Plant Science 153 2000 135 – 144
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 www.elsevier.comlocateplantsci
1. Introduction
Development of recombinant DNA techniques and plant genetic transformation methods facili-
tate introduction of selected genes into plants to obtain transgenic ones with novel phenotypes. A
variety of transgenic plants exhibiting disease re- sistance, 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 impor- tant 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
evaluated. Viral coat protein genes can be used to generate transgenic plants exhibiting virus resis-
tance [4]. The cecropin gene was utilized to gener- ate disease resistant plants because cecropin
contains antibacterial activity against several bac- terial 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 patho-
gens 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 quinquifo-
lia 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-
Abbre6iations
:
CSS, chitinase signal sequence; cv., cultivar; GUS, b-glucuronidase gene; NPT II, neomycin phosphotransferase gene.
Corresponding author. Tel.: + 81-797-742121; fax: + 81-797- 742133.
E-mail address
:
oedasc.sumitomo-chem.co.jp K. Oeda 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 5 4 - X
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
