Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons

Scientia Horticulturae 124 (2010) 360–367

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Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons Mei Gao a,b,*, Makiko Kawabe b, Tatsuya Tsukamoto a, Hiromi Hanada a, Ryutaro Tao b a b

Japan Science and Technology Agency (JST), Regional Joint Research Project of Wakayama Prefecture, Wakayama 649-6261, Japan Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 August 2009 Received in revised form 14 January 2010 Accepted 15 January 2010

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 mM 2,4dichlorophenoxyacetic acid (2,4-D) and 1 mM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 mM a-naphthaleneacetic acid (NAA) and 5 mM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted b-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated ‘‘Sirakaga’’ immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l1 kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Genetic transformation Plant regeneration Prunus Somatic embryos Transgenic plant

1. Introduction Molecular and biotechnological techniques have become powerful tools for plant science. Genetic transformation, for example, is utilized not only for crop breeding but also for genetic and physiological research. As the genus Prunus, which contains many fruit tree species, has a relatively small genome size and a short juvenile period, it is considered to be a model plant for genomic research in fruit trees. However, genetic transformation of Prunus is difficult because it is recalcitrant to in vitro regeneration.

Abbreviations: BA, 6-benzyladenine; CaMV, cauliflower mosaic virus; 2,4-D, 2,4dichlorophenoxyacetic acid; gusA, b-glucuronidase (GUS) gene; IBA, indol-3butyric acid; Km, kanamycin; MEPM, meropenem trihydrate; MS, Murashige and Skoog medium; MUG, 4-methylumbelliferyl-D-glucuronide; NAA, a-naphthaleneacetic acid; nptII, neomycin phosphotransferase II gene; PCR, polymerase chain reaction; SEG, somatic embryogenesis; SEGM, somatic embryo germination medium; SEIM, somatic embryo inducing medium; SEPM, somatic embryo propagation medium; WP, woody plant medium; X-Gluc, 5-brome-4-chloro-3indolyl-b-D-glucuronide. * Corresponding author at: Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Tel.: +81 75 753 6051; fax: +81 75 753 6497. E-mail address: [email protected] (M. Gao). 0304-4238/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.01.021

Although there have been several reports describing the successful transformation of some of the fruit tree species in Prunus (Mante et al., 1991; de Caˆmara Machado et al., 1994; Hammerschlag and Smigocki, 1998; Gutie`rrez-Pesce et al., 1998; Dolgov and Firsov, 1999; Miguel and Oliveira, 1999; Pe´rez-Clemente et al., 2004), the genotypes that can be reliably transformed are quite limited, especially in economically important cultivars or genotypes. The lack of an efficient adventitious regeneration system is a major limiting factor in developing a genetic transformation system for most perennial crops including Prunus fruit trees. Japanese apricot (Prunus mume) is one of the most important fruit crops in Japan. Its fruit is usually harvested when it is unripe and used to make Umeboshi, a Japanese traditional pickled fruit or Umeshu, a kind of sweet flavoured liquor. Both Umeboshi and Umeshu are very popular in Japan for their characteristic flavours. In addition to fruit production, P. mume is renowned as an ornamental tree in China and Japan due to its attractive blossoms in early spring. Because of its economic importance as an ornamental and a fruit tree, there is significant interest in breeding and improvement of this species. Although some attempts at micropropagation of P. mume have been reported (Harada and Murai, 1996), very limited information is available on the

M. Gao et al. / Scientia Horticulturae 124 (2010) 360–367

successful regeneration of this species. Recently, Ning and Bao (2007) and Ning et al. (2007) reported a successful protocol for direct shoot regeneration from immature embryos and cotyledons of P. mume. However, as per our knowledge, no successful genetic transformation system has been reported so far. In this paper, we report a successful protocol for somatic embryogenesis and a plant regeneration system from immature cotyledons of P. mume. Furthermore, we report the establishment of a genetic transformation technique for this species using the regeneration system developed in this study. 2. Materials and methods 2.1. Plant materials Open-pollinated fruits were collected from mature trees of eight cultivars of P. mume, ‘Nanko’ (N), ‘Kensaki’ (K), ‘Kairyou Uchida’ (U), ‘Hachirou’ (H), ‘Kotsubu Nanko’ (KN), ‘Sirakaga’ (S), ‘Kosinoume’ (KU) and ‘Benisasi’ (B) growing in the orchard of Kyoto University on 26 April, 10, 17, and 24 May in 2005. These fruits were surface sterilized in 70% ethanol for 15 min and then rinsed three times with sterile distilled water. Seeds were aseptically taken out and the seed coat was removed (Fig. 1a). Two pieces of immature cotyledon were separated and directly used as explants if they were less than 4 mm in length, however if the cotyledons were over 4 mm, each of them was cut along the midrib (Fig. 1b). Because the embryos that were collected on 26 April were not visible, the whole seeds including albumen and seed coat were cut longitudinally into four pieces and used as explants (Fig. 1c). 2.2. Inducing somatic embryogenesis Two types of basal media were used to induce somatic embryogenesis (SEG): (1) MS medium (Murashige and Skoog, 1962) containing 3% sucrose (SEIM-MS) and (2) WP medium (Lloyd and McCown, 1980) containing 3% sorbitol (SEIM-WP), solidified with 0.7% agar. These media were supplemented with 1 mM 2,4-D and 1 mM BA, and adjusted to pH 5.8 prior to autoclaving at 121 8C for 15 min. The media were dispensed into 90 mm  20 mm Petri dishes (30–35 ml per dish). Four cotyledon explants were placed in each plate, and 100 explants were used in each treatment. These plates were sealed with Parafilm (Pechiney Plastic Packaging Inc., Chicago,

Fig. 1. Making immature cotyledon explants for in vitro culture from the fruits of P. mume (cv. ‘Nanko’). (a) Immature fruit; (b) seed and immature cotyledons; (c) excided seeds of the four sampling dates illustrated the sizes of immature cotyledon. Bar: 5 mm.

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IL, USA) and kept in the dark at 25  1 8C. After 30 days of culture, the frequency of SEG was investigated (primary SEG frequency). The frequency of SEG was defined as the percentage of explants forming somatic embryos per total number of explants cultivated. After investigating the primary SEG frequency, the explants with calli and somatic embryos were subcultured to somatic embryo propagation medium (SEPM), which consisted of the basic medium used for SEG induction but the plant growth regulators were substituted by 0.1 mM NAA and 5 mM BA and solidified with 0.2% gellan gum (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Somatic embryos were propagated by secondary SEG on SEPM. Cultures were kept at 25 8C in the dark. After 30 days of subculture, the SEG frequency was investigated (subculture SEG frequency). Somatic embryos were maintained by subculturing the somatic embryo clusters or embryogenic calli to fresh SEPM-MS every 2 months. 2.3. Germination of somatic embryos and plant regeneration For somatic embryo germination, the somatic embryo clusters of about the same size (1–2 cm diameter) were transferred to a flask (100 ml) containing 40 ml of somatic embryo germination medium (SEGM) composed of WP medium supplemented with 3% sorbitol, 5 mM BA and 0.7% agar, and cultured at 25 8C under a 16-h photoperiod with a light intensity of 60 mmol m2 s1. The germinated somatic embryo clusters were transferred to a fresh medium every 3 weeks until they developed into multiple shoots. When the shoots had grown to 3–5 cm in height, they were transferred to WP basic medium supplemented with 3% sorbitol and 5 mM IBA instead of 5 mM BA, and incubated under light for rooting. When the shoots rooted, they were planted in plastic boxes (6 cm  6 cm  9 cm) containing vermiculite that had been autoclaved and wetted with 1/1000 HYPONeX (HYPONeX Japan Co., Osaka, Japan). They were kept in the same incubator that was used for somatic embryo germination and rooting at 25 8C under a 16-h photoperiod and the covers were gradually opened to allow the plants to become acclimated to ex vitro conditions. 2.4. Genetic transformation using the SEG system Genetic transformation experiments were simultaneously conducted according to the procedure of inducing SEG described in Section 2.2. Immature cotyledons of the eight cultivars of P. mume described above were inoculated with Agrobacterium tumefaciens EHA101 carrying the binary plasmid vector pGUS-INT with the gusAintron and nptII genes in its T-DNA region (Fig. 6) (Gao et al., 2007). The inoculation was carried out by immersing the explants for 15 min in the A. tumefaciens suspension, prepared as described previously (Gao et al., 2000). Then the explants were blotted briefly on a sterile paper towel and placed on a sterilized filter paper on cocultivation medium. Co-cultivation was carried out at 25 8C in the dark for 3 days. The explants were then transferred to disinfection medium containing 50 mg l1 meropenem trihydrate (MEPM) (MEROPEN1, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan). After 7 days of disinfection, they were transferred to a selection medium containing 50 mg l1 MEPM and 25 mg l1 Km. Two types of SEIM (SEIM-MS and SEIM-WP) were used as the basic media for co-cultivation, disinfection and primary selection. The processes of inducing SEG, subculturing and plant regeneration were conducted according to the methods described above, except that all media were supplemented with MEPM and Km. Transformation efficiency was histochemically evaluated by determining transient gusA gene expression (Jefferson, 1987) in three cultivars ‘Nanko’, ‘Kairyou Uchida’ and ‘Hachirou’. Twenty explants from immature cotyledons sampled on 10, 17 and 24 May were inoculated with A. tumefaciens EHA101 carrying the binary

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plasmid vector pGUS-INT and were co-cultivated for 1, 3 and 5 days at 25 8C in the dark. GUS activity was detected by incubating the inoculated explants in X-Gluc solution overnight at 37 8C. After washing with 70% ethanol, the blue spots or blue areas that appeared on the explants were counted. The Agrobacterium infection frequency was defined as the percentage of explants possessing blue spots per the total number of inoculated explants. 2.5. Confirmation of transformation The GUS activity of Km-resistant somatic embryo lines was investigated fluorometrically with MUG as the substrate, as described by Jefferson (1987), using a piece of tissue from the somatic embryo clusters. GUS-positive somatic embryo lines were allowed to germinate. The shoots derived from the GUS-positive lines were subjected to PCR amplification and Southern blot analyses. Total DNA was isolated from the leaves of the shoots according to the modified CTAB protocol (Yamamoto and Hayasi, 2001). For PCR analysis, about 100 ng of the total DNA and two primer pairs were used to amplify the partial gusA gene fragment of 948 bp (forward primer, 50 -GGA AGT GAT GGA GCA TCA GGG CGG30 and reverse primer, 50 -CGG ACG GGT ATC CGG TTC GTT GGC-30 ) and the partial nptII gene fragment of 792 bp (forward primer, 50 ATG ATT GAA CAA GAT GGA TTG CAC GCA-30 and reverse primer, 50 -GAA GAA CTC GTC AAG AAG GCG ATA GA-30 ). PCR amplification was performed in 20-ml reaction volumes with a program

consisting of an initial denaturing step at 94 8C for 2 min, followed by 30 cycles of 45 s at 94 8C, 1 min at 60 8C and 2 min at 72 8C. For Southern blot analysis, 5 mg of the total DNA was digested with HindIII and separated on a 0.8% agarose gel, then transferred to a positively charged nylon membrane (Roche Diagnostics GmbH, Germany). The membrane was hybridized with digoxigenin (DIG)-11-dUTP-labelled probes from partial coding regions of gusA and nptII. The probes were PCR-labelled with a DIG Probe Synthesis Kit (Roche Diagnostics GmbH, Germany) as per the manufacturer’s instructions using the gusA and nptII gene-specific primers described above. Prehybridization and hybridization were performed at 65 8C. Blots were washed twice at 25 8C in 2 saline– sodium citrate (SSC) and 0.1% sodium dodecyl sulphate (SDS) for 5 min and twice at 65 8C in 0.1 SSC and 0.1% SDS for 15 min. Hybridization signals were detected with anti-DIG antibodyalkaline phosphatase and CDP-Star (Roche Diagnostics GmbH, Germany) on X-ray films. GUS expression of the transgenic shoot was histochemically determined with X-Gluc according to the method described above. 3. Results 3.1. Inducing SEG from immature cotyledons The earliest SEG began 3 weeks after the immature cotyledon explants were cultured in vitro. Most of the somatic embryos

Fig. 2. Somatic embryogenesis (SEG) and regeneration of shoot. (a) Somatic embryos formed directly on the adaxial surface of the cotyledons (arrow), and callus formed from the incision surface (triangle); (b) zygotic embryo grew to visible and regenerated somatic embryos and callus from the 26 April explant; (c) secondary SEG (triangle); (d) SEG was induced from the embryogenic callus; (e) leafy or cotyledon-like structures illustrated irregular development of somatic embryos; (f, g and j) normal development of somatic embryos; (h) multi-thallus that has no apical meristem derived from somatic embryo cluster; (i) multiple buds derived from somatic embryo cluster. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)

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Table 1 Frequency of SEG from immature cotyledons of P. mume. Cultivars

N K U H KN S KU B

26 April

10 May

17 May

Primary SEG (%)

Primary SEG (%)

Subculture SEG (%)

Primary SEG (%)

Subculture SEG (%)

Primary SEG (%)

Subculture SEG (%)

MS

WP

MS

WP

MS

WP

MS

WP

MS

WP

MS

WP

MS

WP

3.0 4.0 5.0 5.0 11.3 1.9 6.7 16.7

0.0 1.0 0.0 2.0 0.0 0.0 0.0 16.7

16.7 38.5 40.6 27.8 40.0 33.3 16.7 8.7

2.2 11.9 6.3 3.3 6.3 8.3 8.3 0.0

19.4 38.5 40.6 36.1 40.0 54.2 33.3 21.7

33.3 35.7 50.0 10.0 62.5 25.0 41.7 40.0

2.5 15.0 0.0 0.0 2.5 0.0 0.0 0.0

0.0 7.5 0.0 0.0 0.0 8.5 0.0 0.0

2.5 17.5 0.0 5.0 7.5 6.3 23.3 0.0

0.0 22.5 0.0 0.0 5.0 55.3 0.0 0.0

0 7.5 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 7.5 0 0 0 0 0 0

0 0 0 0 0 0 0 0

formed directly on the adaxial surface of the cotyledons (Fig. 2a). They were white in colour, had smooth and hard globular surfaces and were usually clustered as a somatic embryo mass. Somatic embryos could be distinguished from calli that were formed from the cut surface. The calli were usually loose and yellow or brown in colour. SEG was found in the cotyledons collected on 26 April and 10 May of all the eight cultivars. The primary SEG frequency from the 10 May cotyledons was the highest among the four sampling stages in all the cultivars (Table 1). At this stage, the size of immature cotyledons was about 2–3 mm in length (Fig. 1c). The highest frequency of primary SEG was 40.6% in ‘Kairyou Uchida’ in MS medium, followed by 40.0% in ‘Kotsubu Nanko’ in MS medium (Table 1). Because the immature zygotic embryos in the 26 April seeds were not visible, whole seeds were used as explants. The excised seeds contained seed coat, albumen and invisible embryos at the top of the seeds. After a few days of culture, the albumen had dried up, and the seed coats had shrunk and become brown. However, in some of the explants, the embryos situated at the top of the seeds grew and regenerated somatic embryos or calli (Fig. 2b). Although the SEG frequency of the 26 April explants was lower than that of the 10 May explants in most cultivars, the somatic embryos induced from these explants were more vigorous with more proliferation ability in later subcultures than other cotyledons. The SEG from the cotyledons collected on 17 May was remarkably reduced. The sizes of cotyledons at this stage for most cultivars were over 5 mm (Fig. 1c) except the ‘Kensaki’. The growth of ‘Kensaki’ embryos was slower than other cultivars and the cotyledons on 17 May were about 2–3 mm, which was similar to the 10 May cotyledons of other cultivars in length. The SEG frequency of the 17 May cotyledons of ‘Kensaki’ was 15% and 7.5% in MS and WP media, respectively, and was higher than those of the other cultivars. The cotyledons of most cultivars collected on 24 May became hard and filled the seeds; no SEG was observed from these cotyledons except for ‘Kensaki’. At this stage, the size of cotyledons of ‘Kensaki’ was on average about 5 mm and the SEG frequency was 7.5% in MS medium. The primary SEG frequency in MS medium was higher than that in WP medium in most cultivars (Table 1). However, when the explants were subcultured, somatic embryo formation was stimulated in SEPM-WP and the SEG frequency reached close to or over than that in SEPM-MS (Table 1). 3.2. Propagation of somatic embryos and plant regeneration The somatic embryos that regenerated from the immature cotyledons were friable and the clusters were easily isolated from the cotyledon tissue. When the somatic embryo clusters were transferred to SEPM, some of them proliferated by secondary

24 May

embryogenesis (Fig. 2c), and the secondary embryos rapidly propagated and clustered. Whereas some of the somatic embryo clusters stopped regeneration or germinated into multi-thallus or multi-buds, these clusters graduated into brown then died. In some cases, the somatic embryo cluster dedifferentiated to embryogenic callus, from which SEG could be induced (Fig. 2d). The somatic embryos could be propagated by subculturing the new regenerated somatic embryo clusters to fresh SEPM-MS medium every 2 months. Once in the while, the somatic embryos could develop into mature embryos through the normal embryonic developmental process: globular, heart-shaped, torpedo-shaped and cotyledon stages (Fig. 2f, g and j), and when the mature embryos were cultured in SEGM under the light, they were developed into rooted plantlet (Fig. 3c). When the somatic embryo clusters were transferred to SEGM and cultured under the light, some of them germinated and grew into the multi-thallus, an abnormal structure that has no apical meristem (Fig. 2h), or the normal multiple buds (Fig. 2i). The frequency of germination of the somatic embryo clusters was almost 50% (60 of 119 somatic embryo clusters changed into green and grew to multi-thallus or multi-buds). The other clusters changed into brown in colour or developed abnormal structures, i.e. leafy or cotyledon-like structures (Fig. 2e). Although the multiple buds occasionally developed into normal multiple shoots (Fig. 3a), most of them grew into abnormal structures, such as leaf clump structures consisting of only leaves and no apical meristem or hyperhydrated shoots (Fig. 3b). The normal multiple shoots could be subcultured by transferring them to fresh medium every 3 weeks. When the multiple shoots were about 3–5 cm high, they were transferred to the rooting medium. After approximately 2 months of culture under the light, roots formed from the callus situated on the bottom of multiple shoots (Fig. 3d) and the rooted shoots could be planted in vermiculite for acclimatization (Fig. 3e). 3.3. Transient GUS expression in the immature cotyledons Transient GUS expression after co-cultivation was found mostly at the cut surfaces or wounded regions of the cotyledons (Fig. 4a). The inoculation efficiency of the 10 May explants was lower than those of the 17 and 24 May explants, while there was no difference between the inoculation efficiencies of the 17 and 24 May explants (data not shown). The inoculation efficiency of the 17 May explants of the three cultivars (‘Nanko’, ‘Kairyou Uchida’ and ‘Hachirou’) is shown in Fig. 4b. Although we showed the results of the GUS analysis for three cultivars, there was no cultivar difference in the inoculation efficiency as revealed by the number area of blue spots among all cultivars used in this study. The inoculation efficiency increased with an increase in the co-cultivation period from 1 to 5 days. However, the explants were greatly damaged due to Agrobacterium overgrowth when they were co-cultured for 5 days.

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Fig. 3. Plant regeneration from transgenic somatic embryo clusters derived from the immature cotyledon of P. mume ‘Sirakaga’. (a) Normal multiple shoots; (b) hyperhydrated shoots; (c) plantlet derived from normal somatic embryo development; (d) rooting for the transgenic shoot; (e) acclimation of the transgenic plantlet; (f) expression of gusA gene in the transgenic plant. Bar: 1 cm.

3.4. SEG from the inoculated immature cotyledons The 26 April explants of all eight cultivars were weakened drastically and died during the inoculation, co-cultivation and

primary selection process. No SEG was observed from the 26 April inoculated explants. As in the 10 May non-inoculated explants, the 10 May inoculated cotyledons showed the highest SEG frequency (Table 2) among the four sampling dates in most cultivars. The highest frequency of SEG after 30-day (primary SEG) selection culture was observed in ‘Kensaki’ (46.3%). The SEG frequency of the 17 May cotyledons was remarkably reduced. ‘Kensaki’ alone showed SEG from three different sampling dates. In most cases, the regeneration frequency in MS medium was higher than in WP medium in the primary selection culture. However, when the inoculated explants were subcultured to SEPM containing 50 mg l1 MEPM and 25 mg l1 Km, the SEG increased in the WP medium (Table 2). These results were similar to the noninoculated control treatments. In some cases, the SEG frequency in inoculated explants was higher than that in non-inoculated explants (Tables 1 and 2). This tendency was obvious in the 17 May explants. After 2 months of selection culture, SEGs in the 17 May inoculated explants were observed in all of the treatments (Table 2). 3.5. Confirmation of transgenes and production of transgenic plants

Fig. 4. Investigation of transformation frequency represented by transient expression of gusA gene. (a) Histochemical GUS assays for the immature cotyledons of ‘Nanko’ on 17 May after 3-day co-culture and (b) inoculation frequency of the 17 May immature cotyledons of three cultivars.

We selected only a single somatic embryo cluster for further experiments because a single somatic embryo cluster seemed to be formed from a single primary somatic embryo. In this way, we could avoid treating a mixture of several different transgenic lines as a single transgenic line. From all of the Km-resistant somatic embryo lines, we obtained three GUS-positive activity lines, which were derived from the 10 May cotyledons of ‘Sirakaga’ (Fig. 5a). Most of the somatic embryo lines were not GUS-positive, although they had survived and grown in the selection medium containing Km. However, when the somatic embryos were subcultured in SEPM containing Km for several cycles, they eventually turned

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Table 2 Frequency of SEG from immature cotyledons inoculated with A. tumefaciens. Cultivars

N K U H KN S KU B

10 May

17 May

24 May

Primary SEG (%)

Subculture (%)

SEG

Primary SEG (%)

Subculture (%)

SEG

Primary SEG (%)

Subculture SEG (%)

MS

WP

MS

WP

MS

WP

MS

WP

MS

WP

MS

WP

25.0 46.3 25.0 26.9 25.6 33.3 0.0 0.0

0.0 10.0 0.0 8.3 0.0 0.0 0.0 0.0

25.0 46.3 25.0 65.4 33.3 50.0 8.0 5.9

11.1 22.0 4.2 8.3 14.0 0.0 24.0 11.8

0.0 24.4 2.0 3.3 23.4 0.0 8.7 0.0

0.0 7.5 0.0 0.0 0.0 0.0 5.0 0.0

11.5 11.1 16.0 3.3 29.8 16.3 13.0 4.1

7.7 15.0 6.0 3.3 27.7 10.6 5.0 2.2

0.0 17.1 0.0 0.0 0.0 0.0 5.0 0.0

0.0 6.5 0.0 0.0 0.0 0.0 0.0 0.0

0.0 17.1 0.0 0.0 0.0 0.0 5.0 0.0

0.0 6.5 0.0 0.0 0.0 0.0 0.0 0.0

brown and died. Thus, long-term selection in Km is necessary to eliminate ‘escapes’ (non-transgenic lines). Genomic DNA from the three GUS-positive lines yielded the expected DNA fragments in PCR analysis for the amplification of partial gusA and nptII sequences (Fig. 5b). Southern blot analysis was conducted for one GUS-positive line using gusA and nptII probes; the results indicated that a single copy of T-DNA was integrated in its genome (Fig. 6). The other two GUS-positive lines could not be accepted Southern blot analysis, because they died during subculture process before Southern blot analysis. The transgenic somatic embryos germinated to form shoots and roots under selection with 25 mg l1 Km. Histochemical analysis using X-Gluc showed that these plantlets had GUS activity (Fig. 3f). 4. Discussion This study described a successful regeneration and genetic transformation system of P. mume through SEG from immature cotyledons. Although there were some reports describing plant regeneration systems for P. mume through organogenesis using immature cotyledons (Ning and Bao, 2007; Ning et al., 2007), to our knowledge, this is the first report of a successful SEG and genetic transformation for this species. Ning and Bao (2007) described that callus induced from immature cotyledons of P. mume (cv. ‘Xuemei’ and ‘Lv’e’) could be

Fig. 5. Confirmation of transformation. (a) GUS activity examination for the transgenic somatic embryos derived from the inoculated cotyledon of ‘Sirakaga’. From left: non-transformed line, transgenic line no. 1, no. 2 and no. 3; (b) PCR analyses for gusA (upper) and nptII (lower) gene in leaves of transgenic shoots. From left: plasmid vector used in the transformation, transgenic line no. 1, no. 2 and no. 3.

classified into three types: type 1, light yellow, loose and globular callus; type 2, smooth white, loose and nodular callus and type 3, light yellow, hard and globular callus. In their study, shoot regeneration through organogenesis was observed from the type 2 callus. In our study, we also observed the formation of similar types of callus and occasional shoot regeneration through organogenesis from the type 2 callus. However, in addition to organogenesis, we also observed SEG from the surface of the cultured cotyledons. Structures with white smooth surfaces and a nodular appearance were formed usually from the adaxial surface of the cotyledons. These structures developed into somatic embryos with shoot and root meristem through the globular to cotyledonary stages. The somatic embryo could be detached easily from the surrounding cells, while adventitious buds were attached tightly to the callus. This feature is typical of somatic embryo since the adventitious structure, which has bipolar organization with shoot and root meristem and lacks vascular connection with parent tissue, has been termed as a somatic embryo (Reinert, 1973). The somatic embryos induced from immature cotyledons of P. mume showed great variability in their morphology. In addition to the typical somatic embryos described above, many atypical ones were also differentiated. Tang et al. (2000) described the morphology of typical and atypical somatic embryos derived from immature cotyledons of sour cherry in detail. They found that other adventitious structures such as cotyledon-like structures, leaves, shoots and roots frequently occurred from immature cotyledons. In our study, we also observed that these structures frequently formed from the immature cotyledons of P. mume. The stage of development of the immature cotyledons seemed to be the most important in inducing SEG, which was most frequently induced in young cotyledons. In this study, SEG was found in all the cultivars when the immature cotyledons were 2– 3 mm. However when the sizes of immature cotyledons were over 5 mm, the ability for SEG distinctly decreased regardless of cultivars, although the development of the cotyledons at the same date differed among cultivars. It appeared that SEG was induced more easily in younger cotyledons, regardless of cultivars. The SEG frequency of the 26 April seeds explants was not the highest in the SEG inducing and transformation experiments. We presumed that this might be because the embryos which were not visible were lost in the process of the explants being excised or inoculated with Agrobacterium. On the other hand, for transformation, relatively bigger cotyledons might be appropriate, because if the cotyledons are too small, they are less likely to withstand inoculation and selection with Km. We also found that in the 17 May cotyledons the SEG frequency in inoculated explants was higher than that in noninoculated explants; it seemed that inoculation or selection could probably accelerate SEG in the bigger cotyledon explants. The types of plant growth regulators (PGRs) used in the culture medium greatly influence SEG. In this study, we found that SEG occurred when 1 mM 2,4-D was used in combination with 1 mM BA

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Fig. 6. Southern blot analysis of genomic DNA from leaves of P. mume shoots. Genomic DNA was digested with HindIII and the fragments were allowed to hybridize with gusA (left) and nptII (right) probes. Lane C: non-transformed control line, lane T: no. 1 transgenic line. A physical map of the T-DNA digested with HindIII is illustrated below. The positions of the probes were shown with dark bars below the construction. RB: right border, LB: left border, 35S-50 : CaMV 35S promoter, 35S-30 : CaMV 35S transcription terminator, tml30 : polyadenylation region of tml gene from Agrobacterium tumefaciens, nptII: neomycin phosphotransferase gene, gusA-int: intron-containing chimaeric bglucuronidase gene.

in the initial culture. In our preliminary experiments, however, when NAA was used in place of 2,4-D, no SEG occurred (data not shown). These results were consistent with previous reports on many other plant species, in which 2,4-D was shown to be the most effective auxin for SEG (Michalczuk et al., 1992; Feher et al., 2003). However, continuous use of 2,4-D stimulated somatic embryos to dedifferentiate, forming loose and formless callus resembling the type 1, as described above; this type of callus did not have any regeneration ability. Dedifferentiation of the somatic embryos could be avoided when they were subcultured in SEPM supplemented with 0.1 mM NAA and 5 mM BA. On this medium, secondary SEG from the primary somatic embryos was occasionally observed, although most of the somatic embryo cultures did not grow for a long time. When the somatic embryos were separated from the somatic embryo cluster and individually cultured on SEGM for germination, on rare occasions, they germinated into plantlets with both shoot and root meristem. However, when the somatic embryo clusters were transferred to SEGM and cultured under the light, some of them germinated to form multi-buds, and sometimes the multi-buds grew to give multi-shoots with no roots. Adventitious bud formation seemed to be involved in this type of shoot formation from somatic embryos. It should be noted that somatic embryos subcultured into the medium containing NAA sometimes dedifferentiated to the type 2 callus, which could undergo organogenesis. Although the difference between the induction conditions for SEG and organogenesis are not well understood, Ammirato (1985) described that SEG and organogenesis are two mutually exclusive processes of in vitro differentiation. Our results, however, indicated that SEG and organogenesis occurred under the same conditions. Similar results were obtained in Prunus cerasus (Tang et al., 2000), Prunus avium (De March et al., 1993) and Juglans nigra L. (Long et al., 1995). SEG was more successful on MS medium than WP medium, while the germination of somatic embryos and shoot proliferation

seemed to be stimulated more on WP with 3% sorbitol than MS, indicating that MS is more suitable for SEG than WP, while WP is better for shoot regeneration and proliferation. Similar results have been reported for other Prunus species (Hammatt and Grant, 1998; Neil and Neil, 2000; Tang et al., 2002; Bhagwat and Lane, 2004; Andrea and Johannes, 2005). Km is widely used as a selectable marker for plant genetic transformation (Shaw et al., 1983). Km sensitivity varies with the types of explants and species, and a wide range of concentrations have been used for selection. In this study, non-transformed somatic embryos were found in all the cultivars at 25 mg l1 Km selection. The result indicated that 25 mg l1 Km was not enough to inhibit SEG from immature cotyledons of P. mume. However, these non-transformed somatic embryos could be selected out and eliminated during long-term subculturing on the selection medium. From these results, we considered that it might be possible to improve the transformation efficiency by increasing Km selection pressure especially in early selection step. Histochemical GUS analyses of the transformed shoots revealed a positive GUS expression. However, GUS expression varied among the leaves in the same shoot. We presumed that the transformed shoot might be a chimera, composed of transformed and untransformed cells. Since SEG is generally thought to have a unicellular origin in the epidermis (Polito et al., 1989), regenerated plants derived from somatic embryos should be non-chimeric (Dandekar et al., 1992). Thus, if our transformed shoot was a chimera, the transformation event must have occurred after SEG. Alternatively, in the transformed shoot, expression of CaMV 35S gusA gene could be variable in different leaves. Although the CaMV 35S promoter is regarded as a constitutive promoter, many researchers have reported that the expression of the CaMV 35S promoter is cell-type specific and developmentally regulated (Benfey and Chua, 1989; Williamson et al., 1989; Yang and Christou, 1990; Terada and Shimamoto, 1990). Some reports have suggested that GUS expression in young leaves of the shoot tip is

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