Breakthrough Dividend
Chapter 18. Agriculture/Plants (II)

Plant Diagnostics

This field produced some of Israel's earliest world-class successes. One example involves "Irish" potatoes, one of the most American of all food products, especially as french fries and chips. In fact, America produces over $2.43 billion of potatoes a year.

Since the true sexually-produced seed of potatoes is too small and difficult to grow, new potato plants are started from the sprouting "eyes" of seed potatoes. This vegetative propagation scheme favors the transmission of debilitating viral diseases, such as potato leaf-roll virus (PLRV) from generation to generation, with substantial economic losses. Downgrading U.S. Grade #1 potatoes to U.S. Grade #2 can mean a loss of $400-600/ton to the farmer. Assuring virus-free seed potatoes is thus essential to the profitability of the industry.

Most American seed potatoes are checked for PLRV using an Israeli test developed by BARD (Chapter 9) grantees. Purified PLRV viruses extracted from diseased plants were injected into rabbits and sheep to stimulate the production of antiviral antibodies. These became the basis of an ELISA diagnostic test that detects a broad spectrum of immunologically-different PLRV strains. The new test, both cheaper and more general than its predecessors, is so sensitive it can detect the PLRV viruses carried by a single aphid. The test is now produced in quantity by Washington State University, which distributes it free of charge to the Idaho and Montana Seed Certification Agencies. A commercial diagnostic kit is also sold on the U.S. market. The recent dramatic drop in PLRV infections in the American Northwest -- from more than 40 percent to less than 10 percent during the last decade -- is partly due to this BARD-sponsored biotechnological research. North Carolina, America's leading producer of sweet potatoes (yams), also produces $28 million of Irish potatoes each year.

Other U.S.-Israel BARD grantees developed highly-sensitive ELISA diagnostic tests for cucumber mosaic virus, bean yellow mosaic virus (BYMV), and other viral infections in corms, tubers and bulbs. These tests are already being used to produce virus-free gladiolus breeding stock in both Israel and Florida. More recently, these researchers developed methods for converting the BYMV virus' RNA molecule back into its corresponding DNA molecule (reverse transcription). Specific DNA sequences can then be copied millions of times (amplified) using the polymerase chain reaction to improve their detection. This new test is 1,000-10,000 times more sensitive than their original ELISA test, and can detect a trillionth of a gram of BYMV in a gladiolus leaf.

Other Volcani Center scientists have isolated the tomato yellow leaf curl virus (TYLCV), cloned and sequenced the virus's genetic material, prepared antibodies against its virus coat protein and developed a quick TYLCV diagnostic test that detects infected plants long before visible symptoms appear. YISSUM's most recent list includes no projects falling into this category in its narrowest definition.

Israel only has one company, Agrilab Biotechnology, with a major interest in the development of diagnostics for plant diseases. Agrilab has developed, with researchers at the Volcani Institute and Hebrew University, plant diagnostic kits (ELISA and ANA probes) for a wide range of plant viruses: TYLCV (tomato yellow leaf curl virus), TMV (tobacco mosaic virus), PLRV (potato leaf roll virus), CTV, CaMV, LSN and PFBV.

Although North Carolina does not list any companies explicitly devoted to diagnostic test kits for plant diseases, Biosystems Technologies (Durham) has produced a test for hybrid vigor, and EDITEK (Burlington) produces tests to detect mycotoxins (alfatoxin, ochratoxin, T2 toxin, etc.) in grains.

Plant Protection

This field can be further subdivided into three parts:

a) Transgenic plants endowed with foreign "protective" genes that repel, kill or mitigate the effects of pests. These, for convenience, have already been discussed under Genetic Manipulation (Chapter 17);

b) Traditional biological control, in which whole organism biocontrol agents (competitors, predators, superparasites) or chemical biocontrol agents (naturally-produced toxins) are found, tested and used in integrated pest management schemes. This is considered a closely "associated" technology, rather than biotechnology per se, in some classification schemes. (Is a bacterial insecticidal toxin any more "biotechnological" than penicillin and other natural products?); and

c) The use of biotechnological techniques to improve natural biocontrol agents, their range, persistence and efficacy.

In the area of whole organism biocontrol agents, Israeli researchers have found and, using biotechnology, have improved several important fungal biocontrol agents. For example, Hebrew University investigators have discovered "friendly" Trichoderma fungus species in the soil that can prevent fungus damping-off diseases in emerging seeds and young plants. With American colleagues they have used protoplast fusion techniques (Appendix A) to combine cells from different Trichoderma varieties to produce new "super-strains," effective on a wider range of crops and pests. One such strain has already been registered with the EPA, a necessary prelude to commercial use. Another Hebrew University-U.S. team has studied how Trichoderma works and found that a newly-discovered Pythium fungus species also attacks its pathogenic relatives. The researchers have developed superior culture media and mass production methods for growing such fungi, and used ultraviolet light to produce useful new mutants, which have been patented by both the U.S. and Israeli partners. Conventionally-produced Trichodermas are already FDA approved for commercial use. Other strains of Trichoderma and nonpathogenic Fusarium are effective against Fusarium wilt disease.

As an added bonus, some Trichoderma also produce potent plant growth regulators. One strain increases potato plant growth up to 300 percent, and boosts potato yields by $200 per acre, compared to soil inoculation with unimproved wild Trichoderma species. The growth promoting substance has been extracted and resembles no known plant hormones. Since plant root tip surfaces are colonized by the mutant fungus, the growth-stimulating factor is constantly applied just where it is needed. The fungus also protects both seeds and developing roots from soil-borne pathogens that can stunt plant growth.

Infection by a double-stranded RNA virus apparently gives the pandemic "damping-off" fungus disease, a major cause of young plant death, its ability to spread rapidly. Israeli BARD researchers have found non-virulent strains of the fungus lack this virus, and, in fact, protect wheat, cotton, radish and other seedlings from their more deadly cousins. Of the 107 strains of Rhizoctonia isolated from Israeli soils and tested for wheat protection, one virus-free fungus gave 93 percent protection to wheat seedlings. In field trials, the non-virulent fungi actually promoted plant growth and increased the crop yields of wheat, cotton, carrots, lettuce and radish. The potential market for such products is tremendous. The U.S. produces over $7.71 billion of wheat a year; and North Carolina alone produces over $140 million of cotton.

Turning to naturally-produced chemical biocontrol agents, Israelis have had a particular interest in, and success with, antiviral agents. Consider, for example, tobacco, a plant that sometimes gets bad press, but is an ideal model for biotechnological manipulation (PTC, fusion, gene-transfer, "pharming"). It also produces some unexpected positive natural products. BARD grantees at the ARO Volcani Center and their American collaborators have found that tobacco plants naturally produce very low levels of anti-viral factors (AVF), chemicals that can inhibit viral replication (IVR). Growing tobacco protoplasts in test-tubes produced enough tobacco IVR for further testing. A culture of 100 million protoplasts yields only a millionth of a gram of IVR, but even a hundredth of that amount produces a detectable effect. Simply spraying IVR on plant leaves was sufficient to reduce tobacco mosaic virus (TMV) infections in tobacco and tomato plants, and cucumber mosaic virus (CMV) infections in cucumbers and bell peppers. The preparation was also active against potato viruses X and Y, and other viral diseases in blackeyed peas. As America's largest tobacco producer, with annual sales exceeding $1 billion, North Carolina can particularly benefit from this research.

BARD researchers used immunological biotechnology techniques to produce still higher levels of IVR. Injecting tobacco IVR into mice triggered an allergic reaction that produced highly specific monoclonal antibodies that exactly fit the "foreign" IVR molecule. The antibodies, in turn, were extracted and can be used to make a highly sensitive ELISA test to detect extremely low levels of IVR. Turning to DNA-based technology, the researchers searched their "library" of tobacco DNA fragments for the one that codes for IVR production. They inserted this DNA into genetically-engineered bacteria, and can now produce IVR-like compounds by simple fermentation. They are now inserting copies of this DNA directly into plants, to create virus-resistant transgenic crops. The results could revolutionize U.S. and Israeli agriculture, and private companies are already expressing interest.

YISSUM's most recent set of R&D opportunities include three whole organism biocontrol projects, all in an early stage of R&D:

• The discovery of a new virus that kills locusts;

• The search for improved fungi to control the citrus rust mite; and

• Screening and improving bacteria and actinomycetes to control the fungal pests (pathogens) of plants.

As presented, these are mostly still in the research or "willing to screen" stage.

Two other YISSUM projects focus on extracted, naturally-produced chemical control agents:

• Lytic enzymes excreted by Trichoderm and

• A Streptomyces extract that is highly active against both Lepidoptera (e.g., Heliothis armigera) and mosquitoes (e.g., Aedes aegypti).

These are also still in the research stage.

Israel has no whole-organism R&D-oriented biocontrol companies per se, though both Ecogen (Chapter 10) and Makhteshim Chemicals do develop and sell chemical biocontrol agents. Israel's Bacillus thurengiensis var. israelensis toxin for the biocontrol of blackflies and mosquitoes is particularly well-known (Chapter 9).

Propagation and Tissue Culture

Plant cells from many different species can now be grown and manipulated in plant tissue culture (PTC) and then regenerated into whole plants (Appendix A). Such clonal propagation techniques have long been used to rapidly create large numbers of (usually) identical copies of selected superior plants. One recent YISSUM project on offer uses biotechnology to put a new twist on the old goal of producing inexpensive plant products without bothering to produce the plants.

The idea of making transgenic plant cells produce useful human proteins in tissue culture is not itself new. Although plant cell cultures are simple, inexpensive, and virus free, and although secreted mammalian proteins are easily recovered, previous attempts have foundered on the typically low levels of expression. Hebrew University (HU) investigators have inserted their genetic "messages" into special genetic constructs that greatly boost the production of the desired proteins. Their systems can also glycosylate and phosphorylate proteins after production. These are important post-expression chemical modifications in mammalian cells that are often required to activate proteins. YISSUM reports plant cell cultures that have already produced fully-active human beta-interferon at rates exceeding a million units per liter per day uninterruptedly for at least four months. New constructs designed to boost production another factor of 10 are now under test. Pilot plant scaleups are the next logical step, and could radically alter the economics of this approach.

Two other YISSUM projects seek to simplify and improve the PTC propagation process itself, using large-scale liquid cultures (bioreactors). Since most corm and bulb crops (garlic, gladioli, lilies) are vegetatively propagated, they are particularly vulnerable to the dissemination of viral diseases, etc. Although existing agar-plate PTC propagation methods prevent this, they are labor-intensive. In fact, manual handling amounts to 70-80 percent of the final plant price. Liquid cultures avoid this, but are plagued by the unwanted growth of leaves (at the expense of corms) in culture. The HU investigator has successfully grown bulbs and corms in culture at reasonable efficiency using axillary and adventitious bud starting materials, cytokinins in the medium to enhance bud proliferation and growth inhibitors to reduce leaf elongation. Small "protocorms" 1.2-1.4 cm. in diameter were produced in just 10-12 weeks. Planted directly in soil without prior "hardening," these gladiolus protocorms grew to a marketable 2.5-3.0 cm, 8-10 gram size in 20 months. Roughly similar results were obtained with Brodiaea bulbs and Nerine corms. The technique is appropriate for mass-production in large modified fluidized-bed or air-lift bioreactors.

The same investigator, further back on the R&D continuum, also hopes to use somatic embryogenesis PTC techniques to propagate parental lines for breeding certain hybrid plants. Starting with cucumber cultivars, he will try to develop efficient methods for creating and culturing embryos (in bioreactors), accomplishing in vitro hardening and encapsulating the embryos to create "artificial seeds" for prolonged storage or automated sowing.

Israel has five companies with a major emphasis on plant tissue culture (PTC, Appendix C), mostly as a practical technique for the rapid, accurate propagation of superior plant varieties. Annual sales vary between $1-2 million.

Rahan Meristem, founded in 1974 by Kibbutz Rosh Hanikra in Israel's far North, now employs 150 people. It does R&D in applied "molecular biology and other aspects of PTC," and produces PTC-propagated plant products. Allon Selecta breeds and produces flowers, carnations, poinsettias, geraniums and impatiens, largely for local consumption ($1 million was exported to Europe). It cooperates with the Volcani Center and Hebrew University in research matters. Ben Zur Nursery mass produces and exports PTC-derived plant materials. Vitrostar is a new kibbutz startup that includes some PTC of food crops. The amount of biotechnology R&D in all three is uncertain.

Although several North Carolina companies, such as Ciba Agricultural Biotechnology (RTP), use PTC techniques in their mission-oriented R&D, and would benefit from its improvement, only Weyerhauser Company's small New Bern Laboratories emphasize PTC propagation. Its goal is to develop procedures for the rapid propagation of the Atlantic white cedar, a valuable near-extinct tree species, and the loblolly pine for Weyerhauser's extensive tree planting/harvesting operations. Biosystem Technologies' flowering hormones and growth regulators also promote plant propagation, development and growth.