- Hiroshi Harada, Docteur-es-Sciences,
Professor Emeritus, University of Tsukuba
Home: 5-13-11 Minami-Aoyama, Minato-ku, Tokyo 107, JAPAN
Ed- This is an abridged summary of a plenary lecture given in July 1996 in Tsukuba, at the 4th International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, on the recent developments in plant breeding techniques and the impact on quantitative and qualitative improvement of food production, and also some related problems that we are now facing and we are expected to overcome in near future. To date there have been 84 applications including 42 transgenic plants that have complied with the MAFF Guidelines in Japan. The 15 plant lines of virus resistant-tomatoes, -petunia, -rice, -melon, ripening delayed-tomatoes and herbicide tolerant-canola and -soybean are now released in Japan. The Ministry of Health and Welfare notified the guidelines including the food safety assessment of crop plants derived through biotechnology in February 1996. MAFF notified the guidelines of the safety assessments for animal feed and feed additive derived through biotechnology in April 1996. 7 transgenic crops including herbicide tolerant canola and soybean, and insect resistant potato and corn are now being under review in the Ministries.
Today, many people will share a common idea that deepening basic research and promoting practical application of biotechnology are essential to maintain and even to enhance agricultural production, to conserve natural resources and to improve environmental quality for the present and future life of the people on the earth. This idea has, in fact, been materialized in various areas of plant science, particularly in plant breeding. We can now count dozens of new varieties worldwide that have outstanding unique characteristics introduced by recombinant DNA techniques.
These achievements require us to consider genetically modified plants within the context of genetic changes occurring on the earth, either naturally - which we cannot manage; or artificially - which we can manipulate. This further tells us that it is important to understand and utilize new biotechnological approaches within the comprehensive nature of plant breeding that takes initial, unfinished materials and transforms them into practically useful, new varieties.
My research team at University of Tsukuba was one of the earliest groups which applied for safety evaluation of genetically modified plants in Japan. My involvement in biosafety evaluation is continuing, and I chair the plant subcommittee of MAFF's committee on utilization of genetically modified organisms. These experiences make me more and more confident about the importance to make further progress in plant breeding techniques in which biotechnology is an essential component.
The tremendous genetic improvements in crop plants achieved to date are largely based on sexual hybridization. As emerging cellular and molecular technologies develop further, sexual methods will continue to play a major role in crop improvement. For example, a new rice Japanese variety "Hitomebore", was developed which possesses a strong tolerance to cold injuries and also good eating quality.
Although plant geneticists have frequently been able to manipulate and recombine the genome into agriculturally effective combinations using sexual breeding methods, they are limited by a lack of understanding of interactions among genes.
Genetic manipulation via sexual methods exploits the natural reproductive systems of crop species. More fundamental knowledge of the nature and control of reproductive mechanisms may facilitate their manipulation to increase the efficiency. Many traits of interest in plant breeding are quantitatively inherited. Better understanding on the genetic base of multigenic traits using DNA markers is valuable in establishing a proper breeding strategy and might permit the molecular dissection of these traits.
The ability of plant cells to regenerate a whole plant is called totipotency. Plant breeding by using tissue and cell culture is based on this unique characteristics of plant cells. The expression of totipotency depends on not only the cultural conditions but also plant species or even varieties within the same species. In this slide you can see the difference of shoot regeneration from seed callus between two rice varieties. Somatic embryogenesis is a typical example of totipotency of plant cells. Somatic embryos are formed directly or indirectly from various kinds of tissues.
A great number of studies have been conducted to develop plant regeneration systems from plant cells. The culture conditions to regenerate plants are so variable for each plant material that in most cases rather empirical investigations have been usually required. During the past several years, however, studies are being more and more intensively undertaken to know what exactly is totipotency at the biochemical and molecular levels. These studies are certainly useful for more effective application of totipotency in plant breeding.
For the plant breeding, haploids are an attractive material. They do not have any alleles, therefore a recessive character in a diploid is detectable in a haploid plant. In addition, we can easily get recombinant inbred lines through doubling chromosomes of haploid plants derived from hybrids. Anther and isolated microspore culture are suitable techniques for obtaining a large number of haploid plants, and have been developed in over 250 plant species since the first report in 1964. Up to now, registered varieties of rice, tobacco, Chinese cabbage and other crops have been produced by these methods, in Japan.
For breeding, a large number of doubled haploid plants is necessary. More efficient methods should be developed. Androgenic embryogenesis has been reported in several crops. It seems to be less mutable than callus-formed culture. Haploid plants do not have any alleles. Then, the analysis of genes by molecular genetic methods in haploid is expected to be fruitful. We need efficient transformation systems in haploid plants.
The protoplasts can be fused to obtain somatic hybrid plants whose parents are not able to be cross pollinated or introduce genes from wild species. Several cell fusion and screening methods have been developed; for example, fusion by the treatment with PEG and electric pulse, asymmetrical fusion techniques and screening of fused cells by antibiotics, identification by RFLP and RAPD analysis, and so on. At first, the protoplast fusion was thought to be a miracle method to produce new crops. Unfortunately, few new crops have been obtained because of chromosome elimination, imbalance of sink and source organs, sterility and so on. But now, we know that the fusion is still promising method to introducing genes into vegetatively propagated crops from their allies, re-synthesize polyploid crops with various combination of genome, and introducing cytoplasmic genes.
To clarify the characteristics of genes controlling phenotypical traits and to improve plants by changing the genetic information, identification and isolation of genes corresponding to trait are required. Map-based cloning is one of the methods to perform this goal. In case of rice, precise tagging of target trait is promised using many DNA markers produced by genome analysis. Further, DNA fragment carrying a gene of target trait is identified utilizing a rice physical map produced by genome analysis. Narrowing a target genomic region by increasing a segregation population size and by down-sizing a candidate DNA fragment, we expect to isolate a gene of target trait.
The candidate cDNA or cosmid clones have to be ascertained for their ability to carry a target trait by transformation. In addition, biochemical and physiological analysis of the candidate gene product is indispensable for the final identification of gene corresponding to target trait. The genes isolated and characterized as above are to be utilized for improvement of not only rice but also other crops and the molecular information accompanying with these genes are to be the fundamental knowledge in plant science.
Incorporating foreign genes
Transformation can introduce new useful genes into plants without changing their original basic characters. Because of this, transformation is considered to be an attractive tool in plant breeding. Among the several methods of introducing foreign gene(s) into plants, Agrobacterium-mediated transformation is the most popular. Transformation by Agrobacterium had been applied in many plant species. Recently successful transformation of monocots, for example rice and maize, has also been achieved, thus widening the possibility of this method. To date, several genes had already been introduced into many plants, and the production of new breeding material is underway.
For utilization of transgenic plants in breeding, there are several barriers to overcome. First point is to establish efficient transformation systems in various plant species, second point is to isolate more useful genes, third point is to control gene expression. Finally we must determine the influence of transgenic plants on the environment, the quality of them as foods, and have public acceptance.
As a mechanical method for gene transfer, electroporation is used to species for which protoplast culture system has been established. Microprojectile bombardment employs high velocity metal particles to deliver DNA into plant cells or tissues. A wide range of plant species recalcitrant to Agrobacterium- mediated method or protoplast transformation have been transformed by microprojectile bombardment. It is apparent that the transformation frequency by this method is still low. This makes the experiment labor intensive and rather expensive. However, improvements in hardware design and particle gun availability will certainly improve efficiencies and extend the list of crops that can be transformed using this technology.
Diverse genetic resources of crop species involve the genes needed for future crop improvement. Germplasm collections provide food, feed and fiber security for mankind and materials to answer fundamental questions of biology. These days pressing limitations on assembling and utilizing genetic resources include the rapid decrease in diversity worldwide. The urgency to protect global genetic resources is greater than ever before.
Since 1991, collaborative explorations between Japanese and Russian scientists have been undertaken to collect diverse genetic resources in north Caucasian and Central Asia. The Japanese government has ratified the "Convention of Biological Diversity", which is an international agreement to share the benefits and sustainable use of global genetic resources. Major focus in the research on genetic resources is placed on strengthening the international network for conservation of plant diversity.
Improving food production and environment
Due to the recent explosion in world population and the deterioration of the earth's environment, great concern is mounting over global food supply. In many countries scientific research and innovative agronomical activities have been and are being conducted in order to improve world's food supply and global environment. In this regard, biotechnology, including the measures for the creation and use of genetically modified organisms, is one of the key-technologies. Many researchers are trying to create epoch making plants and microorganisms, such as low-allergen plants and producing bio-degradative plastics, vaccine and other useful industrial materials.
Safety assessment of transgenic plants
However, the introduction of new molecular technologies in the early 1970s initiated discussion on safety in biotechnology. This discussion resulted in a number of national and international recommendations, guidelines or regulations, and legislation. The OECD has worked to set general principles for the safe development of rDNA organisms since 1983, such as "good industrial large-scale practice (GILSP)", "familiarity" and "substantial equivalence" pertaining to microorganism handling, agricultural applications and new foods, respectively. In many countries voluntary guidelines or laws are compatible to the national regulations currently in effect.
Many enterprises are moving rapidly towards the commercialization and marketing of agricultural and industrial products of modern biotechnology. Both public and private sectors have, therefore, identified the need for harmonization of regulatory approaches to assess these products in order to reduce unnecessary duplication, facilitate administrative work, improve public acceptance, thus contributing to safe application of biotechnology, promoting global exchange of biosafety information, and avoiding further trade barrier. Such an international framework should not give negative impacts to research and development in biotechnology and technology transfer. Furthermore, individual technical provisions contained in the framework should be established on the basis of scientific consideration, and considering the speed of development in this field, the framework should be designed in such a way that technical provisions could be revised periodically and the results of the technical development could be incorporated into the framework without delay. We think it is important to establish an internationally harmonized framework for the safe handling of rDNA organisms within a few years.
Present State of agricultural use of transgenic plants in Japan
In Japan overall development, including handling and culture of rDNA organisms are placed under seven guidelines. Of the five covering post -laboratory fields, one by the Ministry of Agriculture, Forestry and Fisheries (MAFF) covers all categories of organisms, including plants, animals and microorganisms, and general release of those. The purpose of these Guidelines is to establish basic requirements concerning the appropriate application of recombinant organisms in agro-industries so as to assure the safe use of recombinant organisms and to achieve sound overall development. The MAFF Guidelines were re-issued in 1995 concerning the handling of recombinant plants developed in foreign countries.
In the case of plants, safety is assessed in four steps, two steps are conducted in green houses for research, other two in fields for practical use. So far, eighty-four applications including forty-two transgenic plants have complied with the MAFF Guidelines by the Minister. The fifteen plant lines of virus resistant-tomatoes, -petunia, -rice, -melon, ripening delayed-tomatoes and herbicide tolerant-canola and -soybean are now released in Japan.
The Ministry of Health and Welfare notified the guidelines including the food safety assessment of crop plants derived through biotechnology in February 1996. Furthermore, MAFF also notified the guidelines of the safety assessments for animal feed and feed additive derived through biotechnology in April 1996. Seven transgenic crops including herbicide tolerant- canola and -soybean ,and insect resistant potato- and corn- are now being under review in the Ministries.
On the other hand, some of the general public are not familiar with rDNA technology, and some seem to feel uncomfortable with biotechnology. In order to promote agricultural biotechnology, it is essential to give precise information about biotechnology, especially rDNA technology, to general public so that they can comfortably accept biotechnology and the products.
Governments and developers should constantly promote activities to build and foster perception and comprehension of general public about rDNA technology and its safety to introduce the products without consumer's misgivings. Lectures and practical experiences on rDNA to train key-persons, and setting a place to actually touch and see the products derived through rDNA techniques for general public are an important process. The MAFF has been implementing a special project to gain public acceptance throughout Japan since 1995.
Our desire is to develop this technology even further so that it will be more useful in the welfare of people all over the world as we try to live in harmony with nature. Therefore, all scientists concerned, especially ourselves gathered here today, should continue the research work in order to secure more stable supply of foods, better environment and necessary energy for the world.
Needs of biotechnology in developing countries
There are various constraints on agricultural production: heat, drought, salinity, pests, diseases, etc. These biotic and abiotic constraints result in the instability of crop yield, which often leads to starvation and malnutrition of a large number of people in developing countries. In addition, the rapidly growing population accelerates the deterioration of natural resources, especially in marginal lands. We are now faced with the need to increase the sustainability of agricultural production, while the lack of materials and infrastructure necessary for agricultural production in most of the developing countries makes it difficult to increase and stabilize their agricultural productivity. Therefore, the role of plant breeding or genetic improvement of crop species for high adaptability will become increasingly important in the agriculture of the developing countries.
Japanese rice breeders have recently developed a new upland rice variety with drought tolerance which was selected from a cross between JC81(local indica variety introduced from IRRI) and Norin-mochi 4 (Japanese upland variety). The former variety is highly tolerant to drought due to the deep root system, a trait which has been successfully incorporated into the new variety. However, the determination of the root depth of progenies was considerably laborious. Biological procedures such as a molecular marker assisted screening method may greatly facilitate plant breeding for drought tolerance.
Moreover, recent progress in biotechnology is rapidly increasing the possibility of modifying crops genetically to make them highly tolerant to various kinds of adverse conditions. Using biotechnology along with genetic resources, it will become possible in the near future to carry out breeding at the molecular level for many important crops for the induction of stress tolerance. These new crops should contribute to the increase and stabilization of agricultural production with lower labor and financial input in developing countries.
Although biotechnology facilities have been recently established in many developing countries, their scientific and technical capability remains still limited compared to that of industrialized countries. Lack of funding and human resources results in the application of limited aspects of biotechnology in most of these countries, where genetic engineering is too costly. It is hardly possible to carry out molecular breeding in the developing countries for the time being, although the farmers in these countries need the benefits of modern high technology.
The private sector now plays an important role in research, development and use of biotechnology in industrialized countries and they invest more in those areas where they expect a profit. Many of the staple or subsistence crops important to developing countries, however, receive too little attention. Thus advanced research institutes in the public sector of industrialized countries may take the initiative to promote shuttle-type of programs for the development of biotechnology techniques adapted to the specific conditions of individual developing countries in collaboration with international organizations.
How to assist developing countries
There are considerable differences in the biotechnological capability among developing countries. In China, Thailand and several other countries, for example, some transgenic crops including tobacco, tomato, etc. are being cultivated in the field, while the majority of developing countries have only a limited capacity in advanced biotechnology. The involvement of the private sector in this field is very limited in most of the developing countries, in contrast to the conditions of industrialized countries.
One of the major constraints on promoting biotechnology research and development in the developing countries is the shortage of appropriately trained researchers and technicians. Some advanced biotechnology research units of industrialized countries and international organizations now offer high-level training programs to researchers and technicians in developing countries. In addition to the training of researchers and technicians, training of research managers and leaders of national agricultural research systems in developing countries is urgently required. Since many researchers in basic scientific fields such as molecular biology are being involved in biotechnological research and development without any experience in agricultural production, biotechnology research planning should be carefully examined and well-organized by sufficiently trained managers and leaders.
Another issue is the enactment of regulations and guidelines necessary for the promotion of biotechnology research, development and use in developing countries. In order to stimulate the private sector to participate in this field in developing countries, intellectual property protection is a prerequisite. In addition , biosafety considerations are necessary to get public acceptance for genetically modified plants and microbes, which we are going to discuss in detail in this symposium. The experiences of industrialized countries in this matter could be helpful to many developing countries.
One of the most important goals we should keep in mind is to create excellent plant varieties which can really provides benefits to the mankind. In fact, development of biotechnology in application to plant breeding has been so successful that more than twenty new varieties with unique characteristics have been brought into practical use. This has been supported in part by continuous improvement in procedures of biosafety evaluation, from rigid to flexible and commonality-pursuing nature. Recent achievement in cross-species commonality in gene sequences of genome would be a valuable addition to earlier research to support this trend of advancement.
There will be two ways to further enhance effective use of outcome of biotechnology, especially biosafety field tests. One is to improve public understanding. Since public attitudes are shaped more by history, culture, and sociological factors than they are by scientific considerations, special efforts to improve public acceptance need to be strengthened by all sectors involved.
Another is to promote international harmonization. This should aim to reduce duplication, contribute to safe application of biotechnology, promote international exchange of information on biosafety and avoid further trade barrier.
These two ways are actually interlinked, widely agreed harmonization will certainly bring an increased assurance to public acceptance. Above all, the most important thing for all of us is to strictly maintain scientific quality and validity under which biosafety issues are evaluated.