Rice Genetics Newsletter, Vol. 17

Rgn17

C.	Report of the Committee on Genetic Engineering (Stages in developing superior transgenic rice plants)
	R. WU, Convener Department of Molecular Biochemistry and Genetics, Cornell University, Itharca, NY 14853, USA

INTRODUCTION

Over the last 15 years, research in rice biotechnology has resulted in very substantial progress by producing transgenic rice plants containing many different potentially beneficial genes. The rapid development of rice biotechnology, largely due to support and guidance from the Rockefeller Foundation, can be arbitrarily divided into five stages. Stage one involves developing methods for transformation of rice protoplasts or cells, followed by regeneration of rice plants. Stage two involves discovering suitable promoters, utilizing available potentially beneficial genes and selectable marker genes for generating transgenic rice plants, and preliminary testing of the desired trait. Stage three involves optimizing transgene expression and sustainability of expression over several generations. Stage four involves discovering additional potentially beneficial genes by advanced molecular techniques. Stage five involves extensive testing of suitable transgenic plants in the greenhouse and then in the fields.

Many scientists have been involved in rice biotechnology research. Since they did not all join in this worldwide effort at the same time, work in the five stages extensively overlaps chronologically. Based on the number and dates of publications, each stage may take 4 to 8 years to reach maximum output, and several additional years to be fully developed.

This article briefly summarizes the achievements to date, followed by proposing future prospects. Due to space limitation, only a selected number of review articles and several original publications are quoted.

FIVE STAGES OF DEVELOPMENT

Stage one started in 1988, when three publications reported the successful introduction of gus-reporter-gene-containing plasmids into rice protoplasts by electroporation or polyethylene-glycol-based procedures, followed by regeneration of transgenic rice plants (see Cao et al. 1991, Ayres and Park 1994). By 1993, many more papers had appeared, and most of them reported the introduction of plasmids into intact rice cells by the biolistic method (see Jenes et al. 1993). Between 1994 and 2000, another wave of publications appeared, largely as a result of using the Agrobacterium-based transformation procedure to introduce different genes into rice cells (Hiei et al. 1994, Roy et al. 2000).

Stage two began in 1990 when two publications reported the usefulness of the rice actin1 and CaMV 35S promoters for driving the expression of transgenes in rice (McElroy et al. 1990, Zhang et al. 1991). Between 1988 and 1993, several selectable marker genes were found useful, including the nptII gene, the hygromycin resistance gene (hyg), and the phosphinothricin phosphotransferase gene (bar). The latter two have been favored by most investigators. Publications that include the use of potentially beneficial genes started in 1993, but did not reach a peak until between 1996 and 2000 (compare the papers in Rice Genetics III, 1996, with those in Rice Genetics IV, 2001). These genes include Bacillus thuringiensis (B.t.) genes (Fujimoto et al. 1993, Cheng et al. 1998), protease inhibitor genes (Duan and Wu 1996) for controlling insects, chitinase and beta-glucanase genes for controlling fungal diseases (Lamb et al. 1997, Thara et al. 1997), viral coat protein genes for controlling viral diseases (Fauquet et al. 1997), and the Xa21 gene for controlling bacterial blights (Song et al. 1993). Publications regarding introducing genes to increase salt and drought tolerance first appeared in 1996. These genes include Hva1 (Xu et al. 1996) and p5CS (Zhu et al. 1998). A paper was published which describes the introduction of a cod gene that increases tolerance to low temperatures (Sakamoto et al. 1998). Publications on transgenic rice plants with improved nutritional value include the synthesis of beta-carotene (Ye et al. 2000), and accumulation of iron (Goto et al. 1999). These areas of development have not yet reached a peak.

In general, potentially beneficial genes have been tested one at a time. Each time, one gene is used and its effect in transgenic rice plants determined. The results of these tests serve to show that a particular gene gives expected positive effects. However, in most cases, the expression of the transgene is far from optimal and the copy number of transgenes is often too high. The effect of the transgene has often been tested for only one or two generations.

Even though over 20 different genes have been individually introduced into rice and transgenic plants obtained, not all of the readily available potentially useful genes have been tested. Thus, stage two research is expected to continue for at least five to ten years.

Stage three involves optimization of transgene expression. This includes several aspects, one of which is to increase the probability of achieving maximum expression levels of the transgene that can be sustained for at least six generations. Choosing plants that harbor only a single copy of the transgene may minimize the possibility of gene silencing (Iyer et al. 2000, Cheng et al. 2000). Including matrix attachment region (MAR) sequences also appears to be beneficial (Cheng et al. 2000).

A second aspect is to use inducible promoters instead of constitutive promoters in certain cases. For example, using an abiotic stress-inducible promoter appears to provide a higher level of beneficial effects than using a constitutive promoter (Cheng et al. 2000). Similarly, using a wound-inducible promoter to drive the transgene expression (Duan et al. 1996) may be better than using a constitutive promoter for insect resistance. A specific pathogen-induced promoter for driving the expression of a gene for the control of a specific disease may also prove to be beneficial.

A third aspect is to introduce several potentially useful genes, which have already been tested, into the same transgenic plant to increase the degree of beneficial effects.

Stage four involves attempts to discover additional potentially beneficial genes. This can be done by map-based cloning (Song et al. 1995), differential display, microarraybased expression profiling, and functional genomics. Even though this work began in 1995, very few additional publications have been reported due to the time-consuming nature of this type of research. However, it is expected that extensive developments will be made within the next 5 to 10 years.

Stage five involves extensive greenhouse testing, followed by field testing of promising transgenic rice plants for the expected benefits. Once the expected benefits are realized, this particular transgenic rice variety can go into production. This stage of work has barely begun, but extensive progress is expected to be made within the next 10 to 20 years.

FUTURE PROSPECTS

One major goal of rice biotechnology is to produce superior transgenic rice plants that are resistant to various diseases and insects, or tolerant to common abiotic stresses (drought, high salinity, low temperature and flooding). Additional superior transgenic rice plants are also expected to produce grains with higher nutritional value or better taste, as well as higher yields. Based on current knowledge, these goals can be best achieved by:

(1)	Making use of a large variety of agronomically beneficial genes that have been individually tested and shown to give the expected benefit.
(2)	Making use of suitable inducible promoters as well as constitutive promoters, to drive the expression of transgenes in transgenic rice plants.
(3)	Making use of regulatory genes, including those that code for transcription factors, to turn on or off of a group of downstream target genes.
(4)	Making use of different matrix attachment region sequences to flank the genes of interest to maximize high-level expression and minimize gene silencing or positional effect.
(5)	Transforming many different elite rice varieties that are suitable for planting in major rice-growing regions in the world, so that crossing with local rice varieties can be minimized or eliminated.
(6)	Choosing those transgenic plants that each harbor only a single copy of the input plasmid and do not contain any rearranged transgene.
(7)	Combining several agronomically beneficial genes and introducing them into the same transgenic plants to maximize the desired effects.
(8)	Testing several plant lines of transgenic plants harboring the same desired gene(s) for at least six generations, and carrying out field tests with only those plant lines that give sustained levels of transgene expression.
(9)	Planting superior transgenic plant lines in fields and monitoring their performance annually.

References

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