Euphytica 121: 123–127, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Field evaluation of insect-resistant transgenic Populus nigra trees

J.J. Hu1, Y.C. Tian2∗, Y.F. Han1, L. Li1 & B.E. Zhang3

1Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, P.R. China; 2Institute of Mi-crobiology, Chinese Academy of Science, Beijing 100080, P.R. China; 3Manas Forest Station, Xinjiang UygurAutonomous Region 832206, P.R. China; (∗author for correspondence)

Received 15 December 1999; accepted 30 October 2000

Key words: cross protection, Cry1Ac gene, field evaluation, insect-resistance, Populus nigra

Summary

The performance of insect-resistant transgenic poplar trees (Populus nigra) expressing a Cry1Ac gene from Bacillusthuringiensis subsp. Kurstaki HD-1 against poplar defoliators was evaluated in the field at the Manas Forest Stationin Xinjiang Uygur Autonomous Region during 1994–1997. The results showed that the average percentage ofhighly damaged leaves on the transgenic trees was 10% while that on the control trees in nearby plantations reached80–90%. The average number of pupae per m2 of soil at 20cm depth in transgenic poplar plantation was 18 whichwas only 20% of that found in the non-transgenic control field. The number of pupae and the leaf-damage ontransgenic trees described above are all far below the threshold set for chemical protection measures. The non-transformed poplar trees grown in the same plantation with the transgenic trees were also protected indicating thatcross protection occurred between these two kinds of trees. Insect-resistant transgenic poplar trees have a potentialapplication value in afforestation.

Introduction

Poplar trees play an important role in the economicdevelopment and construction of protective forestryin the three Northern Regions of China. High growthrate, easy propagation and wide range of usage makepoplar one of the most important tree species for theestablishment of artificial forests. However, the mainthreats to poplar plantations are from defoliators andstem borers (Dai et al., 1988). The damage causedby defoliators, mainly the poplar lopper (Apocheimacinerarius) and gypsy moth (Lymantria dispar), ex-ceeded ten thousand hectares or about 40% of the totalpoplar plantation in China in 1989. The fact that theinsect-resistant trait of trees is controlled by multigenefamilies coupled with the long life cycle of woodyplants makes conventional tree breeding very diffi-cult. Knowledge obtained in plant biotechnology inthe last decade has led to a new approach to solvingthis problem.

The first insect-resistant transgenic poplar plant(Populus alba × P. grandidentata) was obtained in

McCown’s laboratory (McCown et al., 1991) by elec-tric discharge particle acceleration with an expressionvector containing a Cry1Aa gene of Bacillus thuring-iensis (Bt). Field trials of the transgenic poplar treesshowed a nearly complete protection against the lar-vae of gypsy moth and forest tent caterpillar (Kleineret al., 1995). In our work, Insect-resistant trans-genic P. nigra plants were selected by Agrobacterium-mediated transformation with a binary vector contain-ing a Cry1Ac gene from B. thuringiensis (Tian et al.,1993; Wang et al., 1996). Here, results of insect resist-ance from field evaluation of transgenic P. nigra treesare presented.

Materials and methods

Plantation establishment

In the spring of 1994, about one ha of transgenicP. nigra plantation was established at Manas ForestStation, Xinjiang Uygur Autonomous Region, China.Fourteen transgenic P. nigra lines expressing Cry1Ac

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Table 1. The average percentage of leaf damage caused by insects feeding in the transgenic plantation and in non-transgenic plantations

Plantations Clone Number Average percentage of damaged leaves at different leaf damage rate (%)

No. of plants 100% 50% 25% <25% 0%

Transgenic P. × eur. 23 15 14 21 26 24

plantation P. nigra (Ck2) 17 11 15 21 28 25

P. nigra (Ck3) 12 7 12 19 30 32

Non-transgenic Average – 11 14 20 28 27

12 12 12 14 22 31 22

153 12 14 17 23 30 16

192 6 5 13 22 31 29

172 16 11 16 23 27 22

13 11 9 14 21 27 29

139 11 9 11 20 30 30

162 12 9 15 21 29 26

197 11 7 13 20 30 29

208 10 13 16 24 27 20

209 10 11 14 21 29 25

222 6 6 12 20 36 26

254 12 19 19 21 22 18

110 11 8 13 18 27 35

141 11 7 12 19 30 32

Transgenic Average – 10 14 21 29 26

Non-transgenic P. × eur. 30 80 – – – –

poplar plantation P. nigra 30 90 – – – –

gene, two non-transformed P. nigra lines (Ck2 andCk3) and one Populus × euramericana cv.‘Robusta’line were randomly arranged with two repeats andplanted in the same plantation. The lines and numberof plants for each line are listed in Table 1. The totalnumber of transgenic poplar trees was 151 and thatof the non-transgenic poplar trees from the three non-transgenic control lines was 52, accounting for 34.4%of all trees in the plantation. Each tree was grown inthe plantation at a spacing of 3 m × 4 m.

The non-transgenic P. nigra plantation and P. ×euramericana cv. ‘Robusta’ plantation at the same ageas the transgenic one were located 2 km Southeastfrom the transgenic plantation, and 1 km from eachother.

The insect pests (Apocheima cinerarius Erschoffand Orthosia incerta Hufnagel), that damaged thelocal poplar plantations, occurred naturally and seri-ously almost every year in this area during 1995–1997with an average larval density of 40 heads per meterbranch of the poplar trees, especially in the young pop-lar plants. In this case, it was not necessary to inoculatethe plants with the corresponding insect larvae. All the

survey of insect-resistance in this study was carried outon the naturally occurred insects.

Insect larva density survey

At the peak of leaf damage by insect larvae (29 April1997), one-meter long branch samples were collec-ted from the six trees of transgenic lines No.12, 153,172, 192, and non-transgenic P. nigra controls Ck2 andCk3, respectively, and the number of Apocheima ciner-arius Erschoff larvae on these branches was counted.

Damaged leaf rate survey

Just after the occurrence of insect larva damage (13–14 May 1997), leaf samples (100 leaves per tree) in thetransgenic plantation were randomly collected fromdifferent parts of individual trees. According to thedamage degree, leaf samples were sorted into the fol-lowing five classes: 100% damaged, 50% damaged,25% damaged, less than 25% damaged (<25%) andundamaged (0%).

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Figure 1. Insect larvae density on transgenic and control trees. The fine vertical bars represent the standard deviation for each sample.

Insect Pupa survey in the plantation soil

After the larvae of the two main defoliators Apocheimacinerarius Erschoff and Orthosia incerta Hufnagel de-veloped into pupae and inhabited the soil, a pupasurvey was performed in the transgenic as well asin the non-transgenic poplar plantations. Thirty plotsof 1 m × 1 m × 0.2 m were randomly selected tosearch for pupae in transgenic plantation, P. nigra con-trol plantation and P. × euramericana cv. ‘Robusta’control plantation, respectively.

The data collected were statistically analyzed us-ing the SAS System for Windows v6.12 (SAS InstituteInc., 1989–1996). Since the data collected in the firsttwo years (1995, 1996) were not complete, only thedata for last year (1997) were shown in this paper.

Results and discussion

Insect larva density on transgenic poplar trees

The construction of plant expression vector forCry1Ac gene, transformation of Populus nigra plantsand molecular characterization of most of the trans-genic lines described in this paper have been reportedpreviously (Tian et al., 1993; Wang et al., 1996). Theinsect larvae density on one-meter branches of trans-genic tree lines No. 153, 172, 192 and 12 are shownin Figure 1. The insect density on transgenic trees issignificantly lower than that on non-transgenic trees ofCk2 and Ck3 at α = 0.01 level as revealed by statisticalanalysis. There was no significant difference betweenfour transgenic lines as well as between two controls.

Damaged leaf rate between transgenic andnon-transgenic poplar trees

As shown in Table 1, the average damaged leaf rateof transgenic trees (refer to 100% damaged leaves)in the transgenic plantation was 10%, while the dam-aged leaf rate of P. × euramericana cv.‘Robusta’ andthe two non-transformed P. nigra control lines Ck2,Ck3 grown in the same plantation with the transgenicones, were 15%, 11% and 7%, respectively. Theseare much lower than those in P. × euramericana cv.‘Robusta’ plantation (80%) and P. nigra plantation(90%). Similar results about leaf damage rates wereobtained in the previous two years (data not shown).This indicates that transgenic plants not only had astrong ability to resist leaf damage caused by insectlarvae, but could also protect non-transgenic plantsin the same plantation despite the insect density onthese plants being higher than that on the transgenicplants (Figure 1). Therefore, at least in the case ofabove described plantation in which 74% of the treeswere transgenic, non-transgenic trees could also beprotected from insect damage.

The average rate of damaged leaves in the trans-genic plantation described above was much lower thanthat of the protection index (40%) (Xue, 1991), thusapplication of chemical insecticides was not carriedout in the last four years. These results indicate thatthe insect-resistant transgenic plants could be valu-able in integrated pest management of forest and insilviculture.

Insect pupa number in different plantation soil

Results from an investigation of the number of insectpupae in the soil of the transgenic poplar plantation,

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Figure 2. Comparison of insect pupae number between different plantations. The fine vertical bars represent the standard deviation for eachsample.

P. × euramericana cv. ‘Robusta’ plantation and P.nigra plantation are presented in Figure 2. Though twokinds of pupae were observed, the majority of insectpupae are from A. cinerarius. The pupae in the soil ofthe non-transgenic P. × euramericana cv. ‘Robusta’plantation and P. nigra plantation are 4.9 and 4.1 timesof that in the soil of the transgenic plantation. Accord-ing to previous experience of disease and pest controlin Manas Plain Farm, it is predicted that heavy insectpest damage will occur in the coming year if the num-ber of pupae in the soil is over 36 heads per squaremeter. Since there were only 18 heads per square meterin the soil of the transgenic plantation, it would notbe necessary to carry out chemical protection meas-ures in this plantation stand. The low pupa number inthe soil of the transgenic plantation would result in alow insect density in the coming year. Therefore, thelow pupa number together with the toxic effect of thetransgenic plants on the insects may contribute to thecross-protection of the non-transgenic plants growingin the transgenic plantation.

Conclusion

From 1995 to 1997, insect pests, especially A. cin-erarius and O. incerta, occurred on a large scale inManas, and caused serious damage and economiclosses for local poplar plantation and timber forests.Therefore insect-resistant poplar tree clones are badlyneeded in the development of artificial afforestation.Bioassays in the laboratory demonstrated that the de-tached leaves of transgenic P. nigra plants carrying aBt toxin gene had a high level of insecticidal activity

(Tian et al., 1993; Wang et al., 1996). Results fromfield evaluation of these transgenic poplar plants in1997 also showed significant resistance to the two de-foliators. The damaged leaf rate and pupae in the soilwere both lower than protection indexes, and there wasno serious loss in the transgenic plantation, thus nopesticide treatment was needed. This is in contrast tothe loss in non-transgenic P. nigra and P. × euramer-icana cv. ‘Robusta’ plantations where pesticides hadto be used.

In the field trial of insect-resistant transgenicplants, insect larva density, damaged leaf rate and pupanumber per unit area in the soil are good indexes toevaluate insect-resistance of transgenic tree plants. Inthis work, we demonstrated that an insect-resistanttransgenic poplar plantation showed good cross pro-tection from insect damage to other non-trangenicplants, in addition to its protection of the transgenictrees. Therefore it could be beneficial to timber pro-duction, environmental protection and insect controlif these transgenic trees could be applied. Transgenicpoplar could be interplanted with other poplars, oreven with other species for insect control as it wasdone in this field trial. Certainly, for this is a prelim-inary result of field evaluation, the interplanting ratioof different cultivars in a plantation and the long-terminsecticidal effect of these transgenic trees should befurther studied.

Acknowledgements

This project was supported by National Natural Sci-ence Foundation of China (1990–1992), and partly

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by National Key Program in Science and Techno-logy of China (1995) and the World Laboratory of theInternational Centre for Science and Culture (ICSC).

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