Plant Cell Rep (2008) 27:11971206 DOI 10.

1007/s00299-008-0540-y

GENETIC TRANSFORMATION AND HYBRIDIZATION

In planta transformation of Notocactus scopa cv. Soonjung by Agrobacterium tumefaciens

Euna Seol Yuchul Jung Jungjin Lee Changhui Cho Taihyun Kim Yong Rhee Sukchan Lee

Received: 9 December 2007 / Revised: 4 March 2008 / Accepted: 6 March 2008 / Published online: 27 March 2008 Springer-Verlag 2008

Abstract Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum inltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum inltration (2030 cmHg for 15 min) resulted in a transformation efciency of 67100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efciency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efcient and timesaving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species. Keywords Cactus Notocactus scopa cv. Soonjung Agrobacterium tumefaciens In planta transformation

Abbreviations GUS b-Glucuronidase nptII Neomycin phoshotransferase X-Gluc 5-Bromo-4-chloro-3-indolyl-b-D-glucuronic acid YEP Yeast extract peptone MS Murashige and Skoog media

Introduction Notocactus scopa, generally referred to as the Silver ball cactus, is a xerophytic succulent native to Southern Brazil and Uruguay, and is commonly cultivated as a potted plant (Heywood et al. 2007). Due to the white-silver woolly hairs on its attractive globose or cylindroid green body (bulb), the genetic improvement of N. scopa for additional ornamental traits has been a primary eld of interest to cactus researchers in the oriculture industry. However, cactus improvement schemes have been principally dependent on classical breeding methods, which have imposed many limitations on the cactus breeding program. These classical methods include a series of hybridization processes followed by continual selection, limited genetic resources, etc, thereby resulting in the sluggish pace of new cultivar development. Therefore, all of these situations necessitate novel molecular biological approaches, which are hoped to eventually improve the rate of improved cactus development. With regard to the molecular breeding of cactus, a stable transformation system should be established for the introduction of a target gene into the cactus genome. However, cactus transformation techniques are still in their infancy, and the majority of gene delivery approaches have involved in vitro culture systems in which the transformation efciency is

Communicated by J.R. Liu. E. Seol Y. Jung Y. Rhee (&) S. Lee (&) Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea e-mail: rheepd@skku.edu S. Lee e-mail: sukchan@skku.ac.kr J. Lee C. Cho Cactus Research Institute, Gyeonggi-do Agricultural Research and Extension Services, Goyang 411-809, Korea T. Kim Invitroplant Co. Ltd, Suwon 441-814, Korea

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relatively low, 22.7% for Rhipsalidopsis cv. CB5 (Easter cactus) (Al-Ramanmeh et al. 2006) and 3.2% for Opuntia cus-indica L. cv. Villa Nueva (prickly-pear cactus) (SilosEspino et al. 2006), respectively. Moreover, the in vitro culture process requires contamination-sensitive aseptic techniques and a series of time-consuming selections for authentic transformants on putative prospects. The genome stability of transgenic cactus species will be also (epi)genetically challenged during an in vitro tissue culture. It has been generally established that a new cactus bud (clone) can be developed from an areole (areola is a little area or open space in Latin; this term refers to a localized small area with potential meristem) asexually on the mother cactus (Fig. 2b), and this regeneration process can be promoted by physical stresses including cutting and scarring on the body itself (Rowley 2003; Jeong et al. 2004). Thus, the cactus is considered to be a good candidate for in planta transformation in plants. Several strategies have been developed for the simple and efcient delivery of foreign genes directly into the plant itself. In a few previous studies conducted regarding the in planta transformation of Arabidopsis, the plant was immersed in Agrobacterium-harboring inltration medium in a vacuum chamber, and was then placed under vacuum pressure. Gene delivery was completed when the vacuum was released (Bechtold et al. 1993; Ye et al. 1999). Infectious viral DNA was also directly agro-inoculated into Arabidopsis via needle puncturing (pin-pricking) of the plant leaves (Briddon et al. 1989; Stenger et al. 1992; Lee et al. 1994). In the present study, an efcient and reliable in planta Agrobacterium-mediated transformation system for Notocactus scopa cv. Soonjung (its abbreviation, N. scopa cv. Soonjung will be used over all of text) via vacuum inltration and pin-pricking is presented. Additionally, its application to other cactaceae is described herein.

dark). For in planta transformation, the roots were removed from the engrafted cactus in order to prevent bottom rot. Plasmid vector and Agrobacterium tumefaciens preparation The binary pBI-121 plasmid (Jefferson 1987) was utilized for the construction of the transformation vector. The b-glucuronidase (uidA) gene was driven by the cauliower mosaic virus (CaMV) 35S promoter as a reporter gene to conrm transgenic expression. The plasmid vector was then introduced into the cells of Agrobacterium strain LBA4404 via heat-shock transformation. The transformed Agrobacterium was cultured in YEP media (10 g l-1 bacto peptone, 10 g l-1 yeast extract and 5 g l-1 NaCl) containing rifampicin, streptomycin, and kanamycin at 28C. The culture at OD600 (0.60.8) was re-suspended in one volume of transformation buffer (2.2 g l-1 MS salts, 50 g l-1 sucrose, 0.5 g l-1 2-(N-morpholino) ethanesulfonic acid (MES), 200 ll l-1 VAC-IN-STUFF (Sillwet L-77) (LEHLE SEEDS, USA, Cat #VIS-02) and 200 lM acetosyringone (pH 5.7)). Transformation As shown in Fig. 1, four principal approaches were utilized to introduce Agrobacterium harboring the b-glucuronidase (uidA) gene directly into N. scopa cv. Soonjung with modications of the in planta transformation of Arabidopsis thaliana (Bechtold et al. 1993; Ye et al. 1999; Martinez-Trujillo et al. 2004): vacuum inltration (Type I), pin-pricking (Type II), and pin-pricking combined with vacuum inltration on pre-top-cutting-off (Type III) and post-top-cutting-off (Type IV). For vacuum inltration (Type I), the intact cactus bulb was soaked into a transformation buffer mixture with Agrobacterium and was then subjected to a series of vacuum pressures (30, 40, and 50 cmHg) for 30 min each in a tightly sealed vessel. The degassing process released vacuum pressure from the container and completed the forceful uptake of Agrobacterium into the cactus tissue. Another approach involved the pricking of the transformation buffer mixture-soaked cactus bulbs using a ne insect pin (needle) (Elefant, Australia, Cat #26000-25) ve to ten times (Type II). This procedure facilitates the permeation of the buffer mixture with Agrobacterium into the cactus bulb through minute pinholes. The upper one-third of the bulb top was cut to induce the regeneration of cactus buds after each of the two treatments described as methods Type I and Type II. Alternatively, the tops of the pin-pricked cactus bulbs were pre-cut (Type III) and post-cut (Type IV) to inltrate the bulb with a transformation buffer mixture under four vacuum pressure conditions (10, 20, 30, and 50 cmHg) for

Materials and methods Plant material and growth conditions N. scopa cv. Soonjung was developed originally through the inter-crossing of Notocactus scopa cv. Sojungs and the selection of progenies for small bulb size and soft silver-colored needles by the Cactus Research Institute (http://www.nongup.gyeonggi.kr/Web/unit/suninjang/html/ index.jsp, Gyeonggi-do, Korea). The small bulb of N. scopa cv. Soonjung was engrafted on the stock cactus, Hylocereus trigonus (its abbreviation, H. trigonus will be used over all of text), for mass propagation (Fig. 2a), and the plant was grown for 56 weeks at 2230C in a greenhouse under short-day conditions (10 h light/14 h

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Plant Cell Rep (2008) 27:11971206 Fig. 1 In planta transformation methods for N. scopa cv. Soonjung. Two principal technical approaches, vacuum inltration and pin-pricking, were conducted to deliver Agrobacterium into the N. scopa cv. Soonjung in separate (Type I and II, respectively) or combined fashion (Type III and IV). Type I vacuum inltration, Type II pin-pricking, Type III pin-pricking combined with vacuum inltration (pre-topcutting-off), Type IV pinpricking combined with vacuum inltration (post-top-cuttingoff). See the text for more details

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15 min each. Finally, all treated cactus bulbs were dried under dark conditions for 24 h and compelled to produce roots (2530C for 1014 h). Three in planta transformation methods were employed for another cactus cultivar, H. trigonus. Two approaches were premised on the same rationales as the Type I and II methods for the transformation of N. scopa cv. Soonjung. Vacuum inltration was conducted under ve vacuum pressure conditions (10, 20, 30, 40, and 50 cmHg) for 15 min each after H. trigonus was soaked in the transformation buffer mixture. The insect pin-pricking was administered to the cactus prior to the inltration of the transformation buffer mixture into it under a series of vacuum conditions (10, 20, 30, 40, and 50 cmHg for 15 min each). Finally, the Type II method used for N. scopa cv. Soonjung was modied with a syringe injection for the transformation of H. trigonus. The transformation buffer mixture (0.1 ml) was directly injected into the cactus by pricking with a syringe needle 5, 10, and 15 times, respectively. b-glucuronidase (GUS) assay GUS activity was histochemically conducted (Jefferson 1987) in order to assess the uidA gene expression in

transgenic cactus. The transformed cacti were xed in 90% of pre-chilled acetone (-20C) and washed once with staining buffer (50 mM sodium phosphate buffer (pH 7.2), 0.1% Triton X-100, 2.0 mM potassium ferricyanide, 2.0 mM potassium ferrocyanide, and 10 mM EDTA (pH 7.0)). The samples were incubated overnight in staining buffer with 2.0 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid (X-Gluc) at 37C and de-stained with ethanol (70%) at 37C. Genomic DNA isolation Total genomic DNA was isolated via the phenol method (Sambrook et al. 1989). The collected tissues were ground in liquid nitrogen and cell lysis was conducted with 1.0 ml of lysis buffer [0.05 M Tris-HCl (pH 8.0), 0.1 M NaCl, 0.05 M EDTA, 0.5% SDS and 0.01 M 2-hydroxyethylmercaptan (b-mercaptoethanol)]. After the addition of one volume of isopropanol to the supernatant, the samples were subjected to 5 min of centrifugation (14 9 1,000 rpm) to precipitate the DNA pellets at room temperature. The DNA pellet was washed with 70% ethanol and dissolved in 500 ml of TNE buffer (0.01 M Tris-HCl (pH 8.0), 0.1 M NaCl and 1.0 mM EDTA).

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Genomic polymerase chain reaction (PCR) Genomic PCR was conducted in order to verify the presence of the transgene in the transformed cacti. PCR amplications were conducted under the following conditions: 19 PCR buffer, 0.3 mM dNTPs, 0.1 unit Taq polymerase (RexGene Biotech, Korea), 0.5 pmol primers each, 50 ng of genomic DNA. The oligonucleotide primer pairs, 50 -CAACGAACTGAACTGGCAGA-30 and 50 -AG AGGTTAAAGCCGACAGCA-30 were utilized for the amplication of the uidA gene while 50 -GCTTGGG TGGAGAGGCTATT-3 and 50 -GAACTCGTCAAGAAGG CGATA-30 were used for the amplication of the neomycin phosphotransferaseII (nptII) gene. The PCR reaction was conducted under the following conditions: the rst denaturing cycle (denaturation at 94C for 4 min 30 s, annealing at 55C for 30 s and extension at 72C for 1 min), the second amplication cycle (denaturation at 94C for 30 s, annealing at 55C for 30 s and extension at 72C for 40 s with 34 cycles), and 10 min of nal extension at 72C. The PCR products were assessed by 1% agarose gel electrophoresis. Total RNA isolation The tissue samples were collected and freeze-dried immediately in liquid nitrogen, then ground. Total RNA isolation was conducted with TRIZOL reagent (Invitrogen, US, Cat #15596-026). Approximately 50100 mg of ground tissue was mixed with 1 ml of TRIZOL and incubated for 5 min at room temperature. 200 ll of chloroform was then added to each of the samples. The mix was incubated for 2 min at room temperature and centrifuged (14 9 1,000 rpm) for 30 min at 4C. After the supernatant was transferred to a new tube, 1.0 ml of isopropanol was added to the supernatant. The mixture was incubated for 10 min at room temperature and centrifuged (14 9 1,000 rpm) at 4C for 15 min. The RNA pellet was collected and washed with 75% ethanol. The pellet was then dissolved in nuclease-free water. Complementary DNA synthesis and reverse transcription (RT)-PCR Reverse transcription was conducted in order to synthesize the rst strand of complementary DNA according to the following procedure: after 10 min of incubation of 5.0 lg of total RNA with 1.2 pmol of oligo-dT primer in diethylpyrocarbonate (DEPC) treated distilled water at 70C, 0.6 mM dNTPs and 19 moloney murine leukemia virus (MMLV) RT reaction buffer was added to the reaction at 4C. Later, a brief incubation was conducted for 10 min at 37C, and 0.2 units of MMLV reverse

transcriptase (RexGene Biotech, Korea) was subsequently added at 4C. Finally, the reaction was incubated for 1 h at 37C and for 10 min at 70C. PCR transcript analysis was conducted using 4 ll of synthesized cDNA, 19 PCR reaction buffer, 0.3 mM dNTPs, 0.1 unit Taq polymerase (RexGene Biotech, Korea), 0.5 pmol of forward and reverse primers each, and distilled water. The primer sets and PCR conditions utilized were identical to those of the genomic PCR. The PCR products were then analyzed by 1% agarose gel electrophoresis. Northern blot anaysis Total RNA was extracted from fresh cactus tissues by using the TRIZOL reagent (Invitrogen, Life technologies, USA) and all the steps followed the instructions of the manufacturer. RNA transcript analysis was conducted by a standard Northern blotting (Sambrook et al. 1989) with small modications. Total RNA (20 lg) was resolved by electrophoresis on 1.2% agarose gel containing 0.66 mol l-1 formaldehyde in 19 MOPS buffer and blotted onto a Hybond-N+ nylon membrane (GE Healthcare, Amersham, UK) in 109 SSC buffer. The nylon membranes were air-dried and cross-linked by UVC 500 Cross-Linker (Amersham Biosciences, UK). The nylon membranes were then pre-hybridized for 1 h at 42 C and hybridized with P-32 labelled uidA gene probe for 20 h at 42 C, and then washed with 29 SSC and 0.1% SDS for 15 min at 42C. Western blot analysis The transgenic N. scopa cv. Soonjung tissues were ground in liquid nitrogen and each sample (50100 mg) was mixed with 500 ll of protein extraction buffer [100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 5% (w/v) sucrose, 5% (w/v) b-mercaptoethanol]. After vortexing, the sample was placed on ice for 20 min and immediately subjected to 20 min of centrifugation (15 9 1,000 rpm). Finally, the supernatants were collected and Western blot analysis was conducted to conrm the expression of the nptII gene for the transgenic cactus. The total protein (0.6 lg) of the cactus was boiled for 5 min at 95C with 29 protein loading buffer [2.4 g of urea, 20 ll of ampholyte solution (pH 3.510), 100 ll of ampholyte solution (pH 46), 500 ll of 20% (w/v) Triton X-100, 50 ll of b-mercaptoethanol and 200 ll of 1% (w/v) bromophenol blue in total volume of 5 ml] and electrophoresed in 12% SDS polyacrylamide gel. The separated protein was then transferred to a nitrocellulose membrane (Amersham Bioscience, UK, Cat #PRN303F) and immunoblotting was conducted using the commercial primary antibody against the nptII protein (Sigma-Aldrich, USA, Cat #6537) and the secondary

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antibody (Amersham Bioscience, UK, Cat #338280) specic to the nptII protein. For detection, the membrane was soaked with a horseradish peroxidase (HRP) detection kit (PIERCE, UK, Cat #34080) for 10 min and developed on X-ray lm (AGFA, Bel, Cat #79510099).

Results In planta transformation of cactus For all of the transformation approaches utilized here, two criteria were evaluated with regard to their practical applications to cactus transformation (Tables 1, 2). The survival rates were initially measured in terms of the number of surviving cacti (Survival number) over the total number of treated cacti (Execution number). Furthermore, the transformation efciency was calculated on the basis of the percentage of positively GUS-stained cacti over the total number of treated cacti with each approach. In the N. scopa cv. Soonjung transformation, vacuum inltration (Type I) showed the lowest survival rates (00.5%) among the four methods utilized with the cactus in the present study. However, as is shown in Table 1, the transformation efciencies were considerably high, between 75100% for the T0 and R1 cacti. We noted a transitional decrease in transformation efciency from the T0 generation (90%) to the R1 bud generation (72.7%) with

a 71.3% survival rate in the pin-pricking method group (Type II) (Table 1). However, it was also shown that the survival rates (034%) were quite lower than those of the Type II (71.3%) and Type IV method groups (5085%) in the pin-pricking method group combined with vacuum inltration on pre-top-cutting-off (Type III). Transformation efciencies were shown to be decreased from 100 to 66.6% in T0 to R1 generation in the Type III method group (Table 1). The highest survival rate (5085%) for the treated cactus bulbs was obtained from the pin-prickling method combined with vacuum inltration on posttop-cutting-off (Type IV) among the four methods tested for N. scopa cv. Soonjung. However, the transformation frequencies also decreased from T0 (90100%) to R1 (66.675%) at the given vacuum duration time (15 min). H. trigonus was subjected to the treatments employed for N. scopa cv. Soonjung with minor modications (Table 2). The hypodermic syringe injection technique left no surviving transgenic cacti (0% of survival rates) and 90100% of the treated cacti survived in the vacuum inltration and pin-pricking groups as combined with inltration under the given vacuum pressures. However, no transformed cacti were rescued for the vacuum-inltrated cactus under pressures of 10 and 20 cmHg for 15 min each. Thus, transformation efciency was not available for these two conditions at this point. This result is in sharp contrast to the vacuum inltration method, which resulted in 100% transformation efciency in both the T0 and R1 cacti.

Table 1 In planta transformation for N. scopa cv. Soonjung Method Pressure Duration Execution Survival Survival Transgenic cactus (R1) (cmHg) (min) number number rate (%)b Numberc Efciency (%)d Numberc 50 40 30 Pin-pricking (Type II) Pre-top- cutting-off (Type III) 30 30 30 210 210 520 150 20 90 44 68 20 20 20 20 1 0 2 107 0 13 15 11 10 16 17 17 0.5 0.0 0.4 71.3 0.0 14.4 34.0 16.1 50.0 80.0 85.0 85.0 1/1 ND 2/2 4/5 ND 3/3 5/5 3/3 3/3 5/5 5/5 4/5 100 ND 100 90 ND 100 100 100 100 100 100 90 2/2 ND 3/4 8/11 ND 2/3 2/3 2/3 2/3 2/3 3/4 3/4

Efciency (%)d 100 ND 75.0 72.7 ND 66.6 66.6 66.6 66.6 66.6 75.0 75.0

Vacuum inltration (Type I)

510 timesa 50 30 20 10 15 15 15 15 15 15 15 15

Pin-pricking combined with vacuum inltration

Post-top- cutting-off (Type IV) 50 30 20 10

a b c d

Frequency of pin-pricking Survival rate = (Execution number/survival number) 9 100 GUS Stained samples/total number of the samples Transformation efciency = (GUS stained samples/total number of the samples) 9 100

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1202 Table 2 In planta transformation for H. trigonus Method Pressure (cmHg) Duration (min) Execution number Survival number

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Survival Transgenic cactus (T0) Transgenic cactus (R1) rate (%)c Numberd Efciency Numberd Efciency e (%) (%)e ND ND ND 95 100 100 100 100 90 95 95 100 100 ND ND ND 4/4 4/4 4/4 0/2 0/2 4/4 4/4 4/4 4/4 3/4 ND ND ND 100 100 100 0 0 100 100 100 100 75 ND ND ND 3/3 3/3 3/3 0/2 0/2 3/3 3/3 3/3 2/3 2/3 ND ND ND 100 100 100 0 0 100 100 100 67 67

Syringe injection

0.1 mla 0.1 ml

a

5 timesb

10

0 0 0 19 20 20 20 20 18 19 19 20 20

10 timesb 10 15 timesb 10 15 20 15 15 15 15 15 15 15 15 15 20 20 20 20 20 20 20 20 20

Vacuum inltration

0.1 mla 50 40 30 20 10

Pin-pricking combined with vacuum inltration

50 40 30 20 10

a b c d e

The volume of transformation buffer mixture with Agrobacterium Frequency of pin-pricking Survival rate = (Execution number/survival number) 9 100 GUS Stained samples/total number of the samples Transformation efciency = (GUS stained samples/total number of the samples) 9 100

Molecular analysis of transgenic cactus The identication of each transgenic cactus line is provided in order of experimental year, transformation method type, the serial number of the T0 cactus, followed by its R1 bud number. For instance, line 061-1-1 indicates the #1 transgenic R1 bud generated on the #1 T0 cactus transformed by the Type I transformation method (in this case, vacuum inltration) conducted in 2006 (Refer to the Results section for the identication of each R1 bud progeny). Basically, ten of the R1 cactus progenies were selected from the collection of the transgenic N. scopa cv. Soonjung R1 generation obtained by each of four transformation approaches (Type I, II, III and IV); one (061-1-1) from Type I, two (062-4-1, 062-5-2) from Type II, three (063-31, 063-5-3, 063-7-1) from Type III, four (064-2-2, 064-261, 064-34-2, 064-72-2) from Type IV. Additionally, genomic PCR (Fig. 3a) and RT-PCR (Fig. 3b) were conducted in order to conrm the presence of transgenes (uidA and nptII) on the nucleic acid levels in the selected R1 cacti. In fact, all of the transgenic R1 individuals evidenced the expected PCR products for the uidA and nptII genes in genomic PCR and RT-PCR, respectively. Four of the transgenic R1 cacti (061-1-1, 063-3-1, 064-22 and 062-5-2) were further selected from the PCR results for steady-state transcript analysis on the inserted uidA gene by Northern blotting (Fig. 3c). As expected, the uidA

transcript was detected in all of the examined cacti transformants except wild-type. Transgene was nally conrmed at the protein level in the selected transformants. At this time, the nptII protein in the transgene construct of the pBI-121 vector was targeted for Western blotting (Fig. 3d). The nptII protein was detected in all of the selected R1 cacti; in particular, a R1 progeny, 063-3-1, evidenced the most profound nptII protein expression in the four R1 individuals examined, whereas the wild-type (control) N. scopa cv. Soonjung evidenced no nptII protein expression. For H. trigonus, molecular analysis could not be attempted for the transgenic cactus due to the poor extraction of nucleic acids and protein from the tissues containing polysaccharides and phenolic compounds. Histochemical GUS assay All of the transformation techniques in this study evidenced uidA gene expression with variable expression rates by GUS staining in both N. scopa cv. Soonjung and H. trigonus (Tables 1, 2). Two transgenic R1 cacti, 063-3-1 (for the N. scopa cv. Soonjung) and 073-6-1 (for H. trigonus) were analyzed by GUS staining together with the respective control cacti. The results indicated that uidA gene expression was not detected in the control cacti and their R1 cactus (buds) of N. scopa cv. Soonjung (Fig. 2c, e, g) and H. trigonus (Fig. 4b, c) whereas positive b-glucuronidase

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Fig. 2 Histochemical GUS assay on the control cactus and a transgenic N. scopa cv. Soonjung R1 cactus (063-3-1). a The N. scopa cv. Soonjung globose bulb (the upper part) engrafted upon the stock cactus, Hylocereus trigonus (the lower part). b New cactus buds developed asexually from the top-cut cactus bulb. In this case, the small cactus buds (R1) were regenerated from the T0 cactus bulb of the N. scopa cv. Soonjung transformed by Type IV. c, e, g The control N. scopa cv. Soonjung after GUS staining. The b-glucuronidase activity was neither detected in the cactus bulb nor in the new cactus bud regenerated on it (c). After GUS staining, the whole cactus bud (e) and its longitudinal section (g) evidenced no b-glucuronidase activity in the completely regenerated small cactus. d, f, h The transgenic N. scopa cv. Soonjung R1, 063-3-1 after GUS staining. By contrast with the control Notocactus cv. Soonjung with the small cactus bud (c), the primary transformant (T0) evidences partial b-glucuronidase activity, principally in the epidermal area around the areole spot from which a new transgenic cactus (R1) is asexually regenerated (d). The R1 cactus bud (f) and its longitudinal section (h) evidence b-glucuronidase activity overall. (Bars in a, b, c, d 10 mm. Bars in e, f, g, h 5 mm)

Fig. 3 Molecular analysis of the transgenic N. scopa cv. Soonjung R1 cactus. a Genomic PCR was conducted to verify the presence of uidA gene (as the reporter gene) and nptII gene (as the selectable marker gene) in 10 of the transgenic R1 cacti selected for each of the four transformation method types (061-1-1, 062-4-1, 062-5-2, 063-3-1, 063-5-3, 063-7-1, 064-2-2, 064-26-1, 064-34-2, 064-72-2). (): negative control (distilled water), (+): positive control (pBI-121 plasmid DNA). b The expression of uidA gene and the nptII gene was detected by RT-PCR in the same transgenic R1s selected for genomic PCR. c The steady-state RNA transcript was analyzed for the transgene (uidA gene) by Western blotting in four R1 cacti (061-1-1, 062-5-2, 063-3-1, and 064-5-2) selected from ten of the transgenic cacti examined. The uidA transcript was detected in all of four cactus transformants beside wild-type. d The four R1 cacti (061-1-1, 062-52, 063-3-1, and 064-5-2) were further analyzed via Western blotting to detect the transgenic expression on the protein level. In this case, nptII gene expression was assessed using the antibody against the nptII protein

was observed between the control areole (Fig. 4d) and the transgenic R1 cactus areole (Fig. 4g) for H. trigonus.

Discussion In this study, we have reported for the rst time, an in planta Agrobacterium tumefaciens-mediated transformation system for the cactus. The results acquired with GUS staining, genomic PCR, RT-PCR, Northern blotting, and Western blotting conrmed the presence of the uidA gene (Figs. 2d, f, h and 3a, b, c) and nptII gene (Fig. 3a, b, d) in the transgenic cultivar N. scopa cv. Soonjung. The positive

(GUS) activities were detected in both the transgenic T0s and their R1 cacti [063-3 and 063-3-1 for the N. scopa cv. Soonjung (Fig. 2d, f, h), 073-6 and 073-6-1 (Fig 4e, f) for H. trigonus]. In particular, clearly contrasting GUS staining

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Fig. 4 Histochemical GUS assay for H. trigonus (073-6). a Asexually regenerated cactus (buds) from areoles on the mother cactus. b, c, d The regenerated control H. trigonus cactus (R1) after GUS staining. b-glucuronidase activity was detected neither in the inner tissue (transverse section) (b) nor over the cactus body (c). The enlarged areoles also evidence no b-glucuronidase activity (d).

e, f, g A transgenic R1 cactus (073-6-1) after GUS staining. Its transverse tissue section (e) and the whole cactus (f) both evidence b-glucuronidase activity. The areole also evidences quite clear blue color as the positive indicator of b-glucuronidase activity (g). (Bars in a, b, c, e, f 10 mm. Bars in d and g 4 mm)

transformants were also conrmed for H. trigonus via histochemical assay for b-glucuronidase (GUS) activity (Fig. 4e, f, g). The four transformation approaches utilized here (Type I, II, III and IV) were designed on the basis of the previous strategies for in planta agro-transformation (Bechtold et al. 1993; Ye et al. 1999; Martinez-Trujillo et al. 2004) and agro-inoculation (Briddon et al. 1989; Stenger et al. 1992; Lee et al. 1994) of Arabidopsis, and evaluated to determine the optimal protocol in terms of survival rate and transformation efciency for the in planta transformation of N. scopa cv. Soonjung (Fig. 1; Table 1). All of the transformation types evidenced reasonable transformation efciencies of 90100% and 66.6100% in the T0 cactus and R1 cactus (buds), respectively. A positive transformation signal (indicated as blue color for GUS staining) can be generated from both primary transformants (transient b-glucuronidase activity) and authentic ones (stable b-glucuronidase activity) of areoles on the T0 cactus, and the R1 cactus buds and only with GUS activities would be the authentic transformants regenerated from stably transformed areoles on the T0 cactus. Therefore, the number of transformants selected in generation R1 will be smaller than in generation T0, and thus we noted a reduction in net transformation efciency from the T0 to R1 cacti. This may explain transitional differences in transformation efciencies from the T0 to R1 cactus generally detected in the transformation methods used in the present study. The survival rates varied considerably among the tested methods. The vacuum inltration (Type I) has the lowest survival rates of the four methods. More cacti survived in the group subjected to pin-pricking combined with vacuum inltration on post-top-cutting-off (Type IV) than in the group subjected to pin-pricking combined with vacuum

inltration on pre-top-cutting-off (Type III) (Table 1). The pin-pricking method (Type II) yielded a moderate survival rate. It is most probable that the cactus tissue/cells were damaged severely by vacuum inltration (Type I) with longer vacuum duration times (30 min) than the other three methods, and thus only a few specimens were able to overcome the physical stress, even with a large number of treated cacti (Execution number). Physical damage to the cactus cells appeared to be minimized in the group subjected to pin-pricking (Type II) targeted to areole spots on the cactus bulb. Agrobacterium could attack intact totipotent cells via minute holes made around the areoles. In contrast, strong vacuum suction can threaten the cell integrity to crush down the areole cells. This is manifested in the higher survival rate(s) in the pinpricking group (Type II) than in the vacuum inltration group (Type I). However, vacuum inltration (Type I) will provide securer delivery of Agrobacterium into cactus cells than pin-pricking (Type II). Thus, a sizable proportion of stable/ authentic R1 transgenic cacti (buds) will be regenerated from the majority of transient T0 transgenic cacti and higher transformation efciencies can be achieved in the vacuum inltration group (Type I) than in the pin-pricking group (Type II). When pin-pricking is combined with vacuum inltration, the top-cut cactus bulb after pin-pricking but prior to vacuum inltration (Type III) may be more responsive to damage than the specimens subjected to top-cutting after two subsequent treatments (Type IV). Moreover, it was observed that the cut area underwent severe necrosis when exposed to the transformation buffer mixture (data not shown). Ultimately, all of these factors can result in a low

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Plant Cell Rep (2008) 27:11971206 Table 3 Comparison of this study with other previous studies about Agrobacteriummediated cactus transformation

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Transformation Method Efciency Tissue culture Target Months Species

This study In planta vacuum inltration and/or pin-pricking 68.5% Not required Areole \3 months Notocactus spora, Hylocereus trigonus

Silos-Espino group Injection by hypodermic syringe needle 3.2% Required Explants [3 months Opuntia cus-indica

Al-Ramamneh group Co-cultivation of callus 22.7% Required Callus [12 months Rhipsalidopsis gaertneri

number of surviving T0s. In particular, we noted that vacuum pressure of above 30 cmHg might be sufciently high to exert a negative effect on the viability of promising transformed somatic cells around the areoles on the T0 cactus to regenerate the transgenic R1 cactus buds. In fact, no data was available for transformation efciency due to the fact that we noted no T0 survival at 50 cmHg for 15 min. It is most probable that the cactus bulb in Type IV has relatively higher tolerance to physical damages than the Type III bulbs. The Type IV bulbs showed high transformation efciencies with signicant survival rates in comparison with the other three types. The minute pinholes on the areoles could maintain the integrity of areole cells on the cactus surface principally via the absorption of the detrimental impact induced by vacuum-mediated pressurization. It is also possible that the accessibility of Agrobacterium to potential cactus cells was facilitated efciently through holes via pin-pricking of the areoles. Finally, vacuum-mediated pressurization will improve the forceful delivery of Agrobacterium into the cactus cells through the holes. All four treatments considered, it has been proposed that the most promising in planta transformation can be conducted via pin-pricking combined with vacuum inltration (10 and 20 cmHg for 15 min each) prior to bulb top-cutting off (Type IV). This demonstrated transformation efciencies (T0s: 90 and 100%, R1s: 66.7 and 75%) with reasonably high survival rates (up to 85%) for N. scopa cv. Soonjung. In addition, the modied transformation approaches utilized for N. scopa cv. Soonjung were conducted for the in planta transformation of the stock cactus, H. trigonus (Table 2). A hypodermic syringe needle was utilized in the injection of the transformation buffer mixture into areoles on the cactus body. It is believed that the direct injection of Agrobacterium into the areoles via a hypodermic syringe needle exerts harmful effects on cactus tissues. In fact, tissue necrosis occurred around the injected areoles, resulting in the death of the cactus. This is the principal reason for the observed 0% survival rate. However, in the

vacuum inltration group and the combined method of pinpricking and vacuum inltration group, H. trigonus was shown to be more tolerant to physical damage/stress than was the relatively fragile tissue of N. scopa cv. Soonjung. Thus, a large proportion of the tested samples might remain alive after treatment. Two approaches also results in high transformation efciency values, but it is interesting to note that the T0 cactus was not rescued when subjected to vacuum inltration under pressures of 10 and 20 cmHg for 15 min each. Possibly, the transformation buffer mixture used with Agrobacterium was not completely sucked into the areoles of H. trigonus under the vacuum pressures applied. From an anatomical perspective, the thick epidermis of this cactus may function as a protective surface against mild pressure. These results contrast sharply with the results observed with vacuum inltration applied to N. scopa cv. Soonjung. In that case, the transformation efciencies evidenced relatively high values, although the survival rates were quite poor (Table 1). Consequently, it can be speculated that the anatomical advantage of H. trigonus also rendered the cells underneath the areoles tolerant to inltration under majority of the vacuum pressures applied after pin-pricking. Once Agrobacterium was safely delivered into the viable cells of the cactus, the transformation process could proceed further to completion. This may explain the high transformation efciencies with signicant survival rates in the H. trigonus group subjected to pin-pricking combined with vacuum inltration. Early studies about cactus transformation (Al-Ramamneh et al. 2006; Silos-Espino et al. 2006) were dependent on a cell and tissue culture process conducted with contamination-sensitive aseptic in vitro techniques and a series of selection procedures designed to establish authentic transformants. Also, in cases in which the callus stage is involved, genetic delity was not guaranteed for the regenerated cactus transformants, principally because tissue culture is a highly mutagenic process for cultured cells (Kaeppler et al. 2000). Furthermore, the majority of cactus cultivars are recalcitrant to tissue culture, which

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Plant Cell Rep (2008) 27:11971206 thaliana plants. C R Acad Sci Paris Sci La Vie/Life Sci 316:11941199 Briddon RW, Watts J, Markham PG, Stanley J (1989) The coat protein of beet curly top virus is essential for infectivity. Virology 172:628633 Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387405 Heywood VH, Brummitt RK, Culham A, Seberg O (2007) Flowering plant families of the world (Revised edition). Firey books, Ontario, pp 7879 Jeong MI, Cho CH, Lee JM (2004) Production and breeding of cacti for grafting in Korea. Chron Horticult 4(3):710 Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43(23):179188 Lee S, Stenger DC, Bisaro DM, Davis KR (1994) Identication of loci in Arabidopsis that confer resistance to geminivirus infection. Plant J 6:525535 Martinez-Trujillo M, Limones-Briones V, Cabrera-Ponce JL, HerreaEstrella L (2004) Improving transformation efciency of Arabidopsis thaliana by modifying the oral dip method. Plant Mol Biol Rep 22:6370 Rowley G (2003) What is an areole? Br Cactus Succul J 21(1):411 Sambrook J, Fritsh EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, New York n-Cruz Q, Rodrguez-Salazar Silos-Espino H, Valdez-Ortiz A, Rasco E, Paredes-Lopez O (2006) Genetic transformation of pricklypear cactus (Opuntia cus-indica) by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 86:397403 Stenger DC, Davis KR, Bisaro DM (1992) Limited replication of tomato golden mosaic virus DNA in explants of nonhost species. Mol Plant Microbe Interact 5:525527 Ye GN, Stone D, Pang SZ, Creely W, Gonzales K, Hinchee M (1999) Arabidopsis ovule is the target for Agrobacterium in planta vacuum inltration transformation. Plant J 19(3):249257

necessitates optimal media and culture conditions. All of these have presented major hurdles to the prevention of molecular improvements in the cactus. Table 3 shows a brief comparison of the in planta transformation conducted in our study with the other two methods described earlier. In summation, an efcient in planta transformation method (pin-pricking combined with vacuum inltration) was established for N. scopa cv. Soonjung using uidA as a reporter gene, and a relatively high transformation efciency (67100%) was demonstrated with a signicant survival rate (5085%) in additional applications to other cactus cultivars and related species of succulent plants. The system established here overcomes the technical limitations presented by earlier cactus transformation methods, and demonstrates the feasibility of mass transformation technology for the cactaceae.
Acknowledgments This research was supported by grants from the Agricultural Research and Development Promotion Center (ARPC) funded by the Ministry of Science and Technology of the Korean government.

References
Al-Ramamneh EA, Sriskandarajah S, Serek M (2006) Agrobacterium tumefaciens-mediated transformation of Rhipsalidopsis gaertneri. Plant Cell Rep 25:12191225 Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated gene transfer by inltration of adult Arabidopsis

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