ISB News Report
May 1998


Engineering Cold Tolerance Takes a Major Step Forward
A First Step Towards Engineering Improved Phosphorus Uptake
Recombinant Antibodies in Milk Confer Virus Resistance
Commercialization of Genetically Engineered Sinorhizobium meliloti
New Venture Fund for Ag-Biotech Start-ups
Conference: Strategic Partnerships to Successfully Commercialize Agricultural Biotechnology



Those who live in the sub-temperate and temperate regions of the world know only too well the damaging effects that freezing temperatures can have on plants. Cold injury kills crops and often causes billions of dollars in losses to agriculture. Traditional plant breeding has had little success in improving the cold tolerance of crops. Thus, a report on the development of cold-hardy plants by Mike Thomashow and colleagues at Michigan State University is good news to farmers and home gardeners (1). The research shows that several cold tolerant genes can be activated in the plant by introducing a key regulatory gene, thus helping them defend against a cold snap.

Thomashow's research focuses on the phenomenon of "cold acclimation" where plants exposed to gradual low, non-freezing temperatures tolerate subsequent freezing temperatures by expressing a series of "cold-regulated" (COR) genes. Thus, a gradual cooling can help the plants to better prepare for the icy weather while a sudden freeze can kill them. When one such gene, COR15a, was expressed constitutively in Arabidopsis, freezing tolerance of isolated chloroplasts and protoplasts, but not whole plants, was increased (see article in the January 1997 News Report). Thomashow recognized the need to express many such COR genes in order to sufficiently beef up cold tolerance in plants. Current plant transformation protocols, however, do not allow for transfer of many genes together into a plant. In a clever twist, the Thomashow group identified a transcription factor called CBF1 that regulates the expression of many COR genes. Transcription factors bind to the regulatory sequences of genes and can act as a "master switch" to turn on the expression of an array of genes.

Michigan State scientists created transgenic Arabidopsis plants to overexpress the CBF1 gene under the control of a constitutive CaMV 35S promoter. As expected, this switched on the cold-regulated genes. Plants derived from a transgenic line producing large amounts of the transcription factor put four COR genes on a hyperdrive and survived exposure to lethal low temperatures (-5C for two days). The nontransgenic plants which showed no such expression of these genes turned yellow and quickly died. The response of transgenic plants was similar to the cold-acclimated, untransformed plants indicating that the cold tolerance was achieved by tricking the plants to continuously produce COR proteins through transcriptional activation. Electrolyte leakage tests, which measure the amount of ions leaking from plant cell membranes due to cold damage, also indicated that freezing tolerance of transgenic plants was similar to that of cold acclimated nontransgenic plants. These results are "a tour de force for the field" according to Gary Warren of Imperial College of London (2).

The Science report clearly shows the potential of genetic modification of plants to confer cold hardiness and provides hope that crops in the future may be redesigned to brave an occasional arctic express. Charles Guy of the University of Florida says that Thomashow's study "has profound implications for agriculture"(2). While this research was done in a model system and it remains to be seen whether the CBF1 protein will activate the cold tolerant genes in crop plants, there is reason to be hopeful as some of these genes are likely to be conserved across many plant species. According to Thomashow, even a small increment in cold tolerance may save substantial crop losses in the face of frigid weather. Further, since the defense response of the plants against cold and drought are similar, there is a potential to protect the crops against water stress also, according Thomashow. The Michigan group has filed patents for the cold-fighting gene and is finalizing a licensing agreement with Mendel Biotechnology, Inc. (Palo Alto, California) to commercialize this technology.


1. Jaglo-Ottosen, et al. 1998. Arabidopsis CBF-1 overexpression induces COR genes and enhances freezing tolerance. Science 280:104-106.

2. Pennisi, E. 1998. Transferred gene helps plants weather cold snaps. Science 280:36.

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University


Among the trio of major plant nutrients, phosphorous is the most limiting compared to nitrogen and potassium. Many soils are low in phosphorous and even when it is abundant, uptake of this nutrient by plants can be tricky. Bioavailability of phosphorous is very critical in the acidic soils of the Tropics where iron and aluminum interfere with its uptake. Thus, millions of acres of land in developing countries have phosphorous deficiency problems. Calcium-rich soils in the Southeast and Great Plains of the United States are also plagued with a similar problem. Countries such as India spend an enormous amount of their precious foreign exchange in importing phosphate fertilizer which is derived from rock phosphate found in a few areas such as the U.S., Russia, Morocco and Tunisia. Global reserves of high quality rock phosphate are limited and may run out in about 100 years according to one estimate.

A logical way to address this problem is by developing plants which can efficiently draw phosphate (a common form of phosphorous) from soil. Until recently, scientists knew very little about the molecular basis of how plants absorb critical nutrients such as phosphorous, potassium and sulfur. A recent flurry of papers reporting the isolation of ion transporters in plants is improving our understanding of nutrient uptake in plants. A research group led by K. G. Raghothama at Purdue University were the first to clone phosphate transporter genes in plants from Arabidopsis in 1996 (1). More recently they have also shown the existence of such genes in tomato (2).

To clone the phosphate transporter genes, the Purdue group grew roots of Arabidopsis under phosphate-deprived conditions. This led the plants to switch on the phosphate transporter genes which were then isolated using expressed sequence tags (ESTs) as probes to screen a plant cDNA library. These genes were found to be expressed differentially in the roots of phosphate-starved plants. The phosphate transporter genes in tomato isolated by the Purdue group, and those from potato, Medicago and Catharanthus identified by other researchers, appear to be similar in structure and function to the Arabidopsis genes. Predictably, expression of the tomato genes appears to be localized in the root epidermis, the site of phosphate uptake. Changes in the cellular concentration of phosphorous apparently induce the expression of these genes. Raghothama's team is now developing transgenic plants to overexpress transporter genes to test whether this would result in a higher efficiency uptake of phosphorous.

Identification of genes involved in phosphate uptake is a major first step towards the eventual development of plants which can absorb phosphorous from soil in an efficient manner. Strategies like this may play an increasingly important role in the future to deal with the problems of poor soil fertility and to reduce the dependency on fertilizer application. This would be particularly welcome by resource-poor farmers in developing countries.


1. Muchhal, U. S., J. M. Pardo & K. G. Raghothama. 1996. Phosphate transporters from the higher plant Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 93:10519-10523.

2. Liu, C. et al. 1998. Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorous. Plant Physiol. 116:91-99.

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University



The first line of defense against pathogens invading the body at a mucosal surface, for example the intestines, are the antibodies secreted by the cells lining the mucous membranes. These antibodies can adhere to the pathogen and block a subsequent infection. An alternative approach to neutralizing an intestinal pathogen is to supply the antibody orally to develop passive immunity. The protective benefit of antibodies in colostrum, the milk secreted for a few days after birth, is a well known fact. Extending the synthesis of antibodies in milk could provide a longer period of protection to the neonate.

With the development of transgenic animals that synthesize foreign proteins in milk, it is now possible to examine the ability of antibodies synthesized in milk to confer passive immunity on neonates. Researchers in Spain report in the April 1998 issue of Nature Biotechnology the generation of transgenic mice that secrete virus neutralizing antibodies in their milk. These antibodies were directed against transmissible gastroenteritis coronavirus (TGEV).

The transgenic mice expressed a recombinant TGEV neutralizing antibody under the control of a mammary-specific promoter. Antibody concentrations in the milk ranged from 0.005 to 5 milligrams per milliliter. The highest concentration of recombinant antibody was able to reduce virus infectivity over one million-fold. This high level of active recombinant antibody expression is impressive considering that two recombinant gene products (the heavy and light immunoglobulin chains) need to correctly interact to form a functional antibody molecule.

Expression of high concentrations of a recombinant antibody in the milk had no adverse side effects on the transgenic mice and no major effects on the synthesis of other antibodies in milk. In addition, no significant weight loss or early mortality was observed in the neonatal mice suckling milk containing high concentrations of a recombinant antibody.

This study has important implications for improving health and disease resistance in livestock. TGEV infection is an important disease in swine that causes a mortality close to 100% in three week old piglets and severe diarrhea in young pigs. Therefore, the development of transgenic sows which synthesize virus neutralizing antibodies in their milk could reduce the serious effects resulting from TGEV infection in newborn swine.

In the future, transgenic livestock could be developed which secrete an array of virus-neutralizing antibodies into their milk. In this way passive immunity against a variety of intestinal pathogens could be maintained in neonates until their own immune systems are fully developed.


Castilla, J. et al. 1998. Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk. Nature Biotech. 16:349-354.

Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech



The Office of Pollution Prevention and Toxics (OPPT) of the U.S. Environmental Protection Agency approved an intergeneric Sinorhizobium meliloti strain for use as an alfalfa seed inoculant in September 1997. This is the first intergeneric organism approved for release to the environment on a commercial basis. S. meliloti is an indigenous soil bacterium that forms a symbiosis with alfalfa in nodules that develop on the alfalfa roots. The beneficial results of this symbiosis are that the bacteria receive nutrition from the plant and in turn contribute or "fix" nitrogen for the host plant. Fixing nitrogen refers to the procedure whereby the bacteria produce an enzyme nitrogenase, which converts the gaseous nitrogen in the air, which the plant cannot use, into ammonia that the plant can use.

The genus Sinorhizobia is one of a number of genera referred to as "rhizobia." Commercial production of rhizobia inoculants began in Germany in 1895. In 1985, the BioTechnica Company of Cambridge, Massachusetts decided to develop better rhizobia by genetic manipulation. The strategy for improvement was threefold:

Small-scale field trials with genetically modified strains developed according to these strategies were initiated in 1989. All field trials were conducted with the consultation and consent of the EPA and local regulatory authorities. In 1994, Research Seeds asked for the right to commercialize one of the strains known as RMBPC-2. Urbana Laboratories, a division of Research Seeds, Inc. of St. Joseph, MO, had purchased the rhizobia program from BioTechnica in 1991.

RMBPC-2 has an extra copy of the nifA regulatory gene. NifA has a positive regulatory role on the expression of the other genes necessary for nitrogen fixation. A promoter from Bradyrhizobium japonicum, the rhizobium that nodulates soybeans, was inserted upstream of the additional copy of nifA to control and prevent deleterious excess expression. Initial greenhouse work had shown biomass yield decrease if the expression of the second copy of nifA was too high, and it had been observed that this heterologous promoter gave optimum expression. The transport system for C4-dicarboxylates, which uses a single protein permease encoded by the dctA gene, is enhanced by addition of dctB and dctD. These are regulatory genes which activate dctA and which are required for C-4 dicarboxylic acid transport in free-living, but not symbiotic, rhizobia. Thus a dctABD sequence from Rhizobium leguminosarum (the rhizobium that nodulates peas and beans) was added to the RMBPC-2 genome. Finally, resistance to streptomycin and spectinomycin was added so the strain could be tracked during field trials. Bosworth et. al. (1) gives a complete description of the construction.

OPPT produced an extensive risk assessment document for a Biotechnology Science Advisory Committee (BSAC) meeting in January 1995. Antibiotic resistance, possible adverse ecological effects, and what constituted "significant" benefits were the most discussed items. OPPT, in its discussion of the release, concluded that the PC-2 strain would not significantly contribute to the naturally occurring antibiotic resistance gene pool because of the stability of the antibiotic genes in the strain.

With regard to ecological testing, the BSAC panel felt that more small-scale field studies should be conducted to evaluate cross-inoculation to other legumes such as sweet clover and mesquite, which might increase their ability to compete as weeds. However, the EPA concluded that five years of field-testing indicated that the modified strain acted consistently with already commercially available non-engineered Sinorhizobium strains, and therefore presented no additional hazard. Finally, a paper published after the BSAC meeting by Scupham et. al. (2) demonstrated significant yield increases. A fact sheet with more details is available from OPPT, EPA, 401 M Street S.W.,Washington, DC 20460. Phone 202 260 3725, email hyperlink at

1. Bosworth, A. et. al. 1994. Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression. Appl. Environ. Microbiol. 60:3815-3832.

2. Scupham, A. et. al. 1996. Inoculation with Sinorhizobium meliloti RMBPC-2 increases alfalfa yield compared with inoculation with a nonengineered wild-type strain. Appl. Environ. Microbiol. 62:4260-4262.

Tom Wacek
Urbana Laboratories



In 1997, an estimated $5.8 billion in financing flowed into the biotechnology sector, the majority of which went toward funding health care related endeavors (1). The historical preference for early stage funding of drug related companies is reflected in the numbers: 383 biotechnology firms with a primary focus on therapeutic development, in contrast to 90 focusing on plant or animal agriculture (2). The California merchant bank Burrill & Company is looking to improve the opportunities for new ag-biotech ventures with the formation of a $100+ million agricultural biotechnology venture capital fund.

On the initial closing of the Fund, $75 million had been invested, with an expected $35 million to be added in a second closing on or before June 30, 1998. The "Burrill Ag-bio Capital Fund" as it will be known, is being formed in association with Limited Partners Bayer Corporation, Hoechst-Schering AgrEvo GmbH, and Transamerica Business Credit Corporation. A number of other large companies and financial institutions will be participating in the second closing.

The Fund is designed to focus in areas of interest of the Limited Partners, and to identify, evaluate, and access technologies aligned with their strategic needs. By investing in a broad spectrum of ag-biotech companies, it allows the Fund's Limited Partners to potentially gain early access to technologies of specific interest and the opportunity to form strategic alliances with companies that are more established, thus mitigating risk (1).

The Fund expects to invest in companies having biotechnology-based technologies broadly applicable to the agricultural, pharmaceutical and chemical industries at two main stages of development: the early venture capital stage, after proof of technology, and at the later mezzanine round. G. Steven Burrill, CEO of Burrill & Company, believes that with the breakthroughs in genomic research, the success of emerging products from biotechnology, and the demand for technologies that are increasingly safe, healthy and environmentally friendly, there should be a growing emergence of revolutionary new agricultural products. He feels the lack of capital is hindering this growth, and is looking to the creation of the fund to improve the situation and expand what he believes is a current shortage of innovative ag-biotech start-ups.

1. Information Release, Burrill & Company web site (, April 15, 1998.

2. U.S. Companies Database, Institute for Biotechnology Information, April 1998.

William O. Bullock
Institute for Biotechnology Information, LLC
Research Triangle Park, NC



July 23-24, 1998. Sutton Place Hotel, Chicago, Illinois, USA. The conference aims to bring together leaders from across the industry- from genomic companies to food processors- to discuss new technology, legal, and regulatory issues, and developing strategic alliances to help ensure market access for new products. The conference is organized by Global Business Research Ltd.; for more information see their web site at

The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. P.L. Traynor, Editor.

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