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Forest Engineering

Eucalyptus with pea genes

A partnership between Esalq and Cia. Suzano prepares a genetically modified plant that will develop more cellulose

Carlos Alberto Labate, a professor at the Department of Genetics of the Luiz de Queiroz Agricultural College (Esalq), at the University of São Paulo (USP), and his team of researchers are celebrating the first results from a project begun almost three years ago: production of eucalyptus modified with a pea gene. The purpose of pioneering methodology is to obtain trees with greater biomass and a bigger yield of cellulose, which is of direct interest to the Companhia Suzano de Papel e Celulose, the partner in this project within the scope of the FAPESP’s Partnership Program for Technological Innovation (PITE).

“We already have the first genetically modified plants”, says Labate. “At present, we are assessing these genetically modified plants containing the pea gene Lhcb1*2 (Pisum sativum).” In his favor, he is able to rely on the performance of the purpose of the research: the eucalyptus has the advantages of fast growth with high germinating capacity, which makes it possible to obtain a large number of clones in a short time. The species Eucalyptus grandis together with the hybrid between Eucalyptus grandis and Eucalyptus urophylla were used in the experiments.

Labate explains that the project was begun in June 1998 with two essential goals; the first, now achieved, was the development of genetic modification technology, which resulted in the request for a patent for the methodology of modifying the eucalyptus developed at the Esalq. The other goal is the improvement in the plant’s performance. To achieve this, the next steps are the laboratory and greenhouse tests, where the plants grow under controlled conditions. Then, the experiments will be transferred to field-testing. Introducing the plant into the production process should take place in a five- year period. To reach this point, all the stages of the work comply with standards set by the National Technical Biosafety Commission (CTNBio), associated with the Ministry of Science and Technology (MCT). No genetically modified plant will be introduced into the field without all the authorizations requirement by the CTNBi.

Sun and genes
The studies carried out at the Esalq are based on introducing a gene able to improve the capture of sunlight. Through genetic modification, the tree is able increase the its biomass, producing more cellulose per tree. Labate’s experiment began based on earlier work carried out with tobacco, which he calls the “model-system”, much used in the present genetic studies. “Genetically modified tobacco and petunia plants have broader leaves with a greater capacity for photosynthesis in low light conditions”, explains the researcher. In the future, other genes may be added to these, with pest or unfavorable climate resistant functions. “We will even be able to use other genes to alter the quality of the wood fiber,” aims the researcher.

The experiments with tobacco and now with eucalyptus use the bacterium Agrobacterium tumefaciens, which works as a vehicle for transferring the genes. It has the intrinsic ability to transfer part of its genome, the T-DNA (transfer DNA), located in a plasmid (fragment of circular self replicating DNA), to the plant’s genome. The plasmid bears three essential elements: the first is the pea gene Lhcb1*2, responsible for capturing light and increasing the biomass, also known as the CAB gene, the acronym forchlorophyll a/b binding protein, the name of the protein it codes. The other two are those of the region 35S promoting the virus that causes mosaic in cauliflower, which that has the property of expressing a large number of proteins all the time, and the gene nptII isolated from the bacterium Escherichia coli, which gives resistance to antibiotics. The bacterium infects bits of the young leaves and transfers these elements to them.

To know whether the eucalyptus transformation method is working, the researchers introduced the marker known as the reporting gene (uidA), also extracted from the bacterium Escherichia coli, which codes the enzyme betaglucoronidase. The transformed vegetable tissue with this gene turns blue when submitted to histological activity tests (microscopic analysis of vegetable tissue) of this enzyme, enabling the result to be seen. Leaves with blue dots under the microscopic gave the scientist the assurance of the success of the technique.

The right dose
The next step was to cause the plants to regenerate based on the different vegetable structures of the eucalyptus, producing plants containing the new genes. The regeneration enables a whole plant, with root, stem, and leaves, to be obtained from a piece of leaf. “This was one of the most difficult stages of the project”, recalls Labate. It took almost two years. They needed to find the right dose of hormones to bring about the regeneration. During this period, various concentrations were tested until the ideal dosage was found, which Labate is keeping as a trade secret.

Plants that grow from these leaves may be genetically modified or not. To make the selection, the researchers use an antibiotic that inhibits the development only of the non-genetically modified plants, since the modified eucalyptus was given the nptII gene giving it resistance to the drug. It is not as easy as it seems. “Sometimes, certain non-genetically modified cells resist, since the eucalyptus has a natural toleranceto given antibiotics and, for this reason, we are researching more efficient means of selection”, says Labate. Once selected, the genetically modified eucalyptus goes to the growth chambers with controlled temperature and light, with new equipment bought in Canada, using money from the partnership.

Now it is just a question of waiting for the plants to grow. According to the professor, as a first step, we need to examine the physiological performance and the changes to the morphology of the plants, such as, for example, comparing the size of the genetically modified leaf with a non-transgenic one from the same source plant. “An increase in photosynthesis and productivity, that has already taken place in the experiments with tobacco and petunia, is the result that we also expect with the eucalyptus”, he explains. For pulp and paper production, this may mean more biomass, which is essential for increasing the competitiveness of the Brazilian product in the local and international markets.

No hurry
Today, Brazil is the 7th biggest pulp producer in the world – with production of 7.4 million tons in 2000 – and the biggest short-fiber (eucalyptus fiber) producer in the world. But we know that we can – and must – go beyond this. The partnership between Esalq and Suzano is designed for this purpose. That is why it enjoys funding of R$ 125,000 and US$ 163,000 from FAPESP, plus R$ 83,000 in study grants from the Human Resources Strategic Area Training Program (Rhae) of the national Scientific and Technological Development Council (CNPq). The Cia. Suzano is investing R$ 75,000 and expects no immediate return, according to Natural Resources Division manager, Osni Sanchez.

“We cannot afford to take an short-term view. Our most important goal is to extend knowledge”, he says. And this is not just demagogy. Suzano has a long history of partnership with research centers and it knows that the waiting time is fully rewarded. In the 50s, in partnership with the University of Florida, in Gainsville, the United States, the company supported research into the development of technology for manufacturing paper using 100% eucalyptus fiber, gaining worldwide recognition for its pioneering nature.

The company was also one of the founding partners of the Forestry Research and Studies Institute (Ipef), created in the 60’s jointly by Brazilian forestry companies and Esalq’s Department of Forestry Sciences to develop new techniques and promote the sector’s growth. Results were not long in coming. “In the 70s, eucalyptus production was 25 stereos (a piled cubic meter) per hectare per year. Nowadays, on average, 60 stereos are produced. The productivity improvement in these 30 years is due to research into genetic improvement and plant handling”, recognizes Osni. Now he believes the time has come for another leap forward, with genetic engineering.

Strategic technology
Professor Carlos Labate tells that the first contacts with the company took place in 1997, when the agronomist Shinitiro Oda, research manager at Suzano, was at the Esalq to learn about the progress in genetic engineering. “For the company, the development of this technology is strategic”, points out Oda. “There is a worldwide race in this. From the 70s to today, Brazil has developed enviable forestry technology. But nowadays it has a great competitor: Asia. Countries like Malaysia, Indonesia, the Philippines and China have progressed rapidly in this field, benefiting from a tax incentive policy – finance at low, long-term interest rates- which we do not have in Brazil”, he explains.

But it is not just Suzano that gains from the partnership. Labate is enthusiastic about the dynamism that the company has contributed to his laboratory at the Esalq. “I have never stopped doing basic research”, he says happily. “The company is interested in mastering this technology. It has a research department but it would cost a lot to equip it”. The company thus has a system integrated with the university, a common procedure in countries with greater scientific and technological production. “The company is very demanding and as a result we work much faster”, says Labate. Today, the project has 11 exclusively dedicated members: three technicians, one post-doctor, one doctor, one doing his master’s degree and five science students. In addition, the investment enabled the purchase of new equipment with centrifuges, freezers, and controlled-growth chambers.

According to Labate, the connection with the company also brings other benefits to the university. “The technological innovation program gives us the opportunity of showing to the business sector the university’s potential as a partner in technological development and in generating capital. And, we have the ability to create critical mass. We may even be the embryo for the development of future new companies”, believes the researcher.

Legislation for genetically modified organisms

After putting together a collection of genetically modified seedlings in the greenhouse, the next step in professor Carlos Labate’s research will be to assess the plants in the field. For this stage, authorization from the National Technical Bio-safety Commission (CTNBio), associated with the Ministry of Science and Technology (MCT), will be required. The CTNBio is responsible for compliance with Law No. 8,974, of January 1995 – the Bio-safety Law – which lays down the guidelines for controlling activities and products associated with genetically modified organisms (GMOs), such as transgenic plants. The Commission is made up of 36 associates including both full members and replacements: eight biotechnology specialists, representatives of the ministries of Science and Technology, Environment, Education, Foreign Affairs and Agriculture, as well as representatives of consumer protection bodies, the business community and labor health protection organizations.

To guide research institutions and companies as to the bio-safety criteria and procedures, the commission publishes, in the Official Federal Gazette, normative instructions laying down technical guidelines to ensure the safety of these products. Currently, there are 18 of these sets of normative instructions. No. 3 establishes the rules for releasing GMOs into the environment and is made up of a technical questionnaire in which researchers give detailed information on the experiment, bibliographical references, and previous experiments carried out in Brazil or in other countries. If necessary, the CTNBio may require an Environmental Impact Study (EIA) to be carried out and an Environmental Impact Report (Rima) to be prepared.

After careful technical assessment, the vegetable organism is okayed for planting, and this decision is also published in the Official Federal Gazette. But this is still not the final step. After examining countless trials carried out in the field, the CTNBio will then issue a formal opinion regarding the sale of the product, which must also meet the requirements laid down by the ministries of Agriculture, Health, and the Environment. Brazil has 130 institutions certified to carry out research in to GMOs. In the last five years, the CTNBio has assessed more than 800 experiments. Further information: www.ctnbio.gov.br.

The project
Increase in Eucalyptus Biomass by Introducing the Gene CAB via Genetic Modification (nº 98/01394-0)Type Technological Innovation Partnership (PITE); Coordinator Carlos Alberto Labate – USP; Investment R$ 125,057.00 and US$ 163,791.76, from FAPESP, and R$ 75,127.00, from the Cia. Suzano

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