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 se-
quence 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, Kurodago- sun K5 and Nantes Scarlet NS. More than 20
transgenic carrots were regenerated from embryo- genic calli and they were raised to mature plants.
The obtained transgenic plants were tested for disease resistance against Erysiphe heracle pow-
dery 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.
2. Materials and methods
2
.
1
. In 6itro assay against phytopathogens Fungal pathogens, Rhizoctonia and Alternaria,
bacterial ones, Erwinia caroto6ora and Pseu- domonas 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 supple- mented with 0, 30 and 100 mgl of human
lysozyme, respectively. Each hypha length was measured after 24 h.
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 mgl 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 gl 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
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-glu- curonidase 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.
2
.
4
. Transformation and regeneration Explants were inoculated in the bacterial solu-
tion for 5 min, then placed on the co-cultivation medium which consists of MS salts, 4.5 mM 2,4-
dichlorophenoxyacetic acid 2,4-D, 3 gl sucrose and 0.8 gl agar. This medium is basal to produce
embryogenic callus. Infected explants were incu- bated in the dark at 20 – 21°C for 2 – 3 days.
After co-cultivation, explants were rinsed in the liquid basal medium containing 500 mgl cefo-
taxime, and then placed on the basal medium supplemented with 300 mgl cefotaxime for 4
weeks to prevent bacterial growth. Then, the ex- plants were transferred to the basal medium con-
taining
50 mgl
kanamycin and
100 mgl
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
mgl kanamycin, as the second selection. After co-cultivation, the cultures were placed under con-
ditions of 16 h light8 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 main-
tained 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 car-
rot leaves, using cetyltrimethyl ammonium bro- mide [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-GAACGTTGTGAATTGGCCAG- 3 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,
was carried out, and the reaction mixture was subjected to 0.8 agarose gel electrophoresis. Spe-
cific amplification of the 350 base pairs fragment indicates that the human lysozyme gene was in-
serted 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 K
2
HPO
4
, 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 nitrocellu- lose membranes Schleicher Schuell, Dassel Ger-
many. 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 phos- phatase conjugated antibody against goat IgG
from a rabbit Vector Laboratories, CA, USA. The human lysozyme on the membrane was visu-
alized 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-culti- vated 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.
Table 1 Disease rating on powdery mildew
Disease developmental area Disease rating
0.5 0–5
5–10 1
2 10–25
25–50 4
\ 50
8
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
