Adventitious shoot regeneration from hypocotyl slices of mature apricot (Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering

Scientia Horticulturae 128 (2011) 457–464

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Adventitious shoot regeneration from hypocotyl slices of mature apricot (Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering Hong Wang a,b , Nuria Alburquerque b , Lorenzo Burgos b , Cesar Petri b,∗ a b

Institute of Fruit and Floriculture Research, Gansu Academy of Agricultural Sciences, Anning, Lanzhou 730070, China Grupo de Biotecnología de Frutales, Departamento de Mejora Vegetal, CEBAS-CSIC, Campus de Espinardo, Apartado de correos 164, 30100 Espinardo, Murcia, Spain

a r t i c l e

i n f o

Article history: Received 4 November 2010 Received in revised form 25 January 2011 Accepted 24 February 2011 Keywords: Apricot Organogenesis Genetic transformation Antibiotic selection GUS Agrobacterium

a b s t r a c t Adventitious shoot regeneration from hypocotyl slices of mature apricot seeds has been achieved with regeneration percentages of 31.7%, 44.4%, and 46.9% for the cultivars ‘Canino’, ‘Dorada’, and ‘Moniqui’, respectively. Regeneration was significantly affected by the parental origin of the explants (P < 0.05) but not by thidiazuron or 3-indolebutyric acid for any of the three cultivars, at the levels tested. None of the other factors studied (basal medium, 2,4 dichlorophenoxy-acetic acid pulses, dark incubation period, or addition of silver thiosulfate) affected significantly shoot regeneration percentage from ‘Canino’ hypocotyl sections. The effect of paromomycin on regeneration was genotype-dependent and different dose–response curves were obtained for each cultivar. While 40 ␮M paromomycin completely inhibited regeneration from ‘Canino’ sections, some buds were obtained from ‘Dorada’ and ‘Moniqui’ explants. The two aminoglycoside antibiotics tested, kanamycin and paromomycin, showed differing toxicity on ‘Canino’. A lower concentration of kanamycin (20 ␮M) than of paromomycin inhibited totally adventitious regeneration from ‘Canino’ explants. Agrobacterium-mediated transformation experiments, with the nononcogenic strain AGL1 harboring the binary plasmid p35SGUSINT, were performed and GUS assays were carried out after four weeks to determinate stable transformation events. The utilization of paromomycin (10 ␮M) as the selective agent increased significantly both the number of explants that presented at least one transformation event (P < 0.05) and the number of large area or calli expressing the gus gene (P < 0.001), compared with the addition of kanamycin (10 ␮M). Moreover, when 10 ␮M paromomycin was added to the medium some massively transformed explants were observed and a chimerical bud was regenerated. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Adventitious regeneration is a key step in the application of genetic engineering techniques. Prunus are among the most recalcitrant species and in apricot (Prunus armeniaca L.) only three reports on shoot regeneration from immature seed-derived tissues (Goffreda et al., 1995; Lane and Cossio, 1986; Pieterse, 1989) have been published. Regeneration from leaves has been achieved for a few genotypes: clones H.152 and H.146 (Escalettes and Dosba, 1993) and the commercial cultivars ‘Bulida’, ‘Helena’, and ‘Canino’ (Burgos and Alburquerque, 2003; Pérez-Tornero et al., 2000a; Petri et al., 2005b).

Abbreviations: 2,4-D, 2,4 dichlorophenoxy-acetic acid; BA, 6benzylaminopurine; DKW, Driver and Kuniyuki (1984); IBA, 3-indolebutyric acid; MS, Murashige and Skoog (1962); QL, Quoirin and Lepoivre (1977); RM, rooting medium; SRM, shoot regeneration medium; STS, silver thiosulfate; TDZ, thidiazuron; WPM, woody plant medium (Lloyd and McCown, 1980). ∗ Corresponding author. Tel.: +34 968 396200; fax: +34 968 396213. E-mail address: [email protected] (C. Petri). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.02.020

Apricot genetic transformation still remains difficult and inefficient. In 1992, some transgenic apricot lines, from immature cotyledons, were produced (Laimer da Câmara Machado et al., 1992). So far, ‘Helena’ remains the only commercial apricot cultivar that has been genetically modified. A transformation procedure was developed in our laboratory, for leaves of the commercial apricot cultivar ‘Helena’, by means of antibiotic-based selection strategies and transgenic plants expressing the marker genes gfp or uidA and nptII were produced (Petri et al., 2008a,b). More recently, transgenic apricot plants from the cultivar ‘Helena’ have been obtained by using MAT vectors technology, which combines the regeneration-promoting gene ipt and site-specific recombination in order to allow the elimination of the marker genes (LópezNoguera et al., 2009). Nevertheless, the general applicability of the methods is not established since regeneration/transformation protocols are highly genotype-dependent and there are no publications reporting the successful reproduction of these techniques and the generation of apricot plants in other laboratories. Mante et al. (1991) developed an Agrobacterium-mediated transformation protocol for plum hypocotyls. This protocol was

458

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

improved by optimizing the selection (80 mg L−1 kanamycin was added just after co-cultivation), rooting, and acclimatization steps and reached 4.2% transformation efficiency (Gonzalez-Padilla et al., 2003). Recently, the addition of 2,4 dichlorophenoxy-acetic acid (2,4-D) during co-cultivation and the optimization of the timing of each step in the protocol allowed transformation efficiencies of up to 42% and enabled the production of self-rooted transgenic plants in the greenhouse in approximately 6 months (Petri et al., 2008c). The procedure has been successfully transferred to Prunus salicina and some transgenic plants, expressing the nptII and gfp marker genes, have been generated (Urtubia et al., 2008). In the present work we studied the possibility of transferring the plum system to apricot since, to the best of our knowledge, there are no publications detailing the use of this type of explants to produce transgenic apricot plants. Such methodology would allow the confirmation of a hypothesis when there is no need or interest in producing improved cultivars (i.e. testing new constructions or developing protocols for marker genes elimination) and also would be useful for functional genomic studies, by evaluating the activity of genes in this species. Furthermore, it will be of interest for producing transgenic rootstocks with agronomically interesting traits, such as fungal or nematode resistance, since most apricot cultivars are grafted onto apricot seedlings (frequently ‘Canino’). Researchers at the “Instituto Valenciano de Investigaciones Agrarias” (IVIA, Valencia, Spain) have studied the uniformity of seedlings from different apricot cultivars and have selected ‘Canino’ clone 9–7 for its good seed germination and seedling vigor and uniformity (Orero et al., 2004). Currently, most of the nurseries in Valencia use seedlings from this ‘Canino’ clone as apricot rootstocks. 2. Materials and methods 2.1. Plant material and explant preparation Mature seed hypocotyl slices from the apricot cultivars ‘Canino’, ‘Dorada’, and ‘Moniquí’ were used as the source of explants (Fig. 1A). Seed disinfection and explant preparation was performed following previous procedures (Gonzalez-Padilla et al., 2003). Briefly, after the endocarp had been removed with a nutcracker, the seeds were immersed in a 1% sodium hypochlorite solution, containing approximately 20 ␮l Tween-20 per 100 ml solution, for 20 min and rinsed four times with sterile distilled water in a laminar-flow bench. Disinfected seeds were soaked in sterile water overnight at 4 ◦ C and the seed coats removed with a scalpel. The radicle and the epicotyl were discarded, and the hypocotyl was sliced into three cross-sections (0.5–1 mm) (Fig. 1A). 2.2. General regeneration strategy Hypocotyl slices were placed on shoot regeneration medium (SRM), which consisted of 3/4-strength Murashige and Skoog (1962) (MS) salts supplemented with full-strength MS vitamins (Duchefa Biochemie, Haarlem, The Netherlands), 2% (w/v) sucrose, and 0.7% (w/v) purified agar (Laboratorios Conda, Torrejón de Ardoz, Spain. Cat. No. 1806). The growth regulators used to induce shoot regeneration from hypocotyl slices were 7.0 ␮M thidiazuron (TDZ) and 0.25 ␮M 3-indolebutyric acid (IBA). The medium was adjusted with 1 N NaOH to pH 5.8, autoclaved at 121 ◦ C for 20 min, and then dispensed into 9 cm × 1.5 cm sterile plastic Petri dishes (∼25 ml each). After explants were positioned on the medium, the dishes were sealed with Parafilm® and incubated in the dark at 23 ± 1 ◦ C. After 1 week in the dark, explants were transferred to the light with a 16h photoperiod (20–25 ␮mol m−2 s−1 , cool-white fluorescent lamp) at 23 ± 1 ◦ C.

The explants were maintained in the same Petri dish (not transferred to fresh medium) during the entire experiment. This shoot regeneration protocol was followed, with the modifications indicated below, for all of the experimentation. Buds or clusters from ‘Canino’ regeneration experiments were isolated and placed on a specific medium for apricot meristems growth (Pérez-Tornero et al., 1999), composed of QL medium supplemented with 3% sucrose, 6.65 ␮M 6-benzylaminopurine (BA), and 0.05 ␮M IBA. After 2–3 weeks, buds or clusters were transferred to a shoot-growing medium described previously for ‘Canino’ micropropagation (Pérez-Tornero et al., 2000b). Briefly, the shoot elongation and multiplication medium consisted of QL macronutrients, DKW micronutrients and vitamins, 3 mM calcium chloride, 0.8 mM phloroglucinol, 3% (w/v) sucrose, 1.12 ␮M BAriboside, 0.05 ␮M IBA, 2.1 ␮M 6-(3-hydroxybenzylamino) purine (Meta-Topolin), 29.6 ␮M adenine, and 0.7% (w/v) agar (Laboratorios Conda, Torrejón de Ardoz, Spain. Cat. No. 1812). Elongated shoots were placed in rooting medium (RM) (Petri et al., 2008a) when they were 2–3 cm long. The acclimatization and establishment of plantlets in a greenhouse were performed following standard apricot procedures (Pérez-Tornero and Burgos, 2000).

2.3. Study of factors affecting adventitious shoot regeneration To study the effect of different basal media on regeneration, slices from ‘Canino’ hypocotyls were placed on SRM differing in their basal salts and vitamins composition: 3/4-strength MS salts with full-strength MS vitamins (Murashige and Skoog, 1962), fullstrength woody plant medium (WPM) salts and vitamins (Lloyd and McCown, 1980), or full-strength Quoirin and Lepoivre (QL) macronutrients (Quoirin and Lepoivre, 1977) with Driver and Kuniyuki (1984) (DKW) micronutrients and vitamins. The effect of the growth regulator concentrations was studied by placing explants from ‘Canino’, ‘Moniquí’, and ‘Dorada’ on SRM with the following combinations and concentrations of growth regulators: 7.0 ␮M TDZ with 1.0 or 0.25 ␮M IBA, 5.0 ␮M TDZ with 1.0 or 0.25 ␮M IBA. ‘Canino’ explants were pretreated with 2,4 dichlorophenoxyacetic acid (2,4-D). Hypocotyl sections were placed on SRM supplemented with 0, 1.12, 2.25 or 4.5 ␮M of 2,4-D for 3 days and cultured in the dark at 23 ± 1 ◦ C. After 3 days, explants were transferred to SRM without 2,4-D. The effect of the length of the dark incubation period on adventitious regeneration was also studied. ‘Canino’ hypocotyl slices were cultured on SRM under four light and dark treatments: a 16-h photoperiod (20–25 ␮mol m−2 s−1 ) at 23 ± 1 ◦ C or complete darkness for 1, 2, or 3 weeks (at 23 ± 1 ◦ C), before transfer to a 16-h photoperiod (20–25 ␮mol m−2 s−1 ) at 23 ± 1 ◦ C. The addition to the SRM of the ethylene inhibitor silver thiosulfate (STS) was studied by culturing ‘Canino’ explants on SRM supplemented with 0, 30, 60, 90, or 120 ␮M STS prior to autoclaving. The stock solution was prepared as described by Burgos and Alburquerque (2003).

2.4. Regeneration dose–response curves to aminoglycoside antibiotics Explants from ‘Canino’ were cultured on SRM for 3 days and then transferred to SRM with the addition of 0, 10, 20, or 40 ␮M paromomycin sulfate (Duchefa) or kanamycin sulfate (Duchefa). The paromomycin sulfate concentrations tested for ‘Moniquí’ and ‘Dorada’ explants were 0, 20, 30, and 40 ␮M. The antibiotics were filter-sterilized and added to the medium after autoclaving.

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

459

Fig. 1. Shoot regeneration from apricot mature seed hypocotyl slices (A) Apricot hypocotyl slices, where 1, 2, and 3 represent the explants used in this study. The epicotyl (E) and radicle (R) were not used. (B) Adventitious shoot regeneration from ‘Dorada’ explants 6 weeks after explants were placed in the shoot regeneration medium (SRM). (C) Adventitious shoot regeneration as a “crown” from a ‘Moniquí’ explant. The photograph was taken 7 weeks after explants were placed in SRM. (D) Elongated ‘Canino’ shoots. (E) Rooted ‘Canino’ shoot. (F) ‘Canino’ plantlets potted and cultured in a greenhouse. Horizontal bars indicate 1 mm.

2.5. Agrobacterium tumefaciens-mediated transformation The non-oncogenic A. tumefaciens strain AGL1, carrying the binary plasmid p35SGUSINT (Vancanneyt et al., 1990), was used in the transformation experiments. The T-DNA of the plasmid con-

tains the Nospro-nptII-Noster cassette as the selectable marker gene, and the 35Spro-uidA-Noster cassette as the reporter gene. The reporter gene includes an intron for plant-specific expression. The engineered A. tumefaciens strain was cultured and prepared for infection as described by Gonzalez-Padilla et al. (2003). Briefly,

460

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

for co-cultivation, a single colony was inoculated into 10 ml of Luria–Bertani medium with 100 mg l−1 ampicillin and 50 mg l−1 kanamycin, and incubated overnight at 28 ◦ C with constant agitation (175 rpm). Overnight culture growth reached an O.D., at 600 nm, of between 0.2 and 1.0. The cultures were centrifuged at 3000 × g for 15 min and resuspended in 50 ml of bacterial resuspension medium consisting of MS salts, 2% (w/v) sucrose, and 100 ␮M acetosyringone. The culture in bacterial resuspension medium was shaken (175 rpm) at 25 ◦ C for 5 h before use. Slices from ‘Canino’ seed hypocotyls were immersed in resuspended Agrobacterium for about 20 min, blotted briefly on sterile filter paper, and co-cultivated on SRM without antibiotics. After 3 days the explants were washed in a sterile solution of 1/2-strength MS medium with 300 mg l−1 cefotaxime, and blotted briefly on sterile filter paper, and then placed on SRM with 300 mg l−1 cefotaxime and 10 ␮M kanamycin or 10 ␮M paromomycin. GUS assays (Jefferson, 1987) were performed 4 weeks after the infection in order to determine stable transformation. 2.6. Experimental design and data analysis An average of 20 explants per treatment was evaluated for each experiment. Experiments were performed at least twice. Regeneration was evaluated weekly from the 3rd week after the beginning of the experiment until adventitious buds stopped appearing for 2 consecutive weeks, approximately 9–10 weeks from the beginning of the experiment. At this point, final data were collected on the shoot regeneration frequencies and yields. Regeneration and transformation percentages were compared with maximum likelihood ANOVA. The number of shoots per regenerating explant and transformation events were analyzed with an ANOVA (SAS Institute, 1988). 3. Results 3.1. Adventitious regeneration Explants of the three cultivars tested showed a similar response on the SRM. Explants had grown to about three-fold their initial size after the 1-week dark-incubation period. Shoots initially developed along the edges within 2–3 weeks after the culture initiation and after one more week buds were evident. Regeneration rarely appeared as a single bud, but usually as clusters (Fig. 1B) and sometimes like a “crown” all around the explant (Fig. 1C). Regeneration was achieved for all three cultivars tested (Table 1). When ANOVA was performed, no significant effect was observed in relation to the TDZ or IBA levels for all three cultivars, but there were differences in the regeneration percentages among cultivars (P < 0.05) (Table 1). Further analysis revealed significant differences (P < 0.05) among cultivars regarding regeneration percentages for certain growth regulator combinations (Table 1). In average, ‘Canino’ explants showed significantly (P < 0.05) less regeneration ability (26.7%) than ‘Moniqui’ and ‘Dorada’ (37.8% and 36.0%, respectively). Significant differences among cultivars in the number of clusters per regenerating explant were not detected (Table 1). None of the factors studied for ‘Canino’ mature seed hypocotyl slices (dark incubation period, 2,4-D pre-treatment, basal medium, addition of STS) affected significantly the adventitious bud regeneration percentages (Fig. 2). More callus formation was observed as the dark incubation period and the 2,4-D concentration, over 3 days, were increased but, at the same time, there was a slight reduction in regeneration (Fig. 2A and B). Buds started appearing approximately 1 week earlier when explants were cultured with a 16-h photoperiod and a dark period was not applied than when explants were

cultured in the dark for 1, 2, or 3 weeks before transfer to a 16-h photoperiod. No significant effect of the basal salt medium composition on shoot regeneration was observed (Fig. 2C). As mentioned above, addition of the ethylene inhibitor STS did not affect significantly the regeneration percentages. The number of explants that produced shoots decreased as the STS concentration increased (Fig. 2D). Most of the isolated buds regenerated from ‘Canino’ explants developed and elongated successfully (85.7%) (Fig. 1D). Following elongation, shoots were placed in RM and, after 2 weeks, some roots began to grow. Three weeks later, 77.4% of the shoots showed a well-developed root system (Fig. 1E). The acclimatization step was accomplished effectively following our standard procedures (Pérez-Tornero and Burgos, 2000), with practically no loses. 3.2. Effect of aminoglycoside antibiotics on regeneration ‘Canino’ hypocotyl slices were affected differently by the two antibiotics tested. Kanamycin at 20 ␮M totally inhibited adventitious regeneration but 40 ␮M paromomycin was necessary to observe the same effect (Fig. 3A). The effect of paromomycin was genotype-dependent. While 20 ␮M paromomycin drastically reduced regeneration from ‘Canino’ and ‘Moniquí’ explants, compared with the control without the addition of the antibiotic, shoot regeneration from ‘Dorada’ explants was not affected at this concentration (Fig. 3A and B). Paromomycin at 40 ␮M severely reduced regeneration from ‘Dorada’ and ‘Moniquí’ explants (Fig. 3B). 3.3. Tissue transformation Four weeks after infection, stable transformation appeared as spots (Fig. 4A) or large zones or calli (Fig. 4B) and, eventually, the gus gene was expressed throughout the whole explant (Fig. 4C). The transformation rate, based on GUS detection, was significantly (P < 0.05) affected by the aminoglycoside antibiotic added to the medium (Table 2). When paromomycin was used as the selective agent, the number of explants that presented at least one transformation event was significantly higher than when kanamycin was added to the medium at the same concentration (Table 2). Although the number of transformation events per transformed explant was slightly superior (no statistical differences) when kanamycin was used, transformation events usually appeared as multiple spots (Fig. 4A). The number of transgenic large areas and/or calli was significantly higher (P < 0.001) with paromomycin (Table 2) and some completely transformed explants were observed (Fig. 4C). Furthermore, the addition of 10 ␮M paromomycin allowed the regeneration of a chimerical bud (Fig. 4D). 4. Discussion This is the first manuscript reporting regeneration from apricot hypocotyls. Although previous papers reported regeneration and even transformation of apricot seeds, immature cotyledons were used as explants (Laimer da Câmara Machado et al., 1992). The advantage of the methodology reported here is that mature seeds are easily stored in the refrigerator, while immature embryos should be collected at a certain stage of development. The size of the embryo is critical for the subsequent regeneration and the optimum time for fruit collection has to be estimated empirically every year since fruit development is affected by environmental conditions. Moreover, the rate of regeneration from stored immature embryos is considerably lower than from fresh embryos (Scorza and Srinivasan, personal communication). Regeneration percentages reported from apricot immature cotyledon explants, 30% for the cultivar ‘Kecskemeter’ (Laimer da Câmara Machado et al., 1992) are comparable to those reported in this manuscript from mature seed hypocotyl slices.

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

461

Table 1 Adventitious shoot regeneration from hypocotyls of mature apricot seeds. Regeneration (% ± SE)

Treatment TDZ (␮M)

IBA (␮M)

‘Canino’

7.0 7.0a 5.0 5.0

1.0 0.25a 1.0 0.25

21.4 31.7 29.7 24.1

± ± ± ±

No. of clusters per regenerating explant (mean ± SE) ‘Dorada’

5.5 a 5.9 7.5 5.8 a

36.0 37.7 23.9 44.4

± ± ± ±

‘Moniquí’

6.8 b 6.2 6.3 6.8 b

46.9 34.4 36.2 36.6

± ± ± ±

‘Canino’

7.1 b 4.9 6.3 7.5 ab

1.25 1.85 1.27 1.77

± ± ± ±

‘Dorada’

0.13 0.33 0.19 0.32

1.77 1.87 1.54 1.66

± ± ± ±

‘Moniquí’

0.23 0.04 0.25 0.20

1.74 1.72 1.76 1.80

± ± ± ±

0.18 0.14 0.24 0.26

Different letters within each plant growth regulator combination indicate significant differences (P < 0.05) in regeneration percentages among cultivars. For this study, 210 explants of ‘Canino’, 241 explants of ‘Moniquí’, and 211 explants of ‘Dorada’ were used. a Standard regeneration conditions as described in Section 2.

Regeneration rate (%)

50

50

A

B

40

40

30

30

20

20

10

10

0

0 0

1

2

0

3

1

2

3

4

2,4-D (µM)

Weeks cultured in dark 50

50

Regeneration rate (%)

C

D

40

40

30

30

20

20

10

10

0

0 MS

WPM

QL

0

30

60

Basal medium

90

120

STS (µM)

Fig. 2. Study of factors in adventitious shoot regeneration from ‘Canino’ hypocotyl sections. (A) Effect of the length of the dark incubation period. A total of 290 explants were used in this study. (B) Effect of 2,4-D pre-treatment for 3 days at different concentrations. A total of 493 hypocotyl slices were used. (C) Effect of the basal salts medium composition. A total of 144 explants were used in this study. (D) Effect of the addition of the ethylene inhibitor STS to the SRM. A total of 269 slices were used. 45

Regeneration rate (%)

40

45

A

Paromomycin Kanamycin

35

B

Moniquí Dorada

40 35

30

30

25

25

20

20

15

15

10

10

5

5

0 0

10

20

Antibiotic (µM)

30

40

0

10

20

30

40

0 50

Paromomycin (µM)

Fig. 3. Effect of aminoglycoside antibiotics (kanamycin and paromomycin) on adventitious shoot regeneration from apricot hypocotyl slices. (A) Effect of the antibiotics kanamycin and paromomycin on regeneration from ‘Canino’ mature seed hypocotyl sections. (B) Effect of the antibiotic paromomycin on regeneration from ‘Dorada’ and ‘Moniquí’ mature seed hypocotyl explants. A total of 197, 115 and 202 explants from ‘Canino’, ‘Moniquí’, and ‘Dorada’, respectively, were used in this study.

462

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

Fig. 4. GUS detection in ‘Canino’ hypocotyl slices four weeks after infection with the Agrobacterium tumefaciens strain AGL1 harboring the p35SGUSINT plasmid. (A) Transformation events as a pattern of spots (arrows). (B) Explant with several transgenic calli. (C) GUS expression throughout the whole explant surface. (D) Putative chimerical bud displaying GUS activity in some leaves (arrow). Horizontal bar indicates 1 mm.

For any of the combinations tested, TDZ and IBA successfully induced shoot regeneration for all three cultivars used in this study. TDZ has been used to induce regeneration from seed-derived tissues in apricot (Laimer da Câmara Machado et al., 1992) and other Prunus species, such as Prunus avium (Canli and Tian, 2008), Prunus cerasus (Mante et al., 1989), Prunus domestica (Mante et al., 1991, 1989; Tian et al., 2007a), Prunus microcarpa (Nas et al., 2010), Prunus mume (Ning et al., 2007), Prunus persica (Mante et al., 1989), and P. salicina (Canli and Tian, 2009; Tian et al., 2007b). When a range of TDZ concentrations (from 0.5 to 25.0 ␮M with 2.5 ␮M IBA) were tested for shoot induction from P salicina cv. ‘Shiro’ hypocotyl slices, no great differences were observed in the percentage of explants producing shoots, ranging from 9.3% to 14.6% (Tian et al., 2007b). On the other hand, the authors reported significant differences in the percentage regeneration response when 7.5 ␮M TDZ and 2.5 ␮M IBA were added to the SRM, depending on the cultivar, varying from 28.3% for ‘Early Golden’ to no regeneration at all for ‘Redheart’ (Tian et al., 2007b). This agrees with our results, where significant differences were observed according to the parental origin of the explants (P < 0.05) but not due to the levels of growth regulators. When hypocotyl slices of 13 plum (P. domestica) cultivars were placed in a medium containing 7.5 ␮M TDZ and 2.5 ␮M IBA, most of them showed regeneration at frequencies of 20–30%. However, two showed higher regeneration (58% and 57% for ‘Italian’ and ‘Stanley’, respectively) while

one showed poor regeneration competence (10%), indicating, once again, the high genotype dependence of the regeneration process (Tian et al., 2007a). Although the effect of the genotype is widely recognised, this is mitigated when a very-effective procedure is used. For instance, the procedure developed for European plum (Petri et al., 2008c) has been tested in our laboratory and it worked well for all the genotypes tested, the regeneration percentages ranged from 50% to 97%. Additionally, the procedure is being used successfully in different laboratories. In further studies, hypocotyl slices of several apricot cultivars may be tested with combinations of growth regulators in order to determine which cultivars show the highest regeneration capacity. Nevertheless, the regeneration rates from hypocotyl slices reported in this manuscript are comparable to those described for the European plum cultivar ‘Stanley’ (37.5%) and superior to those published for the Japanese cultivars ‘Angeleno’ and ‘Larry Anne’ (11.4% and 19.4%, respectively), which have been successfully transformed (Scorza et al., 1995, 1994; Urtubia et al., 2008). Moreover, the regeneration pattern, as clusters and/or as a “crown” (Fig. 1C and D), was similar to that reported for the high-throughput plum transformation system using mature hypocotyl slices (Petri et al., 2008c). This may increase the probability of the combination of regeneration and transformation events occurring in the same plant cell, especially if transformation occurs massively in the peripheral zone (Fig. 4C) where shoot regeneration is gener-

Table 2 Transformation of ‘Canino’ hypocotyl slices by AGL1 carrying p35SGUSINT based on the GUS assay. Antibiotic (10 ␮M)

Transformation rate (%)

No. of events/transformed explant

No. of transformed zones or calli/transformed explant

Paromomycin Kanamycin

97.8 ± 2.2 70.3 ± 7.5

10.1 ± 1.7 13.2 ± 2.2

3.5 ± 0.4 0.8 ± 0.2

A total of 83 explants were used in this study.

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

ally obtained. Microscopic examination of plum hypocotyl sections indicated that shoots originated de novo from meristematic cells in the sub-epidermal layers (Mante et al., 1991). Surprisingly, none of the factors affecting adventitious regeneration from apricot leaves known to us (Burgos and Alburquerque, 2003; Pérez-Tornero et al., 2000a; Petri et al., 2005b) significantly improved regeneration from ‘Canino’ mature seed hypocotyl sections. These results suggest that these factors or conditions are closely related to the source of the explants used, other than the genotype. Other authors also reported explant type as a key factor in adventitious regeneration in Prunus (Nas et al., 2010; PérezClemente et al., 2004). For example, peach hypocotyl slices did not show regeneration activity while embryo sections yielded up to 49.3% under the same conditions (Pérez-Clemente et al., 2004). However, most of these factors had not been studied previously in apricot or other Prunus species when mature seed hypocotyl slices were used as the source of explants. Only 2,4-D pre-treatments had been reported earlier to stimulate regeneration/transformation from European plum mature hypocotyl slices (Petri et al., 2008c). The length of the dark period affected significantly the adventitious shoot regeneration from apricot leaves. The regeneration was zero or very low with a dark period of 1 or 4 weeks and the best results were obtained with 2 or 3 weeks (Pérez-Tornero et al., 2000a). In our case, a dark period was not necessary to induce shoot regeneration. This agrees with previous reports for P. domestica (Gonzalez-Padilla et al., 2003; Petri et al., 2008c) and P. salicina (Tian et al., 2007b; Urtubia et al., 2008) hypocotyl explants, where dark incubation was not needed. It has been reported that the dark period can influence endogenous levels of growth regulators such as indole-3-acetic acid (IAA) (Lopez-Carbonell et al., 1992). The increase in the endogenous auxin levels during the dark period could have a detrimental effect on shoot regeneration from ‘Canino’ hypocotyl sections cultured in SRM, because it could lead to a low cytokinin:auxin ratio. This may explain also the increase in callogenesis observed with prolonged dark periods and when the 2,4-D concentration was increased in the auxin pre-treatment experiments. In our study, the explants were exposed to the antibiotic 3 days after they were placed in the SRM, simulating the real situation of a transformation experiment, in which explants are co-cultured with the agrobacteria for 3 days in the absence of any antibiotic before selection is applied. Sometimes, the inhibitory concentration is set by placing the explants immediately in the SRM with antibiotic, which could lead to a wrong selection strategy because it is advisable to establish the inhibitory doses in each medium and in each situation of the protocol, as suggested by Padilla and Burgos (2010). Plant susceptibility to antibiotics seems to change broadly among species, genotypes, and plant tissues (see review by Padilla and Burgos, 2010). Although both antibiotics tested were aminoglycosides, different effects were expected on the basis of their differing chemical structures and previous publications in apricot adventitious shoot regeneration. Regeneration from apricot leaves was affected in a different manner according to whether kanamycin or paromomycin was added to the SRM (Burgos and Alburquerque, 2003; Petri et al., 2005a). Regeneration from apricot leaf explants also resulted more sensitive to kanamycin than to paromomycin. While 40 ␮M kanamycin totally inhibited adventitious regeneration, 40 ␮M paromomycin still allowed the generation of some buds (Burgos and Alburquerque, 2003; Petri et al., 2005a). The fact that the transformation percentage was significantly higher and more transgenic calli or large transformed areas were observed when paromomycin was applied agrees with these previous studies, where paromomycin conferred a better advantage to nptII-transformed apricot cells than the other antibiotics tested (kanamycin, streptomycin, and geneticin) and produced a larger increase in fresh weight of the transformed apricot explants (Petri

463

et al., 2005a). Moreover, the use of paromomycin as the selective agent allowed the regeneration of transgenic apricot plants from ‘Helena’ leaf explants, while kanamycin remained inefficient (Petri et al., 2008a,b). Paromomycin has been successfully used in other woody species, such as apple (Norelli and Aldwinckle, 1993) and grape (Mauro et al., 1995; Wang et al., 2005). We have reported in this paper the regeneration of a putative chimerical bud when paromomycin was applied, perhaps due to the low selection pressure (10 ␮M). 5. Conclusions We have detailed a successful regeneration protocol for all the cultivars tested, ‘Canino’, ‘Moniquí’, and ‘Dorada’, from mature seed hypocotyl slices. The system might be transferable, with more or less efficiency, to other apricot cultivars. In addition, inhibition curves for two selective agents have been established, the susceptibility of the tissues to A. tumefaciens infection has been shown, and a putative chimerical plant regenerated. The procedure seems promising as an efficient transformation system for this species. Although transformation of seed-derived material is of limited use for improving vegetatively propagated apricot scion cultivars, it could have an impact on the development of transgenic rootstocks, particularly if using ‘Canino’ seeds. Additionally, an efficient apricot transformation procedure for seeds will allow the approach to be incorporated into proof-of-concept projects, that do not need to produce improved new cultivars, and functional genomics studies. Acknowledgments The authors wish to thank Dr. Ricardo Ordas for kindly providing the AGL1 strain, Dr. José Egea for providing ‘Dorada’ and ‘Moniquí’ fruits, Frutales Mediterraneo, SAT for providing ‘Canino’ seeds, and Dr. David J. Walker for English language correction. W.H. and C.P. acknowledge the financial support of a JAE fellowship and postdoctoral contract, respectively. This research was supported by the CICYT AGL2010-20270 project, co-financed by FEDER funds. References Burgos, L., Alburquerque, N., 2003. Low kanamycin concentration and ethylene inhibitors improve adventitious regeneration from apricot leaves. Plant Cell Rep. 21, 1167–1174. Canli, F.A., Tian, L., 2008. In vitro shoot regeneration from stored mature cotyledons of sweet cherry (Prunus avium L.) cultivars. Sci. Hortic. -Amsterdam 116, 34–40. Canli, F.A., Tian, L., 2009. Regeneration of adventitious shoots from mature stored cotyledons of Japanese plum (Prunus salicina Lind1). Sci. Hortic. -Amsterdam 120, 64–69. Driver, J.A., Kuniyuki, A.H., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19, 507–509. Escalettes, V., Dosba, F., 1993. In vitro adventitious shoot regeneration from leaves of Prunus spp. Plant Sci. 90, 201–209. Goffreda, J.C., Scopel, A.L., Fiola, J.A., 1995. Indole butyric acid induces regeneration of phenotypically normal apricot (Prunus armeniaca L.) plants from immature embryos. Plant Growth Regul. 17, 41–46. Gonzalez-Padilla, I.M., Webb, K., Scorza, R., 2003. Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (Prunus domestica L.). Plant Cell Rep. 22, 38–45. Jefferson, R.A., 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–405. Laimer da Câmara Machado, M., da Câmara Machado, A., Hanzer, V., Weiss, H., Regner, F., Steinkeliner, H., Mattanovich, D., Plail, R., 1992. Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of plum pox virus. Plant Cell Rep. 11, 25–29. Lane, W.D., Cossio, F., 1986. Adventitious shoots from cotyledons of immature cherry and apricot embryos. Can. J. Plant Sci. 66, 953–959. Lloyd, G., McCown, B., 1980. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc. Intl. Plant Prop. Soc. 30, 421–427. Lopez-Carbonell, M., Alegre, L., Prinsen, E., van Onckelen, H., 1992. Diurnal fluctuations of endogenous IAA content in aralia leaves. Biol. Plantarum 34, 223–227. López-Noguera, S., Petri, C., Burgos, L., 2009. Combining a regeneration-promoting gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes. Plant Cell Rep. 28, 1781–1790.

464

H. Wang et al. / Scientia Horticulturae 128 (2011) 457–464

Mante, S., Morgens, P.H., Scorza, R., Cordts, J.M., Callahan, A.M., 1991. Agrobacteriummediated transformation of plum (Prunus domestica L.) hypocotyl slices and regeneration of transgenic plants. Bio/Tech. 9, 853–857. Mante, S., Scorza, R., Cordts, J.M., 1989. Plant regeneration from cotyledons of Prunus persica, Prunus domestica, and Prunus cerasus. Plant Cell Tiss. Org. Cult. 19, 1–11. Mauro, M.C., Toutain, S., Walter, B., Pinck, L., Otten, L., Coutos-Thevenot, P., Deloire, A., Barbier, P., 1995. High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene. Plant Sci. 112, 97–106. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 15, 473–497. Nas, M.N., Bolek, Y., Sevgin, N., 2010. The effects of explant and cytokinin type on regeneration of Prunus microcarpa. Sci. Hortic. -Amsterdam 126, 88–94. Ning, G.G., Bai, S.P., Bao, M.Z., Liu, L., 2007. Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”. In Vitro Cell. Dev. Biol. - Plant 43, 225–230. Norelli, J.L., Aldwinckle, H.S., 1993. The role of aminoglycoside antibiotics in the regeneration and selection of neomycin phosphotransferase-transgenic apple tissue. J. Am. Soc. Hortic. Sci. 118, 311–316. Orero, G., Cuenca, J., Romero, C., Martínez-Calvo, J., Badenes, M.L., Llácer, G., 2004. Selection of seedling rootstocks for apricot and almond. Acta Hort. 658, 529–533. Padilla, I., Burgos, L., 2010. Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols. Plant Cell Rep. 29, 1203–1213. ˜ Pérez-Clemente, R.M., Pérez-Sanjuán, A., García-Férriz, L., Beltrán, J.P., Canas, L.A., 2004. Transgenic peach plants (Prunus persica L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an in vivo marker. Mol. Breed. 14, 419–427. Pérez-Tornero, O., Burgos, L., 2000. Different media requirements for micropropagation of apricot cultivars. Plant Cell Tiss. Org. Cult. 63, 133–141. Pérez-Tornero, O., Burgos, L., Egea, J., 1999. Introduction and establishment of apricot in vitro through the regeneration of shoots from meristem tips. In Vitro Cell. Dev. Biol. - Plant 35, 249–253. Pérez-Tornero, O., Egea, J., Vanoostende, A., Burgos, L., 2000a. Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot. Plant Sci. 158, 61–70. Pérez-Tornero, O., López, J.M., Egea, J., Burgos, L., 2000b. Effect of basal media and growth regulators on the in vitro propagation of the apricot (Prunus armeniaca) cv. ‘Canino’. J. Hortic. Sci. Biotech. 75, 283–286. Petri, C., Alburquerque, N., Burgos, L., 2005a. The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of nptII-transformed leaf tissues. Plant Cell Tiss. Org. Cult. 80, 271–276. Petri, C., Alburquerque, N., Pérez-Tornero, O., Burgos, L., 2005b. Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve

adventitious regeneration from apricot leaves and Agrobacterium-mediated transformation of leaf tissues. Plant Cell Tiss. Org. Cult. 82, 105–111. Petri, C., López-Noguera, S., Alburquerque, N., Egea, J., Burgos, L., 2008a. An antibioticbased selection strategy to regenerate transformed plants from apricot leaves with high efficiency. Plant Sci. 175, 777–783. Petri, C., Wang, H., Alburquerque, N., Faize, M., Burgos, L., 2008b. Agrobacteriummediated transformation of apricot (Prunus armeniaca L.) leaf explants. Plant Cell Rep. 27, 1317–1324. Petri, C., Webb, K., Hily, J.M., Dardick, C., Scorza, R., 2008c. High transformation efficiency in plum (Prunus domestica L.): a new tool for functional genomics studies in Prunus spp. Mol. Breed. 22, 581–591. Pieterse, R.E., 1989. Regeneration of plants from callus and embryos of ‘Royal’ apricot. Plant Cell Tiss. Org. Cult. 19, 175–179. Quoirin, M., Lepoivre, P., 1977. Etude de milieux adaptes aux cultures in vitro de Prunus. Acta Hort. 78, 437–442. SAS Institute, I., 1988. SAS/STAT User’s Guide. Release 6.03. SAS Institute, Inc., Cary, NC. Scorza, R., Levy, L., Damsteegt, V.D., Yepes, L.M., Cordts, J.M., Hadidi, A., Slightom, J., Gonsalves, D., 1995. Transformation of plum with the papaya ringspot virus coat protein gene and reaction of transgenic plants to plum pox virus. J. Am. Soc. Hortic. Sci. 120, 943–952. Scorza, R., Ravelonandro, M., Callahan, A.M., Cordts, J.M., Fuchs, M., Dunez, J., Gonsalves, D., 1994. Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene. Plant Cell Rep. 14, 18–22. Tian, L., Sibbald, S., Subramanian, J., Svircev, A., 2007a. Characterization of Prunus domestica L. in vitro regeneration via hypocotyls. Sci. Hortic. -Amsterdam 112, 462–466. Tian, L., Wen, Y., Jayasankar, S., Sibbald, S., 2007b. Regeneration of Prunus salicina Lindl (Japanese plum) from hypocotyls of mature seeds. In Vitro Cell. Dev. Biol.: Plant 43, 343–353. Urtubia, C., Devia, J., Castro, A., Zamora, P., Aguirre, C., Tapia, E., Barba, P., Dell’Orto, P., 2008. Agrobacterium-mediated genetic transformation of Prunus salicina. Plant Cell Rep. 27, 1333–1340. Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L., Rocha-Sosa, M., 1990. Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220, 245–250. Wang, Q., Li, P., Hanania, U., Sahar, N., Mawassi, M., Gafny, R., Sela, I., Tanne, E., 2005. Improvement of Agrobacterium-mediated transformation efficiency and transgenic plant regeneration of Vitis vinifera L. by optimizing selection regimes and utilizing cryopreserved cell suspensions. Plant Sci. 168, 565–571.