ScienceDirect. In planta Agrobacterium-Mediated Transformation of Rice

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1 Available online at ScienceDirect Rice Science, 2017, 24(3): In planta Agrobacterium-Mediated Transformation of Rice Kumrop RATANASUT, Weerawan ROD-IN, Kawee SUJIPULI (Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand) Abstract: The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog (MS) medium, 5% sucrose, 44 nmol/l benzylaminopurine, and 0.075% surfactant Tween-20 with ph 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD 600 = and the co-cultivation at 25 ºC for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gusa gene by PCR using the gusa specific primers in the T 0 lines, there were 4 transgenic lines from 286 T 0 lines (1.4% transformation efficiency). However, histochemical glucuronidase (GUS) assay demonstrated that only three of four transgenic lines exhibited gusa expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future. Key words: Agrobacterium-mediated transformation; floral-dip; floral-drop; transformation efficiency; in planta; rice Rice transformation has been successfully developed via several techniques including electroporation, polyethylene glycol treatment, particle bombardment and Agrobacteriummediated method since the late 1980s (Chen et al, 2009). Agrobacterium-mediated gene transfer, which is one of the most common rice transformation methods, has been extensively used for developing transgenic rice to study gene function and gain improving agricultural traits, for example, resistance to disease and insect pest, tolerance to drought and salt, and higher quality and yield. In general, Agrobacteriummediated transformation is a tissue culture-based method, which usually requires aseptic condition and a long process to regenerate shoots and roots from the transformed tissues. Moreover, a tissue-culture technique frequently generates transformed plants harbouring undesirable mutations such as somaclonal variation (Clough and Bent, 1998; Bent, 2000). To avoid tissue culture, in planta transformation can directly transfer the interest gene into plant tissues or organs and can be a potential alternative for plant transformation experiments. In planta transformation techniques have been developed in various types including vacuum infiltration, agroinfiltration, sonication, spraying, floral drop and floral dip. The floral-dip transformation is the simplest and the most convenient method with no requirement of any equipment and tissue-culture procedures. The first successful Agrobacteriummediated floral-dip transformation was demonstrated in flowering Arabidopsis plants (Clough and Bent, 1998). This offers a simple, convenient and inexpensive transformation method, which eliminates the risk of unnecessary microbial contamination commonly observed in tissue culture. However, the floral-dip method has low transformation efficiency and requires many flowers and seeds because it has no specific target site of organ transformation. Apart from Arabidopsis, this method has been successfully applied to various crops such as Received: 1 Octorber 2016; Accepted: 28 November 2016 Corresponding author: Kumrop RATANASUT (kumropr@nu.ac.th) Copyright 2017, China National Rice Research Institute. Hosting by Elsevier B V This is an open access article under the CC BY-NC-ND license ( Peer review under responsibility of China National Rice Research Institute

2 182 Rice Science, Vol. 24, No. 3, 2017 radish (Curtis and Nam, 2001), tomato (Yasmeen et al, 2009), wheat (Zale et al, 2009), rapeseed (Li et al, 2010), white sweet clover (Hirsch et al, 2010), camelina (Liu et al, 2012), corn (Mu et al, 2012) and flax (Bastaki and Cullis, 2014). The in planta rice transformation was firstly established by Supartana et al (2005) for japonica varieties by inoculating the embryonic apical meristems of soaked seeds with A. tumefaciens. Lin et al (2009) produced transgenic indica rice by vacuum infiltration of soaked mature seeds pierced by a needle with A. tumefaciens. Recently, Rod-in et al (2014) reported that anthers are a major target of rice transformation using a modified floral-dip method of Arabidopsis (Clough and Bent, 1998) and pollen is also transformed. Here, we report the achievement of transgenic rice production using a modified floral-dip method of Rod-in et al (2014), which can be used to study the role and function of rice genes, and for molecular breeding of rice via genetic engineering. MATERIALS AND METHODS Plant materials and growth conditions Rice (Oryza sativa L.) variety RD41, obtained from the Phitsanulok Rice Research Center, Thailand, was grown in pots with natural light until flowering in a greenhouse. The inflorescences at the growth stage (the beginning of panicle emergence and tip of inflorescence emerged from sheath) (Lancashire et al, 1991) were used for transformation. Agrobacterium-mediated transformation A single colony of A. tumefaciens strains AGL1 or EHA105 harbouring pcambia1304 carrying a gusa gene (pcambia1304 sequence and map are available at daisy/cambia/585.html) was cultured in Luria Bertini (LB) broth medium supplemented with kanamycin (50 mg/l) and rifampicin (40 mg/l) at 28 ºC and 250 r/min shaking for 2 d. One millilitre of the Agrobacterium suspension culture was inoculated in 250 ml LB broth medium without antibiotics at 28 ºC and 250 r/min shaking overnight until reaching the stationary phase (OD 600 = ). Agrobacterium cells were collected by centrifugation at r/min for 3 min. The cell pellets were resuspended in 200 ml standard inoculation medium (SIM) containing Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 5% sucrose, 44 nmol/l benzylaminopurine (BAP) and 0.075% Tween-20, modified from Clough and Bent (1998), and Das and Joshi (2011). The ph of the inoculation medium was adjusted to 5.7. In planta rice transformation The floral-dip method was modified from Rod-in et al (2014). Rice inflorescences were prepared by clipping the tips of the pre-anthesis spikelet located at the middle of the inflorescences. Non-selected spikelet for clipping was removed from the inflorescences, which was subsequently dipped into the SIM containing Agrobacterium for 1 min. For the floral-drop technique, 100 ml SIM containing Agrobacterium were directly dropped into the individual spikelet cup (clipped spikelet) using the 1 ml syringe with a needle. The inoculated inflorescences were covered with plastic bags to maintain humidity and co-cultivated at 25 ºC for 48 h. The Agrobacteriumdipped or Agrobacterium-dropped spikelets were then covered with paper bags until seed collection. Screening for transgenic rice by PCR analysis Genomic DNA was extracted from T 0 transgenic rice leaves using the cetyltrimethylammonium bromide (CTAB) method according to Ahmadikhah (2009). The transgenic lines were verified using specific primers designed from the gusa sequence of pcambia1304 binary vector (GenBank: AF234300). The PCR reaction was performed using a pair of specific primers, gusa-f1: 5 -CAACGAACTGAACTGGCAGA-3 and gusa-r1: 5 -TCTCTTTGATGTGCTGTGCC-3, to amplify the 989 bp fragment, and a pair of specific primers, gusa-f2: 5 -TGCG TCACAGCCAAAAGC-3 and gusa-r2: 5 -CTCGCATTACC CTTACGC-3, to amplify the 361 bp fragment. The PCR cycling conditions were pre-denaturation at 95 ºC for 5 min, 35 cycles of 95 ºC for 30 s, 55 ºC (gusa-f1/gusa-r1) or 51 ºC (gusa-f2/gusa-r2) for 30 s, 72 ºC for s, followed by a final extension at 72 ºC for 5 min. Histochemical glucuronidase (GUS) assay The transformed rice tissues were evaluated through gusa expression according to the method of Jefferson et al (1987). Inoculated spikelet and T 0 leaves were immersed in 90% acetone for 1 h and incubated at 37 ºC overnight in glucuronidase (GUS) staining solution containing 50 mg/ml 5-bromo-4-chloro-3-indolyl-ß-D glucuronidase (X-gluc), 0.5 mmol/l potassium ferrocyanide, 0.5 mmol/l potassium ferricyanide, 1 mol/l Na 2 PO 4, 0.5 mol/l EDTA (ph 7.0), 0.5% Triton X-100 and 20% methanol. The stained tissues were washed once with methanol:acetic acid (3:1) at room temperature (25 ºC 28 ºC ) overnight and then once with 70% ethanol, and subsequently stored at 4 ºC for at least 2 h. Transformation efficiency of spikelet was calculated from percentage of spikelet with GUS-stained floral tissues. Transformation efficiency of anthers was reckoned from percentage of GUS-stained anthers in GUS-positive spikelet. Statistical analysis All experiments were performed in a completely randomized design with three biological replicates (three inflorescences per replicate). Statistical significance was analysed using one-way ANOVA (analysis of variance) followed by Duncan s multiple range tests and t-test for mean comparison using SPSS 17.0 software. Differences in P < 0.05 were considered significant.

3 Kumrop RATANASUT, et al. In planta Agrobacterium-Mediated Transformation of Rice 183 RESULTS AND DISCUSSION Floral-dip versus floral-drop for the effective in planta rice transformation Most pollen in the transformed anthers was successfully transformed (Table 1). The floral-dip method transformed anthers by chance, and the floral-drop transformation was an alternative technique that could directly target anthers of each rice spikelet. Therefore, we examined the efficiency of anther transformation using the floral-drop technique which was modified from the floral-dip method. Instead of dipping the rice panicles into the standard inoculation medium containing A. tumefaciens strain AGL1 carrying pcambia1304, we directly transformed anthers by dropping the inoculation medium into individual clipped rice spikelet and followed the standard protocol of the floral-dip method for rice (Rod-in et al, 2014). Compared to the floral dip transformation, the floral-drop technique gave higher floral-tissue transformation efficiency (Table 1). Although the spikelet transformation efficiencies of the two techniques were significantly different, the anther transformation efficiency based on transformed spikelet was not statistically different (Table 1). This indicated that either the floral-dip or floral-drop techniques could efficiently transform anthers whenever the spikelet was subject to the inoculation medium. However, we observed that the inoculum-dropped flowers, but not the dipped flowers, turned brown and dried within 2 d after inoculation. This symptom might be caused by overexposure to Agrobacterium inoculation medium and could affect the survival of the transformed spikelet and result in failure to generate the transgenic seeds. Our result showed different aspect from the floral-drop transformation in Arabidopsis, which targets ovules and gives 2-fold higher transformation efficiency than the floral-dip method (Martinez-Trujillo et al, 2004). With other drawbacks of the floral-drop technique including tedious work and time consuming in floral dropping, therefore, the floral-dip transformation would be a promising simple and convenient technique in planta for transgenic rice production. Evaluation of a simple and effective inoculation medium recipe for floral-dip transformation of rice To determine the effect of the SIM composition on transformation efficiency, we added, deleted or replaced the medium in nine different compositions of the inoculation medium for the floral-dip transformation with A. tumefaciens strain AGL1 carrying pcambia1304 (Table 2). The inoculation medium with no ph adjustment or no MS slightly reduced the transformation efficiency as well as the substitution of 5% sucrose with 5% glucose. Deletion of sucrose and Tween-20 from the inoculation medium dramatically reduced the transformation efficiency. Although substitution of sucrose with glucose in the inoculation medium could increase the transformation efficiency of Arabidopsis (Clough and Bent, 1998; Das and Joshi, 2011), the results demonstrated that sucrose could not be effectively substituted by glucose in the inoculation medium for the floral-dip transformation of rice. Table 1. Transient transformation efficiency of the floral-dip and floral-drop methods in floral tissues and anthers. % Transformation method Floral tissue Anther Floral-dip ± 1.76 b (94 GUS-stained/837 dipped flowers) ± 7.71 a (84 GUS-stained anthers/94 GUS-stained flowers) Floral-drop ± 0.81 a (117 GUS-stained/784 dropped flowers) ± 5.47 a (96 GUS-stained anthers/117 GUS-stained flowers) GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. Values are expressed as Mean ± SE (n = 9). The different lowercase letters after the numbers in the same column indicate significant differences between treatments by the Duncan s multiple range test (P < 0.05). Table 2. Transient transformation efficiency in floral tissues and anthers at different composition in inoculation medium. % Inoculation media Floral tissue Anther Standard ± 1.76 a (50 GUS-stained/413 dipped flowers) ± 6.77 a (42 GUS-stained anthers/50 GUS-stained flowers) No ph adjustment 9.68 ± 0.06 b (34 GUS-stained/351 dipped flowers) ± 7.35 a (28 GUS-stained anthers/34 GUS-stained flowers) No Murashige and Skoog 8.14 ± 1.52 b (34 GUS-stained/419 dipped flowers) ± ab (26 GUS-stained anthers/34 GUS-stained flowers) No benzylaminopurine 5.51 ± 1.20 c (26 GUS-stained/477 dipped flowers) ± bc (16 GUS-stained anthers/26 GUS-stained flowers) No Tween ± 1.22 d (10 GUS-stained/414 dipped flowers) ± 8.39 bc (7 GUS-stained anthers/10 GUS-stained flowers) No sucrose 3.81 ± 0.93 d (13 GUS-stained/352 dipped flowers) ± c (8 GUS-stained anthers/13 GUS-stained flowers) No sucrose, 5% glucose 9.42 ± 0.91 b (34 GUS-stained/359 dipped flowers) ± 7.53 a (27 GUS-stained anthers/34 GUS-stained flowers) No sucrose, 10% glucose 4.39 ± 1.90 cd (18 GUS-stained/390 dipped flowers) ± 7.70 ab (14 GUS-stained anthers/18 GUS-stained flowers) Add 200 µmol/l AS ± 0.40 ab (36 GUS-stained/353 dipped flowers) ± 6.40 a (30 GUS-stained anthers/36 GUS-stained flowers) GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. AS, Acetosyringone. Values are expressed as Mean ± SE (n = 9). The different lowercase letters after the number in the same column indicate significant differences among treatments by the Duncan s multiple range test (P < 0.05).

4 184 Rice Science, Vol. 24, No. 3, 2017 Tween-20 as a surfactant was required for the transformation. The anther transformation efficiency was also significantly reduced if the inoculation medium lacked sucrose and Tween-20. Clough and Bent (1998) also reported that use of the inoculation medium with no surfactant Silwet-L77 for the floral-dip transformation in Arabidopsis drastically reduced the transformation efficiency whereas increasing the concentration of the surfactant Silwet-L77 dramatically enhanced the transformation efficiency. The addition of 200 µmol/l acetosyringone (AS) gave the transformation efficiency no different from the standard medium (AS free) (Table 2). Clipping of rice spikelet before dipping could produce phenolic compounds, which are signals generally released after wounding and required for agrobacterium attachment and transformation. Our results showed that AS was not necessary for the floral-dip transformation technique developed in rice. For Agrobacterium-mediated transformation of plant tissues with no wounding, AS could enhance the transformation efficiency of both monocot and dicot crops, for example, wheat (Raja et al, 2010), phalaenopsis orchid (Belarmino and Mii, 2000) and cotton (Sunilkumar and Rathore, 2001). Potential of A. tumefaciens strain EHA105 for anther transformation via floral-dip method The anther transformation efficiency of A. tumefaciens strain EHA105 was compared to that of A. tumefaciens strain AGL1 using the floral-dip condition as described in Rod-in et al (2014). The gusa expression analysis demonstrated that the anther transformation efficiency of A. tumefaciens strain EHA105 was slightly higher than AGL1 (Table 3). Yi et al (2000) also reported that EHA105 was more efficient than AGL1 for rice transformation. However, statistically, the anther transformation efficiency of both Agrobacterium strains in rice spikelet was not significantly different. Cho et al (2014) reported that, in maize, AGL1 gives the highest transformation frequency among tested Agrobacterium strains (AGL1, LBA4404, EHA105 and GV3101), whereas EHA105 is ineffective. This suggests that the virulence of Agrobacterium strains AGL1 and EHA105 is different among plant species even those plants are in the same group (monocot or dicot). Transgenic rice production from floral-dip transformation To determine the possibility of transgenic rice generation via the floral-dip transformation, we performed the floral-dip Fig. 1. Analysis of gusa transgenes and expression in T 0 bulk tests Nos. 2, 4, 7 and 13. A, PCR amplification of a 989 bp expected product for positive transformed plants using a pair of primers gusa-f1 and gusa-r1; B, PCR amplification of a 361 bp expected product for positive transformed plants using a pair of primers gusa-f2 and gusa-r2; C, gusa expression analysis of 20 individual T 0 plants from the PCR-positive bulk tests Nos. 2, 4, 7 and 13. M, 1 kb Plus DNA Ladder (Invitrogen, USA). transformation of rice variety RD41 using the modified conditions from Rod-in et al (2014) which co-cultivated for 48 h and let the dipped spikelet set T 0 seeds. The tip-clipped spikelets at the growth stage were subjected to dipping into the standard inoculation medium containing A. tumefaciens strain EHA105 harbouring pcambia1304. The inoculated spikelets were left to set seeds for three weeks. A total of 286 two-week-old T 0 lines were screened for the real transgenic plants. One leaf from each T 0 lines was collected and 20 collected leaves were bulked into one test. The gusa transgene in each bulk sample was screened using mixed gdna extracted from each bulk sample as a PCR template. Four (bulk tests Nos. 2, 4, 7 and 13) out of fifteen bulk tests gave PCR products corresponding to the expected sizes 989 bp and 361 bp for primers gusa-f1/gusa-r1 and gusa-f2/gusa-r2, respectively (Fig. 1-A and -B). These results indicated that there were at Table 3. Transient transformation efficiency in floral tissue and anther of the rice spikelets dipped in two different A. tumefaciens strains, AGL1 and EHA105, harbouring pcambia1304 determined by GUS assay. % A. tumefaciens strain Floral tissue Anther AGL1 EHA ± 0.65 a (106 GUS-stained flowers/866 dipped flowers) ± 4.73 a (82 GUS-stained anthers/106 GUS-stained flowers) ± 3.25 a (132 GUS-stained flowers/904 dipped flowers) ± 1.43 a (107 GUS-stained anthers/132 GUS-stained flowers) GUS-stained flowers indicate flowers with transformed floral tissues. GUS-stained anthers indicate flowers with transformed anthers. Values are expressed as Mean ± SE (n = 9). The same lowercase letter after the numbers in the same column indicates non-significant differences between treatments by the independent sample t-test.

5 Kumrop RATANASUT, et al. In planta Agrobacterium-Mediated Transformation of Rice 185 least 4 transgenic lines from 286 T 0 lines. To confirm these transgenic lines, leaves of plants from four PCR-positive bulk tests were tested by GUS assay. The bulk tests Nos. 2, 4 and 7 carried one GUS-stained leaf each (Fig. 1-C) whereas the bulk test No. 13 had no GUS-stained leaf. Our results demonstrated that the gusa transgenes in three transgenic lines were expressed but no gusa expression occurred in another transgenic line. It is possible that silencing of the gusa transgene was caused by multiple copies of gusa. This event could induce a high level of gusa expression leading to gene silencing. Rearrangement of transgenes in host cells also causes silencing of transgenes. Incomplete expression cassette of gusa transferred into the rice genome could be another possibility of impaired gusa expression. However, these results indicate that the floral-dip transformation could give transgenic rice with at least 1.4% (4 transgenic plants from 286 tested T 0 lines) transformation efficiency. The efficiency of transgenic plant production from the floral-dip transformation was varied in different plant species. For example, in Arabidopsis, the transformation efficiency can be up to 3% (Clough and Bent, 1998), whereas the transformation efficiencies of Brassica napus and B. carinata are 1.86% and 1.49%, respectively (Verma et al, 2008). Mu et al (2012) reported that the floral-dip transformation efficiency of maize is 3.3%. CONCLUSION Acetosyringone was unnecessary whereas sucrose and Tween-20 were required for floral-dip transformation efficiency in rice. Comparing the transformation efficiencies between A. tumefaciens strains AGL1 and EHA105 revealed that EHA105 gave slightly higher transformation efficiency than AGL1, but showed no statistically significant difference. The floral-drop transformation was applied to rice variety RD41 and gave slightly higher anther transformation efficiency than the floral-dip method but not statistically significant difference. However, the Agrobacterium-dropped flowers turned brown and dried within 2 d. Screening of the T 0 seeds derived from the floral-dip transformation using A. tumefaciens strain EHA105 by PCR with the specific primers to the gusa gene revealed that there were at least 4 transgenic lines from 286 T 0 lines (1.4% transformation efficiency). Histochemical GUS assay confirmed that three of four transgenic T 0 lines exhibited gusa expression. No GUS activity was detected in another line. It is possible that, somehow, the gusa transgene in this line was silenced. However, these results indicate that floral-dip transformation is a simple, effective and economical method for production of transgenic rice. ACKNOWLEDGEMENTS We thank Professor Dr. Sompong TE-CHATO from Prince of Songkla University for kindly providing A. tumefaciens strains AGL1 and EHA105 harboring pcambia1304. This work was supported by the research grant (Grant No. R2556B036) from Naresuan University, Thailand. REFERENCES Ahmadikhah A A rapid mini-prep DNA extraction method in rice (Oryza sativa). Afr J Biotechnol, 8(2): Bastaki N K, Cullis C A Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate. J Vis Exp, 94: doi: / Belarmino M, Mii M Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Rep, 19(5): Bent A F Arabidopsis in planta transformation. Uses, mechanisms, and prospects for transformation of other species. Plant Physiol, 124(4): Chen H, Lin Y J, Zhang Q F Review and prospect of transgenic rice research. 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