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Chapter21
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Transformation of Hybrid Aspen for Resistance to
Crown Gall Disease1
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Hiroyasu Ebinuma, Etsuko Matsunaga, Keiko Yamada, and Mikiko Yamakado
Introduction
'Kitakami Hakuyou' are elite clones of hybrid aspen
produced by Nippon Paper Industries (NPij for commercial use. These elite clones were obtained by crossing 5
selected female trees of Populus sieboldii with a male elite
tree of Canadian P. grandidentata, exhibit better growth features than other species or interspecific hybrids belonging
to the section Leuce (currently termed Populus), and express significant heterosis (Takayama 1968). These characteristics make them well suited for afforestation and pulp
wood in Japan. However, in some geographic areas,
'Kitakami Hakuyou' clones are extremely sensitive to
crown gall disease, caused by Agrobacterium tumefaciens.
This disease can cause considerable economical loss to
nurseries growing rosaceous plants, Rubus species, grapevines, or various nut-bearing trees. The phytopathogenic
bacterium, A. tumefaciens, infects a wide variety of dicotyledonous plants and induces tumors on the infected plants
(Moore and Warren 1979). Because this bacterium provides
a useful method to introduce desirable genes into plants,
we have used biotechnology in an attempt to improve characteristics of 'Kitakami Hakuyou.' Our objective was to
use an antisense DNA method to improve crown gall disease resistance in aspen clones (Ebinuma et al. 1991, 1992).
In this paper, we report on the construction of plasmids
containing antisense DNA, and on a highly efficient transformation procedure for hybrid aspens.
Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.~ Carman, R.C.; Eskew, L.G., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO:
U.S. Department of Agriculture, Forest Service, Rocky Mountain
Research Station. 326 p.
1
Construction of Vectors
Containing Resistance Genes
to Crown Gall Disease
A. tumefaciens introduces genes located on the transfer
DNA (T-DNA) of the tumor-inducing plasmid (TI-plasmid;
Kim et al. this volume) into the plant genomic DNA, and
induces tumors in the infected regions (Bevari and Chilton
1982). In the tumors, plant hormones, auxin and cytokinin, are over produced due to the expression of 3 integrated
oncogenes (iaaH, iaaM, and ipt) in the plant genome. We
used the 1i plasmid of the pathogenic A. tumefaciens strain
P022, which was isolated from a natural crown gall on
hybrid aspen, to make antisense DNA constructs (Wabiko
et al. 1989). We constructed the binary vector based on the
plasmid pBI121, which contained the 1.1-kilobase (kb},
EcoRI fragment 34 of the iaaH gene and the 0.7-kb, Hindiii
fragment 41 of the iaaM gene in the sense (S) or antisense
(A) orientation under the control of the cauliflower mosaic virus 355 promoter (figure 1). The plasmid was transferred to A. tumefaciens strain LBA4404 and used to
transform tobacco.
A dramatic inhibition of gall formation was observed
after infection by a virulent A. tumefaciens strain in 1 of the
5 transgenic tobacco plants expressing a part of iaaM gene
in the sense orientation (41s4} (table 1, figure 2). Another
of the 5 transgenic tobacco plants with the antisense orientation (41a3) showed weak, but distinct, resistance. We
investigated the copy number and expression of the integrated genes in transgenic tobacco plants by the Southern
and northern hybridization analyses (Sambrook et al.
1989). Southern analysis revealed that the resistant
transgenic plant 41s4 contained 4 copies of the sense insert, the nonresistant plant 41s2 had 1 copy, and 41s3 had
6 copies. The amount of transcript in the resistant plant
(41s4} was lower than that in the nonresistant plants (41s2
and 41s3); however, no correlation between transcriptional
activity and resistance was detected. Although the molecu161
Section IV Biotic and Abiotic Resistance
iaaH
E
iaaM
E
H
H
pBI121
GUS-
345
iaaH-
34A
-Haai
415
iaaM -
41A
-Maai
Control
Figure 1. Plasmid construction. iaaH=indoleacetamide
hydrolase gene; iaaM=tryptophan monooxygenase gene; GUS=glucuronidase gene;
NPT //=neomycin phosphotransferase gene;
35S=35S promoter of cauliflower mosaic virus;
N=promoter of nopaline synthase gene;
T=polyadenylation signal of nopaline synthase
gene; E= EcoRI restriction site; H=Hindll l restriction sites.
Table 1. Bioassay of tumorigenicity using leaf discs of
transgenic tobacco.
Number of
leaf discs inoculated 2
Percentage of discs
producing tumors 2
C1
15
93
Lc2
15
73
41a3
15
41s4 3
15
80
0
Tobacco
clones'
1
C1 =normal tobacco plants
Lc2 =transgenic tobacco plants transformed by p81121
41 a3 = transgenic tobacco plants transformed with antisense DNA fragment 34
41 s4 = transgenic tobacco plants transform ed with
sense DNA fragment 41
2
Leaf discs were inoculated with pathogenic
Agrobacterium tumefaciens and placed on agar plates (5
leaf discs/plate, total of 3 plates) and gall development was
examined on the 15 leaf discs.
3
Nine little shooty teratomas developed on leaf discs.
162
41s4
Figure 2. Leaf disc tests of transgenic tobacco.
Control=stem segments test;
41 s4=stem infection test.
lar mechanism is not fully understood, our findings indicate that crown gall disease in hy~rid aspen may be red uced by the 0.7-kb, Hindiil fragment 41 of the iaaM gene
in the sense orientation under the control of the 355 p romoter.
Highly Efficient Transformation
Procedures for Hybrid Aspens
To improve the genetic transformation o f hybrid aspens, we developed improved regenera tion procedures.
Beca use shoo t regene ration frequencies can vary from
0 to 100 percent d epending on the va riety of Popu l us
sp p ., commercia lly useful trees in the gen us Populus
freque ntly exhib it lower regeneration frequencies than
model trees used in resea rch. To help overcom e this
problem, we ap plied an Agrobacterium trans fo rmation
method, developed for tobacco, to ou r commercial hybrid as pen clones (M atsunaga et al. 1992). This tra nsfo rm atio n procedure requires 2 steps, induction and
growth of transgenic ca llus from infected stem segments, and s hoot regen eration from the callus. This
method has 3 potential prob lems: 1) the ca llus growth
stage p resents a high risk of gene rating genetic mutan ts;
2) a relative ly long time is required for shoot regene ra-
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Transformation o f Hybrid Aspen for Resistance to Crown Gall Disease
tion; and 3) the efficiency of obtaining transgenic trees
with the desired gene is very low. To allev iate these
problems, we developed an improved tissue cu lture
method that increases the frequency of ad ventitiou s
shoot differentiation of our commercial hybrid aspen
clones.
By investigating the composi tion of the nitrogen
source in the tissue culture medium, we found tha t concentrations of both nitrogen sources, ammonium and
nitrate, influence the physiological s tate that induces
differentiation of adventitious buds. Molar ratios from
1:2 to 1:5 of ammonium to nitrate were tested in the
cult~re medium. The ratio of 1:3 was particularl y effective for highly efficient regeneration of adventitious
buds. By u sing a culture medium w ith this nitrogen
composition, we could directly diffe rentiate adventitious buds from infected internodal segments of hybrid
aspen clones, without employing a prelimina ry s tep for
inducing and growing callus, and subsequently regenerate plantlets. This method should reduce risks o f genetic mutations during culturing, and requires a s horter
regeneration time because extra steps are no t required
to induce and grow callus.
The binary vector plasmid, pBI121, which contained the
0.7-kb, Hindiii fragment 41 of iaaM gene in the sense (S)
orientation under control of the 355 promoter, was transferred to A. tumefaciens strain LBA4404. Under aseptic conditions,.stems of flask-grown plants of hybrid aspen clone
'Y63' (P. sieboldii x P. grandidentata) were cut into internodal segments of 5 mm in lerygth. Stem segments were
further cut longitudinally into 2 pieces, then inoculated
with the A. tumefaciens vector strain. Infected stem segments were trans ferred to modified Murashige and Skoog
(MS) (Murashige and Skoog 1962) agar solid medium (2
percent w I v sucrose, 0.5 mg /1 zeatin, 500 mg/1 ca rbenicillin, 100 mg / 1kanamycin, and 0.8 percent w /v aga r) in
which nitrogen was supplied as 10 mM ammonium and
30 mM nitrate. After culturing for 2 months at 25 oc under 3,000 lux, 20 to 40 percent of the inoculated stem segments regenerated adventitious buds. After culturing an
additional month under similar conditions, the foliage
frcm each bud had g rown to a length of 2 to 3 em. These
stems were aseptically excised and subcultured to a modified 2/3-strength MS medium, in which zeatin was replaced by 0.05 mg /1 indole-3-bu tyric acid (IBA) for
rooting. The rooting culture continued and 85 plantlets
were obtained after 1 month.
Resistance of transgenic aspens to crown gall disease
was evaluated by bioassay tests of tumorigenicity. Stem
segments of transgenic and control hybrid aspens were
inocula ted with a pathogenic A. tumefaciens strain P022
with the same method used for transformation. Inoculated
s tem segments were placed on MS agar med ium to
observe gall formation. In resistant clones, we observed
the development of teratoma tous shoots (figure 3a). For
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
a) stem segments test
Control
Transgenic Aspen
b) stem infection test
Control
Transgenic Aspen
Figure 3. Bioassays of transgenic hybrid aspen.
Transgenic aspen with sense DNA fragment 41 .
a) Internodal stem segments were infected with
pathogenic Agrobacterium tumefaciens strain
P022 and placed on agar plates. b) Internodes
of stems were wounded with a needle and
inoculated with A. tumefaciens strain P022 .
a nother bioassay, we wounded 5 internodes of g reenhouse-grown, transgenic aspens with a needle a t 1 point
pe r inte rnod e, and inocu lated w ith A. tumefaciens strain
P022. In approxim ate ly 10 percent of the transgenic
plants, chara cteristic sy mptoms of crown gall disease
were inhibited (figure 3b); however, consid erable variability in gall development was ap pare nt. Although
several aspects need in tensive investi gation, these resu lts provide interesting p rospects for use of an
antisense Dt A method to improve crown gall disease
resistance in aspen clones.
163
Section IV Biotic and Abiotic Resistance
Acknowledgments
The authors want to thank Drs. H. Sano and H. Wabiko
for their collaborative works to produce a transgenic tobacco resistant to crown gall disease.
Literature Cited
Bevan, M.W.; Chilton, M.-D. 1982. T-DNA of the
Agrobacterium Ti and Ri plasmids. Ann. Rev. Genet. 16:
357-384.
Ebinuma, H.; Wabiko, H.; Ohsima, K.; Hata, K.; Sano, H.
1991. The genetic engineering of poplar trees.- Construction of resistant genes to crown gall disease. Forest Breeding. 4: 14-17.
Ebinuma, H.; Wabiko, H.; Ohshima, K.; Hata, K.; Sano, H.
1992. The genetic engineering of poplar trees.- A first
practical application of a homology-based interaction
(Matzke effect) for protection against the plant disease.
In: Proceedings, 1992 Fifth international conference on
164
biotechnology in the pulp and paper industry: Tokyo,
Japan. Uni Publishers Co., LTD.: 467-472.
Matsunaga, E.; Ebinuma, H.; Yamada, K.; Ohsima, K.; Hata,
K. 1992. Introduction of resistant genes into hybrid aspen and analysis. In: Abstract of Papers, 1992 Second
tree molecular biology symposium: 72-77.
Moore, L.W.; Warren, G. 1979. Agrobacterium radiobacter
strain 84 and biological control of crown gall. Annu. Rev.
Phytopathol. 17: 163-179.
Murashige, T.; Skoog, F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiol. Plant. 15: 473-497.
Sambrook, J.; Fritsch, E.F.; Maniatis, T., eds. 1989. Molecular cloning: A laboratory manual. 2nd edition. Cold
Spring Harbor, NY, U.S.A.: Cold Spring Harbor Laboratory Press.
Takayama, Y. 1968. Studies on the breeding of aspens (I).
-Height growth in the early stage of F1 seedlings of
Populus sieboldii Miq. x P. grandidentata Michx. Nichirinshi. 50(9): 267-273.
Wabiko, H.; Kagaya, M.; Kodama, I.; Masuda, K.; Kodama,
Y.; Yamamoto, H.; Shibano, Y.; Sano, H. 1989. Isolation
and characterization of diverse nopaline type Ti plasmids of Agrobacterium tumefaciens from Japan. Arch.
Microbial. 152: 119-124.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.