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(American Journal of Botany. 2001;88:1704-1706.)
© 2001 Botanical Society of America, Inc.

Brief Communication

Bt corn has a higher lignin content than non-Bt corn1

Deepak Saxena and G. Stotzky2

Laboratory of Microbial Ecology, Department of Biology, New York University, New York, New York 10003 USA

Received for publication December 21, 2000. Accepted for publication April 19, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Bt corn has been genetically modified to express the Cry1Ab protein of Bacillus thuringiensis to kill lepidopteran pests. Fluorescence microscopy and staining with toluidine blue indicated a higher content of lignin in the vascular bundle sheaths and in the sclerenchyma cells surrounding the vascular bundle in all ten Bt corn hybrids, representing three different transformation events, studied than of their respective non-Bt isolines. Chemical analysis confirmed that the lignin content of all hybrids of Bt corn, whether grown in a plant growth room or in the field, was significantly higher (33–97% higher) than that of their respective non-Bt isolines. As lignin is a major structural component of plant cells, modifications in lignin content may have ecological implications.

Key Words: Bacillus thuringiensis • Bt corn • Cry1Ab protein • lignin • pleiotropic effects • transgenic plants


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Bt corn is maize (Zea mays L.) that has been genetically modified to express the cry1Ab gene from Bacillus thuringiensis (Bt) and produce a larvicidal toxin to kill lepidopteran pests, especially the European corn borer (ECB; Ostrinia nubilalis). There is concern that genetically engineered crops may pose risks to natural and agricultural ecosystems (e.g., Rissler and Mellon, 1996 ; Conway, 2000 ; Hails, 2000 ; Stotzky, 2000 ). Transformation with the cry1Ab gene may lead to pleiotropic effects that could have ecological implications. Here we show that Bt corn has a higher lignin content than isogenic non-Bt corn.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
A freshly collected Riverhead sandy loam soil from East Marion, Long Island, New York, USA, was sieved through a 15-mm screen, then through a 5-mm screen, and mixed thoroughly; 4.5 kg was placed in each of 20 plastic pots (18 cm diameter, 21 cm deep). Some physicochemical characteristics of the soil are the following: pH 5.2; 0.92% carbon and 0.07% nitrogen; 58% sand, 41% silt, and 1% clay. Seeds of ten different Bt corn hybrids (Table 1), representing three transformation events (Bt11, MON810, and 176), and their respective non-Bt isolines were planted (3 seeds/pot), and the pots were kept in a plant-growth room at 26 ± 2°C, with a 12-h light-dark cycle (light intensity of ~110 µmol from Cool-White, Sylvania F96T12, 215W fluorescent lamps; Ontario, Canada). Seeds of eight Bt hybrids (Table 2) and their non-Bt isolines were also planted in a Haven sandy soil in another site in East Marion, Long Island, New York, USA. Some features of the soil are the following: pH 7.1; 5.24% carbon and 0.25% nitrogen; 94% sand, 5% silt, and 1% clay. Both soils are classified as mesic typic dystochrepts. Plants were irrigated but not fertilized. The plants (Bt hybrids or isolines) were harvested after 97 d of growth in the plant growth room and after 90 d of growth in the field. Uniform free-hand sections of fresh corn stems between the 3rd and 4th node from the surface of the soil (thickness was ~11 mm for plants grown in the plant growth room and ~18 mm for field-grown plants) were examined for lignin by fluorescence microscopy at 400 nm (Hu et al., 1999 ) and by staining with 0.01% toluidine blue (Sylvester and Ruzin, 1994 ). The content of lignin of the same portion of the stems (oven-dried, ground, and passed through an 80-mesh sieve) was determined by the acetyl bromide method (Hatfield et al., 1999 ).


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  		Table 1. Lignin content (% ± SEM) in different hybrids of corn, grown in a plant growth room, with (Bt+) and without (Bt–) the cry1Ab gene

 

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  		Table 2. Lignin content (% ± SEM) in different hybrids of corn, grown in the field, with (Bt+) and without (Bt–) the cry1Ab gene

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
A higher content of lignin was observed by fluorescence microscopy in the vascular bundle sheath and in the sclerenchyma cells surrounding the vascular bundle of all Bt corn hybrids than of their respective non-Bt isolines grown in the plant growth room (Fig. 1A, B), which was confirmed by staining the sections with toluidine blue (Fig. 1C, D). The average diameter of the vascular bundle and surrounding lignified cells in Bt corn was 21.5 ± 0.84 µm, whereas that of non-Bt corn was 12.4 ± 1.14 µm (Fig. 1C, D). Similar results were obtained with plants grown in the field.



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  		Fig. 1. Fluorescence microscopy of a transverse stem section of the third internode from the soil surface from (A) non-Bt and (B) Bt corn grown in a plant growth room. Lignin autofluorescence was visualized following ultraviolet excitation at 400 nm (x250). The same transverse sections from (C) non-Bt and (D) Bt corn were stained with toluidine blue. More lignified cells and large vascular bundles were observed in Bt corn than in non-Bt corn (x250)

 
The content of lignin of the same portion of the stems, determined by the acetyl bromide method, was significantly higher (33–97% higher) in all hybrids of Bt corn than in their respective non-Bt isolines, whether grown in the plant growth room (Table 1) or in the field (Table 2). The lignin content of field-grown plants was higher than that of plants grown in the plant growth room, which were smaller. There was a significantly higher lignin content (P < 0.002) in plants transformed by event Bt11 (7.4 ± 0.10 and 6.7 ± 0.12% for field- and growth room-grown plants, respectively) than by event MON810 (6.9 ± 0.07 and 6.2 ± 0.10% for field- and growth room-grown plants, respectively). There were no significant differences in the lignin content of isogenic non-Bt plants (P > 0.67 for field-grown plants and P > 0.30 for growth room-grown plants). The lignin content of the only available hybrid transformed by event 176 was lower than that of hybrids transformed by events Bt11 and MON810. These results differ from those reported by Faust (1999) , which indicated no significant differences in lignin content between the dried biomass of whole plants of Bt (event MON810) and non-Bt corn but which indicated that Bt corn had a higher moisture content and a lower level of ammonia than non-Bt corn (P < 0.05). However, Masoero et al. (1999) reported a 16% higher lignin content in Bt than in non-Bt corn.

Lignin is a major structural component of plant cells that confers strength, rigidity, and impermeability to water. Any modifications in lignin content could have ecological implications (Halpin et al., 1994 ). For example, the increase in lignin content in Bt corn may be beneficial, as it can provide greater resistance to attack by second-generation ECB (Ostrander and Coors, 1997 ), reduce susceptibility to molds (Masoero et al., 1999 ), and retard litter degradation and decomposition by microbes (Reddy, 1984 ; Tovar-Gomez et al., 1997 ). The addition of biomass from Bt corn to soil resulted in a significantly lower gross metabolic activity (i.e., CO2 evolution) in soil than did the addition of non-Bt corn (S. Flores, D. Saxena, and G. Stotzky, New York University, unpublished data; Stotzky, 2000) , which may be beneficial, as the organic matter derived from Bt corn may persist and accumulate longer and at higher levels in soil, thereby improving soil structure and reducing erosion (James et al., 1998 ), or it may be detrimental, as the longer persistence of the biomass of Bt corn may extend the time that the toxin is present in soil and, thereby, may enhance the hazard to nontarget organisms and result in the selection and enrichment of toxin-resistant target insects. Moreover, lignin is relatively indigestible and reduces the ability of herbivores to digest plant material, and its increase in forages might affect rates of feeding and population dynamics of defoliators (Barriere and Argillier, 1993 ; Jung and Allen, 1995 ; Gardner et al., 1999 ). Additional studies are necessary to clarify the environmental impacts of a higher lignin content, especially in Bt corn, as about 8.1 million hectares (20 million acres) of Bt corn (26% of total corn acreage) were planted in the United States alone in 1999 (U.S. Environmental Protection Agency, 2000) .


   FOOTNOTES
 
1 The authors thank Saul Flores for his valuable suggestions. These studies were supported, in part, by grant R826107-01 from the U.S. Environmental Protection Agency. The opinions expressed herein are not necessarily those of the Agency. Back

2 Author for reprint requests (tel: 01-212-998-8268; FAX: 01-212-995-4015; gs5{at}nyu.edu ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Barriere Y. O. Argillier 1993 Brown-midrib genes of maize: a review. Agronomie 13: 865-876[CrossRef][ISI]

Conway G. 2000 Genetically modified crops: risks and promise. Conservation Ecology 4: 2

Faust M. A. 1999 Research update on Bt corn silage. In Proceedings of the Four-State Applied Nutrition and Management Conference. MWPS-4SD5. Iowa State University, Ames, Iowa, USA

Gardner P. T. T. J. Wood A. Chesson T. Stuchbury 1999 Effect of degradation on the porosity and surface area of forage cell walls of differing lignin content. Journal of the Science of Food and Agriculture 79: 11-18[CrossRef][ISI]

Hails R. S. 2000 Genetically modified plants—the debate continues. Trends in Ecology and Evolution 15: 14-18

Halpin C. M. E. Knight G. A. Foxon M. M. Campbell A. M. Boudet J. J. Boon B. Chabbert M. Tollier W. Schuch 1994 Manipulation of lignin quality by down regulation of cinnamyl alcohol dehydrogenase. Plant Journal 6: 339-350[CrossRef][ISI]

Hatfield R. D. J. Grabber J. Ralph K. Brei 1999 Using the acetyl bromide assay to determine lignin concentration in herbaceous plants: some cautionary notes. Journal of Agricultural and Food Chemistry 47: 628-632[CrossRef][ISI][Medline]

Hu W. S. A. Harding J. Lung J. L. Popko J. Ralph D. D. Stokke C. Tsai V. L. Chiang 1999 Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nature Biotechnology 17: 808-812[CrossRef][ISI][Medline]

James R. R. S. P. Difazio A. M. Brunner S. H. Strauss 1998 Environmental effects of genetically engineered woody biomass crops. Biomass and Bioenergy 14: 403-414[CrossRef]

Jung H. G. M. S. Allen 1995 Characteristics of plant-cell walls affecting intake and digestibility of forages by ruminants. Journal of Animal Science 73: 2774-2790[Abstract]

Masoero F. M. Moschini F. Rossi A. Prandini A. Pietri 1999 Nutritive value, mycotoxin contamination and in vitro rumen fermentation of normal and genetically modified corn (Cry1A(B)) grown in northern Italy. Maydica 44: 205-209[ISI]

Ostrander B. N. J. G. Coors 1997 Relationship between plant composition and European corn borer resistance in three maize populations. Crop Science 37: 1741-1745[Abstract/Free Full Text]

Reddy C. A. 1984 Physiology and biochemistry of lignin degradation. In M. J. Klug and C. A. Reddy [eds.], Current perspectives in microbial ecology: proceedings of the Third International Symposium on Microbial Ecology, 558–571. American Society for Microbiology, Washington, D.C., USA

Rissler J. M. Mellon 1996 The ecological risk of engineered crops. Massachusetts Institute of Technology Press, Cambridge, Massachusetts, USA

Stotzky G. 2000 Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA bound on clays and humic acids. Journal of Environmental Quality 29: 691-705[Abstract/Free Full Text]

Sylvester A. W. S. E. Ruzin 1994 Light microscopy I: dissection and microtechnique. In M. Freeling and V. Walbot [eds.], The maize handbook, 83–95. Springer-Verlag, New York, New York, USA

Tovar-Gomez M. R. J. C. Emile B. Michalet-Doreau Y. Barriere 1997 In situ degradation kinetics of maize hybrid stalks. Animal Feed Science and Technology 68: 77-88[CrossRef]

U.S. Environmental Protection Agency. 2000 Preliminary risk and benefit assessments for Bt plant-pesticide. U.S. Environmental Protection Agency, Washington, D.C., USA




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