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Transgenic plants from fragmented shoot tips of apple (Malus baccata (L.) Borkhausen) via agrobacterium-mediated transformation
Scientia Horticulturae 128 (2011) 450–456
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Transgenic plants from fragmented shoot tips of apple (Malus baccata (L.) Borkhausen) via agrobacterium-mediated transformation Yongjie Wu a , Yunhe Li b , Yaqin Wu a , Hehe Cheng a , Yin Li c , Yanhua Zhao a , Yusheng Li a,∗ a b c
Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli Town 066600, China South Subtropical Crop Research Institute, Chinese Academy of Tropical Agricultural Science, Zhanjiang 524091, China School of Life Sciences, Sun Yat-sen University, No. 135, West Xin-gang Road, Guangzhou 510275, China
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 28 January 2011 Accepted 19 February 2011 Keywords: Apple Agrobacterium tumefaciens Fragmented shoot tips GFP Transformation
a b s t r a c t Transgenic apple (Malus baccata (L.) Borkhausen) plants were obtained via Agrobaterium-mediated transformation of fragmented shoot tips. Our results showed that without wounding treatment or with wounding treatment (II, cutting shoot tips vertically into two parts), shoots generally regenerated from meristem tissues directly and adventitious shoot regeneration was rarely observed. Otherwise, when shoot tips were cut vertically into four parts, a high percentage of callus formation (89.2%) and of adventitious shoot regeneration (60.8%) was observed. Under 20 mg l−1 kanamycin selection pressure, over 51.7% fragmented shoot tips developed callus and seven transgenic plants with GFP (Green fluorescent protein) expression were obtained from about 120 explants (efficiency of 5.8%). No transgenic plant was obtained from agrobacteria mediate transformed leaves, even though 23.2% of which formed callus after co-cultivation and selection. Molecular analysis (PCR and RT-PCR) of the transformed lines with GFP expression confirmed integration and transcription of the transgene. Under fluorescence microscopy, areas with high density of transgenic cells were observed at the cutting edges of fragmented shoot tips, which indicated that shoot regeneration from transgenic cells should be a major factor inhibiting transformation efficiency. Our experiments also showed that with moderate or low selection pressure, transgenic shoots were obtained generally accompanied by a high numbers of chimeric shoots. While by using fluorescence microscopy observation of GFP expression, the transgenic and chimeric shoots could be detected and separated precisely for further transgenic plats regeneration or multiplication. This may be very useful for apple genetic breeding, as large numbers of transgenic plants could be obtained in a short time. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Many commercial or new apple cultivars with unique quality traits are generally nonadaptable to varied plant conditions or accompanied by undesirable deleterious characteristics. Thus further improvement of target traits is necessary. However, introduction or deletion of target genes by means of conventional hybridization is generally costly, of low efficiency and a long-term process because of the high heterozygocity and long juvenile period of the apple plants. Transformation technologies offer promising tools to modify established cultivars by introducing one or a few traits without altering their existing genetic constitution.
Abbreviations: AS, acetosyringone; BA, 6-benzyladenine; GFP, green fluorescent protein; NAA, a-naphthaleneacetic acid; TDZ, thidiazuron. ∗ Corresponding author. Fax: +86 335 2023417. E-mail address: showersound [email protected] (Y. Li). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.02.013
Since the first report of transgenic apple plants (James et al., 1989), significant progress has been made on apple transformation. Cultivars such as Delicious (Sriskandarjah et al., 1994), Royal Gala (Yao et al., 1995), Golden Delicious (Puite and Schaart, 1996), Marshall McIntosh (Bolar et al., 1998), McIntosh Wijcik (Song et al., 2000), Elstar (Szankowski et al., 2003), Fuji (Seong et al., 2005), Jonagold (De Bondt et al., 1996), Orin (Kanamaru et al., 2004), as well as the rootstock M26, M9 and M29 (Lambert and Tepfer, 1992; Maheswaran et al., 1992; Zhu et al., 2001) have been transformed with different functional genes. Although Agrobacterium-mediated transformation has been recognized as the preferred system for transforming apple plants, the transformation efficiency appears to be low and highly genotype-dependent (James and Dandekar, 1991; Trifonova and Atanassov, 1998; Hanke, 2004), especially for cultivars which are recalcitrant to shoot regeneration or sensitive to antibiotic selection. A number of factors affect the efficiency of Agrobacteriummediated apple transformation. Among which, shoot regeneration ability is a crucial prerequisite for successful transformation.
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Because of the high level of tolerance to drought and low temperature, Malus baccata (L.) Borkhausen, is a traditional and main apple rootstock in the north of China. However the M. baccata (L.) Borkhausen plants are too vigorous to reduce grafted tree size for high yield production. Thus there is a great interest to obtain dwarf rootstock by means of genetic transformation. Preliminary results showed that shoot regeneration from leaves of M. baccata (L.) Borkhausen is low and very sensitive to antibiotic selection (Wang, 2005) and thus it is better to select other suitable materials for shoot regeneration and transformation. Besides the young leaves (Dandekar et al., 1990), internodal explants from etiolated apple shoots (Liu et al., 1998) have been used to improve shoot regeneration ability. Results of these study indicated that the closer to the apical meristem, the higher the regeneration ability. In fact, fragmented tissues of grape and banana shoot apices have been used for rapid multiplication of plant material (Barlass and Skene, 1980; Salami et al., 2005; Mbanaso et al., 2006). Recently Dutt et al. (2007) established a stable transformation protocol using fragmented grape shoot tips and confirmed that wounded shoot tips co-cultivated with agrobacteria could reconstruct complete meristems and ultimately produce non-chimeric transgenic plants. This implies that cells of apical meristems could be an alternative material for apple transformation. The aims of this research were: (1) to study the shoot regeneration ability of fragmented apple shoot tips and the effect of wounding treatments; (2) to establish a genetic transformation of apple (M. baccata (L.) Borkhausen) using fragmented shoot tips; and (3) to confirm the target gene transformation and expression by fluorescence observation and polymerase chain reaction (PCR) or RT-PCR. 2. Materials and methods 2.1. Plant materials Apple (M. baccata (L.) Borkhausen) plantlets were established from field-grown plants and multiplied in vitro for 2 years before the transformation experiments. Shoots were maintained by subculturing every 4–5 weeks on proliferation medium containing MS basal medium (Murashige and Skoog, 1962) 3% (w/v) sucrose and 0.7% (w/v) plant agar, pH 5.8) supplemented with 2.0 M BA and 0.6 M NAA at 25 ± 2 ◦ C with a 16 h photoperiod (supplied by fluorescent cool-white lamps at an intensity of 40 mol m−2 s−1 ). Shoot tips approximately 1 cm in length, composed of the apical meristem young leaves and a short basal stem, were harvested from 3to 4-week-old micropropagated shoots and transferred on proliferation medium. Etiolation was promoted by placing shoots in the dark for 2 weeks according to Liu et al. (1998). In experiment 2, the three youngest top leaves from 3- to 4-week-old micropropagated shoots were dissected and scored transversally two-to-four times (depending on the size of the leaf) with a scalpel for transformation.
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medium (MS basal medium supplemented with 12.0 M BA and 0.6 M NAA) and cultured for 4 weeks in the dark at 25 ± 2 ◦ C. The percentage of explants that formed callus was recorded and the explants were then transferred on the same fresh medium for shoot regeneration in the light. The number of regenerated shoots were counted and the percentage of explants that formed shoots were determined after 4–5 weeks culture in the light.
2.3. Plant regeneration from fragmented shoot tips For plant regeneration, fragmented shoot tips with different wounding treatments were transferred on regeneration medium (MS basal medium supplemented with 0.6 M NAA and 4.0, 12.0 or 20.0 M BA, or with 10.0 M TDZ and 0.6 M NAA) and cultured for about 4 weeks in the dark at 25 ± 2 ◦ C. The percentage of explants that formed callus was recorded and the explants were then transferred on the same fresh medium for shoot regeneration in the light. The number of regenerated shoots were counted and the percentage of explants that formed shoots were determined after 4–5 weeks culture in the light.
2.4. Agrobacterium-mediated transformation Agrobacterium-mediated transformation was conducted as previously described by James et al. (1993) and Norelli et al. (1996). Agrobacterium strain EHA105 harbouring the modified binary vector, pCAMBIA-1302, that incorporates the GFP reporter gene and the kanamycin resistance gene (npt II) for plant selection was used for transformation experiments Fragmented shoot tips (with wounding treatment III) or scored leaves were inoculated with agrobacteria that had been grown overnight in yeast extract broth (YEB) medium (Van Larebeke et al., 1977), centrifuged and resuspended in liquid MS medium (pH 5.2) to an OD600 of 0.8 for 10 min. The explants were then blotted and transferred to co-culture medium (MS basal medium supplemented with 12.0 M BA, 0.6 M NAA and 100 M AS). After co-cultivation for three days with Agrobacterium in the dark, they were placed on selection medium (MS basal medium supplemented with 12.0 M BA and 0.6 M NAA for fragmented shoot tips or 10.0 M TDZ and 0.6 M NAA for leaves) containing 200 mg l−1 cefotaxime and 20 mg l−1 kanamycin). The explants were transferred to fresh selection medium every 4–5 weeks. In experiment 4, the influence of kanamycin selection at 10 mg l−1 on transgenic shoot regeneration of fragmented shoot tips were observed. The influence of kanamycin selection on the proliferation of transformed cells was determined 4 weeks after co-cultivation by measuring the number of calli expressing GFP. The regenerated shoots were observed under a fluorescence microscope and GFP-expressing shoots were counted before excision and further transferred to proliferation medium (MS basal medium supplemented with 2.0 M BA, 0.6 M NAA, 200 mg l−1 cefotaxime and 20 mg l−1 kanamycin) in the light.
2.2. Wounding treatments prior to genetic transformation Etiolated shoot tips were further dissected to obtain 1–3 mm long explants, composed of the apical meristem with a few leaf primordia. Explants were placed on a Petri dish and subjected to the following fragmentation treatments: (I) no wounding; (II) cut vertically into two equal portions; (III) cut vertically into four equal portions; or (IV) cut into small pieces (approx 6–10 portions depending on the size of shoot tips). In experiment 1, the effect of different wounding treatments on callus formation and shoot regeneration were compared. The fragmented shoot tips with different wounding treatments were transferred on regeneration
2.5. GFP expression observation The fluorescent images were observed using an Olympus BX51 microscope equipped with an epifluorescence attachment, a 100 W mercury light source, and an Olympus DP70 digital microscope camera. A 450–490 nm excitation filter was used with a 520–560 mm emission filter that transmitted only green light. The regenerated calli were observed under the fluorescence microscope and those showing GFP fluorescence in all cells were measured and marked as transgenic calli.
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Table 1 Effect of wounding treatments on callus formation and shoot regeneration from fragmented shoot tips. Fragment treatments
Percent of explant forming calli
Percent of explant regenerated shoot
I II III IIII
1.7 ± 2.9c 48.3 ± 10.4b 89.2 ± 7.6a 100a
100 78.3 60.8 20.8
± ± ± ±
No. of shoots per explant
0a 8.8b 12.6b 10.4c
1.0 1.2 2.3 1.6
± ± ± ±
0c 0.04c 0.06a 0.27b
Data are presented as means ± S.E. The figures followed by different letters in each column differ significantly at P ≤ 0.05.
2.6. Statistic analyses Each treatment was replicated three times and a replicate consisted of 20 shoot tips (wounding treatments I and II) or of 40 fragmented shoot tip explants (wounding treatments III and IIII). The data are presented as means ± S.E. and were subjected to statistical analysis (ANOVA and Duncan’s Multiple Range Test) using the SPSS 13.0 for windows software (SPSS Inc., Chicago, IL, USA).
Regeneration percent (%)
120
60 40
3.1. Effect of wounding treatments on callus formation and shoot regeneration from fragmented shoot tips Significant differences were observed on the callus formation and shoot regeneration from fragmented shoot tips with different wounding treatments (Table 1). Without wounding treatment (treatment I) or with wounding treatment II, only 1.7% or 48.3% fragmented shoot tips formed callus after about 4 weeks of culture on the regeneration medium. While, a much higher percentage of callus formation (89.2% or 100%) was observed on the fragmented shoot tips with wounding treatments III or IV. Though shoots regeneration on the fragmented shoot tips with wounding treatments I and II was significant different (100 and 78.3%, respectively), these shoots were generally regenerated from the meristematic tissue directly and few adventitious shoots were observed. Compared to that, about 20.8% or 60.8% fragmented shoot
d
d
b a
20 0
3. Results
c
80
2.7. PCR and RT-PCR analysis Genomic DNA was extracted from transgenic and nontransgenic apple leaves, using the cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1990). PCR amplification of the gfp gene was carried out using the forward primer (GFP-S1), (5 TAG ATG GTG ATG TTA ATG GGC 3 ) and the reverse primer (GFP-SA1), (5 GCT GTT ACA AAC TCA AGA AGG 3 ). Total RNA was isolated from transgenic and nontransgenic apple leaves using TRNzol Total RNA Reagent (Tiangen Biotech Co., Beijing, China) according to the manufacturer’s instructions. RNA samples were treated with Dnase I to remove contaminating genomic DNA. The cDNAs were synthesized from 0.2 g of total RNA using the Quant cDNA reverse transcription system (Tiangen Biotech Co.) with an oligo (dT) primer. PCR was performed using the same gfp-specific primers as above and the PCR products were run in 1% (w/v) agarose gels and visualized with a UV transilluminator after staining with 0.5 g ml−1 ethidium bromide.
percent of explant forming calli percent of explant regenerated shoots
100
CK
BA4.0
BA12.0
BA20.0
TDZ10.0
Growth regulator concentration (μM) Fig. 1. Effect of BA concentration and TDZ on callus formation and adventitious shoot regeneration from fragmented shoot tips. (CK: without plant growth regulators. Medium with different BA concentration or TDZ was all supplemented with 0.6 M NAA. The figures on each column differ significantly at P ≤ 0.05.)
tips with wounding treatment IV or III formed shoots separately and most of which were adventitious shoots. What’s more, more than two adventitious shoots were obtained on each fragmented shoot tips with wounding treatment III. 3.2. Effect of BA concentration and TDZ on the callus formation and shoot regeneration of fragmented shoot tips For plant regeneration from fragmented shoot tips, high BA concentration was shown to be important to stimulate callus formation and adventitious shoot regeneration (Fig. 1). With regeneration medium containing 12.0 M BA, about 87% fragmented shoot tips formed callus and 62% of which produced shoots. Increasing the BA concentration to 20.0 M, all the fragmented shoot tips formed callus while the percentage of normal shoots regenerated decreased to 38%. Substituting growth regulator BA with 10.0 M TDZ did not increase the percentage of shoot regeneration (43%) on fragmented shoot tips, though 100% of which formed callus. 3.3. Effect of kanamycin selection on shoot regeneration of fragmented shoot tips and scored leaves Callus formation and shoot regeneration of fragmented shoot tips or scored leaves with kanamycin selection were compared by quantification and GFP expression observation (Table 2). Without kanamycin selection, 71.1% scored leaves formed callus and 47.7% developed shoots, which was lower than that of fragmented shoot tips (85.0% and 61.7%, separately). The difference increased
Table 2 Effect of kanamycin selection pressure on regeneration activity and shoots regeneration ability of fragmented shoot tip or scaled leave cells. Fragmented shoot tips
Percent of explant formed calli Percent of transgenic calli Percent of explant formed shoots
Scored leaves
CK
kana-selection
CK
85.0 ± 13.2a
51.7 ± 8.8b 87.5 ± 4.9 5.8 ± 3.8c
71.1 ± 7.7a
61.7 ± 12.6a
47.7 ± 11.2b
kana-selection 23.2 ± 12.8c 95.4 ± 4.2 0d
CK: fragmented shoot tips or scored leaves were cultured on regeneration medium for shoot regeneration without inoculation, co-cultivation and selection. Data are presented as means ± S.E. The figures followed by different letters in each line differ significantly at P ≤ 0.05.
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Fig. 2. Recovery of transgenic apple plants and GFP expression observation during transformation. (A) Fragmented apple shoot tips with wounding treatment III (bar = 13 mm). (B) Nontransgenic shoots regenerated on fragmented shoot tips (CK) (bar = 11 mm). (C) Cells with GFP fluorescence were observed at the cutting edge of fragmented apple shoot tips. (D) Transgenic calli with strong GFP fluorescence were obtained under 20 mg l−1 kanamycin selection. (E and F) Chimeric transgenic shoots with limit areas of GFP fluorescence were obtained under 10 mg l−1 kanamycin selection. (G) Transgenic shoots regenerated from fragmented shoot tips on selection medium (bar = 11 mm). (H) GFP expression observation of transgenic shoots.
significantly when fragmented shoot tips and scored leaves were cultured on selection medium with 20 mg l−1 kanamycin. About 51.7% fragmented shoot tips formed callus and 87.5% of the calli were transgenic with GFP expression. After 8–9 weeks culture under kanamycin selection, 5.8% fragmented shoot tips devel-
oped transgenic shoots. Otherwise, only 23.2% scored leaves formed callus on selection medium and nontransgenic shoots were obtained after 8–9 weeks culture on kanamycin selection medium, though 97% of the calluses were transgenic with GFP expression.
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amplification bands were detected for the non-transformed plants without GFP fluorescence.
4. Discussion
Fig. 3. PCR and RT-PCR analysis of the gfp gene of non-transformed control shoots and transgenic clones of apple. (A) PCR results. (B) RT-PCR results. T represents transgenic and 1–4 are clones numbers. C represents nontransgenic control shoots.
3.4. GFP expression observation of transgenic shoots regeneration of fragmented shoot tips Adventitious shoots generally developed on callus that originated from the fragmented shoot tips (control) after 3–4 weeks culture on regeneration medium (Fig. 2B) and no fluorescent image was observed on nontransgenic apple plants under fluorescence microscope. When fragmented shoot tips were inoculated with agrobacterium and co-cultivated for 3 days, transgenic cells were observed after cultured on selection medium for further 3 days and areas with high density of transgenic cells were localized at the cutting edges of fragmented shoot tips (Fig. 2C). These cells could develop into small calli that with strong GFP fluorescence (Fig. 2D) and adventitious buds differentiated from these transgenic calli after about 6–8 weeks of culture on regeneration medium containing 20 mg l−1 kanamycin (Fig. 2G and H). Transfer of the differentiated shoots to light could inhibit the development of hyperhydricity and promote the growth of normal shoots. With selection medium containing 20 mg l−1 kanamycin, all the transgenic calli showed strong GFP fluorescence (Fig. 2D). While many chimeric transgenic shoots were obtained when the fragmented shoot tips were cultured on regeneration medium with 10 mg l−1 kanamycin for about 8 weeks (Fig. 2E and F). It is interesting to note that these chimeric shoots could be detected precisely under the fluorescence microscope and isolated for further multiplication and transgenic plants regeneration. 3.5. PCR and RT-PCR confirmation of GFP expression Seven clones of putative transgenic plants with GFP expression were obtained from about 120 explants (fragmented shoot tips, efficiency of 5.8%) after about 8 weeks culture under 20 mg l−1 kanamycin selection. The PCR and RT-PCR analysis of the four clones verified the results of GFP expression observed. Amplification with GFP primers gave rise to a 539 bp band for the transformed plants that showed GFP expression (Fig. 3A). Proof of transcription of the gfp gene in the transgenic plants was also obtained by RT-PCR amplification (Fig. 3b). No PCR or RT-PCR
Up to date, the successful apple transformation generally based on the selection of transformed cells with antibiotics (Broothaerts et al., 2001). As many apple cultivars are recalcitrant in terms of adventitious organ formation from leaves (Sriskandarajah et al., 1982; Welander, 1988) or sensitive to antibiotic selection, the frequency of apple transformation is generally low and genotype dependent highly (James and Dandekar, 1991; Maheswaran et al., 1992; Norelli and Aldwinckle, 1993). Various factors that affect the transformation and shoot regeneration efficiency have been studied (Huetteman and Preece, 1993; Sriskandarajah and Goodwin, 1998). Results showed that high percentage of adventitious shoots formation could be obtained when the explants closest to the meristem were used (Liu et al., 1998; Fasolo et al., 1989). Although several experiments on shoot regeneration from apple stem explants showed that the morphogenetic response of these explants was low (James et al., 1984; Welander, 1988; Belaizi et al., 1991). These studies only tested the internode stems that without meristematic tissues. Liu et al. (1998) found that, compared to the distal internodal explants and leaves, the first internodal stems (with meristems) produced high percentage of adventitious shoots. Our experiments confirmed that adventitious shoot regeneration could be obtained on apple (M. baccata (L.) Borkhausen) fragmented shoot tips. Although it is pointed that excessive wounding treatment may favor for the rapid clonal multiplication (Salami et al., 2005; Mbanaso et al., 2006), this was not true on apple (M. baccata (L.) Borkhausen) and the high percentage of shoot regeneration (78.3% or 60.8% separately) were obtained only from the fragmented shoot tips with moderate wounding treatment (II and III). What is more, it is noted that unlike the shoot direct regeneration on fragmented shoot tips with wounding treatment II, more than two adventitious shoots were obtained from the calli that formed on fragmented shoot tips with wounding treatment III. As until now, the only way to obtain apple transformed plants is adventitious shoot formation from transformed callus (De Bondt et al., 1994; Puite and Schaart, 1996), we think that it is better to use the fragmented shoot tips with wounding treatment III (cut vertically into four parts) as an alternative materials to improve the efficiency of apple shoot regeneration transformation. Previous studies have pointed that apple leaves are generally competent for regeneration, but susceptible to antibiotic selection (De Bondt et al., 1996). Mezzetti et al. (2002) found that although grape leaves were hypersensitive to antibiotic selection, the meristematic tissues of which were more resistant to the selection pressure and regeneration ability of transgenic cells was unaltered. Our results showed that apple (M. baccata (L.) Borkhausen) leaf cells were also very sensitive to kanamycin selection. However, the cells of fragmented shoot tips were rather resistant to kanmycin selection. Under 20 mg l−1 kanamycin selection pressure, 51.7% explants of fragmented shoot tips formed callus and 5.8% of which developed transgenic plants, compared to that 23.2% scaled leaves formed callus and nontransgenic plants was obtained. This implies that, characterized as a high regeneration ability and the resistance to antibiotic selection, fragmented shoot tips could be used as an alternative materials to improve the transformation efficiency of apple cultivars which until now have been recalcitrant to transformation. For successful apple transformation, the shoot regeneration from transgenic cells was an essential prerequisite. It was demonstrated that care must be taken on the analysis of early events for optimization of apple transformation protocol (De Bondt et al., 1994). However results of this study showed that cells with GFP
Y. Wu et al. / Scientia Horticulturae 128 (2011) 450–456
expression could be observed within less than 3 days after culture on selection medium and that areas with high density of transgenic cells were observed at the cutting edge of fragmented shoot tips. These cells could further developed into transgenic calli, while the efficiency of transgenic shoot regeneration was still low (5.8%). This indicated that studies should be focused on the factors that affect shoot regeneration from transgenic cells to improve the transformation efficiency. Although high antibiotic selection pressure is generally necessary for successful Agrobacterium-mediated transformation, it did inhibit the shoot regeneration of most transformed and non-transformed cells (Dominguez et al., 2004). Decreasing the selection pressure promotes the regeneration of escapes and chimeric shoots and makes the available transformation procedures rather inefficient (Matthews et al., 1998; Flachowsky et al., 2008; Dominguez et al., 2004). The results obtained here showed that with high kanamycin selection pressure (20 mg l−1 ), the transgenic calli and plants obtained were visualized with strong GFP fluorescence under microscope. Otherwise, many chimeric regenerated shoots were obtained when fragmented shoot tips were exposed to moderate selection pressure (10 mg l−1 ). Recent studies have shown that gfp gene could be used as a sensitive and noninvasive marker to visualize and precisely detect the transformed cells and plants (Maximova et al., 1998; Hily and Liu, 2009). This makes it possible to improve the transformation efficiency through decreasing or even omitting the antibiotic selection pressure. Now it is interesting to note that GFP fluorescence signals have been used to isolate the transgenic cells to improve the efficiency of transgenic plant regeneration (Flachowsky et al., 2004), and a simple and sensitive high-throughput GFP screener (Hily and Liu, 2009) has been developed. Thus we think that, strong emphasis should therefore be given towards using GFP expression scanning to overcome the current boundaries on apple genetic transformation. Acknowledgement Financial support from the Hebei Province Natural Science Foundation (project number CD2007000968) and the Scientific Research Fund for Doctor of Academic of Hebei Agriculture and Forestry science are acknowledged. We are grateful to Dr. Engelmann Florent for the careful remarks and revisions on this paper. References Barlass, M., Skene, G.M., 1980. Studies on the fragmented shoot apex of grapevine. I. The regenerative capacity of leaf primordial fragments in vitro. J. Exp. Bot. 31, 483–488. Belaizi, M., Paul, H., Sangwan, R.S., Sangwan-Norreel, B.S., 1991. Direct organogenesis from internodal segments of in vitro grown shoots of apple cv. Golden Delicious. Plant Cell Rep. 9, 47l–474. Bolar, J.P., Brown, S.K., Norelli, J.L., Aldwinckle, H., 1998. Factors affecting the transformation of ‘Marshall McIntosh’ apple by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 55, 31–38. Broothaerts, W., Wiersma, P.A., Lane, W.D., 2001. Multiplex PCR combining transgene and S-allele control primers to simultaneously confirm cultivar identity and transformation in apple. Plant Cell Rep. 20, 349–353. De Bondt, A., Eggermont, K., Penninckxt, I., Goderis, I., Broekaert, W.E., 1996. Agrobacterium mediated transformation of apple (Malus domestica Borkh): an assessment of factors affecting regeneration of transgenic plant. Plant Cell Rep. 15, 549–554. De Bondt, A., Eggermont, K., Druart, P., Vil, M., De Goderis, I., Vanderleyden, J., Broekaert, W.F., 1994. Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps. Plant Cell Rep. 13, 587–593. Dandekar, A.M., Uratsu, S.L., Matsuta, N., 1990. Factors influence virulence in agrobactrium-mediated transformation of apple. Acta Hort. 280, 483–494. Dominguez, A., Cervera, M., Perez, R.M., Romero, J., Carmen, C., Cubero, J., Lopez, M.M., Juarez, J.A., Navarro, L., Pena, L., 2004. Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol. Breed. 14, 171–183. Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
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Transgenic plants from fragmented shoot tips of apple (Malus baccata (L.) Borkhausen) via agrobacterium-mediated transformation Yongjie Wu a , Yunhe Li b , Yaqin Wu a , Hehe Cheng a , Yin Li c , Yanhua Zhao a , Yusheng Li a,∗ a b c
Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli Town 066600, China South Subtropical Crop Research Institute, Chinese Academy of Tropical Agricultural Science, Zhanjiang 524091, China School of Life Sciences, Sun Yat-sen University, No. 135, West Xin-gang Road, Guangzhou 510275, China
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 28 January 2011 Accepted 19 February 2011 Keywords: Apple Agrobacterium tumefaciens Fragmented shoot tips GFP Transformation
a b s t r a c t Transgenic apple (Malus baccata (L.) Borkhausen) plants were obtained via Agrobaterium-mediated transformation of fragmented shoot tips. Our results showed that without wounding treatment or with wounding treatment (II, cutting shoot tips vertically into two parts), shoots generally regenerated from meristem tissues directly and adventitious shoot regeneration was rarely observed. Otherwise, when shoot tips were cut vertically into four parts, a high percentage of callus formation (89.2%) and of adventitious shoot regeneration (60.8%) was observed. Under 20 mg l−1 kanamycin selection pressure, over 51.7% fragmented shoot tips developed callus and seven transgenic plants with GFP (Green fluorescent protein) expression were obtained from about 120 explants (efficiency of 5.8%). No transgenic plant was obtained from agrobacteria mediate transformed leaves, even though 23.2% of which formed callus after co-cultivation and selection. Molecular analysis (PCR and RT-PCR) of the transformed lines with GFP expression confirmed integration and transcription of the transgene. Under fluorescence microscopy, areas with high density of transgenic cells were observed at the cutting edges of fragmented shoot tips, which indicated that shoot regeneration from transgenic cells should be a major factor inhibiting transformation efficiency. Our experiments also showed that with moderate or low selection pressure, transgenic shoots were obtained generally accompanied by a high numbers of chimeric shoots. While by using fluorescence microscopy observation of GFP expression, the transgenic and chimeric shoots could be detected and separated precisely for further transgenic plats regeneration or multiplication. This may be very useful for apple genetic breeding, as large numbers of transgenic plants could be obtained in a short time. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Many commercial or new apple cultivars with unique quality traits are generally nonadaptable to varied plant conditions or accompanied by undesirable deleterious characteristics. Thus further improvement of target traits is necessary. However, introduction or deletion of target genes by means of conventional hybridization is generally costly, of low efficiency and a long-term process because of the high heterozygocity and long juvenile period of the apple plants. Transformation technologies offer promising tools to modify established cultivars by introducing one or a few traits without altering their existing genetic constitution.
Abbreviations: AS, acetosyringone; BA, 6-benzyladenine; GFP, green fluorescent protein; NAA, a-naphthaleneacetic acid; TDZ, thidiazuron. ∗ Corresponding author. Fax: +86 335 2023417. E-mail address: showersound [email protected] (Y. Li). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.02.013
Since the first report of transgenic apple plants (James et al., 1989), significant progress has been made on apple transformation. Cultivars such as Delicious (Sriskandarjah et al., 1994), Royal Gala (Yao et al., 1995), Golden Delicious (Puite and Schaart, 1996), Marshall McIntosh (Bolar et al., 1998), McIntosh Wijcik (Song et al., 2000), Elstar (Szankowski et al., 2003), Fuji (Seong et al., 2005), Jonagold (De Bondt et al., 1996), Orin (Kanamaru et al., 2004), as well as the rootstock M26, M9 and M29 (Lambert and Tepfer, 1992; Maheswaran et al., 1992; Zhu et al., 2001) have been transformed with different functional genes. Although Agrobacterium-mediated transformation has been recognized as the preferred system for transforming apple plants, the transformation efficiency appears to be low and highly genotype-dependent (James and Dandekar, 1991; Trifonova and Atanassov, 1998; Hanke, 2004), especially for cultivars which are recalcitrant to shoot regeneration or sensitive to antibiotic selection. A number of factors affect the efficiency of Agrobacteriummediated apple transformation. Among which, shoot regeneration ability is a crucial prerequisite for successful transformation.
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Because of the high level of tolerance to drought and low temperature, Malus baccata (L.) Borkhausen, is a traditional and main apple rootstock in the north of China. However the M. baccata (L.) Borkhausen plants are too vigorous to reduce grafted tree size for high yield production. Thus there is a great interest to obtain dwarf rootstock by means of genetic transformation. Preliminary results showed that shoot regeneration from leaves of M. baccata (L.) Borkhausen is low and very sensitive to antibiotic selection (Wang, 2005) and thus it is better to select other suitable materials for shoot regeneration and transformation. Besides the young leaves (Dandekar et al., 1990), internodal explants from etiolated apple shoots (Liu et al., 1998) have been used to improve shoot regeneration ability. Results of these study indicated that the closer to the apical meristem, the higher the regeneration ability. In fact, fragmented tissues of grape and banana shoot apices have been used for rapid multiplication of plant material (Barlass and Skene, 1980; Salami et al., 2005; Mbanaso et al., 2006). Recently Dutt et al. (2007) established a stable transformation protocol using fragmented grape shoot tips and confirmed that wounded shoot tips co-cultivated with agrobacteria could reconstruct complete meristems and ultimately produce non-chimeric transgenic plants. This implies that cells of apical meristems could be an alternative material for apple transformation. The aims of this research were: (1) to study the shoot regeneration ability of fragmented apple shoot tips and the effect of wounding treatments; (2) to establish a genetic transformation of apple (M. baccata (L.) Borkhausen) using fragmented shoot tips; and (3) to confirm the target gene transformation and expression by fluorescence observation and polymerase chain reaction (PCR) or RT-PCR. 2. Materials and methods 2.1. Plant materials Apple (M. baccata (L.) Borkhausen) plantlets were established from field-grown plants and multiplied in vitro for 2 years before the transformation experiments. Shoots were maintained by subculturing every 4–5 weeks on proliferation medium containing MS basal medium (Murashige and Skoog, 1962) 3% (w/v) sucrose and 0.7% (w/v) plant agar, pH 5.8) supplemented with 2.0 M BA and 0.6 M NAA at 25 ± 2 ◦ C with a 16 h photoperiod (supplied by fluorescent cool-white lamps at an intensity of 40 mol m−2 s−1 ). Shoot tips approximately 1 cm in length, composed of the apical meristem young leaves and a short basal stem, were harvested from 3to 4-week-old micropropagated shoots and transferred on proliferation medium. Etiolation was promoted by placing shoots in the dark for 2 weeks according to Liu et al. (1998). In experiment 2, the three youngest top leaves from 3- to 4-week-old micropropagated shoots were dissected and scored transversally two-to-four times (depending on the size of the leaf) with a scalpel for transformation.
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medium (MS basal medium supplemented with 12.0 M BA and 0.6 M NAA) and cultured for 4 weeks in the dark at 25 ± 2 ◦ C. The percentage of explants that formed callus was recorded and the explants were then transferred on the same fresh medium for shoot regeneration in the light. The number of regenerated shoots were counted and the percentage of explants that formed shoots were determined after 4–5 weeks culture in the light.
2.3. Plant regeneration from fragmented shoot tips For plant regeneration, fragmented shoot tips with different wounding treatments were transferred on regeneration medium (MS basal medium supplemented with 0.6 M NAA and 4.0, 12.0 or 20.0 M BA, or with 10.0 M TDZ and 0.6 M NAA) and cultured for about 4 weeks in the dark at 25 ± 2 ◦ C. The percentage of explants that formed callus was recorded and the explants were then transferred on the same fresh medium for shoot regeneration in the light. The number of regenerated shoots were counted and the percentage of explants that formed shoots were determined after 4–5 weeks culture in the light.
2.4. Agrobacterium-mediated transformation Agrobacterium-mediated transformation was conducted as previously described by James et al. (1993) and Norelli et al. (1996). Agrobacterium strain EHA105 harbouring the modified binary vector, pCAMBIA-1302, that incorporates the GFP reporter gene and the kanamycin resistance gene (npt II) for plant selection was used for transformation experiments Fragmented shoot tips (with wounding treatment III) or scored leaves were inoculated with agrobacteria that had been grown overnight in yeast extract broth (YEB) medium (Van Larebeke et al., 1977), centrifuged and resuspended in liquid MS medium (pH 5.2) to an OD600 of 0.8 for 10 min. The explants were then blotted and transferred to co-culture medium (MS basal medium supplemented with 12.0 M BA, 0.6 M NAA and 100 M AS). After co-cultivation for three days with Agrobacterium in the dark, they were placed on selection medium (MS basal medium supplemented with 12.0 M BA and 0.6 M NAA for fragmented shoot tips or 10.0 M TDZ and 0.6 M NAA for leaves) containing 200 mg l−1 cefotaxime and 20 mg l−1 kanamycin). The explants were transferred to fresh selection medium every 4–5 weeks. In experiment 4, the influence of kanamycin selection at 10 mg l−1 on transgenic shoot regeneration of fragmented shoot tips were observed. The influence of kanamycin selection on the proliferation of transformed cells was determined 4 weeks after co-cultivation by measuring the number of calli expressing GFP. The regenerated shoots were observed under a fluorescence microscope and GFP-expressing shoots were counted before excision and further transferred to proliferation medium (MS basal medium supplemented with 2.0 M BA, 0.6 M NAA, 200 mg l−1 cefotaxime and 20 mg l−1 kanamycin) in the light.
2.2. Wounding treatments prior to genetic transformation Etiolated shoot tips were further dissected to obtain 1–3 mm long explants, composed of the apical meristem with a few leaf primordia. Explants were placed on a Petri dish and subjected to the following fragmentation treatments: (I) no wounding; (II) cut vertically into two equal portions; (III) cut vertically into four equal portions; or (IV) cut into small pieces (approx 6–10 portions depending on the size of shoot tips). In experiment 1, the effect of different wounding treatments on callus formation and shoot regeneration were compared. The fragmented shoot tips with different wounding treatments were transferred on regeneration
2.5. GFP expression observation The fluorescent images were observed using an Olympus BX51 microscope equipped with an epifluorescence attachment, a 100 W mercury light source, and an Olympus DP70 digital microscope camera. A 450–490 nm excitation filter was used with a 520–560 mm emission filter that transmitted only green light. The regenerated calli were observed under the fluorescence microscope and those showing GFP fluorescence in all cells were measured and marked as transgenic calli.
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Table 1 Effect of wounding treatments on callus formation and shoot regeneration from fragmented shoot tips. Fragment treatments
Percent of explant forming calli
Percent of explant regenerated shoot
I II III IIII
1.7 ± 2.9c 48.3 ± 10.4b 89.2 ± 7.6a 100a
100 78.3 60.8 20.8
± ± ± ±
No. of shoots per explant
0a 8.8b 12.6b 10.4c
1.0 1.2 2.3 1.6
± ± ± ±
0c 0.04c 0.06a 0.27b
Data are presented as means ± S.E. The figures followed by different letters in each column differ significantly at P ≤ 0.05.
2.6. Statistic analyses Each treatment was replicated three times and a replicate consisted of 20 shoot tips (wounding treatments I and II) or of 40 fragmented shoot tip explants (wounding treatments III and IIII). The data are presented as means ± S.E. and were subjected to statistical analysis (ANOVA and Duncan’s Multiple Range Test) using the SPSS 13.0 for windows software (SPSS Inc., Chicago, IL, USA).
Regeneration percent (%)
120
60 40
3.1. Effect of wounding treatments on callus formation and shoot regeneration from fragmented shoot tips Significant differences were observed on the callus formation and shoot regeneration from fragmented shoot tips with different wounding treatments (Table 1). Without wounding treatment (treatment I) or with wounding treatment II, only 1.7% or 48.3% fragmented shoot tips formed callus after about 4 weeks of culture on the regeneration medium. While, a much higher percentage of callus formation (89.2% or 100%) was observed on the fragmented shoot tips with wounding treatments III or IV. Though shoots regeneration on the fragmented shoot tips with wounding treatments I and II was significant different (100 and 78.3%, respectively), these shoots were generally regenerated from the meristematic tissue directly and few adventitious shoots were observed. Compared to that, about 20.8% or 60.8% fragmented shoot
d
d
b a
20 0
3. Results
c
80
2.7. PCR and RT-PCR analysis Genomic DNA was extracted from transgenic and nontransgenic apple leaves, using the cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1990). PCR amplification of the gfp gene was carried out using the forward primer (GFP-S1), (5 TAG ATG GTG ATG TTA ATG GGC 3 ) and the reverse primer (GFP-SA1), (5 GCT GTT ACA AAC TCA AGA AGG 3 ). Total RNA was isolated from transgenic and nontransgenic apple leaves using TRNzol Total RNA Reagent (Tiangen Biotech Co., Beijing, China) according to the manufacturer’s instructions. RNA samples were treated with Dnase I to remove contaminating genomic DNA. The cDNAs were synthesized from 0.2 g of total RNA using the Quant cDNA reverse transcription system (Tiangen Biotech Co.) with an oligo (dT) primer. PCR was performed using the same gfp-specific primers as above and the PCR products were run in 1% (w/v) agarose gels and visualized with a UV transilluminator after staining with 0.5 g ml−1 ethidium bromide.
percent of explant forming calli percent of explant regenerated shoots
100
CK
BA4.0
BA12.0
BA20.0
TDZ10.0
Growth regulator concentration (μM) Fig. 1. Effect of BA concentration and TDZ on callus formation and adventitious shoot regeneration from fragmented shoot tips. (CK: without plant growth regulators. Medium with different BA concentration or TDZ was all supplemented with 0.6 M NAA. The figures on each column differ significantly at P ≤ 0.05.)
tips with wounding treatment IV or III formed shoots separately and most of which were adventitious shoots. What’s more, more than two adventitious shoots were obtained on each fragmented shoot tips with wounding treatment III. 3.2. Effect of BA concentration and TDZ on the callus formation and shoot regeneration of fragmented shoot tips For plant regeneration from fragmented shoot tips, high BA concentration was shown to be important to stimulate callus formation and adventitious shoot regeneration (Fig. 1). With regeneration medium containing 12.0 M BA, about 87% fragmented shoot tips formed callus and 62% of which produced shoots. Increasing the BA concentration to 20.0 M, all the fragmented shoot tips formed callus while the percentage of normal shoots regenerated decreased to 38%. Substituting growth regulator BA with 10.0 M TDZ did not increase the percentage of shoot regeneration (43%) on fragmented shoot tips, though 100% of which formed callus. 3.3. Effect of kanamycin selection on shoot regeneration of fragmented shoot tips and scored leaves Callus formation and shoot regeneration of fragmented shoot tips or scored leaves with kanamycin selection were compared by quantification and GFP expression observation (Table 2). Without kanamycin selection, 71.1% scored leaves formed callus and 47.7% developed shoots, which was lower than that of fragmented shoot tips (85.0% and 61.7%, separately). The difference increased
Table 2 Effect of kanamycin selection pressure on regeneration activity and shoots regeneration ability of fragmented shoot tip or scaled leave cells. Fragmented shoot tips
Percent of explant formed calli Percent of transgenic calli Percent of explant formed shoots
Scored leaves
CK
kana-selection
CK
85.0 ± 13.2a
51.7 ± 8.8b 87.5 ± 4.9 5.8 ± 3.8c
71.1 ± 7.7a
61.7 ± 12.6a
47.7 ± 11.2b
kana-selection 23.2 ± 12.8c 95.4 ± 4.2 0d
CK: fragmented shoot tips or scored leaves were cultured on regeneration medium for shoot regeneration without inoculation, co-cultivation and selection. Data are presented as means ± S.E. The figures followed by different letters in each line differ significantly at P ≤ 0.05.
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Fig. 2. Recovery of transgenic apple plants and GFP expression observation during transformation. (A) Fragmented apple shoot tips with wounding treatment III (bar = 13 mm). (B) Nontransgenic shoots regenerated on fragmented shoot tips (CK) (bar = 11 mm). (C) Cells with GFP fluorescence were observed at the cutting edge of fragmented apple shoot tips. (D) Transgenic calli with strong GFP fluorescence were obtained under 20 mg l−1 kanamycin selection. (E and F) Chimeric transgenic shoots with limit areas of GFP fluorescence were obtained under 10 mg l−1 kanamycin selection. (G) Transgenic shoots regenerated from fragmented shoot tips on selection medium (bar = 11 mm). (H) GFP expression observation of transgenic shoots.
significantly when fragmented shoot tips and scored leaves were cultured on selection medium with 20 mg l−1 kanamycin. About 51.7% fragmented shoot tips formed callus and 87.5% of the calli were transgenic with GFP expression. After 8–9 weeks culture under kanamycin selection, 5.8% fragmented shoot tips devel-
oped transgenic shoots. Otherwise, only 23.2% scored leaves formed callus on selection medium and nontransgenic shoots were obtained after 8–9 weeks culture on kanamycin selection medium, though 97% of the calluses were transgenic with GFP expression.
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amplification bands were detected for the non-transformed plants without GFP fluorescence.
4. Discussion
Fig. 3. PCR and RT-PCR analysis of the gfp gene of non-transformed control shoots and transgenic clones of apple. (A) PCR results. (B) RT-PCR results. T represents transgenic and 1–4 are clones numbers. C represents nontransgenic control shoots.
3.4. GFP expression observation of transgenic shoots regeneration of fragmented shoot tips Adventitious shoots generally developed on callus that originated from the fragmented shoot tips (control) after 3–4 weeks culture on regeneration medium (Fig. 2B) and no fluorescent image was observed on nontransgenic apple plants under fluorescence microscope. When fragmented shoot tips were inoculated with agrobacterium and co-cultivated for 3 days, transgenic cells were observed after cultured on selection medium for further 3 days and areas with high density of transgenic cells were localized at the cutting edges of fragmented shoot tips (Fig. 2C). These cells could develop into small calli that with strong GFP fluorescence (Fig. 2D) and adventitious buds differentiated from these transgenic calli after about 6–8 weeks of culture on regeneration medium containing 20 mg l−1 kanamycin (Fig. 2G and H). Transfer of the differentiated shoots to light could inhibit the development of hyperhydricity and promote the growth of normal shoots. With selection medium containing 20 mg l−1 kanamycin, all the transgenic calli showed strong GFP fluorescence (Fig. 2D). While many chimeric transgenic shoots were obtained when the fragmented shoot tips were cultured on regeneration medium with 10 mg l−1 kanamycin for about 8 weeks (Fig. 2E and F). It is interesting to note that these chimeric shoots could be detected precisely under the fluorescence microscope and isolated for further multiplication and transgenic plants regeneration. 3.5. PCR and RT-PCR confirmation of GFP expression Seven clones of putative transgenic plants with GFP expression were obtained from about 120 explants (fragmented shoot tips, efficiency of 5.8%) after about 8 weeks culture under 20 mg l−1 kanamycin selection. The PCR and RT-PCR analysis of the four clones verified the results of GFP expression observed. Amplification with GFP primers gave rise to a 539 bp band for the transformed plants that showed GFP expression (Fig. 3A). Proof of transcription of the gfp gene in the transgenic plants was also obtained by RT-PCR amplification (Fig. 3b). No PCR or RT-PCR
Up to date, the successful apple transformation generally based on the selection of transformed cells with antibiotics (Broothaerts et al., 2001). As many apple cultivars are recalcitrant in terms of adventitious organ formation from leaves (Sriskandarajah et al., 1982; Welander, 1988) or sensitive to antibiotic selection, the frequency of apple transformation is generally low and genotype dependent highly (James and Dandekar, 1991; Maheswaran et al., 1992; Norelli and Aldwinckle, 1993). Various factors that affect the transformation and shoot regeneration efficiency have been studied (Huetteman and Preece, 1993; Sriskandarajah and Goodwin, 1998). Results showed that high percentage of adventitious shoots formation could be obtained when the explants closest to the meristem were used (Liu et al., 1998; Fasolo et al., 1989). Although several experiments on shoot regeneration from apple stem explants showed that the morphogenetic response of these explants was low (James et al., 1984; Welander, 1988; Belaizi et al., 1991). These studies only tested the internode stems that without meristematic tissues. Liu et al. (1998) found that, compared to the distal internodal explants and leaves, the first internodal stems (with meristems) produced high percentage of adventitious shoots. Our experiments confirmed that adventitious shoot regeneration could be obtained on apple (M. baccata (L.) Borkhausen) fragmented shoot tips. Although it is pointed that excessive wounding treatment may favor for the rapid clonal multiplication (Salami et al., 2005; Mbanaso et al., 2006), this was not true on apple (M. baccata (L.) Borkhausen) and the high percentage of shoot regeneration (78.3% or 60.8% separately) were obtained only from the fragmented shoot tips with moderate wounding treatment (II and III). What is more, it is noted that unlike the shoot direct regeneration on fragmented shoot tips with wounding treatment II, more than two adventitious shoots were obtained from the calli that formed on fragmented shoot tips with wounding treatment III. As until now, the only way to obtain apple transformed plants is adventitious shoot formation from transformed callus (De Bondt et al., 1994; Puite and Schaart, 1996), we think that it is better to use the fragmented shoot tips with wounding treatment III (cut vertically into four parts) as an alternative materials to improve the efficiency of apple shoot regeneration transformation. Previous studies have pointed that apple leaves are generally competent for regeneration, but susceptible to antibiotic selection (De Bondt et al., 1996). Mezzetti et al. (2002) found that although grape leaves were hypersensitive to antibiotic selection, the meristematic tissues of which were more resistant to the selection pressure and regeneration ability of transgenic cells was unaltered. Our results showed that apple (M. baccata (L.) Borkhausen) leaf cells were also very sensitive to kanamycin selection. However, the cells of fragmented shoot tips were rather resistant to kanmycin selection. Under 20 mg l−1 kanamycin selection pressure, 51.7% explants of fragmented shoot tips formed callus and 5.8% of which developed transgenic plants, compared to that 23.2% scaled leaves formed callus and nontransgenic plants was obtained. This implies that, characterized as a high regeneration ability and the resistance to antibiotic selection, fragmented shoot tips could be used as an alternative materials to improve the transformation efficiency of apple cultivars which until now have been recalcitrant to transformation. For successful apple transformation, the shoot regeneration from transgenic cells was an essential prerequisite. It was demonstrated that care must be taken on the analysis of early events for optimization of apple transformation protocol (De Bondt et al., 1994). However results of this study showed that cells with GFP
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expression could be observed within less than 3 days after culture on selection medium and that areas with high density of transgenic cells were observed at the cutting edge of fragmented shoot tips. These cells could further developed into transgenic calli, while the efficiency of transgenic shoot regeneration was still low (5.8%). This indicated that studies should be focused on the factors that affect shoot regeneration from transgenic cells to improve the transformation efficiency. Although high antibiotic selection pressure is generally necessary for successful Agrobacterium-mediated transformation, it did inhibit the shoot regeneration of most transformed and non-transformed cells (Dominguez et al., 2004). Decreasing the selection pressure promotes the regeneration of escapes and chimeric shoots and makes the available transformation procedures rather inefficient (Matthews et al., 1998; Flachowsky et al., 2008; Dominguez et al., 2004). The results obtained here showed that with high kanamycin selection pressure (20 mg l−1 ), the transgenic calli and plants obtained were visualized with strong GFP fluorescence under microscope. Otherwise, many chimeric regenerated shoots were obtained when fragmented shoot tips were exposed to moderate selection pressure (10 mg l−1 ). Recent studies have shown that gfp gene could be used as a sensitive and noninvasive marker to visualize and precisely detect the transformed cells and plants (Maximova et al., 1998; Hily and Liu, 2009). This makes it possible to improve the transformation efficiency through decreasing or even omitting the antibiotic selection pressure. Now it is interesting to note that GFP fluorescence signals have been used to isolate the transgenic cells to improve the efficiency of transgenic plant regeneration (Flachowsky et al., 2004), and a simple and sensitive high-throughput GFP screener (Hily and Liu, 2009) has been developed. Thus we think that, strong emphasis should therefore be given towards using GFP expression scanning to overcome the current boundaries on apple genetic transformation. Acknowledgement Financial support from the Hebei Province Natural Science Foundation (project number CD2007000968) and the Scientific Research Fund for Doctor of Academic of Hebei Agriculture and Forestry science are acknowledged. We are grateful to Dr. Engelmann Florent for the careful remarks and revisions on this paper. References Barlass, M., Skene, G.M., 1980. Studies on the fragmented shoot apex of grapevine. I. The regenerative capacity of leaf primordial fragments in vitro. J. Exp. Bot. 31, 483–488. Belaizi, M., Paul, H., Sangwan, R.S., Sangwan-Norreel, B.S., 1991. Direct organogenesis from internodal segments of in vitro grown shoots of apple cv. Golden Delicious. Plant Cell Rep. 9, 47l–474. Bolar, J.P., Brown, S.K., Norelli, J.L., Aldwinckle, H., 1998. Factors affecting the transformation of ‘Marshall McIntosh’ apple by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 55, 31–38. Broothaerts, W., Wiersma, P.A., Lane, W.D., 2001. Multiplex PCR combining transgene and S-allele control primers to simultaneously confirm cultivar identity and transformation in apple. Plant Cell Rep. 20, 349–353. De Bondt, A., Eggermont, K., Penninckxt, I., Goderis, I., Broekaert, W.E., 1996. Agrobacterium mediated transformation of apple (Malus domestica Borkh): an assessment of factors affecting regeneration of transgenic plant. Plant Cell Rep. 15, 549–554. De Bondt, A., Eggermont, K., Druart, P., Vil, M., De Goderis, I., Vanderleyden, J., Broekaert, W.F., 1994. Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps. Plant Cell Rep. 13, 587–593. Dandekar, A.M., Uratsu, S.L., Matsuta, N., 1990. Factors influence virulence in agrobactrium-mediated transformation of apple. Acta Hort. 280, 483–494. Dominguez, A., Cervera, M., Perez, R.M., Romero, J., Carmen, C., Cubero, J., Lopez, M.M., Juarez, J.A., Navarro, L., Pena, L., 2004. Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol. Breed. 14, 171–183. Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
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