Plant Cell Reports
Plant Cell Reports (1994) 14: 141-144
~j Springer-Verlag1994
Production of virus resistant and insect tolerant transgenic tobacco plants Xiao-you Liang 1,z, Yu-xian Zhu l, Jing-jiu Mi 2, and Zhang-liang Chen t ' The National Laboratory of Protein Engineering and Plant genetic Engineering, Peking University, Beijing 100871, China 2 Laboratory of Molecular Genetics, College of Biology, Beijing Agricultural University, Beijing 100094, China Received 25 January 1994/Revised version received 1 July 1994 - Communicated by R.N. Beachy
Abstract:
The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis 6-endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with A~robacterium tumefaciens A12 carrying recombinant pE14. Transgenic R 0 and R 1 tobacco plants expressing CMVCP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering. v
Abbreviations: CMV, cucumber mosaic virus; Bt toxin, Bacillus thuringiensis 6-endotoxin; CaMV, cauliflower mosaic virus; NOS, nopaline synthase; Kan, Kanamycin; Spe, spectinomycin; Carb, Carbenicillin.
Introduction With the development of biotechnology, production of transgenic plants became a routine practice for many species. Scientists applied these newly-acquired techniques to obtain individual plant lines with favorable traits such as resistance to virus (Abel et al., 1986; Nelson et al~, 1988), insects (Barton et al~, 1987) and fungi (Broglie et al., 1991), detoxification of pathogen toxin (Anzai et al.__~., 1989) or protection against certain abiotic stresses (Tarczynski et al., 1993). However, since crops are often damaged by more than a single pest or disease, transgenic plants with multiple resistance are needed for improved crop protection. CMV is the causal agent for virus diseases affecting a variety of important crops including tobacco, tomato and pepper. At least 775 plant species representing 85 families are natural hosts of this virus (Douine et al~, 1979). The coat protein (CP) gene from a Chinese isolate was cloned using Polymerase Chain Reaction
Correspondence to."Y. Zhu
(PCR) techniques in our lab (Hu et al., 1990). We have previously demonstrated that transgenic tomato plants carrying the CMV-CP gene are resistant to virus infection (Yang et al., in press). Transgenic plants which expressed a Bt toxin gene were reported to reduce insect damage (Barton et al., 1987; Perlak et al., 1991). Since both CMV and tobacco hornworm (Manduca sexta) cause severe damage to Chinese tobacco production, we initiated the current work to produce transgenic plants resistant to both CMV and .hornworm.
Materials and Methods Experimental material Agrobacterium ~mefaciens A12 and plasrnid E3 were given to us by R.N. Beachy of the Scripps Research Institute. Plasmid E9 and pQC20 were constructed in the lab (Hu et al., 1990; Dou, 1992). To facilitate expression of the Bt toxin gene in plants, we introduced plant preferred codons by the megaprimer PCR method. CMV inocula as well as insects used for bioassays were provided to us by the Institute of Plant Protection, Chinese Academy of Agriculture. Seeds of tobacco (Nicotiana tabacum) cultivar G28 were surface sterilized with 10% NaOCI solution for 20 min, rinsed in sterile distilled water for several times and finally placed for germination in sterile 500ml glass bottles containing 50ml MS basal medium (Murashige and Skoog, 1962) supplemented with 15g/1 sucrose, 0.8% W/V agar, pH 5.8 under a 12-h per-day photoperiod. Leaf-discs for transformation was obtained from tobacco seedlings 5-6 weeks after germination.
Construction of expression vectors for plant transformation The coding sequence for the CMV-CP together with the CaMV 35S promoter and NOS terminator was digested with BamH1 .from pE9 and cloned into the Hind III site of pE3, through blunt end ligation (Fig. 1). A Bt 6-endotoxin gene from Kurstaki HD-1 was inserted into the K p n I site via blunt
142 end ligation, between a 35S promoter and termination sequence of the above construct, resulting in the plasmid El4 (Fig. 1).
5'3~.S__~C~--C-P~os~ 3' 5'L E}ttoxin 13, \X
"X
/
"N ~.
Kpnl
z/
~/ ///
"V"
I
Hindlll
J
v
sequences. For western immuno blot analysis, protein samples prepared from 0.5g leaf tissue of either R0 or R 1 plants were fractionated by SDS-PAGE (in 7 % polyacrylamide) and transferred onto nitrocellulose membrane. A rabbit anti-CMV-CP antibody and alkaline 'phosphatase conjugated anti-rabbit IgG (Promega) were used as primary and secondary antibodies respctively to detect the expression of CMV-CP in transgenic plants.
Bioassays with virus and insects Various R 1 tobacco seedlings as well as controls at the 3-1eaf-stage were inoculated with CMV-D strain (1.25 pg/ml) through gentle rubbing on the leaf surface with carborundum two weeks after transplanting in soil. Tobacco hornworm (Manduca sexta) eggs were hatched on mature wild-type tobacco plants, and were allowed to feed for 2 days prior to transfer to test plants. Two-day-old larvae were placed directly on leaves of test plants, usually 5 larvae per plant per test, six plants per group. In addition, tests were conducted using excised leaves from the same group of R 1 plants in petri dishes, with 10 hornworm larvae per dish. In this assay, weights of larvae were recorded at initiation and termination of tests. Feeding trials usually lasted for 2 to 3 days, with daily monitoring of reductions in feeding and larval deaths.
Fig. 1. Construction of plant expression vector pE14 containing both
Results and Discussion
CMV-CP and Bt toxin genes.
Transformation of tobacco plants with CMV-CP and Bt toxin genes The presence of the two genes in the l~lasmid El4 was verified by Southern blot hybridization and the correct orientations were ascertained by various restriction endonuclease digestions (data not shown). One of the recombinants was named pE14 (Fig. 1). Agrobacterium tumefaciens harbouring the recombinant pE14 was used to transform G28 tobacco leaf discs. About 35 putative primary transformants were regenerated on MS medium containing 100~tg/ml kan in a single transformation experiment. PCR assays were carried out with both CMV-CP and Bt toxin gene specific primers using genomic DNA extracted from leaves of the putative transformants. Seeds collected from 22 PCR-positive plants were germinated in a glasshouse under natural lighting in the spring and summer seasons. R0 plants were used for western immuno blot assay of CMV-CP expression and R 1 plants were used for northern blot analyses of the levels of both CMV-CP and Bt gene tr.anscription.
Plant transformation and regeneration Plasmid E14 was conjugated into Agrobacterium A12 according to the procedure described by Rainer and Lothar (1988). Tobacco was transformed using the leaf disc transformation method of Horsch et al.(1985). Transgenic tobacco plants were regenerated and rooted on MS medium containing 100mg/l Kan. PCR, Northern blot and Western blot analysis DNA was extracted from leaves by the CTAB method (Murray and Thompson, 1980), and amplified under the following conditions: denaturating at 94~ for 30 sec, annealing at 55~ for i min and extension at 72~ for 1 min for amplification of the CMV-CP gene, 2 min for amplification of Bt toxin gene, with a total of 35 cycles. Primers used for amplification of the two genes were the same as those used for molecular cloning (Dou, 1992; Hu et al., 1990). Northern blot analysis was done following the standard procedures (Sambrook et al. 1989). 20tag of total RNA, prepared using the guanidium thiocyanate method (Chomezynski and Sacchim, 1987) was electrophoresed in formaldehyde-containing gels, and blotted onto nitrocellulose membrane. The presence of CMV-CP and Bt toxin mRNA were detected using radioactive labeled probes specific for the two
Expression of CMV-CP and Bt genes in transgenic plants Immuno blot analysis of CMV-CP gene expression in PCR-positive R0 transgenic tobacco plants were carried out and 17 of them showed various degrees of immuno reactivity. The result with line BC20 is shown in Fig.2. A specific immuno reactive polypeptide with molecular
143 weight about 25 kDa (Lane A) comigrates with the standard C M V coat protein (Lane C). This is not observed in plant line 42, which was transformed with the vector alone (Lane B). We performed northern blot hybridization analyses to monitor the expression of the Bt gene in transgenic tobacco due to the unavailability of antisera against Bt toxin. R N A samples extracted from 12 out of 17 R 1 plant lines descended from western-blot-positive primary transformants showed various levels of hybridization with the probe. Fig.3 shows representative hybridization patterns with different R N A samples prepared from plant BC2010 (Lane C), and BC1881 (Lane B); both plants were descended from line BC20. Lane A contains RNA extracted from descendants of the vector-only control, line 42. The strong hybridization band in LanesB andC correspond to an R N A molecular of 1.8kb which is the expected Bt transcript. In a parallel experiment, CMVCP transcript levels were also analyzed from BC2010 (Lane D), BC1881(Lane E) and line 42 descendant (Lane F).
(transgenic); lane D, RNA sample extracted from BC20; lane E, RNA sample extracted from BCI881; lane F, the same as in lane A; Lower arrow indicates migration of 0.6kb; upper arrow indicates 2. lkb. A, B, C were hybridized with a probe for Bt gene transcript; D~ E, F, were hybridized with a probe for the CMV-cp gene transcript.
Plants transgenic for CMV-CP and Bt toxin are resistant to CMV and hornworm Plants germinated from seeds of all 22 PCR-positive primary transformant lines were screened for their resistance to C M V infection and hornworm feeding. Fifteen of these plant line showed statistically significant (P < 0.05) levels of anti-viral activity with descendants of line BC20 showing least symptom development as reported in Table 1. Within 25 days, severe systemic infections were observed on control plants while only 2540% of the transgenic plants showed infection. Table 1. Transgenic plants that contain both CMV-CP and Bt genes were protected against systematic viral infection. % of plants infected Plant type
Fig. 2. Western immunoblot of rabbit anti-CMV-CP against protein samples extracted from different plant lines, lane A, sample from primary transformant BC20; lane B, samples from the vector-only control, line #42; lane C, CMV coat protein.-,~--.indicates migration of 25.2 kDa CMV-CP.
Fig. 3. Northern blot analysis of CMV-CP and Bt gene transcripts in BC2010, BC1881 and the vector control line #42(R1 generation). lane A, RNA sample extracted from vector-only control, descendants of line #42; lane B, RNA sample extracted from BCI881 (transgenic); lane C, RNA sample extracted from BC2010
Days after inoculation 10 25
G281 95+6 100 92+_5 100 #42 21-+3 26+4. CMV-CP+,Bt + 1"G28, nontransgenic parental tobacco cultivar; #42, the vector-only control line; CMV-CP+, Bt+, descendants of CMV-CP and Bt gene containing plant line BC20; DAI, days after inoculation. Thirty-six individual plants were treated as one group and the experiment were repeated 3 times. Meanwhile, a similar set of plants were tested for their ability to resist insect feeding. Assays were carried out both on leaf discs and on intact plants grown in the greenhouse. Surprisingly, only 6 different primary transformant lines showed various degrees of insect tolerance with line BC20 being the most effective plants followed by line BC06(Table 2). One pair of glasshouse grown plants with dead tobacco hornworms and much reduced feeding on transgenic plants are shown in Fig.4. The rate of insect mortality was high and larvae weight gain was low for those fed on offspring of BC20 as well as all other 5 transgenic lines when compared to those fed on control plants. In general, our results indicate that transgenic tobacco plants expressing chimeric Bt toxin a~ad C M V - C P genes to certain level could provide resistance to both hornworm and cucumber mosaic virus (Tables 1 and 2; Fig.4). The insecticid~il activity of transgenic plants descended from line BC20 with both C M V - C P and Bt toxin genes was comparable to that reported in the literature expressing only a Bt toxin gene (Perlak et al., 1991; Barton et al., 1987).
144 Table 2. Insect toxicity of transgenic plants containing CMV-CP and BVtoxingenes Plant type
Insect mortality (out of 50)* 24h
48h
72h
Total larvae weight (mg)
Barton KA, Whiteley HR, Yang NS (1987) Plant Physiol. 85: "1103-1109
Beginning
Broglie K, Chet I, HoUiday M, Cressman R, Biddle P, "Knowlton S, Mauvais CJ, Broglie R (1991) Science 254:1194-1197 Chomezynski P, Sacchim N (1987) Anal.Biochem: 162:156-159 Delannay X, La Valle BJ, Proksch RK, Fuchs RL, Sims SR, Greenplate JT, Marrone PG, Dodson RB, Augustine JJ, Layton JG, Fischhoff DA (1989) Bio/Technology 7:1265-1269 Douine L, Quint JB, Marchoux G, Archange P (1979) Ann.phytopath. 11: 439-475 Dou QC (1992) Master's thesis, Peking University Hu TH, Wu L, Liu W, Mi JJ, Pan NS, Chen ZL (1990) Chinese Science Bulletin 35(14): 1209-1214 Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraly RT(1985) Science 227:1229-1231 John ME, John MC, Ashley P, MacDonald RJ, Simpson ER, Waterman MR(1984) Proc. Nail. Acad. Sci. USA 81:5628-5632 Mizukami H, Ellis BE (1991) Plant Cell Reports 10:321-324 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Murray MG, Thompson WF (1980) Nucleic Acids Res. 8:4321-4325 Nelson RS, McCormick SM, Delannay X, Dube P, Layton J, Anderson EJ, Kaniewska M, Proksch RK, Horsch RB, Rogers SG, Fraley RT, Beachy RN (1988) Bio/Technology 6:403-409 Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischhoff DA (1991) Biochemistry 88:3324-3328 Rainer H, Lothar W (1988) Nucleic Acids Research 16:9877 Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor'Lab. Press, Cold Spring Harbor. Tarczynski MC, Jensen RG, Bohnert HJ (1993) Science 259:508-510 Yang NC, Xu HL, Pan NS, Chen ZL (1994) Horticulture Sciences (Chinese), in press
end(or when dead)
G28 0 0 1 492 1536 #42 0 0 3 515 1611 BC20 18 39 45 489 882 BC06 4 11 19 501 1242 *Each data pointrepresenm results of 5 Jetri dishes which contain leaves collected from 5 different BC20 and BC06 descendents.
The existance and expression of two foreign genes within the same transgenic plant in our experiment did not have major deleterious effects on growth characteristics, fertility or physical appearance of the plants (Fig.4). Our results suggest that we could develop new plant lines with multiple resistances to insect pests and pathogens via Agrobacterium mediated transformation by constructing plant expression vectors containing more than one resistant genes. Transgenic plants with multiple resistances are underway in the lab. Acknowledgements We thank Miss Qun Jing and Xiaohua Li for their excellent work in preparing the manuscript. This work was supported in part by a grant from the National Eighth Five-Year-Planning Committee to Y-X Zhu. Literature Cited Abel PP, Nelson RS, De B, Hoffmann, N, Rogers SG, Fraley RT, Beachy RN (1986) Science. 232:738-743 Anderson JM, Palukaitis P, Zaitlin M (1992) Proc. Natl. Acad, Sci, USA 89:8759-8763 Fig. 4. Comparison of insect toxicity of the greenhouse grown BC2010 plant and the vector control, progeny of line 42. Picture was taken 4 days after initiation of the bioassay. Note dead larvae and little damage on transgenic plant.
Anzai H, Yoneyama K, Yamaguchi I (1989) Mol.Gen.genet. 219: 492-494
Plant Cell Reports (1994) 14: 141-144
~j Springer-Verlag1994
Production of virus resistant and insect tolerant transgenic tobacco plants Xiao-you Liang 1,z, Yu-xian Zhu l, Jing-jiu Mi 2, and Zhang-liang Chen t ' The National Laboratory of Protein Engineering and Plant genetic Engineering, Peking University, Beijing 100871, China 2 Laboratory of Molecular Genetics, College of Biology, Beijing Agricultural University, Beijing 100094, China Received 25 January 1994/Revised version received 1 July 1994 - Communicated by R.N. Beachy
Abstract:
The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis 6-endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with A~robacterium tumefaciens A12 carrying recombinant pE14. Transgenic R 0 and R 1 tobacco plants expressing CMVCP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering. v
Abbreviations: CMV, cucumber mosaic virus; Bt toxin, Bacillus thuringiensis 6-endotoxin; CaMV, cauliflower mosaic virus; NOS, nopaline synthase; Kan, Kanamycin; Spe, spectinomycin; Carb, Carbenicillin.
Introduction With the development of biotechnology, production of transgenic plants became a routine practice for many species. Scientists applied these newly-acquired techniques to obtain individual plant lines with favorable traits such as resistance to virus (Abel et al., 1986; Nelson et al~, 1988), insects (Barton et al~, 1987) and fungi (Broglie et al., 1991), detoxification of pathogen toxin (Anzai et al.__~., 1989) or protection against certain abiotic stresses (Tarczynski et al., 1993). However, since crops are often damaged by more than a single pest or disease, transgenic plants with multiple resistance are needed for improved crop protection. CMV is the causal agent for virus diseases affecting a variety of important crops including tobacco, tomato and pepper. At least 775 plant species representing 85 families are natural hosts of this virus (Douine et al~, 1979). The coat protein (CP) gene from a Chinese isolate was cloned using Polymerase Chain Reaction
Correspondence to."Y. Zhu
(PCR) techniques in our lab (Hu et al., 1990). We have previously demonstrated that transgenic tomato plants carrying the CMV-CP gene are resistant to virus infection (Yang et al., in press). Transgenic plants which expressed a Bt toxin gene were reported to reduce insect damage (Barton et al., 1987; Perlak et al., 1991). Since both CMV and tobacco hornworm (Manduca sexta) cause severe damage to Chinese tobacco production, we initiated the current work to produce transgenic plants resistant to both CMV and .hornworm.
Materials and Methods Experimental material Agrobacterium ~mefaciens A12 and plasrnid E3 were given to us by R.N. Beachy of the Scripps Research Institute. Plasmid E9 and pQC20 were constructed in the lab (Hu et al., 1990; Dou, 1992). To facilitate expression of the Bt toxin gene in plants, we introduced plant preferred codons by the megaprimer PCR method. CMV inocula as well as insects used for bioassays were provided to us by the Institute of Plant Protection, Chinese Academy of Agriculture. Seeds of tobacco (Nicotiana tabacum) cultivar G28 were surface sterilized with 10% NaOCI solution for 20 min, rinsed in sterile distilled water for several times and finally placed for germination in sterile 500ml glass bottles containing 50ml MS basal medium (Murashige and Skoog, 1962) supplemented with 15g/1 sucrose, 0.8% W/V agar, pH 5.8 under a 12-h per-day photoperiod. Leaf-discs for transformation was obtained from tobacco seedlings 5-6 weeks after germination.
Construction of expression vectors for plant transformation The coding sequence for the CMV-CP together with the CaMV 35S promoter and NOS terminator was digested with BamH1 .from pE9 and cloned into the Hind III site of pE3, through blunt end ligation (Fig. 1). A Bt 6-endotoxin gene from Kurstaki HD-1 was inserted into the K p n I site via blunt
142 end ligation, between a 35S promoter and termination sequence of the above construct, resulting in the plasmid El4 (Fig. 1).
5'3~.S__~C~--C-P~os~ 3' 5'L E}ttoxin 13, \X
"X
/
"N ~.
Kpnl
z/
~/ ///
"V"
I
Hindlll
J
v
sequences. For western immuno blot analysis, protein samples prepared from 0.5g leaf tissue of either R0 or R 1 plants were fractionated by SDS-PAGE (in 7 % polyacrylamide) and transferred onto nitrocellulose membrane. A rabbit anti-CMV-CP antibody and alkaline 'phosphatase conjugated anti-rabbit IgG (Promega) were used as primary and secondary antibodies respctively to detect the expression of CMV-CP in transgenic plants.
Bioassays with virus and insects Various R 1 tobacco seedlings as well as controls at the 3-1eaf-stage were inoculated with CMV-D strain (1.25 pg/ml) through gentle rubbing on the leaf surface with carborundum two weeks after transplanting in soil. Tobacco hornworm (Manduca sexta) eggs were hatched on mature wild-type tobacco plants, and were allowed to feed for 2 days prior to transfer to test plants. Two-day-old larvae were placed directly on leaves of test plants, usually 5 larvae per plant per test, six plants per group. In addition, tests were conducted using excised leaves from the same group of R 1 plants in petri dishes, with 10 hornworm larvae per dish. In this assay, weights of larvae were recorded at initiation and termination of tests. Feeding trials usually lasted for 2 to 3 days, with daily monitoring of reductions in feeding and larval deaths.
Fig. 1. Construction of plant expression vector pE14 containing both
Results and Discussion
CMV-CP and Bt toxin genes.
Transformation of tobacco plants with CMV-CP and Bt toxin genes The presence of the two genes in the l~lasmid El4 was verified by Southern blot hybridization and the correct orientations were ascertained by various restriction endonuclease digestions (data not shown). One of the recombinants was named pE14 (Fig. 1). Agrobacterium tumefaciens harbouring the recombinant pE14 was used to transform G28 tobacco leaf discs. About 35 putative primary transformants were regenerated on MS medium containing 100~tg/ml kan in a single transformation experiment. PCR assays were carried out with both CMV-CP and Bt toxin gene specific primers using genomic DNA extracted from leaves of the putative transformants. Seeds collected from 22 PCR-positive plants were germinated in a glasshouse under natural lighting in the spring and summer seasons. R0 plants were used for western immuno blot assay of CMV-CP expression and R 1 plants were used for northern blot analyses of the levels of both CMV-CP and Bt gene tr.anscription.
Plant transformation and regeneration Plasmid E14 was conjugated into Agrobacterium A12 according to the procedure described by Rainer and Lothar (1988). Tobacco was transformed using the leaf disc transformation method of Horsch et al.(1985). Transgenic tobacco plants were regenerated and rooted on MS medium containing 100mg/l Kan. PCR, Northern blot and Western blot analysis DNA was extracted from leaves by the CTAB method (Murray and Thompson, 1980), and amplified under the following conditions: denaturating at 94~ for 30 sec, annealing at 55~ for i min and extension at 72~ for 1 min for amplification of the CMV-CP gene, 2 min for amplification of Bt toxin gene, with a total of 35 cycles. Primers used for amplification of the two genes were the same as those used for molecular cloning (Dou, 1992; Hu et al., 1990). Northern blot analysis was done following the standard procedures (Sambrook et al. 1989). 20tag of total RNA, prepared using the guanidium thiocyanate method (Chomezynski and Sacchim, 1987) was electrophoresed in formaldehyde-containing gels, and blotted onto nitrocellulose membrane. The presence of CMV-CP and Bt toxin mRNA were detected using radioactive labeled probes specific for the two
Expression of CMV-CP and Bt genes in transgenic plants Immuno blot analysis of CMV-CP gene expression in PCR-positive R0 transgenic tobacco plants were carried out and 17 of them showed various degrees of immuno reactivity. The result with line BC20 is shown in Fig.2. A specific immuno reactive polypeptide with molecular
143 weight about 25 kDa (Lane A) comigrates with the standard C M V coat protein (Lane C). This is not observed in plant line 42, which was transformed with the vector alone (Lane B). We performed northern blot hybridization analyses to monitor the expression of the Bt gene in transgenic tobacco due to the unavailability of antisera against Bt toxin. R N A samples extracted from 12 out of 17 R 1 plant lines descended from western-blot-positive primary transformants showed various levels of hybridization with the probe. Fig.3 shows representative hybridization patterns with different R N A samples prepared from plant BC2010 (Lane C), and BC1881 (Lane B); both plants were descended from line BC20. Lane A contains RNA extracted from descendants of the vector-only control, line 42. The strong hybridization band in LanesB andC correspond to an R N A molecular of 1.8kb which is the expected Bt transcript. In a parallel experiment, CMVCP transcript levels were also analyzed from BC2010 (Lane D), BC1881(Lane E) and line 42 descendant (Lane F).
(transgenic); lane D, RNA sample extracted from BC20; lane E, RNA sample extracted from BCI881; lane F, the same as in lane A; Lower arrow indicates migration of 0.6kb; upper arrow indicates 2. lkb. A, B, C were hybridized with a probe for Bt gene transcript; D~ E, F, were hybridized with a probe for the CMV-cp gene transcript.
Plants transgenic for CMV-CP and Bt toxin are resistant to CMV and hornworm Plants germinated from seeds of all 22 PCR-positive primary transformant lines were screened for their resistance to C M V infection and hornworm feeding. Fifteen of these plant line showed statistically significant (P < 0.05) levels of anti-viral activity with descendants of line BC20 showing least symptom development as reported in Table 1. Within 25 days, severe systemic infections were observed on control plants while only 2540% of the transgenic plants showed infection. Table 1. Transgenic plants that contain both CMV-CP and Bt genes were protected against systematic viral infection. % of plants infected Plant type
Fig. 2. Western immunoblot of rabbit anti-CMV-CP against protein samples extracted from different plant lines, lane A, sample from primary transformant BC20; lane B, samples from the vector-only control, line #42; lane C, CMV coat protein.-,~--.indicates migration of 25.2 kDa CMV-CP.
Fig. 3. Northern blot analysis of CMV-CP and Bt gene transcripts in BC2010, BC1881 and the vector control line #42(R1 generation). lane A, RNA sample extracted from vector-only control, descendants of line #42; lane B, RNA sample extracted from BCI881 (transgenic); lane C, RNA sample extracted from BC2010
Days after inoculation 10 25
G281 95+6 100 92+_5 100 #42 21-+3 26+4. CMV-CP+,Bt + 1"G28, nontransgenic parental tobacco cultivar; #42, the vector-only control line; CMV-CP+, Bt+, descendants of CMV-CP and Bt gene containing plant line BC20; DAI, days after inoculation. Thirty-six individual plants were treated as one group and the experiment were repeated 3 times. Meanwhile, a similar set of plants were tested for their ability to resist insect feeding. Assays were carried out both on leaf discs and on intact plants grown in the greenhouse. Surprisingly, only 6 different primary transformant lines showed various degrees of insect tolerance with line BC20 being the most effective plants followed by line BC06(Table 2). One pair of glasshouse grown plants with dead tobacco hornworms and much reduced feeding on transgenic plants are shown in Fig.4. The rate of insect mortality was high and larvae weight gain was low for those fed on offspring of BC20 as well as all other 5 transgenic lines when compared to those fed on control plants. In general, our results indicate that transgenic tobacco plants expressing chimeric Bt toxin a~ad C M V - C P genes to certain level could provide resistance to both hornworm and cucumber mosaic virus (Tables 1 and 2; Fig.4). The insecticid~il activity of transgenic plants descended from line BC20 with both C M V - C P and Bt toxin genes was comparable to that reported in the literature expressing only a Bt toxin gene (Perlak et al., 1991; Barton et al., 1987).
144 Table 2. Insect toxicity of transgenic plants containing CMV-CP and BVtoxingenes Plant type
Insect mortality (out of 50)* 24h
48h
72h
Total larvae weight (mg)
Barton KA, Whiteley HR, Yang NS (1987) Plant Physiol. 85: "1103-1109
Beginning
Broglie K, Chet I, HoUiday M, Cressman R, Biddle P, "Knowlton S, Mauvais CJ, Broglie R (1991) Science 254:1194-1197 Chomezynski P, Sacchim N (1987) Anal.Biochem: 162:156-159 Delannay X, La Valle BJ, Proksch RK, Fuchs RL, Sims SR, Greenplate JT, Marrone PG, Dodson RB, Augustine JJ, Layton JG, Fischhoff DA (1989) Bio/Technology 7:1265-1269 Douine L, Quint JB, Marchoux G, Archange P (1979) Ann.phytopath. 11: 439-475 Dou QC (1992) Master's thesis, Peking University Hu TH, Wu L, Liu W, Mi JJ, Pan NS, Chen ZL (1990) Chinese Science Bulletin 35(14): 1209-1214 Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraly RT(1985) Science 227:1229-1231 John ME, John MC, Ashley P, MacDonald RJ, Simpson ER, Waterman MR(1984) Proc. Nail. Acad. Sci. USA 81:5628-5632 Mizukami H, Ellis BE (1991) Plant Cell Reports 10:321-324 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Murray MG, Thompson WF (1980) Nucleic Acids Res. 8:4321-4325 Nelson RS, McCormick SM, Delannay X, Dube P, Layton J, Anderson EJ, Kaniewska M, Proksch RK, Horsch RB, Rogers SG, Fraley RT, Beachy RN (1988) Bio/Technology 6:403-409 Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischhoff DA (1991) Biochemistry 88:3324-3328 Rainer H, Lothar W (1988) Nucleic Acids Research 16:9877 Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor'Lab. Press, Cold Spring Harbor. Tarczynski MC, Jensen RG, Bohnert HJ (1993) Science 259:508-510 Yang NC, Xu HL, Pan NS, Chen ZL (1994) Horticulture Sciences (Chinese), in press
end(or when dead)
G28 0 0 1 492 1536 #42 0 0 3 515 1611 BC20 18 39 45 489 882 BC06 4 11 19 501 1242 *Each data pointrepresenm results of 5 Jetri dishes which contain leaves collected from 5 different BC20 and BC06 descendents.
The existance and expression of two foreign genes within the same transgenic plant in our experiment did not have major deleterious effects on growth characteristics, fertility or physical appearance of the plants (Fig.4). Our results suggest that we could develop new plant lines with multiple resistances to insect pests and pathogens via Agrobacterium mediated transformation by constructing plant expression vectors containing more than one resistant genes. Transgenic plants with multiple resistances are underway in the lab. Acknowledgements We thank Miss Qun Jing and Xiaohua Li for their excellent work in preparing the manuscript. This work was supported in part by a grant from the National Eighth Five-Year-Planning Committee to Y-X Zhu. Literature Cited Abel PP, Nelson RS, De B, Hoffmann, N, Rogers SG, Fraley RT, Beachy RN (1986) Science. 232:738-743 Anderson JM, Palukaitis P, Zaitlin M (1992) Proc. Natl. Acad, Sci, USA 89:8759-8763 Fig. 4. Comparison of insect toxicity of the greenhouse grown BC2010 plant and the vector control, progeny of line 42. Picture was taken 4 days after initiation of the bioassay. Note dead larvae and little damage on transgenic plant.
Anzai H, Yoneyama K, Yamaguchi I (1989) Mol.Gen.genet. 219: 492-494
