247
Molecular Breeding 3: 247–255, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.
Transgenic sugarcane plants resistant to stem borer attack Ariel Arencibia, Roberto I. V´azquez, Dmitri Prieto, Pilar T´ellez, Elva R. Carmona, Alberto Coego, L´azaro Hern´andez, Gustavo A. De la Riva & Guillermo Selman-Housein Plant Division, Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana 10600, Cuba ( author for correspondence; e-mail:
[email protected]) Received 29 February 1996; accepted in revised form 6 March 1997
Key words: Bacillus thuringiensis, Diatraea, -endotoxin, sugarcane, transgenic plant
Abstract A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis -endotoxin was expressed in transgenic sugarcane plants (Saccharum officinarum L.) under the control of the CaMV 35S promoter. Genetic transformation was accomplished by electroporation of intact cells. The levels of recombinant toxin were established and biological activity tests were performed against neonate sugarcane borer (Diatraea saccharalis F.) larvae. Transgenic sugarcane plants showed significant larvicidal activity despite the low expression of CryIA(b). Abbreviations: BT, Bacillus thuringiensis; BTK; Bacillus thuringiensis var. kurstaki; CaMV 35S, cauliflower mosaic virus promoter for the 35S polyprotein; ICP, insecticidal crystal protein; Tnos, nopaline synthetase gene terminator from Agrobacterium tumefaciens; SCSB, sugarcane stem borer (Diatraea saccharalis F.). tcryIA(B), truncated version of the BT cryIA(b) gene; TMV, tobacco mosaic virus. Introduction Sugarcane (Saccharum officinarum L.) is a monocot plant widely spread and economically important in many regions around the world. The stem borer (Diatraea saccharalis F., Lepidoptera, SCSB) is the most important pest of this crop, causing extraordinary agricultural and industrial losses annually [3]. Control of SCSB is very difficult and expensive due to the typical feeding behavior of the larvae into the sugarcane stem. This fact brings about inaccessibility of the conventional pesticides to the target insect. For many years, entomophages and entomopathogens like Trichogramma sp. and Beauveria basiana have been unsuccessfully tried to control the SCSB in field conditions [16]. The microorganism Bacillus thuringiensis (BT), a gram-positive, spore-forming soil bacterium, produces a crystalline parasporal body during sporulation, which shows biocidal activity against some invertebrate orders such as lepidopteran, dipteran, and coleopteran insects at larval stage, as well as against nematodes [9]. BT is commonly used as a source of biopesti-
*136615*
cide products. The active component of BT consists of insecticidal crystal protein (ICP; Cry proteins or -endotoxins) subunits. ICPs are solubilized and proteolytically activated in the larval midgut. The biocidal activity spectrum of these toxins is very narrow because of their mode of action, which is based on specific receptor-recognition and membrane insertion of the active Cry proteins bringing about the paralysis of the transepithelial transport. [18]. The advantages of biodegradability and total safety both for humans and the environment have led to an increase in their use to control agricultural pests. However, the wide use of BT-based insecticides is limited for two main reasons: variable and ineffective field persistence and difficulty for the ICP toxin to reach the target pest at the site where it feeds. Heterologous expression of several truncated versions of cry genes from BT in transgenic crop plants represents an alternative strategy to overcome the disadvantages of commercial BT formulations. Such plants would increase the selectivity of control since only pest insects that attack the plant will be affected,
GR: 201009189, Pips nr. 136615 BIO2KAP molb28us.tex; 10/07/1997; 15:19; v.7; p.1
248 and continuous control can be achieved. Furthermore, the toxin will reach any target insect in spite of its feeding behavior. The feasibility of this technology has been demonstrated by the production of insectresistant transgenic tobacco [26], tomato [11], potato [1], cotton [23], and maize [19] plants. In this article, we establish the methodology to produce transgenic sugarcane plants resistant to SCSB attack by expressing a truncated version of the cryIA(b) gene (tryIA(b)) from BTK HD-1. A genetic construct containing the tcryIA(b) gene driven by the cauliflower mosaic virus (CaMV) 35S promoter was introduced into sugarcane by intact cell electroporation and transgenic plants regenerated. Molecular characterization and insect resistance data analysis from five transgenic sugarcane lines were performed.
Materials and methods Plant materials The commercial sugarcane variety Ja 60-5 was obtained from the germplasm collection of the Experimental Sugarcane Station, Jovellanos, Cuba. Sugarcane embryogenic calli were used in the electroporation experiment according to the methodology reported by Payan et al. [22]. Briefly, meristematic tissue from sugarcane was excised, disinfected and placed on callus induction medium (P+) containing MS salt, 100 mg/ml myo-inositol, 0.8 mg/l thiamineHCl, 500 mg/l casein hydrolysate, 20 g/l sucrose, 4 mg/l 2,4-D, 7 g/l agar-agar, pH 5.6. The culture was mantained in the dark at 25 C for one month. Subsequently, friable embryogenic calli were transferred to fresh medium for an additional subculture and used for transformation experiments.
protein expression in Escherichia coli. This expression system was also used for the biological activity tests (see below). The levels of CryIA(b) in the E. coli lysates were determined by a DAS-ELISA immunoassay system, as reported previously [29]. Finally, in order to express the tcryIA(b) gene in plants it was subcloned downstream the untranslated leader sequence from TMV, between the CaMV 35S promoter and Tnos terminator. The resulting plasmid was called pBPF 4. The pBI 221.1 selecting plasmid [14] was used in cotransformation experiments. SCSB colonies and test for CryIA(b) larvicidal activity SCSB colonies were established using semi-artificial diet: 33 g/l maize flour, 45 g/l wheat germ powder, 16.5 g/l sugarcane leaf powder, 42.5 g/l dry yeast, 4.3 g/l ascorbic acid, 1.32 g/l boric acid, 3 g/l methyl parabene, 10% formaldehyde [6]. Soluble proteins from E. coli expressing the tcryIA(b) gene were assayed for lethality to neonate SCSB larvae by incorporation into the diet. CryIA(b) concentrations were determined by DAS-ELISA [29]. The evaluation of larvicidal activity was carried out against 1-day old, third-instar SCSB larvae by putting them onto the artificial diet [28]. Ten larvae were used for each toxin dose. A diet, suplemented with lysates obtained from pUC 19transformed E. coli culture, was used as negative control. Mortality was scored after 7 days and LC50 was calculated. Sugarcane transformation
The generation of transgenic sugarcane lines was carried out introducing the pBPF 4 and pBI 221.1 plasmids into sugarcane intact cells by electroporation [2]. Prior to the electroporation experiments, cell aliCryIA(b) gene cloning and genetic constructions quots of 1 ml were dispensed in Eppendorf tubes and pBPF 4 and pBI 221.1 (1:10 molar ratio) plasA 2.1 kb DNA fragment encoding the first 654 mids were added. Electroporation was performed using amino acid residues of CryIA(b) ICP, an active toxcuvettes of 0.4 cm path length in an electroporator of in of B. thuringiensis var. kurstaki HD-1 (Dipel) was exponential pulse EPE-010 (CIGB, Cuba) at 880 F amplified by polymerase chain reaction (PCR) from and 800 V/cm according to previously established prototal bacterial DNA. The oligonucleotides 50-TGGATC tocols. Transformed sugarcane cell clusters were selecCTATGGATAACAATCCGAACATCAA-30 and 50 ted by non-destructive histochemical staining with XGGGATCCCTCATTAGTTTGGATCTTGAAGTA-30 were Gluc. When a blue color began to appear, GUS-positive used as forward and reverse primers, respectively. The transformed cell clusters were rescued and transferred PCR product was BamHI-digested and cloned into the to fresh callus induction medium for four additionBamHI site of pUC19. The truncated cryIA(b) gene al weeks. Putative transgenic calli were then placed was analyzed by DNA sequencing and heterologous onto regeneration medium [2]. Regenerated sugarcane
molb28us.tex; 10/07/1997; 15:19; v.7; p.2
249 plants were micropropagated for three subcultures on a medium containing MS salt, 100 mg/ml myo-inositol, 0.8 mg/l thiamine-HCl, 500 mg/l casein hydrolysate, 30 g/l sucrose, 0.3 mg/l BAP, 1.3 mg/l IAA, 0.86 mg/I kinetin, 7 g/l agar-agar, pH 5.6.
SCSB feeding observed in less than 20% of scored stem segments), medium (tunnels observed in more than 20% segments; no visible damages in the apical zone of the scored stalks) and high (more than 80% of the scored segments tunneled by SCSB or the apical zone destroyed by feeding larvae).
Early bioassay selection Sugarcane plants regenerated from GUS-positive calli [14] were challenged with SCSB larvae. The plants were transferred to sterile glass tubes containing 3 ml destilled water. Two neonate SCSB larvae were applied to the leaves of each plant. The assays were maintained at 28 C with 16 h light of 2000 lux. After three days, plant damage (low, medium, high) was scored. Transgenic sugarcane clones selected in the first bioassay were adapted to soil in greenhouse conditions. The soil-adapted plants were then planted on a small experimental parcel [21]). Biological risk management To avoid dissemination of SCSB from the experimental parcel, thus preventing the apparition of CryIA(b)resistant borer lines in the natural population, the experiment was surrounded by plants of the POJ 2878 sugarcane cultivar highly susceptible to borer attack. Evaluation of plant damage was done before the end of the larval phase of SCSB developmental cycle to prevent the escape of adult insects from the experimental plot [15]. Controlled biological activity test in small parcel conditions Two rows of plants belonging to selected individual transgenic lines were alternated with two rows of non-transgenic sugarcane plants cv. Ja 60-5 used as negative controls. Agriculture managements were performed following the technical instructions for sugarcane crops. When the plants reached 0.7–1 m in height, 25 SCSB neonate larvae were inoculated onto different sites of the plant stalks (2–3 larvae per stalk). The experiment was evaluated after 50 days for three randomly selected plants from each line. After the plants were harvested, each stalk was carefully split and the numbers of tunneled stem segments per plant line were scored. Percentage of segments affected by SCSB tunneling damage was taken as evaluation criterion. Plant damage was assessed, falling into three non-parametric categories: [6]: low (tunnels due to
Detection of recombinant CryIA(b) protein in transgenic plants Qualitative and quantitative detections were performed using western blot analysis and immunoradiometric assays, respectively. Leaf extracts were prepared from parcel-grown plants by grinding leaf tissue with a Polytron (Kinematika, Littau) homogenizer in the presence of extraction buffer (0.1 mM Na2 CO3 pH 9.5, 1% PVP, 1 mM leupeptin, 10 mM PMSF, 0.05 mM EDTA). The extract was clarified by centrifugation and neutralized with 1 M HCl to further assay. Total soluble protein was estimated by the Bradford [4] method, using BSA as standard protein. For western blot analysis, 100 g of extracted proteins were resolved by 10% acrylamide SDS-PAGE [20] and blotted to Hybond C membranes. CryIA(b) protein was detected using immunopurified rabbit anti-CryIA polyclonal antibodies and anti rabbit IgG-phosphatase conjugate reaction with insoluble substrate (X-phosphate). The amount of CryIA(b) protein was monitored by one-step immunoradiometric assay [27]. Microtiter plates (Titertek, Flow Laboratories, Netherlands) were coated with 100 l of a solution, containing 10 mg/ml immunopurified antibody in coating buffer (carbonatebicarbonate buffer, pH 9.6) at 37 C for 2 h. The plates were then washed three times with PBST (PBS saline buffer, 0.05% Tween-20) buffer and remaining binding sites blocked using 100 l 1% BSA in PBST at 37 C for 1 h. After blocking, the plates were washed two times with PBST buffer and stored at 4 C. In antiCryIA coated plates, 50 l neutralized extract (25 g total soluble proteins from standards and samples) and 5 106 cpm of the 125 I-labeled antibody were mixed into each well and incubated at 4 C for 12 h. The plates were then washed five times and bound radioactivity in each well was determined by a CliniGamma -counter (LKB, Bromma, Sweden) after cutting the well from the plate. PCR and Southern blot analysis Total genomic DNA was prepared from parcel-grown sugarcane plants essentially as described by Dellaporta
molb28us.tex; 10/07/1997; 15:19; v.7; p.3
251 Table 1. Self-pesticide activity of the transgenic sugarcane plant lines and CryIA(b) levels in the leaf tissue.
Clone
Total number of stem segments1
Fraction of damaged stem segments (%)
Resistance to SCSB larvae2
Cry IA(b) concentration (ng/mg soluble proteins)4
Standard deviation n=4
Cry IA(b) level in the leaves (ng/g foliar tissues)4
4 48 56 65 209 202 146 12 68 85 171 NC3
329 185 270 258 488 45 91 59 219 77 70 311
9 6 0 3 4 33 49 36 30 62 56 62
High High High High High Medium Medium Medium Medium Low Low Low
0.59 1.35 0.49 0.62 0.69 ND5 ND ND ND ND ND 0.00
49 100 30 32 50 ND ND ND ND ND ND –
11.82 27.23 9.89 12.75 13.24 ND ND ND ND ND ND 0.00
1
Stem segments per clone. SCSB resistance data according to the following criteria: Low: more than 80% stem segments damaged or apical zone destroyed by the larvae; Medium: 20–80% stem segments damaged and intact apical zone; High: less than 20% stem segments damaged; intact apical zone. 3 NC: Negative control. 4 The assays were performed with the leaf extracts from four randomly selected plants per clone using 10 g foliar tissue per plant. 5 ND: Not determined. 2
[9]. The PCR was performed using oligonucleotides homologous to positions 673–709 and 1387–1414 of the BT gene as forward and reverse primers, respectively. PCR products were transferred onto Hybond N+ membranes and analyzed by Southern blot hybridization using a 32 P probe derived from a BamHI-digested DNA fragment containing the tcryIA(b) gene. In order to demonstrate the insertion of the tcryIA(b) gene into the sugarcane genomes, 10 g of total DNA was then processed for Southern blotting using standard procedures.
Results Cloning and sequencing of the tcryIA(b) gene A 2.05 kb DNA fragment encoding the toxic Nterminal segment of the CryIA(b) protein from BTK HD-1, was amplified by PCR from total DNA using oligonucleotide primers homologous to the ,8 to +19 and +2054 to +2085 positions of the native gene and the product was cloned in the pUC19 vector. The entire truncated gene was sequenced by the Sanger method,
resulting identical to the first 2 kb of the native cryIA(b) gene sequence reported by Fischhoff et al. [11]. Heterologous expression in E. coli and biological activity of the recombinant CryIA(b) protein against SCSB larvae Expression of tcryIA(b) gene in E. coli yielded a 70 kDa polypeptide, detected by western blot using immunopurified antisera (data not shown), raised against CryIA ICP from BTK HD-1. The recombinant protein was quantified by DAS-ELISA and the in vivo biological activity against third instar SCSB larvae was evaluated. The protein showed to be toxic with an LC50 = 1:2 g/ml when mixed in the diet. The result is in accord with a previously reported value [28]. Production of transgenic plants As a result of the co-electroporation experiment, 239 GUS-positive cell clusters (15.2% of all electroporated clusters) were selected by histochemical staining and transferred to regeneration conditions. Only 36 selected clusters became embryogenic and 298 plants
molb28us.tex; 10/07/1997; 15:19; v.7; p.5
252
Figure 2. Southern blot analysis of genomic DNA from transgenic sugarcane lines. A. Lanes 1–5: BamHI-digested genomic DNA from lines 4, 48, 56, 65 and 209. Lanes 7–11: integration pattern analyzed by KpnI digestion of genomic DNA from lines 4, 48, 56, 65, 209. Lanes 6 and 12: untransformed plant genomic DNA digested with BamHI and kpnI respectively (negative controls). Lanes 13, 14: 10 and 20 pg tcryIA(b) 2. 1 kb DNA fragment from pBPF 4 plasmid. Lane 15: molecular weight markers. B. Map of the pBPF 4 genetic vector containing the tcryIA(b) gene. The gene was subcloned downstream of the CaMV35S promoter and the TMV untranslated leader, and upstream of the Agrobacterium tumefaciens Tnos terminator.
were regenerated and maintained as single clones. 75 regenerated plants turned out to be whole or partially GUS-positive (Figure 1). Ultimately, 2390 plants were obtained by micropropagation and submitted to the selection by bioassay. Selection of tcryA(b)-transgenic plants Each micropropagated plant was challenged with 2 SCSB larvae as detailed in Materials and methods. None of the 50 un-transformed in vitro cultured plants used as negative controls survived the challenge under the same conditions. In contrast, 41 transgenic plants (0.92% of all evaluated plants) belonging to 22 clones resisted the challenge with low or medium damage level and thus were selected for further assays. tcryIA(b)-PCR assays were performed with all the 22 plants to confirm they were transformed. All the plants resulted positive by PCR (data not shown).
The 41 selected plants were adapted to soil in a greenhouse in order to test their SCSB resistance under small parcel conditions, following the biological risk assessment rules. When the plants reached ca. 1 m height in controlled parcel conditions, they were infested with 25 SCSB larvae onto different sites of the plant stalks. After 50 days the numbers and percentages of tunneled stem segments were scored and the plants classified into three non-parametric cathegories according to Collazo [6]. Only five plant lines showed significative resistance levels (Table 1) and were further caracterizated by molecular methods. Notoriously, no living insects were found inside the tunelled stem segments of these five lines, while in non-resistant lines the larvae grew at a nearly-normal rate. In negative controls, as well as in discarded transgenic clones, ‘dead hearth’, a typical fingerprint of SCSB damage consisting in the destruction of the stem apical zone, was frequently observed (Figure 1).
molb28us.tex; 10/07/1997; 15:19; v.7; p.6
253 Discussion
Figure 3. Western blot analysis of protein extracts from transgenic sugarcane lines expressing the CryIA(b) toxin. Lane 1: 100 ng purified and activated ICP from BTK HD-1. Lanes 2–6: 100 g total soluble protein from high-resistance lines 4, 48, 56, 65, 209. Lane 7: 100 g total soluble protein from a medium-resistance line 202. Lane 8: 100 g total soluble protein from non-transformed sugarcane plant.
Molecular characterization of the selected transgenic plant lines The five selected plant lines were characterized by Southern blot, western blot and immunoradiometric assay (IRMA). Southern blot experiments with genomic DNA from the five selected trangenic lines defined the presence of more than two tcryIA(b) transgene copies per genome in all plants (Figure 2). A different insertion pattern was observed in each line. All of them corresponded to the intact gene within the insertion cassette used in the transformation. Western blot assays were performed to examine the integrity of the CryIA(b) protein produced by the transgenic plants (Figure 3). This experiment gave nearly similar results for all the transgenic lines. A well-defined band was observed at 70 kDa, a molecular weight value similar to the one predicted from the DNA sequence of the corresponding gene. However, some weaker bands of lower molecular weight were also noted, which may indicate that the CryIA(b) protein undergoes proteolytic processing. To quantify the toxin produced by the transgenic plants, ELISA analysis [12] were tried, but without satisfactory results. Thus, we designed a modified IRMA system, described extensively elsewere [27]. The assays were performed with four replicas per clone, using leaves from randomly selected sugarcane plants grown under small parcel conditions. The amounts of the recombinant protein detected were extremely low (Table 1).
The aim of this work was to obtain a model of cryIA(b) gene expression and insect resistance in sugarcane, the most economically important crop in Cuba. Transgenic sugarcane plants expressing a truncated version of the BTK HD-1 cryIA(b) gene driven by the CaMV 35S promoter were obtained by a transformation procedure described elsewhere [2]. The methodology was modified in order to introduce two plasmids into intact cells in suspension at the same time. Molar ratio between pBPF 4 and pBI221.1 was 10:1 in the pulsed electroporation DNA mixture. The higher ratio of pBPF 4 plasmid favored the recovery of plants containing the cryIA(b) transgene from histochemically stained calli, and these transgenic plants were obtained from the regenerated GUS-positive cell clusters as expected. Co-transformation has been reported for particle bombardment [17, 24, 7] and protoplast electroporation [25]. We suggest that this approach may be useful in any direct transformation method. The regenerated plants were multiplied and challenged with SCSB larvae under in vitro culture conditions as described. We believed that only insect resistance should be used as an efficient selectable character to screen the transgenic clones. This early test for conferred biocidal activity allows a fast and efficient selection of the most promising clones by eliminating a significant number of non-transgenic, chimaeric and low-expressing transgenic lines. The efficiency of this approach is supported by the fact that the chosen survivors were cryIA(b)-PCR-positive. A second bioassay was performed by infesting the plant stems with SCSB larvae under small parcel conditions, where only five clones exhibited significant protection against SCSB. These lines were subsequently characterized by Southern blot, showing more than one copy per genome with different insertion patterns of the tcryIA(b) transgene. Direct transformation methods are known to produced complex events in which multiple copies of the introduced DNA become integrated at one or several loci in the recipient genome [25, 5]. As foliar buds are the major initial targets of SCSB attack and its entry port to the stalk, we used leaf tissue to quantify the CryIA(b) toxin. Some degradation of the recombinant protein was observed, as evidenced by western blot. The expression levels of the toxin transgene were determined by an IRMA-system because it was not possible to detect the heterologous protein by any of the ELISA techniques reported up to date [1]. The amount of recombinant protein produced by the
molb28us.tex; 10/07/1997; 15:19; v.7; p.7
254 plants was extremely low. This result is in concordance with the low efficiency of the expression system employed in this work, particularly because cryIA(b) gene sequence modifications [23] and the use of a promoter stronger than CaMV 35S in monocot plants are known to be required to achieve higher expression levels in vivo. These results suggest the possibility that this BT toxin may be effective against SCSB even with very low amounts of the detectable protein. The obtained data are in close agreement with the previously observed facts concerning the hypereffectiveness of BT toxins expressed by transgenic plants under field conditions, when compared with the biological effects assessed in artificial diet feeding tests using the same proteins at similar concentrations [11, 26]. Preliminary assays under field conditions (Vidal et al., unpubl.) have shown that the CryIA(b) toxin is expressed by the transgenic sugarcane lines at low level, but in amounts sufficient to control the SCSB attack. This fact supports the validity of generating SCSB-resistant sugarcane by transformation with BT cryIA(b) gene. To our knowledge, no previous reports about SCSB-resistant sugarcane plants have been published so far. In contrast with resistance to fungal and viral diseases, that may be approached in sugarcane by conventional breeding and cytogenetic methods (i.e. somaclonal variation), the resistance to SCSB seems to be unreachable by any classical technology because of the lack of the corresponding genes in the genetic pool of the sugarcane population. However, despite the fact that at least 5 lines of transgenic sugarcane plants were completely selfpesticide under small parcel conditions, the low expression level of the unmodified truncated cryIA(b) gene version may result in rising of CryIA(b)-resistant SCSB lines in a natural insect population under real field conditions. New transgenic sugarcane lines with high tcryIA(b) gene expression are being developed in our laboratory. A more effective promoter system and an mRNA stabilizing region, both with remarkable activity in monocots, as well as gene modifications in order to get a better codon usage and eliminate destabilizing sequences, are being tried to enhance the transgenic expression.
Ohio, USA) for critical revision of the manuscript. We thank Orlando L. Pardo (Vaccine Division, CIGB) for his help with the English version and Joel Gonzalez (School of Biology, Havana University) for technical assistance.
References 1.
2.
3. 4.
5. 6. 7.
8. 9. 10. 11.
12.
13. 14. 15. 16.
17.
Acknowledgements This work was financially supported by the Council of State of the Republic of Cuba (grant 3031/243). We are grareful to Dr Donald H. Dean (Ohio State University,
18.
Adang M, Brody MS, Cardineau G, Eagan N, Roush RT, Shewmaker CK, Jones A, Oakes JV, McBride KE: The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants. Plant Mol Biol 21: 1131–1145 (1993). Arencibia A, Molina P, De la Riva G, Selman-Housein G: Production of transgenic sugarcane (Sacharum officinarum L.) plants by intact cell electroporation. Plant Cell Rep 14: 305– 309 (1995). Bennett FD: Estudios sobre el barrenador de la ca˜na de az´ucar Diatraea sp. Generalidades y control. Mexico (1977). Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976). Christou P: Genetic transformation of crop plants using microprojectile bombardment. Plant J 2: 275–281 (1992). Collazo D: Revision de la literatura mundial sobre el borer de la ca˜na de az´ucar. Editorial Cientifico T´ecnica, Havana. (1990). Cooley J, Ford T, Christou P: Molecular and genetic characterization of elite transgenic rice plants produced by electricdischarge particle acceleration. Theor Appl Genet 90: 97–104 (1995). Dekeyser R, Claes B, De Rycke R, Habets M, Van Montagu M, Caplan A: Plant Cell 2: 591–602 (1990). Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1: 19–21 (1983). Feitelson JS, Payne J, Kim L: Bacillus thuringiensis: insects and beyond. Bio/technology 10: 271–276 (1992). Fischhoff DA, Bowdish KS, Perlak FJ, Marrone PG, McCormick MS, Niedermeyer JD, Dean DA, KusanoKretzmer K, Mayer EJ, Rochester DE, Rogers SG, Fraley RT: Insect tolerant transgenic tomato plants. Bio/technology 5: 807–813 (1987). Flegg CL, Clark MF: The detection of apple chlorotic leafspot virus by modified procedure of enzyme-linked immunosorbent assay. Ann Appl Biol 91: 61–65 (1979). H¨ofte H, Whiteley H: Insecticidal crystal proteins of Bacillus thurningiensis. Microbiol Rev 53: 242–255 (1989). Jefferson RA: Genetic Engineering: Principles and Methods, vol 10, Plenum Publishing Corporation, Setlow JK (1988). Kidd G: The Bt. working group really does work. Bio/technology 12: 577–582 (1994). King EG, Martin DF, Miles LR: Advances in rearing of Lixophaga diatraea (Dip.: Tachinidae). Enthomophaga 20: 307– 311 (1975). Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P: Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci USA 85: 8502–8505 (1988). Knowles BH, Dow JAT: The crystal -endotoxins of Bacillus thuringiensis: models for their mechanism of action on the insect gut. Bioessays 15: 83–91 (1993).
molb28us.tex; 10/07/1997; 15:19; v.7; p.8
255 19. Koziel MG, Beland GL, Bowman C, Carrozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill MS, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV: Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/technology 11: 194–200 (1993). 20. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680– 685 (1970). 21. Martin JR, Galv´ez G, De Armas Urquiza R, Espinosa R, Hernandez V, Le´on A: La ca˜na de az´ucar en Cuba. Editorial Cientifico T´ecnica, Habana (1987). 22. Payan FA, Carmen H, Tasc´on G: Tecnicas para la micropropagaci´on de la ca˜na de az´ucar (Saccharum officinarum L.) mediante el cultivo de tejidos y yemas. Acta Agron 27 (1977). 23. Perlak FJ, Deaton RW, Armstrong TA, Fuch RT, Sims SR, Greenplate JT, Fischhoff DA: insect resistant cotton plants. Bio/technology 8: 939–943 (1990). 24. Russel JA, Roy MK, Sanford JC: Major improvement in biolistic transformation of suspention-cultured tobacco cells. In Vitro Cell Devel Biol 28P: 97–105 (1992).
25. 26.
27.
28.
29.
Saul MW, Potrykus Y: Direct gene transfer to protoplasts: Fate of the transferred genes. Devel Genet 11: 176–181 (1990). Vaeck M, Reynaerts A, H¨ofte H, Jansens S, De Beukeleer M, Dean DA, Zabeau M, Van Montagu M, Leemans J: Transgenic plants protected from insect attack. Nature 328: 33–37 (1987). V´azquez RI, Prieto D, De la Riva, G., Selman-Housein G: Develoment of an immunoradiometric assay for quantitative determination of CryIA(b) protein in transgenic sugarcane plants. Journal of Immunological Methods 196: 33–39 (1996). V´azquez RI, Prieto D, Mart´ınez A, De la Riva G: P210k: the putative binding protein for Bacillus thuringiensis CryIA(c) -endotoxin in the midgut of the lepidopteran Diatraea saccharalis. Biotecnol Apl 13: 117 (1996). V´azquez RI, Prieto D, Ol´oriz MI, De la Riva G, SelmanHousein G: Heterologous expression of recombinant endotoxins from Bacillus thuringiensis in Escherichia coli: comparative study in three expression systems. Rev Lat-Am Microbiol 37: 237–244 (1995).
molb28us.tex; 10/07/1997; 15:19; v.7; p.9
