Two Insect-Resistant Genes were Transferred into Poplar Hybrid and Transgenic Poplar Shew Insect-Resistance

Molecular Breeding of Woody Plants N. Morohoshi and A. Komamine, editors. 9 2001 Elsevier Science B.V. All rights reserved.

239

TWO INSECT-RESISTANT GENES WERE TRANSFERRED INTO P O P L A R H Y B R I D A N D T R A N S G E N I C P O P L A R S H E W INSECT-RESISTANCE H o n g y u Rao 1, Ningfeng Wu 2, Minren H u a n g I , Yunliu Fan 2& Mingxiu Wang ~

1 The Laborato~ of Forest Genetics & Gene Engineering. Nan/ing Forestty University. Nan/in~ 210037. ('hina : Institute of Biotechnolog~v Research. ('hinese Academv of Agricultural Sciences. Bei/ing 100081. ( 'hina

ABSTRACT Modified Bt CryIA gene and cowpea trypsin inhibitor (CpTI) gene were cointegrated into the genome of clone NL-80106, P. deltoides Marsh. X P.. simonii Carr. by co-infection with Agrobactermm tumefaciens LBA4404. PCR and PCR-Southern blotting analysis showed that both Bt CryIA and CpTI genes were integrated into the transgenic poplar. Insect bioassay indicated that two-gene transgenic poplar were significantly resistant to the larvae of Lymano'ia dispar, whereas the untransformed control plants were sensitive to them. It caused a decreased leaf consumption by larvae, a lower larval weight and a higher larval motility. ELISA analysis of the transgenic clones proved that Bt and CpTI genes were expressed. Bt and CpTI transgenic poplar plants can be used prospectively as the material for selecting highly and long-term insect-resistant poplar clones in the future.

KEY W O R D S l: deltoides X P. simonii, Bacillus thurinxensis (Bt) toxin gene, cowpea trypsin

inhibitor (CpTI) gene, insect-resistance

INTRODUCTION Poplar is an important woody species of artificial forests, with the quantities of fast growth rate, short rotation cycle, and easy vegetative propagation. It is the primary source of wood in many countries and in timber and paper industries. However, diseases and insect pests occur easily in poplar artificial plantations because of large

240 areas and simple diversity. Conventional breeding of poplar takes long time and can not work effective on the genetic improvement of insect-resistance due to the lack of gene resources on poplar genetic background. The method of plant genetic engineering, with the ability to introduce new desirable traits in plant, could contribute to the expansion of the gene pool of poplar species by transferring the useful genes such as those for resistance to disease, insect pest and herbicides into poplar. Insect-resistant transgenic poplar plants were produced by McCown BH et al (1991), Tian Y-C et al (1993) by introducing Cry l A gene from Bacillus thurmgiensis (Bt) into poplar. Confalonieri et al (1999) recently reported that the expressing of a soybean Kunitz proteinase inhibitor gene (KTi3) in transgenic Populus nigra L. did not show any detectable resistance to the tested insects, although transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant ( Duan X et al., 1996). The development of resistance to Bt toxins in laboratory and field populations of pests were reported (McGaughey et al., 1992; Tabashnik, 1994). The use of more than a single resistance gene has been presented as one of the resistance management strategies proposed for transgenic plant (Roush, 1997). The technology for introducing and expressing multiple transgenes in crops is available (Hua X et al., 1993; Chen et al., 1998). Hua et al (1993)and Zhao et al (1995) proved respectively that the transgenic tobacco co-expressing Bt and CpTI genes showed stronger resistant to the tested insect compared to the one only expressing Bt gene, which means that the two difference resistance mechanisms are compatible. We introduced CpTI gene and partially modified ('tyl A gene into poplar hybrid clone NL-80106 (Populus deltoides L. • Popuhls simonii Carr.) by A~'obacterium mmefacien-mediated transformational system. Transgenic poplar plants co-expressing two genes showed strong insect-resistance to the larva of Lymautria di,spar by fed with the leaves of in vitro transgenic plants. MATERIALS

& METHODS

Plant materials hi riO'o-grown plantlets of poplar hybrid clone NL-80106 (Populus deltoides L. • Popuhls simonii Cart.) are maintained in MS medium at our lab. Agrobacterium strains and plasmids

Agrobacterium tumefaciens LBA4404 was gifted by Institute of Biotechnology Research, Chinese Academy of Agricultural Sciences. Binary plasmid pFWZ10, harboring partially modified ('ryl A gene, and pFWY1, harboring CpTI gene, were cloned and constructed by the laboratory of Prof Fan Yunliu, Institute of Biotechnology Research, Chinese Academy of Agricultural Sciences. (Fig. 1). The two plasmids were respectively transferred into A. tume]Cacien,s' LBA4404 by the freezemelt method to construct the strains of LBA4404 pFWZ10 and LBA4404 pFZY1 for the use of poplar transformation.

241 RB

Nos-P

NPT-II

Nos-P

2CaMV35S-P

J--C---l-4--~--t

~

h~serted site

1~',

Nos-T

Nos-T

Ous

35S-P

LB

..... ~ F - - I - - - C E ~ ~ t - - - F - - - - ~ - Tet f

Fig. 1 The scheme of the binars' plasmids pFWZ 10 and pFZY 1 Bt toxin gene and CpTI gene were respectively inserted to the site of 'Inserted site' to construct the binao ,~plasmids of pFWZ 10 and pFZY 1

Transformation by Agrobacterium tumefaciens Leaf discs (5 • 10 mm) obtained from m vitro plantlets ofNL-80106 (P.de#oides L. • t: simonii Cart.) were pre-cultured, co-cultivated and transferred to selective regeneration medium using the conditions of Rao HY et al (2000). Two strains of LBA4404 pFWZ10 and LBA4404 CpTI were respectively cultured and re-suspended by MS liquid medium at the same density of OD~(,,,=0.3-0.4, then mixed completely and used in poplar transformation.

Molecular analysis Extraction of DNA from poplar leaves, PCR analysis, PCR-Southern blotting, extraction of leaf soluble protein and ELISA analysis were performed as described by Wu et al (2000).

Insect bioassay analysis Artificially raised Lymantria di,spar larva from the Institute of Forestry Protection, Chinese Academy of Forest Sciences were hatched at the same time and uniform in the first-instar tbr use of insect bioassay. Detached leaves from ill viO'o control hybrid poplar N1-80106 or transgenic plantlets, placed in 10 cm plates, were used to feed the first-instar larva of Lymano'ia dispar by 5 larva per plate and two plates each clone. The plates were incubated at room temperature (25-30~ and fresh leaves were exchanged to each plate every two days. The data of died and survived larva and the larva developmental stage were observed every two days. RESULTS

Transformation and plant regeneration The rooting of NL-80106 was sensitive to km levels, l0 mg/L km in the rooting medium could inhibit its rooting thoroughly (Rao HY et al., 2000). In order to get intact transformational plantlets, kin-free rooting medium were used to get the rooted km-resistant buds after they were sub-cultured on the selection medium with km 100 mg/L for 60 days. 154 putative transgenic plant lines were obtained from 867 cocultivated leaf explants with a transformation frequency of 17.8% (Fig 2).

242

A Fig2

Transformation of poplar infected with LBA4404 carrying Bt and CpTI A. Km-resistance buds got from selection medium. B. Rooting ofkm-resistance shoots.

Molecular analysis of transgenic plants DNAs from the different regeneration poplar plants were tested by PCR, using gene specific double primers, which were designed in order to bind an internal part of the Clyl A gene or CpTI gene, thus selectively amplifying 146 bp and 300 bp fragments. 9 of these lines were transformed both with LBA4404 pFWZ10 and LBA4404 CpTI, some respectively transformed, out of 121 plants tested (Fig.3).

600bp

500bp 400bp

300bp

300bp

146bp

lOObp 1

2

3

4

5

6

7

8

9

10

11

12

Fig 3. PCR results of km-resistance poplars 1.100bp ladder: 2. pFZY01 (CpTI); 3. pFWZ 10(Bt); 3-11 .km-resistance poplar lines; 12. Untransformed poplar, NL-80106.

243 PCR products were subsequently blotted and hybridized respectively using 1.8 kbp fragment of Cryl A gene and 430 bp of CpTI gene, as probes (Fig. 4). All 9 lines with both PCR products showing hybridized signals were proved to be the transgenic plants by two genes.

300 bp 146 bp

,.:

1

2

3

4

5

6

1

2

3

4

5

6

7

Fig 4. PCR-Southern blotting A. 1.pFWZ 10(Bt); 2.Untransformed poplar, NL-80106; 3-6. km-resistance poplar lines. B. 1. pFZY01(CpTI); 2. Untransformed poplar; 3-7. km-resistance poplar lines.

Expression of Cryl A and CpTI proteins

The soluble protein extraction from 3 lines of double gene transformants and 2 lines of untransformed control poplar plants and Cry l A protein extracted and purified from Bacil&s thuringiensis were tested by immune dot blotting with specific antibody of Cry l A or CpTI protein. 3 lines giving a positive reaction of the two antibodies were proved to have the expression of Cry 1 A and CpTI protein. Sandwich ELISA was used to detect the quantity of Cryl A protein in the soluble protein of the leaves. Cryl A protein levels in the soluble protein extraction of leaves based on a Cry l A standard curve made at the same time are shown on Table 1. The expression level of lines BC5, BC27, BC55 respectively reached of the total soluble protein. This expression level is higher than the reports by Perlak et al (1991), Van der Salm et al (1994) and Tian Y et al (2000). Table 1

Determination of Bt toxin from transgenic poplars by ELISA

Plant line.

Bt protein/total soluble protein of transgenic poplars (%)

BC5

O.O48

BC27

{).{)37

BC55

O.09O

Control

()

Blank

0

244 Insect Bioassays

Preliminary insect bioassay on the leaves of in vitro-grown plantlets BC5, BC27 and BC55 carried on the artificially raised Lymantria dispar larvae (Fig.5). The results on the 12th day of testing are reported in Table 2. Larval mortality was mainly observed in the first week of treatment. According to the preliminary results, these three lines of two-gene transgenic poplar showed obviously stronger resistance to L. dispar larva with the larval mortality of 76.7%, 67.1%,89.3% compared to the controls of 5.0% and 9.6%. Survived larva growth were inhibited with the lower larval weight and delayed larval development. Further insect bioassay on L. dispar and Clostera anchoreta (Fabricius), which heavily destroy poplar in China will be carried out when the transgenic poplar lines are moved to grow in the green house to get enough leaves for testing.

a

b

Fig 5. Lymantria di,spar larvae fed with leaves of transgenic poplar line BC5 (a) and with the leaves of untransformed poplar NL-80106 (b).

Table 2 The bioassay carried on Lymantria dispar larvae fed with leaves of three different transgenic poplars line for 12 days Plant lille

Larval mortality (%)

Survived larva weight (mg)

BC5

76.7

3.9

15.7

BC27

67.1

7.9

9.8

Developmental time from first-instar to second -instar

BC55

89.3

3.0

10.8

Control 1

5.0

18.1

7.7

Control 2

9.6

15.4

9.5

(day)

245 DISCUSSION The development of resistance to Bt toxins in laboratory and field populations of pests were reported (McGaughey et al., 1992; Tabashnik, 1994; De Maagd et al., 1999). Poplar has much longer life cycle compared to most of crops. Insect pests of poplar with short life cycle and large population would be easier to induce the resistance to Bt toxins under continual Bt-toxin pressure caused by Bt transgenic poplar. It has been proved to be able to delay insect resistance to Bt toxins by introducing two or more than two insect resistance genes with different insect resistance mechanisms into plants (Zhao et al, 1998). Bt toxin gene and plant proteinase inhibitor gene were integrated to different plants, including cotton, tobacco and poplar and transgenic plants of these two kind of genes showed stronger resistance to insects. Transgenic hybrid poplar transformed by Bt toxin gene and CpTI gene obtained in this paper will be carried out on further study of the effectiveness and durability of resistance to insect pests.

ACKNOWLEDGEMENTS Thanks are given to Dr Zhao Jun, Chen Rumei and Zhang Chunyi from the Institute of Biology, CAAS; Chen Ying, Wang Guangping and Pan Huixin from Nanjing Forestry University, China for their helps to some parts of this project.

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