g/ml)

Hm500 (␮g/ml)

1 7 10

100 50–100 50–100

>200 >200 >200

4. Discussion 4.1. Strategy for selecting plant cells In a plant cell suspension, the level of target gene expression, which is determined by the balance among several factors, depends on each transformed cell in long-term cultivation. The inserted genes are often inactivated by genetic modification such as methylation [5,40–44] or co-suppression [43–46]. The expression level is also affected by the position at which the insert is integrated (positioning effect), since the surrounding chromatin structure, which determines the accessibility of transcriptional machinery to the inserted gene, differs with each insertion. Actually the expression level of the introduced gene in each independent callus was expected to vary by three orders of magnitude, as observed elsewhere [2,30]. Thus, we tried to select BY-2 cell suspensions that highly expressed the target gene out of genetically heterogeneous BY-2. The CaMV 35S promoter has several cis-acting enhancer domain [47], and works in the orientation and distance dependent manner [48,49]. We developed the co-selection system with two 35S promoters fused to each other in a head to head manner so that the two linked genes

To measure the relationship between the linked genes, we used two reporter genes, GUS and GFP. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been a widely used marker for gene expression and protein localization studies [54–56]. Wild type GFP gene was modified by removing cryptic intron and adding optimal codon usage for human to produce sGFP(S65T), which expressed one hundred times brighter in plant cells [39]. Though wild type GFP could not be assayed quantitatively by a fluorometer due to its faint emission signal [57], the modified GFP has been recently applied to quantitative fluorometric assay [58–61]. Thus, we used the modified GFP. At least in the earlier stage after transformation, we observed well-coordinated expression of the linked GUS gene and GFP gene (Fig. 2). 4.3. The possible mechanisms for co-selection We suppose two possible mechanisms for our co-selection system; exclusion of the lowly expressing cells by strong antibiotic pressure and co-amplification by the increased gene copy number due to homologous recombination. The frequent homologous recombination, which is often observed in plant cell suspensions, would lead to the generation of lowly expressing cells. Therefore, the population of highly expressing cells would be formed only when lowly expressing cells are selectively excluded. It was recently reported that the palindromic sequence of promoter region caused the methylation of promoter sequence in a sequence specific manner, and caused gene silencing at transcriptional level [62]. Therefore, the palindromic structure of two 35S promoters (Fig. 1b) would cause inactivation of both the uidA and hph gene if aberrant recombination occurred. In our system, these silenced cells would efficiently be excluded under the strong antibiotic pressure. As a result, we observed the amplification of GUS enzyme activity (Fig. 4, Hm500), indicating the highly GUS-expressing cells were selected and propagated dominantly in our selection system by 500 ␮g/ml of the hygromycin. 4.4. Conditions required for selecting highly expressing cells in a long-term culture In this study, 100 ␮g/ml of the hygromycin concentration was not sufficient as a selection pressure for the co-introduced uidA gene (Fig. 4). The similar decrease in GUS activity as in Hm100 or Hm0 shown in Fig. 4 was also

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observed in pearl millet callus [5], transgenic rice calli [6], tall fescue suspension [7], ryegrass suspension [8], and rice cell suspensions [9]. The selectable gene appears to express preferably than nonselectable gene [10]. To continuously exclude cells of which T-DNA region was recombined to inactivate GUS, a consecutive selection system, or high concentration of selection pressure, was needed (Fig. 4, compare Hm500 with Hm100). However, once the GUS activity was lost, the activity was not restored by high pressure of hygromycin B (Fig. 5), although the cells showed the resistance to the high concentration of hygromycin B and grew well (data not shown). Strong correlation of linked genes in the initial transformed calli would be lost in the early period of cultivation. In contrast, the cells maintained in the presence of hygromycin appeared to acquire the higher resistance with time. In order to obtain the cells that highly express a target gene, both early stage selection and strong selective pressure may be required for co-selection in a long-term culture of plant cells.

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