Arabidopsis in Planta Transformation. Uses, Mechanisms, and Prospects for Transformation of Other Species1

Update on Plant Transformation Arabidopsis in Planta Transformation. Uses, Mechanisms, and Prospects for Transformation of Other Species1 Andrew F. Bent* Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706 The ability to move DNA into an organism and Wright, 1999). Other methods such as electropora- thereby alter its phenotype is central to both basic tion, microinjection, or delivery by virus have also and applied molecular biology. Transformation is a been exploited. To allow physiological selection of simple task with Escherichia coli or Saccharomyces cer- cells that have been successfully transformed, the evisiae, but is usually more difficult with multicellular DNA of interest is typically cloned adjacent to DNA eukaryotes and can be particularly challenging with for a selectable marker gene such as nptII (encoding some important plant species. However, for Arabi- kanamycin antibiotic resistance). dopsis, in planta transformation methods have been Genetic transformation can be transient or stable, developed that are incredibly simple. Attempts to and transformed cells may or may not give rise to apply in planta transformation methods to other gametes that pass genetic material on to subsequent plant species have often failed. This may be due in generations. Transformation of protoplasts, callus part to a poor understanding of the mechanisms that culture cells, or other isolated plant cells is usually underlie the successful Arabidopsis transformation straightforward and can be used for short-term stud- method. Studies of Arabidopsis transformation have ies of gene function (Gelvin and Schilperoort, 1998). accordingly been pursued, and three groups have Transformation of leaf mesophyll cells or other cells recently published relevant findings. Successful in within intact plants may in some cases broaden the planta transformation of the legume Medicago trunca- utility of single-cell assays (e.g. Tang et al., 1996). tula was also reported recently, showing that the Exciting new approaches such as virus-induced gene method can be adapted to other species. The cellular silencing may also be applicable for some studies target for transformation of M. truncatula may differ (Baulcombe, 1999). In the era of genomics these short- somewhat from the target in Arabidopsis. The above term assays will become increasingly important. findings may guide future efforts to improve trans- However, in many cases it is desirable or necessary to formation of other plant species. produce a uniformly transformed plant that carries This update opens by briefly reviewing transfor- the transgene in the nuclear genome as a single Men- mation protocols that avoid tissue culture, and their delian locus. impressive utility. Recent findings concerning Arabi- The generation of genetically homogeneous plants dopsis and M. truncatula transformation are then de- carrying the same transformation event in all cells scribed. The review closes by commenting on possi- has typically presented two separate hurdles: trans- ble avenues for improvement of transformation in formation of plant cells and regeneration of intact, other plant species. reproductively competent plants from those transformed cells (Birch, 1997; Hansen and Wright, 1999). Although many successful plant regeneration meth- BACKGROUND ods have been developed, these methods often re- quire a great deal of protocol refinement and the Genetic transformation of plants occurs naturally focused effort of expert practitioners. It is unfortu- (Hooykaas and Schilperoort, 1992). Scientists have nate that plant regeneration from single transformed been able to carry out controlled plant transforma- cells often produces mutations ranging from single tion with specific genes since the mid-1970s. The base changes or small rearrangements to the loss of most common methods for introduction of DNA into entire chromosomes. In addition, significant epige- plant cells use Agrobacterium tumefaciens bacteria or netic changes (for example, in DNA methylation) can rapidly propelled tungsten microprojectiles that have also occur (Phillips et al., 1994). It is often necessary been coated with DNA (Birch, 1997; Hansen and to generate and screen a dozen or more independent plant lines transformed with the same construct to 1 Plant transformation research in the author’s laboratory was find lines that have suffered minimal genetic damage supported by the North Central Soybean Research Program. and that carry a simple insertion event (Birch, 1997; * E-mail [email protected]; fax 608–263–2626. Hansen and Wright, 1999). Transformation is feasible 1540 Plant Physiology, December 2000, Vol. 124, pp. 1540–1547, www.plantphysiol.org © 2000 American Society of Plant Physiologists Uses and Mechanisms of in Planta Transformation in many plant species, but has required acceptance of screened for mutant phenotypes of interest and the the above limitations. mutated gene responsible for the phenotype could often be identified by isolation of the Arabidopsis chromosomal DNA flanking the previously known TRANSFORMATION METHODS THAT AVOID T-DNA (transferred DNA from Agrobacterium). TISSUE CULTURE Other laboratories later succeeded in generating transformed Arabidopsis lines by “clip ‘n squirt” A number of laboratories have pursued plant trans- methods (Chang et al., 1994; Katavic et al., 1994). formation methods that avoid tissue culture or regen- Reproductive inflorescences were clipped off, eration. In many cases these methods have targeted Agrobacterium was applied to the center of the plant meristems or other tissues that will ultimately give rosette, new inflorescences formed a few days later rise to gametes (Chee and Slighton, 1995; Birch, were again removed, Agrobacterium was re-applied, 1997). The same is true of popular tissue culture- and plants were then allowed to develop and set based transformation methods for corn, rice, wheat, seed. Transformants were obtained more reliably and soybean, which target young apical meristems than with the seed treatment method, but the meth- for transformation (Birch, 1997). For those methods, ods were only marginally more productive than tra- excised or partially disrupted meristems are trans- ditional tissue-culture approaches to Arabidopsis formed, subjected to antibiotic or herbicide selection, transformation (e.g. Valvekens et al., 1988). and then carried through tissue culture to regenerate A third, crucial stage of the revolution in Arabi- shoots and roots from the transformed tissues. For dopsis transformation came when Georges Pelletier, non-tissue culture approaches, Agrobacterium or Nicole Bechtold, and Jeff Ellis reported success at tungsten particles have been used in a number of transformation by “vacuum infiltration” (Bechtold et species to transform cells in or around the apical al., 1993). Arabidopsis plants at the early stages of meristems that are subsequently allowed to grow flowering were uprooted and placed en masse into a into plants and produce seeds (Chee and Slighton, bell jar in a solution of Agrobacterium. A vacuum was 1995; Birch, 1997). However, transformed sectors applied and then released, causing air trapped within have typically not persisted into gametes at reason- the plant to bubble off and be replaced with the able frequencies, or the methods have been difficult Agrobacterium solution. Plants were transplanted to reproduce (Birch, 1997). Injection of naked DNA back to soil, grown to seed, and in the next genera- into ovaries has also been reported to produce trans- tion stably transformed lines could be selected using formed progeny (Zhou et al., 1983). Variations of this the antibiotic or herbicide appropriate for the select- method and “pollen tube pathway” delivery of DNA are still practiced in China (Hu and Wang, 1999). able marker gene. Transformation rates often ex- Electroporation-mediated gene transfer into intact ceeded 1% of the seeds tested. Variations of this meristems in planta and a variety of pollen transfor- extremely simple new method (Fig. 1) have been mation procedures have also been reported (Chow- widely adopted by Arabidopsis researchers. Tissue rira et al., 1995; Touraev et al., 1997 and refs. therein). culture and plant regeneration are no longer neces- However, most of these methods have been difficult sary and the associated high rates of mutation are to reproduce and have not gained widespread avoided. acceptance. THE UTILITY OF AN ACCESSIBLE ARABIDOPSIS TRANSFORMATION WITHOUT TRANSFORMATION METHOD TISSUE CULTURE The impact of the vacuum infiltration method on Early stages of the revolution that transformed Arabidopsis research has been remarkable. Genera- Arabidopsis transformation were carried out by Ken tion of transformed lines is simple and routine (Fig. 1; Feldmann and David Marks. They applied Agrobac- Bechtold and Pelletier, 1998; Clough and Bent, 1998). terium to Arabidopsis seeds, grew plants to maturity First and foremost, barriers to in planta testing of a in the absence of any selection, then collected prog- gene of interest have been dramatically lowered. eny seeds and germinated them on antibiotic- With minimal effort and in a matter of 3 to 6 months, containing media to identify transformed plants multiple transgenic plant lines can be constructed (Feldmann and Marks, 1987; Feldmann, 1992). Al- and numerous DNA constructs can be tested. though the procedure was difficult to reproduce con- A second example of this method’s utility can be sistently, successful rounds produced

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