Plant Science 135 (1998) 25–30 Production of transgenic deepwater indica rice plants expressing a synthetic Bacillus thuringiensis cryIA ( b ) gene with enhanced resistance to yellow stem borer Mohammad Firoz Alam, Karabi Datta, Editha Abrigo, Alelie Vasquez, Dharmawansa Senadhira, Swapan K. Datta * Plant Breeding, Genetics and Biochemistry Di6ision, International Rice Research Institute, P.O. Box 933 , Manila, 1099 , Philippines Received 14 October 1997; received in revised form 17 December 1997; accepted 23 February 1998 Abstract Yellow stem borer has been identified as a major insect pest of deepwater rice, causing severe yield losses. Bt gene(s) from Bacillus thuringiensis have been proven very effective in pest resistance program. The use of transgenic plants expressing Bt gene(s) is now occupied effective approach to control insect infestation. We have successfully introduced a synthetic cryIA ( b ) gene into embryogenic calli of a deepwater indica rice variety, Vaidehi, by using the biolistic method of transformation. The presence and expression of the Bt gene in regenerated plants were confirmed by Southern and Western blot analyses. Inheritance of the transgene was confirmed in the T 1 generation. Insect bioassays showed an enhancement of resistance against the yellow stem borer. This is the first report of lowland rice, engineered with Bt gene. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Transgenic deepwater rice; cryIA ( b ) gene; Yellow stem borer resistance 1. Introduction Deepwater rice (DWR) is grown on about 9 million hectares of flooded lands in Asia (the river basins of Ganges– Brahmaputra of India and Bangladesh, the Irrawaddy of Myanmar, the Mekong of Vietnam and Cambodia, and the Chao Phraya of Thailand) and West Africa (up- per and middle basins of the Niger River) [1]. Farmers in these areas are very poor and follow the traditional system of rice cultivation [2]. DWR is grown in areas usually flooded deeper than 50 cm (sometimes up to 400 cm) for 1 month or longer during the growing season [3]. Conse- Abbre6iations : 2,4-D, 2,4-Dichlorophenoxy acetic acid; NAA, 1-h Napthalene acetic acid. * Corresponding author. Tel.: +63 2 8450563, 8450569; fax: + 63 2 7612406; 8450606; e-mail: sdatta@irri.cgnet.com 0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0168-9452(98)00053-3 M. Firoz Alam et al. / Plant Science 135 (1998) 25 – 30 26 quently, only traditional tall and elongating rice cultivars can be grown in these areas. The yield of these cultivars is generally low (1–2 t ha −1 ) and very often, reduced further by insect attack. Stem borers are important pests of rice because symptoms of injury can be found from sowing to harvest in all rice ecosystems. At the vegetative stage, stem borer feeding inside the stem can result in the death of the youngest leaf whorl, causing ‘deadhearts’. During the reproductive stage, stem borer feeding inside the panicle stalk can lead to grain remaining unfilled, a condition called ‘whiteheads’ [4]. Several species of stem borer attack DWR, but the major one is undoubt- edly the yellow stem borer (YSB). It is dominant in the flood season, and no other species is well adapted to aquatic conditions. It seriously dam- ages DWR, causing yield losses estimated at 15– 20% (on the average) and reaching up to 60% during severe outbreaks [5]. No DWR germplasm has been found to possess acquired resistance to YSB [6]. Additionally, no feasible biological or cultivation method has been devised to control this key pest in DWR field [5]. The application of insecticides to DWR possess many problems. Ordinary ground applications are limited to the pre –flood period and spraying is not possible when the water is deeper than 50 cm. Moreover, pesticides could affect beneficial natu- ral predator and cause fish mortality [6]. Fish harvested from DWR fields is a major source of protein for people living in these areas. The devel- opment of YSB-resistant varieties of DWR will help farmers in flood-prone ecosystems significantly. The crystal protein or l -endotoxin from Bacil- lus thuringiensis has been found very effective in controlling lepidopteran insects including YSB [7]. Several crop species including rice have been transformed with cryIA ( b ) /cryIAc gene from B. thuringiensis and were shown to be very effective against stem borers including the YSB [8– 19]. The development of transformation techniques presented an opportunity to incorporate this novel bacterial gene into the rice genome, which is not possible by conventional breeding methods. Earlier literature dealing with Bt-rice on japon- ica [10,19] and indica [16,18] cultivars for irrigated ecosystem are not suitable for Bt-management in DWR. Hence, the selected transgenic Bt-rice for deep water conditions with enhanced resistance against yellow stem borer will provide a unique opportunity of environmentally friendly pest con- trol avoiding use of insecticides and other chemi- cals in aquatic conditions. Therefore, this study was undertaken to develop transgenic rice with YSB resistance for deepwater rice conditions. 2. Materials and methods 2.1. Genotype and plasmids A popular deepwater indica rice cultivar, Vaidehi (TCA48) of Bihar State, India, was used. It is a traditional, tall, photoperiod-sensitive vari- ety with a yield potential of more than3tha −1 [20]. Plasmid pCIBBt1 (Fig. 1) carrying the syn- thetic cryIA ( b ) Bt gene with the CaMV 35SP was cotransformed with plasmid pGL2 [21] carrying the hph gene also driven by the 35SP. 2.2. Transformation Embryogenic callus (EC) was induced from scutellar tissues of mature seeds on MS medium [22] supplemented with 2 mg 2,4-D l −1 ,30g maltose l −1 solidified with 8 g agar l −1 (pH 5.8). The EC were bombarded using the biolistic PDS- 1000/He (Bio-Rad, USA) at 1300 or 1500 psi, following the manufacturer’s instructions and the protocol published earlier for cereals [11,23–26]. Selection pressure was applied 16–20 h after bom- bardment on fresh callus induction medium con- taining 50 mg hygromycin B l −1 as described earlier [24]. Selection was maintained for 10–12 weeks with a change of medium every 2 weeks. Surviving callus (embryogenic portion) was placed Fig. 1. Physical map of plasmid pCIBBt1 carrying the syn- thetic cryIA ( b ) gene driven by the CaMV35S constitutive promoter. M. Firoz Alam et al. / Plant Science 135 (1998) 25 – 30 27 on MS plant preregeneration medium containing 2 mg kinetin l −1 , 0.1 NAA l −1 , 30 g maltose l −1 , 50 mg hygromycin B l −1 and 8 g agar l −1 ,pH 5.8. Cultures were kept in the dark for 1–2 weeks. The rest of the steps for plant regeneration of T 0 plants were carried out as described by Alam et al. [27]. 2.3. DNA extraction and Southern blot analysis Genomic DNA was extracted by an improved CTAB method based on the procedure described by Murray and Thompson [28]. Five mg/l DNA of each sample, estimated by fluorometry and treated with RNaseA, was digested with BamHI and BstEII restriction endonucleases in a final volume of 50 ml. The digested DNA was then electrophoresed on 1% (w/v) agarose gel and transferred to hybond N + nylon membrane (Amersham, Arlington Heights, IL) according to manufacturer’s instructions. DNA fragment of the Bt endotoxin coding sequence from plasmid digested with the same enzyme and labeled with h- 32 P dCTp using the rediprime labeling kit (Amersham Arlington Heights, IL) was used as hybridization probe. 2.4. Protein extraction and immunoblot analysis About 0.5 –0.8 g fresh leaf or stem tissues of both transgenic and nontransgenic control plants were ground in the presence of 1.0–1.5 ml 0.05 M Tris– HCl (pH 7.0) and 10% (v/v) glycerol mixed with 0.1 mM phenylmethylsulphonylfluoride (PMSF) at 4°C. Centrifugation at 13000 rpm for 10 min followed by 5 min was carried out two times and supernatants were collected. After de- termining the concentration of total soluble protein using the Bicinchoninic acid (BCA) protein assay reagent (PIERCE), each protein extract was boiled together with sample buffer (12.5 mM Tris pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 0.001% (w/v) bromophenol blue, 2% (v/v) or 0.3 M 2-ME) for 5 min. A total of 50 mg soluble protein was loaded per lane in 10% (w/v) SDS-Polyacrylamide gels. Separated polypeptides were blotted onto nitrocellulose membrane [29] in the semi-dry transblot SN transfer cell (Bio-Rad, Hercules, CA, USA). After overnight blocking with 5% (w/v) TBST-milk, Bt proteins were probed with rabbit anti-BtK protein serum at room temperature for 20–24 h and detected using the procedure described by Lin et al. [30]. 2.5. Insect bioassay Plants (T 0 and T 1 ) positive in Southern or Western blot analysis were tested for resistance against the YSB. A single stem cutting (about 8 cm long) with at least one node (YSB prefer nodes for sucking food) from a plant at the booting stage was placed on a moistened filter paper in a petri dish (100×20 mm). Six neonate larvae of YSB (Scirpophaga incertulas) were placed on the stem, and the petri dish was sealed using masking tape. Incubation was performed for 96 h at 25°C. After 96 h, the number of larvae was determined. Similar infestation was carried out for control plants. Each treatment was repli- cated three times. Mortality rates were expressed as the proportion of dead larvae to applied larvae (%). Missing larvae were grouped within the mor- tality category (i.e. as dead larvae). 3. Results and discussion 3.1. Assessment of T 0 transformants From three experiments, out of the small num- ber of T 0 transgenic plants (30) produced, two independently transformed lines, VA6 and VA10, were recovered and presented in this study. South- ern blot analysis (Fig. 2) showed both transfor- mants had the 1.8 kb coding sequence of the cryIA ( b ) gene (plasmid pCIBBt1; Fig. 1). Multiple bands in VA6 indicated that recombination and rearrangement of the transgene had occurred. Re- arrangements of the transgene in T 0 transgenic plants were also observed in different crops [11,13,14,16,31,32]. Both higher and low molecu- lar weight bands than the expected 1.8 kb size were observed. On the other hand, in VA10, several rearrangement of transforming DNA were observed. However, both transformants were fer- tile and produced sufficient seeds. Expression of M. Firoz Alam et al. / Plant Science 135 (1998) 25 – 30 28 Fig. 2. Southern blot analysis of T 0 plants and some of selected T 1 progeny derived from them. From banding pat- terns two independent transformation events (VA10 and VA6) were identified. Both events showed on 1.8 kb coding sequence of cryIA ( b ) gene. Nontransformed (NT) plant showed no bands. Except for VA10–8, all progenies showed banding similar to that of their respective T 0 mother plants. served in control nontransformed (NT) plants. In addition to the expected 65 kDa band, two addi- tional bands (between 46 and 30 kDa) were also observed. This could be due to post-transcrip- tional and post-translational changes of the gene. Plants transformed with cryIA ( b ) /cryIAc gene showed multiple banding patterns in Western blot analysis [10,13,14,16]. About 0.01 –0.1% of Bt protein was estimated in both transformants, de- termined by comparing the intensity of the band with known 60 kDa pure 10 ng cryIA(b) protein (Fig. 3). Bioassay showed correlation with both Southern and Western blot analysis. No larvae were found alive on either transformants. Consid- ering the bioassay data, it was clear both transfor- mants produced a sufficient amount of Bt protein for complete protection from YSB infestation. Low levels of protein expression may help insects to develop resistance against Bt toxin, which will eventually destroy the utility of Bt transgenic plants. In addition, proper managements will be required to prevent the YSB to become resistant to Bt-positive plants. Transgenic plants with high levels of Bt-protein expression could be poten- tially effective when grown in conjunction with untransformed plants to serve as refuges. 3.2. E6aluation of T 1 progeny Bt positive T 1 progeny from both transformants were identified by Southern or western blot analy- ses. Results of Southern blot analyses of selected progeny from both transformation events are shown in Fig. 4. Except in plant (VA10-8), progeny plants showed Southern banding patterns similar to T 0 plants. The different banding pattern in VA10-8 indicate that a rearrangement of the transgene may occur in successive generations. This may be due to deletion, addition, or translo- cation of the transgene. However, further study on this aspect is needed. Multiple generations and additional progeny analysis may provide useful information regarding such rearrangements. Goto et al. [33] showed differences in banding patterns among T 2 progenies of transgenic rice plants. Nayak et al. [18] also reported different Southern patterns among progenies of specific transforma- tion event they analyzed. the cryIA ( b ) gene in both transformants was confirmed by Western blot analysis (Fig. 3). In both transformants, the expected 65 kDa Bt protein was detected. No such bands were ob- Fig. 3. Immunoblot analysis of protein from T 0 plants (VA10 and VA6) and one T 1 progeny (VA6-4) of VA6. All three plants show bands expressed from cryIA ( b ) Bt gene. Besides the expected 65 kDa, two additional bands between 46 and 30 kDa were also observed. The amount of Bt protein (1–0.1%) over total soluble protein was estimated by comparing the intensity of the band with known 60 kDa pure 10 ng CryIA(b) protein. M. Firoz Alam et al. / Plant Science 135 (1998) 25 – 30 29 Fig. 4. Immunoblot analysis of protein from some selected T 1 progenies of VA10 and VA6. All progenies show banding pattern similar to that of T 0 plants. The nontransformed (NT) control plant does not show any band. 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