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 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 trans- formed 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 . 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 transformants seen in positional cloning projects, in which a gene of at a high enough rate that thousands of transformed unknown structure is isolated based on its genetic lines were produced in a matter of a few years. These map position (e.g. Clough et al., 2000). Once a gene “insertional mutagenesis” lines helped speed gene has been mapped to a genetic interval of a few cen- cloning by the Arabidopsis community (Azpiroz- tiMorgans, an Arabidopsis walk can be Leehan and Feldmann, 1997). The lines could be accelerated by the use of publicly available bacterial

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tagging T-DNAs can be used that enhance the ex- pression level of genes near the T-DNA insert, or one can use T-DNAs that place ␤-glucuronidase (GUS) or other marker genes under the control of host pro- moter or enhancer elements that may flank the T-DNA at the site of insertion (Weigel et al., 2000). Forward genetic approaches study phenotype first and then genotype, whereas reverse genetic strate- gies start with a DNA sequence and then seek a plant line mutated in that gene. Efficient transformation methods have facilitated reverse genetic screening in plants. Public collections have been created that al- low PCR-based screening of ordered pools of DNA from thousands of transgenic lines (Krysan et al., 1999). Once the subpool of DNA carrying an insert in the gene of interest has been identified, progeny seed corresponding to that pool can be requested. It is important to note that many of these insertional mu- tagenesis methods can also be accomplished using transposon mutagenesis (e.g. Tissier et al., 1999). A goal for many T-DNA and transposon-mutagenized seedbank collections is to obtain and compile in da- tabases a small stretch of sequence data for the DNA that flanks each insertion (Tissier et al., 1999). This will allow researchers to simply request plant lines that carry a mutation within any DNA sequence in the genome. The above strategies could be extremely useful in research with other plant species. For some species, Figure 1. It’s really that simple. For floral dip transformation of the current transformation protocols are close to be- Arabidopsis, plants are grown to a stage when they have just started ing sufficient (witness the recent production of more to flower (A), plants are dipped briefly in a suspension of Agrobac- terium, Suc, and surfactant (B), plants are maintained for a few more than 18,000 fertile transgenic rice lines to form an weeks until mature and then progeny seeds are harvested (C), and insertionally tagged population; Jeon et al., 2000). For seeds are germinated on selective medium (e.g. containing kanamy- many important species, however, pursuit of the cin) to identify successfully transformed progeny (D). above strategies would be greatly facilitated by the availability of high-throughput/non-tissue culture transformation methods. After the success of the Ara- artificial chromosome collections. Bacterial artificial bidopsis vacuum infiltration protocol, a number of chromosome clones containing insert DNAs that laboratories tried to use Agrobacterium vacuum infil- span the genomic region can be subcloned into a tration with other plant species, but failed to obtain transformation-competent binary vector, moved into transformants. Why? In the absence of a suitable answer, study of the successful Arabidopsis methods Agrobacterium, and used for “focused shotgun comple- was a logical next step. mentation” of a mutant plant line. With the genome sequence available and anchored to genetic maps, re- searchers may even choose to target their effort to specific candidate genes. The gene of interest is iden- HOW DO IN PLANTA ARABIDOPSIS tified by screening sets of transformed plants for in- TRANSFORMATION PROCEDURES WORK? dividuals that exhibit a corrected phenotype. What is the cellular target of transformation? For Another important use of simple, high-throughput the Arabidopsis seed transformation and vacuum transformation returns to the insertional mutagenesis infiltration methods, it was shown early on that most methods pioneered by Feldmann and others. Collec- primary transformants carry hemizygous T-DNA in- tions of Arabidopsis containing tens of thousands of sertion events (Feldmann, 1992; Bechtold et al., 1993). independent transformed lines are now available for The presence of the T-DNA on only one of two screening (Azpiroz-Leehan and Feldmann, 1997; homologous chromosomes implies that productive Krysan et al., 1999). Researchers alternatively can transformation occurs late in floral development, pursue insertional mutagenesis of a unique plant after the divergence of male and female germ line, for example to carry out screens for genetic lines (Arabidopsis self-pollinates within individual suppressors of a particular mutation. Activation- flowers, and if transformation occurred earlier, self-

1542 Plant Physiol. Vol. 124, 2000 Uses and Mechanisms of in Planta Transformation fertilization would be expected to give rise to some OVULES ARE THE PRIMARY TARGET homozygous transformants due to presence of the FOR TRANSFORMATION same T-DNA insert in pollen and embryo sac cells). Returning to the question of the cellular target of The transformation target is further defined in that transformation, three research groups worked in par- transformants obtained from a given plant usually allel to address this issue and have now published carry independent T-DNA insertion events (Feld- their results (Ye et al., 1999; Bechtold et al., 2000; mann, 1992; Bechtold et al., 1993). This suggests that Desfeux et al., 2000). Given that transformation can transformation occurs after the divergence of individ- occur by mere dipping of flowers in Agrobacterium ual pollen or lineages within a flower. A solution and that anthers and pollen are exposed developmental endpoint for the typical target of trans- whereas ovules are not, it seemed likely that the male formation can also be postulated. Although the result germ-line would be the target of transformation. is not as well established, typical primary transfor- However, all three groups found that the female mants apparently carry the transgene in all parts of the germ-line is the primary target of transformation. plant, suggesting that transformation occurred before In one set of experiments, transformants were pro- the cell divisions in a fertilized embryo that establish duced by outcrossing after Agro-inoculation of only independent meristems and other distinct adult plant the pollen donor or pollen recipient. No transfor- cell lineages. Hence, transformation seems to occur in mants were observed among more than 14,000 seeds developing flowers after individual gametophyte cell produced following inoculation of the pollen donor, lineages form, but before extensive development of but 71 transformants were recovered out of roughly the embryo. The next question was: Does transforma- 14,800 seeds produced following inoculation of the tion occur primarily in pollen, ovules, fertilized em- pollen recipient (Fig. 2; Desfeux et al., 2000). Ye and bryos, or any of the three? colleagues observed zero and 15 transformants, re- It is an interesting historical sidelight that rather spectively, in a similar study (Ye et al., 1999). These than addressing this key question, Arabidopsis re- findings seemingly to rule out transformation of pol- searchers in the mid-1990s focused on empirical len as it develops within anthers, but do not preclude transformation protocol improvement. Practical mo- the possibility that pollen is transformed after it ger- tivation to proceed with the generation of trans- formants was understandably paramount, and over- all satisfaction with the new transformation method delayed efforts to understand how it worked. Nev- ertheless, protocol modifications, ideas, and anec- dotal observations were shared widely through meetings, word-of-mouth, and the Arabidopsis elec- tronic newsgroup (http://www.bio.net/hypermail/ Arabidopsis/). Significant findings resulting from this community effort included the discoveries that (a) Plants did not need to be uprooted, treated with Agrobacterium, and re-planted. Transformants could be obtained by treating only the protruding inflorescences; (b) inclu- sion of Silwet L-77, a strong surfactant that shows relatively low toxicity to plants, often enhanced transformation reliability; and (c) many different Arabidopsis ecotypes were transformable and many different Agrobacterium strains could be used, al- though notable differences in efficiency were ob- served. Most important, the popular name “vacuum infiltration” was superceded when a number of groups found that plants could be transformed when dipped in Agrobacterium solution with no vacuum infiltration. Some workers subsequently moved to spray application of Agrobacterium rather than dip- ping. A number of other mechanistic clues and pro- Figure 2. Bar graph showing the number of Arabidopsis transfor- cedural tips were shared (see http://www.bio.net/ mants obtained by crossing Agro-inoculated pollen recipients with noninoculated pollen donors or vice versa. Breakdown by days prior hypermail/Arabidopsis/; Clough and Bent, 1998). to anthesis at time of inoculation (days after inoculation) shows that A simplified protocol for “floral dip” transforma- no transformants were obtained unless Agrobacterium was applied to tion of Arabidopsis is available at http://plantpath. relatively young pollen-recipient flowers that were 5 or more d away wisc.edu/wisc.edu/ϳafb/protocol.html. from anthesis (Desfeux et al., 2000).

Plant Physiol. Vol. 124, 2000 1543 Bent minates on the stigmatic surface of the pollen (Ye et al., 1999; Bechtold et al., 2000; Desfeux et al., recipient. 2000). Ovule transformation was convincingly demon- strated when constructs containing a GUS marker gene were used to document sites of delivery of GUS STAINING AND TRANSFORMANT T-DNA (Fig. 3; Ye et al., 1999; Desfeux et al., 2000). GENERATION: SALIENT DETAILS 35S and other standard promoters are poorly ex- In the above experiments GUS staining was often pressed in gametophyte tissues, so additional pro- observed only within the embryo sac of ovules, indi- moters used for GUS fusions were Arabidopsis cating a time of transformation late in megagameto- ACT11 (Desfeux et al., 2000), an oilseed rape Skp1- phyte development (Fig. 3c; Ye et al., 1999; Desfeux et like promoter (Bechtold et al., 2000), or a Figwort al., 2000). Late transformants were also obtained, mosaic virus promoter (Ye et al., 1999). Staining was albeit at a very low frequency, from inoculation of observed in ovules in mature flowers and in younger flowers that were sufficiently developed to contain flowers that had not yet reached pollination (Ye et al., trinucleate pollen and embryo sacs at the four-nuclei 1999; Desfeux et al., 2000). Desfeux et al. (2000) and or mature stage of development (Bechtold et al., Bechtold et al. (2000) did not observe GUS staining of 2000). Uniform blue staining of entire ovules was anthers or pollen (except in stably transformed pos- sometimes observed, suggesting that transformation itive controls), providing another line of evidence can also occur earlier in the formation of the megaga- that pollen transformation is not common. Ye et al. metophyte cell lineage (Fig. 3b). However, earlier (1999) reported frequent GUS staining of ovules and transformation events that give rise to larger trans- pollen, but also concluded that ovules are the pri- formed sectors encompassing multiple ovules seem mary target for transformation. It is curious that unlikely. Arabidopsis transformants from the same Bechtold et al. (2000) did not observe staining of plant (Bechtold et al., 1993; Ye et al., 1999) or even ovules or embryos in their work. from the same silique (seed pod; Desfeux et al., 2000) A third, very different line of evidence also points are usually independent. This latter point contrasts to ovules as the primary site of productive transfor- with recent results from the legume M. truncatula mation. Genetic linkage analysis with a marked chro- (discussed below). mosome demonstrated that most transformants (25 Desfeux and colleagues tracked the presence of of 26 tested) carry T-DNA on the maternally derived Agrobacterium by using GUS constructs that are ex- chromosome set (Bechtold et al., 2000). For the one of pressed within Agrobacterium (Desfeux et al., 2000). 26 events associated with the paternal chromosome Floral dip inoculation produced staining along the set, the most likely origin was pollen transformation stigmatic surface and in various crevices of the or integration of T-DNA within the diploid genome flower (not shown), but also produced examples in of a fertilized embryo. However, the aggregate mes- which closed locules were filled with blue stain (Fig. sage from the efforts of these three labs seems con- 3d), suggesting that locules can harbor substantial vincing: developing ovules are the primary target of colonies of Agrobacterium. productive transformation in the Arabidopsis floral Transformants have been obtained from siliques dip or vacuum infiltration transformation procedures located at multiple sites across the inflorescence (Bechtold et al., 1993; Ye et al., 1999). However, trans- formants in one study were not randomly (Poisson) distributed on a per silique or per plant basis (Bech- told et al., 2000). In another study roughly one-half of the transformant-bearing siliques contained more than one and up to seven transformants (Desfeux et al., 2000). In addition, although most siliques on in- oculated plants showed no GUS staining, a few sil- iques showed multiple stained embryos (Desfeux et al., 2000). It is apparent that some flowers are partic- ularly amenable to transformation.

Figure 3. ACT11-GUS staining of Arabidopsis ovules, embryo sacs, TIME OF INOCULATION RELATIVE TO FLOWER and entire locules due to inoculation with Agrobacterium. A, View of DEVELOPMENT IS CRUCIAL an entire flower, with staining present in multiple ovules. B, Ovules If productive transformation events occur in the in a segment of a dissected flower, showing no staining, localized staining, or complete staining of individual ovules. C, Ovules with female germ-line, one is forced to wonder how staining of embryo/embryo sac rather than entire ovule. D, Staining Agrobacterium gains access to ovules that develop of an entire locule cavity due to a bacterially expressed GUS con- within enclosed locules. However, Arabidopsis lo- struct within Agrobacterium that has colonized the locule interior. cules are not always closed. The ovary develops as a Some panels from Desfeux et al. (2000). ring of cells that protrude from the floral meristem,

1544 Plant Physiol. Vol. 124, 2000 Uses and Mechanisms of in Planta Transformation extending to form a vase-shaped structure that is The occurrence of sibling transformants is not de- open at the top. It is only late in floral development, sirable for applications such as insertional mutagen- roughly 3 d prior to anthesis, that locules become esis, but the overall high rates of transformation ob- sealed by formation of the stigma as a cap at the top tained (Trieu et al., 2000) indicate that this method of this vase. This timeline correlates strikingly with will be of tremendous utility to researchers who the outcrossing study (Fig. 2; Desfeux et al., 2000) in study M. truncatula. which siliques that gave transformants were all from flowers inoculated 5 to 10 d prior to anthesis, a time WILL THE ABOVE INFORMATION HELP WITH when the locule is open. No transformants were ob- TRANSFORMATION OF OTHER SPECIES? tained from flowers that were4dorfewer away from anthesis at the time of Agro-inoculation. Further- Transformation by infiltration of adult plants with more, GUS staining of ovules occurred only in flow- Agrobacterium has also been reported for pakchoi (Liu ers that were inoculated 5 or more d prior to anthesis et al., 1998) and has been informally reported for (Desfeux et al., 2000). Agrobacterium apparently en- other beyond pakchoi and Arabidopsis. ters Arabidopsis locules prior to their closure. Alter- Thus multiple plant species have now been success- native interpretations of these timing-of-inoculation fully transformed using Agrobacterium in planta ap- results are that it takes Agrobacterium a number of proaches. In addition, although the methods have not days to build up sufficient numbers and/or to adapt been widely reproduced or adapted, Agrobacterium- to the plant and activate transformation. In addition, mediated shoot apex transformation and related the finding of Bechtold and colleagues (2000) that methods that minimize tissue culture have been re- transformants can be obtained at a low frequency ported for a number of other plant species (Chee and from inoculation of relatively mature flowers sug- Slighton, 1995 and refs. therein). Development of gests that some transformation events may arise from robust in planta transformation protocols for other Agrobacterium delivered by other means; for example, plant species should be within reach. via pollen tubes. On the whole, however, it is clear Transformation technology development has been that Agrobacterium must gain access to ovules or to regarded by some as art as much as science, but ovule-progenitor tissue for the high-efficiency Arabi- success is most likely to come from efforts informed dopsis transformation procedures to succeed. by the scientific literature and past experiences (e.g. see the reviews of Hansen and Wright, 1999 and M. TRUNCATULA: SOMETIMES A DIFFERENT Birch, 1997). This must be coupled with a willingness TRANSFORMATION TARGET? to try different approaches and to tolerate failures along the way. In a finding that suggests that improved transfor- It is very probable that success will be easier to mation of many plant species is within reach, suc- achieve with some species and than with others, but cessful transformation of M. truncatula by Agrobacte- what are some of the criteria that may contribute to rium vacuum infiltration has been reported (Trieu et success? Arabidopsis and M. truncatula are relatively al., 2000). As with Arabidopsis, the investigators had small plants with rapid generation times, but that success inoculating flowering plants or younger may enhance ease of effort more than ultimate suc- seedlings. However, amazing transformation rates cess at transformation. A high seed set is also likely were reported for M. truncatula, ranging from 2.9% to to help, but is not an ultimate determinant: although 76% of all seeds tested (Trieu et al., 2000). It is in- single Arabidopsis plants commonly produce 5,000 triguing that when flowering M. truncatula plants to 10,000 seeds, only 33 or fewer seeds per Agro- were inoculated, transformants homozygous for the inoculated plant were collected in the successful transgene were observed and the majority of trans- transformation of M. truncatula (Trieu et al., 2000). formants from a given plant were siblings derived Aspects of experimental design such as planting con- from the same T-DNA integration event. M. trunca- figuration and mode of Agro-inoculation (i.e. inocu- tula transformation can apparently occur at earlier lating seedlings as opposed to flowering plants) can stages of plant development than in Arabidopsis. allow dramatic shifts in the number of specimens However, five of nine and six of seven transformants processed. Large numbers can be important if rates of examined in two seedling inoculation experiments success are low, but can also be a trap if quality of were judged to be independent, showing that multi- treatment is more important than quantity. Prime ple transformation events can arise on a plant even if examples of this are the low success rates and/or it is inoculated as a seedling (Trieu et al., 2000). The poor reproducibility that were achieved with Arabi- cellular targets of these transformation events are not dopsis in planta transformation procedures until in- known, and determination of these targets should be oculation of plants in full flower was attempted. The a high priority for future research. need for vernalization of M. truncatula to achieve In another intriguing, but unexplained result, Trieu efficient transformation offers another example. Try- and colleagues (2000) obtained transformants only if ing a larger variety of approaches may be more pro- seedlings were subjected to a 4°C/14 d vernalization ductive than trying very large-scale attempts with a treatment that induced earlier flowering. narrow set of methods.

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The discovery of the ovule as the site of productive LITERATURE CITED Arabidopsis transformation produces specific sug- Azpiroz-Leehan R, Feldmann KA (1997) T-DNA insertion gestions for floral transformation efforts with other mutagenesis in Arabidopsis: going back and forth. Trends species. Application of Agrobacterium to flowering Genet 13: 152–156 tissues very early in their development and prior to Baulcombe DC (1999) Fast forward genetics based on locule closure is likely to be important. In an alternate virus-induced gene silencing. Curr Opin Plant Biol 2: manner, with some species it may be possible to 109–13 deliver Agrobacterium by microinjection of ovaries, or Bechtold N, Ellis J, Pelletier G (1993) In planta by shooting Agrobacterium into flowers using micro- Agrobacterium-mediated gene transfer by infiltration of projectiles or high-pressure air guns (e.g. see U.S. adult plants. C R Acad Sci Paris Life patent no. 5,994,624). As a further alternative, plant Sci 316: 1194–1199 lines such as the Arabidopsis CRABS-CLAW mutants Bechtold N, Jaudeau B, Jolivet S, Maba B, Vezon D, that bear a more accessible locule may provide an Voisin R, Pelletier G (2000) The maternal chromosome improved target for transformation (Desfeux et al., set is the target of the T-DNA in the in planta transfor- 2000). Work with M. truncatula and Arabidopsis sug- mation of Arabidopsis thaliana. Genetics 155: 1875–1887 gests that younger plants can also be treated, al- Bechtold N, Pelletier G (1998) In planta Agrobacterium- though onset of flowering soon after Agro- mediated transformation of adult Arabidopsis thaliana inoculation appears to be preferable. plants by vacuum infiltration. Methods Mol Biol 82: In numerous plant transformation systems, the 259–266 choice of host genotype and/or Agrobacterium geno- Birch RG (1997) Plant transformation: problems and strat- type has been an important parameter (Birch, 1997). egies for practical application. Annu Rev Plant Physiol If recent findings with Arabidopsis are any indica- Plant Mol Biol 48: 297–326 tion, surveys for compatible host and bacterial geno- Chang SS, Park SK, Kim BC, Kang BJ, Kim DU, Nam HG types might best be focused on assays that monitor (1994) Stable genetic transformation of Arabidopsis thali- transformation of ovules or ovule progenitor tissues. ana by Agrobacterium inoculation in planta. Plant J 5: With some plant species the use of anti-oxidants or 551–558 other necrosis-reducing approaches has improved Chee PP, Slighton JL (1995) Transformation of soybean transformation rates, and many other modifications (Glycine max) via Agrobacterium tumefaciens and analysis can be considered (Birch, 1997; Hansen and Wright, of transformed plants. In KMA Gartland, MR Davey, eds, 1999). A better understanding of T-DNA transfer and Agrobacterium Protocols: Methods in Molecular Biology, other aspects of Agrobacterium/plant interactions Vol 44. Humana Press, Totowa, NJ, pp 101–119 (e.g. Hooykaas and Schilperoort, 1992; Mysore et al., Chowrira GM, Akella V, Lurquin PF (1995) Electropo- 2000) may also allow engineering of better host/ ration-mediated gene transfer into intact nodal meris- bacteria combinations. tems in planta: generating transgenic plants without in Other substantially different transformation meth- vitro tissue culture. Mol Biotechnol 3: 17–23 ods also must be kept in mind (e.g. Chowrira et al., Clough SJ, Bent AF (1998) Floral dip: a simplified method 1995; Chen et al., 1998). Who would have dreamed, for Agrobacterium-mediated transformation of Arabidopsis 20 years ago, that coating DNA on to little metal thaliana. Plant J 16: 735–743 particles and then shooting it into plants (Klein et al., Clough SJ, Fengler KA, Yu I-c, Lippok B, Smith RK, Bent 1987) could be so successful? AF (2000) The Arabidopsis dnd1 “defense, no death’ gene Agrobacterium floral transformation procedures encodes a mutated cyclic nucleotide-gated ion channel. have been a tremendous success with Arabidopsis, Proc Natl Acad Sci USA 97: 9323–9328 and similar success now seems likely for M. trunca- Desfeux C, Clough SJ, Bent AF (2000) Female reproduc- tula. Such successes, along with the recent informa- tive tissues are the primary target of Agrobacterium- tion about the targets for Arabidopsis transforma- mediated transformation by the Arabidopsis floral-dip tion, should inspire a renewal of efforts to adapt method. Plant Physiol 123: 895–904 these methods to the transformation of other plant Feldmann K (1992) T-DNA insertion mutagenesis in Ara- species. The benefits are clear: transformation with- bidopis: seed infection transformation. In C Koncz, N-H out tissue culture can provide a high throughput Chua, J Schell, eds, Methods in Arabidopsis Research. method that requires minimal labor, expense, and World Scientific, Singapore, pp 274–289 expertise. Rates of unintended mutagenesis are re- Feldmann KA, Marks MD (1987) Agrobacterium-mediated duced. More important, simplified transformation transformation of germinating seeds of Arabidopsis thali- protocols facilitate positional cloning, insertional mu- ana: a non-tissue culture approach. Mol Gen Genet tagenesis, and other transformation-intensive proce- 208: 1–9 dures, reducing the effort required to test any given Gelvin SB, Schilperoort RA (1998) Plant Molecular Biol- DNA construct within plants. ogy Manual. Kluwer Academic Publishers, Dordrecht, Received September 5, 2000; accepted September 21, 2000. The Netherlands

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