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Transgenic carrots with enhanced resistance against two major pathogens, Erysipheheraclei and Alternariadauci
Plant Science 153 (2000) 135 – 144 www.elsevier.com/locate/plantsci
Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci Miyuki Takaichi, Kenji Oeda * Biotechnology Laboratory, Sumitomo Chemical Co. Ltd., 2 -1, 4 -chome, Takatsukasa, Takarazuka, Hyogo 665 -8555, Japan Received 17 June 1999; accepted 3 December 1999
Abstract In vitro assay indicated that the human lysozyme has lytic activity against phytopathogenic fungi and bacteria. A human lysozyme gene was placed under control of the constitutive CaMV 35S promoter and the resulting expression plasmid was introduced into two cultivars (cv.) of carrot, Kurodagosun (K5) and Nantes Scarlet (NS), by Agrobacterium tumefaciens-mediated method. Seven and fourteen transgenic plants of cv. K5 and cv. NS were regenerated, respectively, and the obtained transgenic carrots of T0 generation was tested for disease resistance against Erysiphe heraclei, a pathogenic fungi causing powdery mildew. Among the tested lines, the transgenic plant No. 12-1 and 8-1 of cv. NS showed a fairly strong resistance against E. heraclei. The strong disease resistance was also confirmed in T1 generation. Disease resistance against another pathogen of leaf blight, Alternaria dauci, were also tested using T1 transgenic lines. Significant enhanced resistance was observed in the No. 12-1 of cv. NS. Accumulation of synthesized human lysozyme protein was observed in this line, a finding consistent with observed disease resistance. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human lysozyme; Transgenic carrots; Agrobacterium tumefaciens; Disease resistance
1. Introduction Development of recombinant DNA techniques and plant genetic transformation methods facilitate introduction of selected genes into plants to obtain transgenic ones with novel phenotypes. A variety of transgenic plants exhibiting disease resistance, herbicide resistance [1], delayed ripening [2], and alteration of protein composition in seeds [3] has been generated and some were tested in the field trials. Disease resistance is one of the important targets of plant breeding, because significant yield losses due to pathogenic attack limits crop productivity. Therefore, new disease control strategies to produce transgenic plants exhibiting enhanced disease resistance are now being widely Abbre6iations: CSS, chitinase signal sequence; cv., cultivar; GUS, b-glucuronidase gene; NPT II, neomycin phosphotransferase gene. * Corresponding author. Tel.: + 81-797-742121; fax: +81-797742133. E-mail address: [email protected] (K. Oeda)
evaluated. Viral coat protein genes can be used to generate transgenic plants exhibiting virus resistance [4]. The cecropin gene was utilized to generate disease resistant plants because cecropin contains antibacterial activity against several bacterial pathogens [5]. Both glucanase and chitinase also exhibited antifungal activity. Transgenic plants of tobacco [5–7], rice [8], cucumber [9,10] expressing glucanase or chitinase genes exhibited enhanced disease resistance against fungal pathogens. Lysozymes are basic bacteriolytic proteins widely distributed in nature. A protective and defensive role against bacterial or fungal pathogens has been suggested for lysozymes of plant origin [11], and these lysozymes were purified from latex of papaya [12], fig [13], cultured Rubus hispidus cells [14], cultured Parthenocisis quinquifolia cells [15], turnip roots [16] and wheat germ [11]. Lysozymes from human [17] and hen egg [18] were also purified, and both lysozymes exhibited bacte-
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riolytic function. Human lysozyme cleaves the b(1–4) glycosidic bond of peptidoglycan in the bacterial cell wall and of chitin in the fungal cell wall, other classes of animal lysozymes cannot hydrolyze chitin [19]. Therefore, the human lysozyme might have potential to protect plants from both bacterial and fungal diseases. In the present work, the gene cassette coding for human lysozyme [20] and a chitinase signal sequence (CSS) of Azuki bean [21] was placed under a strong constitutive CaMV 35S promoter, to obtain the transgenic plants expressing the human lysozyme. The resulting construct was introduced into two different cultivars of carrot, Kurodagosun (K5) and Nantes Scarlet (NS). More than 20 transgenic carrots were regenerated from embryogenic calli and they were raised to mature plants. The obtained transgenic plants were tested for disease resistance against Erysiphe heracle (powdery mildew) and Alternaria dauci (leaf blight). Consequently, transgenic carrots of cultivar (cv.) K5 and cv. NS showed enhanced disease resistance against these pathogens. Especially, the No. 12-1 of cv. NS showed distinct disease resistance.
Bacterial cultures (50 ml) of E. caroto6ora or P. syringae, were prepared by dilution of overnight culture, and mixed with the equal volume (50 ml) of 0, 30, 100 mg/l of human lysozyme solution, respectively. These mixtures were incubated for 1 h at room temperature, and spread on agar plates composed of PDA medium. The number of colonies was measured after 24 h.
2.2. Plant materials Carrot (Daucus carota L.) cultivars used in this study are K5 and NS. Seeds were surface-sterilized with 70% ethanol for 30 s and disinfected with 1% chloric acid for 10 min. The seeds were rinsed 3 times in sterile distilled water and then germinated on modified MS medium (half strength Murashige and Skoog [22] salts, 2 g/l sucrose and 0.8% agar) at 25°C in the dark. Hypocotyls were dissected from 1-week-old seedlings and cut into segments of 5–10 mm long. The explants were used for transformation experiments of carrots.
2.3. Agrobacterium tumefaciens strains and the used plasmid 2. Materials and methods
2.1. In 6itro assay against phytopathogens Fungal pathogens, Rhizoctonia and Alternaria, bacterial ones, Erwinia caroto6ora and Pseudomonas syringae, were used to test the effect of human lysozyme (Sigma, USA). Rhizoctonia or Alternaria spores were inoculated on agar plates composed of PDA medium (Nissui, Japan) supplemented with 0, 30 and 100 mg/l of human lysozyme, respectively. Each hypha length was measured after 24 h.
A. tumefaciens strains LBA4404 and C58C1 containing the binary plasmid pNGL2 were used to transform carrots. The plasmid pNGL2 consists of a neomycin phosphotransferase gene (NPT II), a b-glucuronidase gene (GUS) and the CaMV 35S promoter fused to a human lysozyme gene. The gene fragment coding for the CSS of Azuki bean [21] was also placed upstream of the human lysozyme gene (Fig. 1). The signal sequence is thought to be useful for efficient transportation of the synthesized protein into the intercelluar space of the plant cell.
Fig. 1. Structure of the constructed expression plasmid pNGL2. NPT II, neomycin phosphotransferase gene; GUS, b-glucuronidase gene; HLY, human lysozyme gene; CSS, chitinase signal sequence of Azuki bean; 35S, CaMV 35S promoter; NOS-P and NOS-T, Nos promoter and terminator, respectively.
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2.4. Transformation and regeneration Explants were inoculated in the bacterial solution for 5 min, then placed on the co-cultivation medium which consists of MS salts, 4.5 mM 2,4dichlorophenoxyacetic acid (2,4-D), 3 g/l sucrose and 0.8 g/l agar. This medium is basal to produce embryogenic callus. Infected explants were incubated in the dark at 20–21°C for 2–3 days. After co-cultivation, explants were rinsed in the liquid basal medium containing 500 mg/l cefotaxime, and then placed on the basal medium supplemented with 300 mg/l cefotaxime for 4 weeks to prevent bacterial growth. Then, the explants were transferred to the basal medium containing 50 mg/l kanamycin and 100 mg/l cefotaxime for 4 –6 weeks as the first selection. The obtained embryogenic callus exhibiting kanamycin resistance was subcultured on hormone free MS medium, without kanamycin. After 2–4 weeks, the seedlings germinated from embryogenic calli were transferred to MS medium containg 100 mg/l kanamycin, as the second selection. After co-cultivation, the cultures were placed under conditions of 16 h light/8 h dark at 25°C. Transgenic seedlings were regenerated from embryogenic calli and they were examined for disease resistance. Insertion of the human lysozyme gene into the plant genome was confirmed by polymerase chain reaction (PCR) analysis and the transgenic carrots were acclimatized to soil and grown at 20–23°C in a greenhouse. Transgenic carrots with auxetic roots were transferred to a cold room and maintained at 5°C for at least 2 months. After this exposure to a low temperature, transgenic carrots were bolted at 20 –23°C and self-pollinated seeds were obtained.
2.5. PCR analysis Genomic DNA was isolated from cultured carrot leaves, using cetyltrimethyl ammonium bromide [23]. PCR for the human lysozyme gene was performed with the genomic DNA of carrot plants using the synthetic oligonucleotide primers. The primers of 5%-GAACGTTGTGAATTGGCCAG3% and 5%-GTTTTGACAGCGGTTACGCC-3% was designed to hybridize the 5% and 3% regions of human lysozyme gene, respectively. The 42 cycle reaction, which consists of heat denaturation (95°C), annealing (55°C) and extension (72°C),
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was carried out, and the reaction mixture was subjected to 0.8% agarose gel electrophoresis. Specific amplification of the 350 base pairs fragment indicates that the human lysozyme gene was inserted into the genome of the transgenic carrot plants.
2.6. Western blot analysis Young leaves of carrots grown in a greenhouse were homogenized in phosphoric buffer (0.1 M K2HPO4, 2.5 mM EDTA, 0.1% ascorbic acid, 1% mercaptoethanol and 0.5 mM PMSF), and the crude extract was separated by centrifugation for 10 min at 4°C (15 000×g). The supernatant (10 mg total soluble protein) was subjected to 10% SDS-polyacrylamide gel electrophoresis, and the resolved proteins were transferred onto nitrocellulose membranes (Schleicher Schuell, Dassel Germany). The human lysozyme on the membrane was detected by enzyme-linked immunostaining. The membrane was exposed to anti-human lysozyme serum from a goat (Nordic Immunology, Tilburg, The Netherlands), then to alkaline phosphatase conjugated antibody against goat IgG from a rabbit (Vector Laboratories, CA, USA). The human lysozyme on the membrane was visualized by reaction with 5-bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium.
2.7. Biological test for resistance against Erysiphe heraclei Among 23 plants of transgenic carrots, 21 were transferred to the soil, and finally, 13 plants grew to the same stage of development. After 1 month of acclimatization, the obtained transgenic carrots were examined for disease resistance against E. heraclei, the phytopathogen of powdery mildew. The diseased carrots previously infected with E. heraclei were randomly placed among the tested transgenic and non-transgenic plants, and co-cultivated in a greenhouse at 21–23°C for 1 week. Disease rating was classified into six ranks based on the average area of disease, as defined in Table 1. The disease rating was first measured 1 week after inoculation, to observe early symptoms of diseased plants. Then, the disease ratings were successively measured twice every week, and the development of powdery mildew was investigated. Non-transgenic plants were also tested as negative control.
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Table 1 Disease rating on powdery mildew Disease rating
Disease developmental area (%)
0 0.5 1 2 4 8
0 0–5 5–10 10–25 25–50 \50
T1 progeny were also examined for disease resistance against E. heraclei. Self-pollinated seeds derived from T0 plants were sowed on soil, and the germinated seedlings were examined by PCR. Seedlings without the lysozyme gene were discarded. Five to ten plants for each line were grown in a greenhouse. Four weeks later, these T1 progenies were infected with E. heraclei, and disease rating scored as described above. A disease index (%) was also calculated by the following formula, according to the method of the Agrochemical Society of Japan. The disease index = {%(disease rating from 0 to 8) /(8 × a number of plants)} × 100
2.8. Biological test for resistance against A. dauci T1 lines of No. 12-1 and non-transgenic plants, were tested for disease resistance against another carrot pathogen, A. dauci, using a detached petiole
assay [9]. Five plants for each line were grown in vitro up to the same developmental stage, the leaves were trimmed off, and petioles were cut into 8 cm in length. Four segments were prepared from one plant and used for the biological test. Petiole fragments from transgenic and non-transgenic plants were stood upright in actively growing colony layers of the fungal pathogen on PDA medium (Nissui, Japan). The fungal pathogen, which was precultured for 7 days after inoculation, was used. The lesion length developed from the base of the petiole fragment was measured after 14 days of incubation. Lesion length on each petiole was measured.
3. Results and discussions
3.1. Lytic acti6ity of human lysozyme against phytopathogens Fungal pathogen, Rhizoctonia and Alternaria, and bacterial ones, E. caroto6ora and P. syringae, were used to test the effect of human lysozyme. Hypha length of Rhizoctonia and Alternaria were inhibited to 30 and 50% with 30 mg/l of human lysozyme, respectively (Table 2). Similarly, about 65 and 100% of bacteria, E. caroto6ora and P. syringae, lyzed during 1 h incubation with 100 mg/l of human lysozyme, respectively (Table 2). These results indicated that the human lysozyme was effective against phytopathogenic bacteria and fungi [19].
Table 2 Growth inhibition of Rhizoctonia and Alternaria, and reduction of colony formation of Erwinia caroto6ora and Pseudomonas syringae by treatment of human lysozyme Human lysozyme (mg/l)
Rhizoctonia a Hypha length (mm)
Ratio (%)
Hypha length (mm)
Ratio (%)
No. of colonies
Ratio (%)
No. of colonies
Ratio (%)
0 30 100
11.0ac 3.5b 3.0b
100 32 27
4.5a 2.3b 2.0b
100 51 44
236a 111b 82b
100 47 35
222a 21b 0b
100 9 0
a
Alternaria a
Erwinia b
Pseudomonas b
Each spore of Rhizoctonia and Alternaria was inoculated on agar plates composed of PDA medium supplemented with human lysozyme. The hypha length (mm) was measured after 24 h incubation. b Each bacterial culture of E. caroto6ora and P. syringae, and human lysozyme solution was mixed, and incubated for 1 h. These mixtures were spread on agar plates composed of PDA medium. c Within each pathogen, numbers followed by the same letter are not significantly (0.05%) different according to LSD test.
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Table 3 Experimental conditions for carrot transformation Carrot cultiver
Agrobacterium strain
No. of explants
No. of transformants
Transformation efficiency (%)
Kurodagosun
LBA4404 C58C1 LBA4404 C58C1
86 90 85 99
1 6 10 6
1.2 6.7 11.8 6.1
Nantes Scarlet
Fig. 2. Integration of the human lysozyme gene into the carrot genome. Integration of the human lysozyme gene into the carrot genome of T0 and T1 generation was confirmed by polymerase chain reaction (PCR) analysis. (a) Specific amplifications of 350 base pairs fragment of human lysozyme gene were indicated in all genomic DNA samples of T0 plants. (b) The 350 base pairs fragment was also detected in T1 plants. M was molecular marker. Plasmid pNGL2 was a positive control. All samples were cultivar (cv.) Nantes Scarlet (NS).
3.2. Regeneration and culti6ation of transgenic carrots The expression plasmid pNGL2 was constructed to express the human lysozyme gene in carrots (Fig. 1). The plasmid pNGL2 contains the CaMV 35S promoter and the human lysozyme gene. The obtained construct was introduced into two cultivars of carrot, K5 and NS. Both carrot cultivars and strains of A. tumefaciens used for transformation procedure had considerable effects on the transformation efficiency of carrots (Table 3). When A. tumefaciens strain LBA4404 was used, only one transgenic plant was selected with cv. K5, while ten transgenic plants were obtained in the case of cv. NS. The calculated transformation efficiencies were 1.2 and 11.8%, respectively. Six transgenic plants were obtained for both cv. K5 and cv. NS, respectively, when A. tumefaciens strain C58C1 was used. The efficiencies were estimated to be 6.7% and 6.1%, respectively. The A. tumefaciens-mediated method and electroporation [24] can be used for transformation of carrots. However, the A. tumefaciensmediated method is now widely used for carrot transformation [25–28], because this method is
simple and is economical of time. The efficiencies of transformation were, therefore, thought to depend on the combination of A. tumefaciens strains and carrot cultivars. The other carrot cultivar ‘Nanco’ was often used as a major host cultivar because the transformation efficiency was reported to be highest among many cultivars, over 10% [25,27]. When cv. K5 and cv. NS were used in this experiment, the mean efficiency was calculated to be 6.4%, slightly lower than cv. Nanco. Petiole, cotyledon, hypocotyl, root of carrot seedling and even callus could be used for transformation experiments, but the transformation efficiency was also greatly affected by used explants and the developmental stage [25–28]. In this experiment, hypocotyls of 1-week-old seedlings were used as explants because the highest regeneration efficiency was obtained. Insertion of the human lysozyme gene into the carrot genome was tested by PCR analysis (Fig. 2a). Consequently, 7 and 16 independent lines from cv. K5 and cv. NS, respectively, were found to have the amplified human lysozyme DNA fragments. These plants were acclimatized to soil and T1 seeds were produced. Five transgenic plants of cv. K5 were successfully self-pollinated and T1
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Table 4 The number of regenerated, acclimatized and self-pollinated transgenic carrots Carrot cultiver
No. of transformants Regeneration Acclimatization
Kurodagosun 7 Nantes 16 Scarlet
7 14
Selfpollination 5 10
seeds were produced. Similarly, 14 transformants of cv. NS were acclimatized to obtain mature plants. Finally, 10 independent transgenic plants of cv. NS self-pollinated and T1 seeds were produced (Table 4). The amplified DNA bands were also observed in these T1 plants (Fig. 2b).
3.3. Western blot analysis Mature plants of each line were subjected to Western blot analysis to evaluate accumulation of the synthesized human lysozyme. A specific signal of the 14 kDa protein band was detected in almost all transgenic plants. The signal for the human lysozyme was detected in almost all samples from the T0 carrots of cv. K5 and NS. Expression levels of the human lysozyme were relatively high in samples of No. 8-1, 8-2 and 12-1 of cv. NS (Fig. 3a). The transgenic plant No. 7-1, which contained the human lysozyme gene, did not produce human lysozyme. This might be caused by a strong co-suppression [29]. This line was also used for further experiments as one of the negative controls.
Self-pollinated seeds were obtained from transgenic carrots of cv. K5 and cv. NS, respectively. These seeds were germinated and five to ten seedlings for each line were grown in a greenhouse. T1 transgenic plants were also subjected to Western blot analysis to determine expression level of the human lysozyme. Especially, expression level was high and stable in samples of lines No. 8-1 and 12-1 of cv. NS. (Fig. 3b).
3.4. Resistance against pathogens 3.4.1. E. heraclei 3.4.1.1. T0 generation. To observe disease resistance of the obtained transgenic carrots of cv. NS, the resistance against E. heraclei was measured. The disease rating was first measured 1 week after inoculation, to observe early symptom of diseased plants. The disease rating was successively measured twice every week, and the development of powdery mildew was investigated. Symptoms in all transgenic plants were moderate (disease index lower than 20%) compared to that of non-transgenic plants (disease index of 50%), when disease rating was scored 1 week after inoculation. However, symptoms of powdery mildew developed on the upper leaves at a later stage, and the degree of symptoms varied among tested plants. When disease rating was scored 3 weeks later after inoculation, transgenic carrots No. 8-1, 8-2 and 12-1 showed a disease index of 30%, while other transgenic plants showed 50–80% of the disease index. At that time, non-transgenic plants and the transgenic plant No. 7-1, the negative control line, exhibited disease index of about 80% (Fig. 4a).
Fig. 3. Detection of the human lysozyme by Western blot analysis in T0 and T1 generations of cultivar (cv.) Nantes Scarlet (NS). H, 1 mg protein of human lysozyme as a positive control. Ten mg of total soluble protein of each sample was analyzed by Western blot. M, molecular marker. (a) The No. 8-1, 8-2 and 12-1 of T0 generation exhibited the synthesized human lysozyme. The non-specific protein band with lower molecular weight was observed, besides the synthesized human lysozyme. (b) The No. 8-1 and 12-1 also produced high level of human lysozyme on T1 generation.
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8-1 showed about 40% of the disease index, while the No. 12-1 exhibited a very low disease index (15%) (Figs. 4b and 5). A difference of disease development was observed at early stages of pathogen inoculation as found in two tests of non-transgenic plants (Fig. 4a,b). This fluctuation at early stages might be caused by different timing of pathogen infections. However, disease development at later stages was basically the same. Interestingly, the disease index of powdery mildew differed among the carrot cultivars. In cv. K5 development of powdery mildew was slightly delayed compared to findings in cv. NS (data not shown). It was also reported that the process of disease development varied in the carrot cultivars, when transgenic carrots expressing chitinase gene were tested [9].
Fig. 4. Disease index against powdery mildew of transgenic lines of cultivar (cv.) Nantes Scarlet (NS). After 1 month of acclimatization, the obtained transgenic carrots were examined for disease resistance against powdery mildew. The diseased carrots previously infected with Erysiphe heraclei were randomly placed among the tested transgenic and non-transgenic plants, and co-cultivated in a greenhouse at 21 – 23°C for 1 week. Disease rating was classified into six ranks based on the average area of disease in plants, as defined in Table 1. The disease rating was first measured 1 week after inoculation, to observe early symptoms of diseased plants. The disease ratings were successively measured twice every week, and the development of powdery mildew was investigated. (a) T0 generation, investigated on 1 week ( ), 2 weeks ( ) and 3 weeks (b) after the pathogen inoculation. (b) T1 generation, investigated on 1 week ( ), 2 weeks ( ) and 3 weeks (b). The disease index test of T1 generation of transgenic line No. 8-2 was not performed, because T0 generation of this line did not set seeds. Disease index (%) ={(Disease rating from 0 to 8)/(8×number of plants)} ×100. Mean values obtained for five to ten plants per line. Within each generation and line, numbers followed by the same letter are not significantly (0.05%) different according to LSD test.
3.4.1.2. T1 progeny. The disease indexes of all T1 lines were less than 10%, when disease rating was scored just after inoculation. Transgenic lines No. 7-1, showed 80% of the disease index, and the No.
3.4.2. A. dauci The detached petiole assay was done to evaluate disease resistance of T1 generation of the line No. 12-1 and non-transgenic plants against A. dauci. Five plants for each line were grown in vitro up to the same developmental stage, the leaves were trimmed off, and petioles were cut into 8 cm in length. Four segments were prepared from one plant and used for the biological test. The No. 12-1 was selected based on data concerning the disease index against powdery mildew. This line exhibited very high disease resistance against powdery mildew, and also showed high level and stable accumulation of the human lysozyme (Figs. 3 and 4). The lesion lengths of non-transgenic plants were dispersed between 10 and 60 mm at 2 weeks after inoculation. On the contrary, the transgenic line No. 12-1 was mostly collected below 25 mm of lesion development (Fig. 6). The detached petiol assay was repeated twice, and the obtained results were basically the same between two tests (data not shown). Thus, development of pathogen was considered to be inhibited in the No. 12-1 in comparison with the non-transgenic plants. Thus, the No. 12-1 exhibited the disease resistance against pathogens, both E. heraclei and A. dauci. In this resistant line, the production level of the human lysozyme was high and stable compared with those of other lines, which is consistent with the acquired resistance against these pathogens. Taken together, the human lysozyme gene will prove useful for production of transgenic crops
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and vegetables to enhance disease resistance against both bacterial and fungal pathogens. A human lysozyme has strong bacteriolytic activity against bacteria and fungi [30]. In a previous work, T0 generations of transgenic tobacco plants expressing the human lysozyme showed enhanced disease resistance against Erysiphe cichoracearum and P. syringae [20]. In the present work, the stable production of the human lysozyme was further confirmed in T1 as well as T0 generations of the transgenic carrots and also usefulness of the human lysozyme gene to confer the enhanced disease resistance. Leaf extracts of transgenic tobacco plants expressing a hen egg white lysozyme (HEWL) contained only about 30 ng of HEWL per mg of leaf tissue (0.003%) [31]. In transgenic potato plants expressing a bacteriophage T4 lysozyme, the syn-
thesized lysozyme level was estimated to be about 0.001% of total soluble proteins [32]. On the contrary, the production level of the human lysozyme in the transgenic line No. 12-1 was calculated to be about 0.5% per total soluble protein. Thus, the expression level of human lysozyme was very high compared with those of the above cases. The gene fragment coding for the CSS of Azuki bean [21] was placed upstream of the human lysozyme gene. The CSS might cause highly efficient transportation of the synthesized protein to extracellular spaces. Differences of the expression level were observed between the obtained transgenic carrot lines, which is mainly caused by two factors, the positional effects of the inserted gene and the copy number. It is very difficult to discriminate between each contribution of these two factors to the ob-
Fig. 5. Biological test for disease resistance against Erysiphe heraclei (powdery mildew) in T1 generation of cultivar (cv.) Nantes Scarlet (NS). (a) Transgenic line No. 12-1. High level expression of the human lysozyme was confirmed in this transgenic line as described in Fig. 3. Growth of the pathogen was retarded and obvious disease symptom was not observed. (b) Non-transgenic line (wild type control). Distinct disease symptom was observed.
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Acknowledgements We thank T. Chikahisa and S. Sakamoto for technical assistance and M. Ohara for critical comments on the manuscript. References
Fig. 6. Biological test for disease resistance against Alternaria dauci. T1 lines of No. 12-1 ( ) and non-transgenic plants lines (wild type control, "), were tested for A. dauci, using a detached petiole assay. Five plants for each line were grown in vitro up to the same developmental stage, the leaves were trimmed off, and petioles were cut into 8 cm in length. Four segments were prepared from one plant and used for this test. Petiole fragments from transgenic and non-transgenic plants were stood upright in actively growing colony layers of the fungal pathogen on PDA medium. The fungal pathogen, which was pre-cultured for 7 days after inoculation, was used. The lesion length on each petiole developed from the base of the petiole fragment was measured after 14 days of incubation. Each dot in the figure represented the obtained individual data.
tained expression level. However, stable accumulations of the human lysozyme were detected in T0 plants of the No. 8-1, 8-2 and 12-1, and the disease ratings of powdery mildew were inhibited. In the T1 generations of the No. 8-1 and 12-1, expression levels of the human lysozyme were also noted alike the T0 generation, and the disease ratings of powdery mildew were significantly inhibited, in comparison with the other lines. Thus, the increase of disease resistance against powdery mildew correlated with the production level of human lysozyme in both T0 and T1 generations. The highest GUS activity [33] was noted in leaf tissues of the No. 12-1 (data not shown). Therefore, the GUS reporter gene of the No. 12-1 was also expressed at a higher efficiency compared with those of other lines. Some plants of the T1 generation exhibited higher disease resistance compared with those of the T0 generation. These strong resistant plants are thought to be homozygous lines for introduced human lysozyme gene.
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Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci Miyuki Takaichi, Kenji Oeda * Biotechnology Laboratory, Sumitomo Chemical Co. Ltd., 2 -1, 4 -chome, Takatsukasa, Takarazuka, Hyogo 665 -8555, Japan Received 17 June 1999; accepted 3 December 1999
Abstract In vitro assay indicated that the human lysozyme has lytic activity against phytopathogenic fungi and bacteria. A human lysozyme gene was placed under control of the constitutive CaMV 35S promoter and the resulting expression plasmid was introduced into two cultivars (cv.) of carrot, Kurodagosun (K5) and Nantes Scarlet (NS), by Agrobacterium tumefaciens-mediated method. Seven and fourteen transgenic plants of cv. K5 and cv. NS were regenerated, respectively, and the obtained transgenic carrots of T0 generation was tested for disease resistance against Erysiphe heraclei, a pathogenic fungi causing powdery mildew. Among the tested lines, the transgenic plant No. 12-1 and 8-1 of cv. NS showed a fairly strong resistance against E. heraclei. The strong disease resistance was also confirmed in T1 generation. Disease resistance against another pathogen of leaf blight, Alternaria dauci, were also tested using T1 transgenic lines. Significant enhanced resistance was observed in the No. 12-1 of cv. NS. Accumulation of synthesized human lysozyme protein was observed in this line, a finding consistent with observed disease resistance. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human lysozyme; Transgenic carrots; Agrobacterium tumefaciens; Disease resistance
1. Introduction Development of recombinant DNA techniques and plant genetic transformation methods facilitate introduction of selected genes into plants to obtain transgenic ones with novel phenotypes. A variety of transgenic plants exhibiting disease resistance, herbicide resistance [1], delayed ripening [2], and alteration of protein composition in seeds [3] has been generated and some were tested in the field trials. Disease resistance is one of the important targets of plant breeding, because significant yield losses due to pathogenic attack limits crop productivity. Therefore, new disease control strategies to produce transgenic plants exhibiting enhanced disease resistance are now being widely Abbre6iations: CSS, chitinase signal sequence; cv., cultivar; GUS, b-glucuronidase gene; NPT II, neomycin phosphotransferase gene. * Corresponding author. Tel.: + 81-797-742121; fax: +81-797742133. E-mail address: [email protected] (K. Oeda)
evaluated. Viral coat protein genes can be used to generate transgenic plants exhibiting virus resistance [4]. The cecropin gene was utilized to generate disease resistant plants because cecropin contains antibacterial activity against several bacterial pathogens [5]. Both glucanase and chitinase also exhibited antifungal activity. Transgenic plants of tobacco [5–7], rice [8], cucumber [9,10] expressing glucanase or chitinase genes exhibited enhanced disease resistance against fungal pathogens. Lysozymes are basic bacteriolytic proteins widely distributed in nature. A protective and defensive role against bacterial or fungal pathogens has been suggested for lysozymes of plant origin [11], and these lysozymes were purified from latex of papaya [12], fig [13], cultured Rubus hispidus cells [14], cultured Parthenocisis quinquifolia cells [15], turnip roots [16] and wheat germ [11]. Lysozymes from human [17] and hen egg [18] were also purified, and both lysozymes exhibited bacte-
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riolytic function. Human lysozyme cleaves the b(1–4) glycosidic bond of peptidoglycan in the bacterial cell wall and of chitin in the fungal cell wall, other classes of animal lysozymes cannot hydrolyze chitin [19]. Therefore, the human lysozyme might have potential to protect plants from both bacterial and fungal diseases. In the present work, the gene cassette coding for human lysozyme [20] and a chitinase signal sequence (CSS) of Azuki bean [21] was placed under a strong constitutive CaMV 35S promoter, to obtain the transgenic plants expressing the human lysozyme. The resulting construct was introduced into two different cultivars of carrot, Kurodagosun (K5) and Nantes Scarlet (NS). More than 20 transgenic carrots were regenerated from embryogenic calli and they were raised to mature plants. The obtained transgenic plants were tested for disease resistance against Erysiphe heracle (powdery mildew) and Alternaria dauci (leaf blight). Consequently, transgenic carrots of cultivar (cv.) K5 and cv. NS showed enhanced disease resistance against these pathogens. Especially, the No. 12-1 of cv. NS showed distinct disease resistance.
Bacterial cultures (50 ml) of E. caroto6ora or P. syringae, were prepared by dilution of overnight culture, and mixed with the equal volume (50 ml) of 0, 30, 100 mg/l of human lysozyme solution, respectively. These mixtures were incubated for 1 h at room temperature, and spread on agar plates composed of PDA medium. The number of colonies was measured after 24 h.
2.2. Plant materials Carrot (Daucus carota L.) cultivars used in this study are K5 and NS. Seeds were surface-sterilized with 70% ethanol for 30 s and disinfected with 1% chloric acid for 10 min. The seeds were rinsed 3 times in sterile distilled water and then germinated on modified MS medium (half strength Murashige and Skoog [22] salts, 2 g/l sucrose and 0.8% agar) at 25°C in the dark. Hypocotyls were dissected from 1-week-old seedlings and cut into segments of 5–10 mm long. The explants were used for transformation experiments of carrots.
2.3. Agrobacterium tumefaciens strains and the used plasmid 2. Materials and methods
2.1. In 6itro assay against phytopathogens Fungal pathogens, Rhizoctonia and Alternaria, bacterial ones, Erwinia caroto6ora and Pseudomonas syringae, were used to test the effect of human lysozyme (Sigma, USA). Rhizoctonia or Alternaria spores were inoculated on agar plates composed of PDA medium (Nissui, Japan) supplemented with 0, 30 and 100 mg/l of human lysozyme, respectively. Each hypha length was measured after 24 h.
A. tumefaciens strains LBA4404 and C58C1 containing the binary plasmid pNGL2 were used to transform carrots. The plasmid pNGL2 consists of a neomycin phosphotransferase gene (NPT II), a b-glucuronidase gene (GUS) and the CaMV 35S promoter fused to a human lysozyme gene. The gene fragment coding for the CSS of Azuki bean [21] was also placed upstream of the human lysozyme gene (Fig. 1). The signal sequence is thought to be useful for efficient transportation of the synthesized protein into the intercelluar space of the plant cell.
Fig. 1. Structure of the constructed expression plasmid pNGL2. NPT II, neomycin phosphotransferase gene; GUS, b-glucuronidase gene; HLY, human lysozyme gene; CSS, chitinase signal sequence of Azuki bean; 35S, CaMV 35S promoter; NOS-P and NOS-T, Nos promoter and terminator, respectively.
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2.4. Transformation and regeneration Explants were inoculated in the bacterial solution for 5 min, then placed on the co-cultivation medium which consists of MS salts, 4.5 mM 2,4dichlorophenoxyacetic acid (2,4-D), 3 g/l sucrose and 0.8 g/l agar. This medium is basal to produce embryogenic callus. Infected explants were incubated in the dark at 20–21°C for 2–3 days. After co-cultivation, explants were rinsed in the liquid basal medium containing 500 mg/l cefotaxime, and then placed on the basal medium supplemented with 300 mg/l cefotaxime for 4 weeks to prevent bacterial growth. Then, the explants were transferred to the basal medium containing 50 mg/l kanamycin and 100 mg/l cefotaxime for 4 –6 weeks as the first selection. The obtained embryogenic callus exhibiting kanamycin resistance was subcultured on hormone free MS medium, without kanamycin. After 2–4 weeks, the seedlings germinated from embryogenic calli were transferred to MS medium containg 100 mg/l kanamycin, as the second selection. After co-cultivation, the cultures were placed under conditions of 16 h light/8 h dark at 25°C. Transgenic seedlings were regenerated from embryogenic calli and they were examined for disease resistance. Insertion of the human lysozyme gene into the plant genome was confirmed by polymerase chain reaction (PCR) analysis and the transgenic carrots were acclimatized to soil and grown at 20–23°C in a greenhouse. Transgenic carrots with auxetic roots were transferred to a cold room and maintained at 5°C for at least 2 months. After this exposure to a low temperature, transgenic carrots were bolted at 20 –23°C and self-pollinated seeds were obtained.
2.5. PCR analysis Genomic DNA was isolated from cultured carrot leaves, using cetyltrimethyl ammonium bromide [23]. PCR for the human lysozyme gene was performed with the genomic DNA of carrot plants using the synthetic oligonucleotide primers. The primers of 5%-GAACGTTGTGAATTGGCCAG3% and 5%-GTTTTGACAGCGGTTACGCC-3% was designed to hybridize the 5% and 3% regions of human lysozyme gene, respectively. The 42 cycle reaction, which consists of heat denaturation (95°C), annealing (55°C) and extension (72°C),
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was carried out, and the reaction mixture was subjected to 0.8% agarose gel electrophoresis. Specific amplification of the 350 base pairs fragment indicates that the human lysozyme gene was inserted into the genome of the transgenic carrot plants.
2.6. Western blot analysis Young leaves of carrots grown in a greenhouse were homogenized in phosphoric buffer (0.1 M K2HPO4, 2.5 mM EDTA, 0.1% ascorbic acid, 1% mercaptoethanol and 0.5 mM PMSF), and the crude extract was separated by centrifugation for 10 min at 4°C (15 000×g). The supernatant (10 mg total soluble protein) was subjected to 10% SDS-polyacrylamide gel electrophoresis, and the resolved proteins were transferred onto nitrocellulose membranes (Schleicher Schuell, Dassel Germany). The human lysozyme on the membrane was detected by enzyme-linked immunostaining. The membrane was exposed to anti-human lysozyme serum from a goat (Nordic Immunology, Tilburg, The Netherlands), then to alkaline phosphatase conjugated antibody against goat IgG from a rabbit (Vector Laboratories, CA, USA). The human lysozyme on the membrane was visualized by reaction with 5-bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium.
2.7. Biological test for resistance against Erysiphe heraclei Among 23 plants of transgenic carrots, 21 were transferred to the soil, and finally, 13 plants grew to the same stage of development. After 1 month of acclimatization, the obtained transgenic carrots were examined for disease resistance against E. heraclei, the phytopathogen of powdery mildew. The diseased carrots previously infected with E. heraclei were randomly placed among the tested transgenic and non-transgenic plants, and co-cultivated in a greenhouse at 21–23°C for 1 week. Disease rating was classified into six ranks based on the average area of disease, as defined in Table 1. The disease rating was first measured 1 week after inoculation, to observe early symptoms of diseased plants. Then, the disease ratings were successively measured twice every week, and the development of powdery mildew was investigated. Non-transgenic plants were also tested as negative control.
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Table 1 Disease rating on powdery mildew Disease rating
Disease developmental area (%)
0 0.5 1 2 4 8
0 0–5 5–10 10–25 25–50 \50
T1 progeny were also examined for disease resistance against E. heraclei. Self-pollinated seeds derived from T0 plants were sowed on soil, and the germinated seedlings were examined by PCR. Seedlings without the lysozyme gene were discarded. Five to ten plants for each line were grown in a greenhouse. Four weeks later, these T1 progenies were infected with E. heraclei, and disease rating scored as described above. A disease index (%) was also calculated by the following formula, according to the method of the Agrochemical Society of Japan. The disease index = {%(disease rating from 0 to 8) /(8 × a number of plants)} × 100
2.8. Biological test for resistance against A. dauci T1 lines of No. 12-1 and non-transgenic plants, were tested for disease resistance against another carrot pathogen, A. dauci, using a detached petiole
assay [9]. Five plants for each line were grown in vitro up to the same developmental stage, the leaves were trimmed off, and petioles were cut into 8 cm in length. Four segments were prepared from one plant and used for the biological test. Petiole fragments from transgenic and non-transgenic plants were stood upright in actively growing colony layers of the fungal pathogen on PDA medium (Nissui, Japan). The fungal pathogen, which was precultured for 7 days after inoculation, was used. The lesion length developed from the base of the petiole fragment was measured after 14 days of incubation. Lesion length on each petiole was measured.
3. Results and discussions
3.1. Lytic acti6ity of human lysozyme against phytopathogens Fungal pathogen, Rhizoctonia and Alternaria, and bacterial ones, E. caroto6ora and P. syringae, were used to test the effect of human lysozyme. Hypha length of Rhizoctonia and Alternaria were inhibited to 30 and 50% with 30 mg/l of human lysozyme, respectively (Table 2). Similarly, about 65 and 100% of bacteria, E. caroto6ora and P. syringae, lyzed during 1 h incubation with 100 mg/l of human lysozyme, respectively (Table 2). These results indicated that the human lysozyme was effective against phytopathogenic bacteria and fungi [19].
Table 2 Growth inhibition of Rhizoctonia and Alternaria, and reduction of colony formation of Erwinia caroto6ora and Pseudomonas syringae by treatment of human lysozyme Human lysozyme (mg/l)
Rhizoctonia a Hypha length (mm)
Ratio (%)
Hypha length (mm)
Ratio (%)
No. of colonies
Ratio (%)
No. of colonies
Ratio (%)
0 30 100
11.0ac 3.5b 3.0b
100 32 27
4.5a 2.3b 2.0b
100 51 44
236a 111b 82b
100 47 35
222a 21b 0b
100 9 0
a
Alternaria a
Erwinia b
Pseudomonas b
Each spore of Rhizoctonia and Alternaria was inoculated on agar plates composed of PDA medium supplemented with human lysozyme. The hypha length (mm) was measured after 24 h incubation. b Each bacterial culture of E. caroto6ora and P. syringae, and human lysozyme solution was mixed, and incubated for 1 h. These mixtures were spread on agar plates composed of PDA medium. c Within each pathogen, numbers followed by the same letter are not significantly (0.05%) different according to LSD test.
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Table 3 Experimental conditions for carrot transformation Carrot cultiver
Agrobacterium strain
No. of explants
No. of transformants
Transformation efficiency (%)
Kurodagosun
LBA4404 C58C1 LBA4404 C58C1
86 90 85 99
1 6 10 6
1.2 6.7 11.8 6.1
Nantes Scarlet
Fig. 2. Integration of the human lysozyme gene into the carrot genome. Integration of the human lysozyme gene into the carrot genome of T0 and T1 generation was confirmed by polymerase chain reaction (PCR) analysis. (a) Specific amplifications of 350 base pairs fragment of human lysozyme gene were indicated in all genomic DNA samples of T0 plants. (b) The 350 base pairs fragment was also detected in T1 plants. M was molecular marker. Plasmid pNGL2 was a positive control. All samples were cultivar (cv.) Nantes Scarlet (NS).
3.2. Regeneration and culti6ation of transgenic carrots The expression plasmid pNGL2 was constructed to express the human lysozyme gene in carrots (Fig. 1). The plasmid pNGL2 contains the CaMV 35S promoter and the human lysozyme gene. The obtained construct was introduced into two cultivars of carrot, K5 and NS. Both carrot cultivars and strains of A. tumefaciens used for transformation procedure had considerable effects on the transformation efficiency of carrots (Table 3). When A. tumefaciens strain LBA4404 was used, only one transgenic plant was selected with cv. K5, while ten transgenic plants were obtained in the case of cv. NS. The calculated transformation efficiencies were 1.2 and 11.8%, respectively. Six transgenic plants were obtained for both cv. K5 and cv. NS, respectively, when A. tumefaciens strain C58C1 was used. The efficiencies were estimated to be 6.7% and 6.1%, respectively. The A. tumefaciens-mediated method and electroporation [24] can be used for transformation of carrots. However, the A. tumefaciensmediated method is now widely used for carrot transformation [25–28], because this method is
simple and is economical of time. The efficiencies of transformation were, therefore, thought to depend on the combination of A. tumefaciens strains and carrot cultivars. The other carrot cultivar ‘Nanco’ was often used as a major host cultivar because the transformation efficiency was reported to be highest among many cultivars, over 10% [25,27]. When cv. K5 and cv. NS were used in this experiment, the mean efficiency was calculated to be 6.4%, slightly lower than cv. Nanco. Petiole, cotyledon, hypocotyl, root of carrot seedling and even callus could be used for transformation experiments, but the transformation efficiency was also greatly affected by used explants and the developmental stage [25–28]. In this experiment, hypocotyls of 1-week-old seedlings were used as explants because the highest regeneration efficiency was obtained. Insertion of the human lysozyme gene into the carrot genome was tested by PCR analysis (Fig. 2a). Consequently, 7 and 16 independent lines from cv. K5 and cv. NS, respectively, were found to have the amplified human lysozyme DNA fragments. These plants were acclimatized to soil and T1 seeds were produced. Five transgenic plants of cv. K5 were successfully self-pollinated and T1
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Table 4 The number of regenerated, acclimatized and self-pollinated transgenic carrots Carrot cultiver
No. of transformants Regeneration Acclimatization
Kurodagosun 7 Nantes 16 Scarlet
7 14
Selfpollination 5 10
seeds were produced. Similarly, 14 transformants of cv. NS were acclimatized to obtain mature plants. Finally, 10 independent transgenic plants of cv. NS self-pollinated and T1 seeds were produced (Table 4). The amplified DNA bands were also observed in these T1 plants (Fig. 2b).
3.3. Western blot analysis Mature plants of each line were subjected to Western blot analysis to evaluate accumulation of the synthesized human lysozyme. A specific signal of the 14 kDa protein band was detected in almost all transgenic plants. The signal for the human lysozyme was detected in almost all samples from the T0 carrots of cv. K5 and NS. Expression levels of the human lysozyme were relatively high in samples of No. 8-1, 8-2 and 12-1 of cv. NS (Fig. 3a). The transgenic plant No. 7-1, which contained the human lysozyme gene, did not produce human lysozyme. This might be caused by a strong co-suppression [29]. This line was also used for further experiments as one of the negative controls.
Self-pollinated seeds were obtained from transgenic carrots of cv. K5 and cv. NS, respectively. These seeds were germinated and five to ten seedlings for each line were grown in a greenhouse. T1 transgenic plants were also subjected to Western blot analysis to determine expression level of the human lysozyme. Especially, expression level was high and stable in samples of lines No. 8-1 and 12-1 of cv. NS. (Fig. 3b).
3.4. Resistance against pathogens 3.4.1. E. heraclei 3.4.1.1. T0 generation. To observe disease resistance of the obtained transgenic carrots of cv. NS, the resistance against E. heraclei was measured. The disease rating was first measured 1 week after inoculation, to observe early symptom of diseased plants. The disease rating was successively measured twice every week, and the development of powdery mildew was investigated. Symptoms in all transgenic plants were moderate (disease index lower than 20%) compared to that of non-transgenic plants (disease index of 50%), when disease rating was scored 1 week after inoculation. However, symptoms of powdery mildew developed on the upper leaves at a later stage, and the degree of symptoms varied among tested plants. When disease rating was scored 3 weeks later after inoculation, transgenic carrots No. 8-1, 8-2 and 12-1 showed a disease index of 30%, while other transgenic plants showed 50–80% of the disease index. At that time, non-transgenic plants and the transgenic plant No. 7-1, the negative control line, exhibited disease index of about 80% (Fig. 4a).
Fig. 3. Detection of the human lysozyme by Western blot analysis in T0 and T1 generations of cultivar (cv.) Nantes Scarlet (NS). H, 1 mg protein of human lysozyme as a positive control. Ten mg of total soluble protein of each sample was analyzed by Western blot. M, molecular marker. (a) The No. 8-1, 8-2 and 12-1 of T0 generation exhibited the synthesized human lysozyme. The non-specific protein band with lower molecular weight was observed, besides the synthesized human lysozyme. (b) The No. 8-1 and 12-1 also produced high level of human lysozyme on T1 generation.
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8-1 showed about 40% of the disease index, while the No. 12-1 exhibited a very low disease index (15%) (Figs. 4b and 5). A difference of disease development was observed at early stages of pathogen inoculation as found in two tests of non-transgenic plants (Fig. 4a,b). This fluctuation at early stages might be caused by different timing of pathogen infections. However, disease development at later stages was basically the same. Interestingly, the disease index of powdery mildew differed among the carrot cultivars. In cv. K5 development of powdery mildew was slightly delayed compared to findings in cv. NS (data not shown). It was also reported that the process of disease development varied in the carrot cultivars, when transgenic carrots expressing chitinase gene were tested [9].
Fig. 4. Disease index against powdery mildew of transgenic lines of cultivar (cv.) Nantes Scarlet (NS). After 1 month of acclimatization, the obtained transgenic carrots were examined for disease resistance against powdery mildew. The diseased carrots previously infected with Erysiphe heraclei were randomly placed among the tested transgenic and non-transgenic plants, and co-cultivated in a greenhouse at 21 – 23°C for 1 week. Disease rating was classified into six ranks based on the average area of disease in plants, as defined in Table 1. The disease rating was first measured 1 week after inoculation, to observe early symptoms of diseased plants. The disease ratings were successively measured twice every week, and the development of powdery mildew was investigated. (a) T0 generation, investigated on 1 week ( ), 2 weeks ( ) and 3 weeks (b) after the pathogen inoculation. (b) T1 generation, investigated on 1 week ( ), 2 weeks ( ) and 3 weeks (b). The disease index test of T1 generation of transgenic line No. 8-2 was not performed, because T0 generation of this line did not set seeds. Disease index (%) ={(Disease rating from 0 to 8)/(8×number of plants)} ×100. Mean values obtained for five to ten plants per line. Within each generation and line, numbers followed by the same letter are not significantly (0.05%) different according to LSD test.
3.4.1.2. T1 progeny. The disease indexes of all T1 lines were less than 10%, when disease rating was scored just after inoculation. Transgenic lines No. 7-1, showed 80% of the disease index, and the No.
3.4.2. A. dauci The detached petiole assay was done to evaluate disease resistance of T1 generation of the line No. 12-1 and non-transgenic plants against A. dauci. Five plants for each line were grown in vitro up to the same developmental stage, the leaves were trimmed off, and petioles were cut into 8 cm in length. Four segments were prepared from one plant and used for the biological test. The No. 12-1 was selected based on data concerning the disease index against powdery mildew. This line exhibited very high disease resistance against powdery mildew, and also showed high level and stable accumulation of the human lysozyme (Figs. 3 and 4). The lesion lengths of non-transgenic plants were dispersed between 10 and 60 mm at 2 weeks after inoculation. On the contrary, the transgenic line No. 12-1 was mostly collected below 25 mm of lesion development (Fig. 6). The detached petiol assay was repeated twice, and the obtained results were basically the same between two tests (data not shown). Thus, development of pathogen was considered to be inhibited in the No. 12-1 in comparison with the non-transgenic plants. Thus, the No. 12-1 exhibited the disease resistance against pathogens, both E. heraclei and A. dauci. In this resistant line, the production level of the human lysozyme was high and stable compared with those of other lines, which is consistent with the acquired resistance against these pathogens. Taken together, the human lysozyme gene will prove useful for production of transgenic crops
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and vegetables to enhance disease resistance against both bacterial and fungal pathogens. A human lysozyme has strong bacteriolytic activity against bacteria and fungi [30]. In a previous work, T0 generations of transgenic tobacco plants expressing the human lysozyme showed enhanced disease resistance against Erysiphe cichoracearum and P. syringae [20]. In the present work, the stable production of the human lysozyme was further confirmed in T1 as well as T0 generations of the transgenic carrots and also usefulness of the human lysozyme gene to confer the enhanced disease resistance. Leaf extracts of transgenic tobacco plants expressing a hen egg white lysozyme (HEWL) contained only about 30 ng of HEWL per mg of leaf tissue (0.003%) [31]. In transgenic potato plants expressing a bacteriophage T4 lysozyme, the syn-
thesized lysozyme level was estimated to be about 0.001% of total soluble proteins [32]. On the contrary, the production level of the human lysozyme in the transgenic line No. 12-1 was calculated to be about 0.5% per total soluble protein. Thus, the expression level of human lysozyme was very high compared with those of the above cases. The gene fragment coding for the CSS of Azuki bean [21] was placed upstream of the human lysozyme gene. The CSS might cause highly efficient transportation of the synthesized protein to extracellular spaces. Differences of the expression level were observed between the obtained transgenic carrot lines, which is mainly caused by two factors, the positional effects of the inserted gene and the copy number. It is very difficult to discriminate between each contribution of these two factors to the ob-
Fig. 5. Biological test for disease resistance against Erysiphe heraclei (powdery mildew) in T1 generation of cultivar (cv.) Nantes Scarlet (NS). (a) Transgenic line No. 12-1. High level expression of the human lysozyme was confirmed in this transgenic line as described in Fig. 3. Growth of the pathogen was retarded and obvious disease symptom was not observed. (b) Non-transgenic line (wild type control). Distinct disease symptom was observed.
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Acknowledgements We thank T. Chikahisa and S. Sakamoto for technical assistance and M. Ohara for critical comments on the manuscript. References
Fig. 6. Biological test for disease resistance against Alternaria dauci. T1 lines of No. 12-1 ( ) and non-transgenic plants lines (wild type control, "), were tested for A. dauci, using a detached petiole assay. Five plants for each line were grown in vitro up to the same developmental stage, the leaves were trimmed off, and petioles were cut into 8 cm in length. Four segments were prepared from one plant and used for this test. Petiole fragments from transgenic and non-transgenic plants were stood upright in actively growing colony layers of the fungal pathogen on PDA medium. The fungal pathogen, which was pre-cultured for 7 days after inoculation, was used. The lesion length on each petiole developed from the base of the petiole fragment was measured after 14 days of incubation. Each dot in the figure represented the obtained individual data.
tained expression level. However, stable accumulations of the human lysozyme were detected in T0 plants of the No. 8-1, 8-2 and 12-1, and the disease ratings of powdery mildew were inhibited. In the T1 generations of the No. 8-1 and 12-1, expression levels of the human lysozyme were also noted alike the T0 generation, and the disease ratings of powdery mildew were significantly inhibited, in comparison with the other lines. Thus, the increase of disease resistance against powdery mildew correlated with the production level of human lysozyme in both T0 and T1 generations. The highest GUS activity [33] was noted in leaf tissues of the No. 12-1 (data not shown). Therefore, the GUS reporter gene of the No. 12-1 was also expressed at a higher efficiency compared with those of other lines. Some plants of the T1 generation exhibited higher disease resistance compared with those of the T0 generation. These strong resistant plants are thought to be homozygous lines for introduced human lysozyme gene.
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