High-Efficiency Transformation Techniques

CHAPTER 5

High-Efficiency Transformation Techniques Yuji Ishida, Yukoh Hiei, Toshihiko Komari Plant Innovation Center, Japan Tobacco Inc., Iwata, Japan

Contents 5.1 Introduction 5.2 Transformation Methods in Cereals 5.3 Wheat 5.3.1 Wheat Tissues 5.3.2 Tissue Culture 5.3.3 Selection Markers 5.3.4 Strains of A. tumefaciens 5.3.5 Genotype of Wheat 5.4 Barley 5.4.1 Barley Tissues and Culture Techniques 5.4.2 Selection Markers 5.4.3 Strains of A. tumefaciens 5.4.4 Genotype of Barley 5.5 Other Small Grain Cereals 5.5.1 Oat 5.5.2 Rye 5.5.3 Triticale 5.6 Future Tasks in Transformation of Small Grain Cereals 5.6.1 Means to Overcome Genotype Differences 5.6.2 Gene Editing 5.6.3 Development of Useful Traits and Commercialization of Biotechnology Crops 5.7 Conclusion Acknowledgments References

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5.1 INTRODUCTION Small grain cereals, such as wheat, barley, oat, and rye, comprise a unique group of crops originating from the Middle East, including the Fertile Crescent. These crops historically supported the earliest civilizations in Mesopotamia and Ancient Egypt, and are presently still among the most Applications of Genetic and Genomic Research in Cereals https://doi.org/10.1016/B978-0-08-102163-7.00005-3

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important staples for human beings. They are also major feed crops for livestock, and the grains are important materials for beverage production and other industrial applications. Human population on the Earth is currently 7.3 billion and is projected to surpass 10 billion by 2050 (United Nations World Population Prospects 2017, https://esa.un.org/unpd/wpp/Publications/Files/WPP2017_KeyFindings. pdf). If the general pattern of human diet is unchanged, we may assume that 37% more food will be necessary. Unfortunately, further economic growth of developing countries will create a demand for more meat and consequently for much more grain. Yet, even the current level of agricultural capacity is threatened by soil erosion, climate change, and other environmental problems. It is critical that we try to employ every means to sustain and elevate food production, and biotechnology is an indispensable piece in the endeavor. Because the small grain cereals generally exhibit good stress tolerance and are adapted to relatively cool and dry areas, they are also key crops in grain production for the future. On the other hand, the progress of biotechnology in these species is considerably behind that in maize and rice. A key factor in this aspect is gene transfer technologies. Rice is the first cereal crop for which efficient transformation methods were developed, and many genotypes of rice can be routinely transformed now (Hiei and Komari, 2008). Maize quickly followed and was used in the commercialization of biotechnology crops; transgenic varieties are now grown in more than 90% of the cornfield in North America. Compared with rice and maize, the number of reports on transformation methods published in the last 20 years is much smaller for other cereals. Therefore, scientists are urged to develop and improve gene transfer methods for the small grain cereals. In this chapter, the attempts and progress in the transformation technologies in wheat, barley, rye, oat, and triticale, which is a hybrid of wheat and rye, are discussed.

5.2  TRANSFORMATION METHODS IN CEREALS In every step from basic studies in plant molecular biology to the development of commercial varieties, gene transfer technology plays a key role. The dissection of biological processes in plants, examination of the gene effects, proper regulation of transgenes, and robust generation of transgenic events for commercialization in a crop of interest could all be effectively conducted if given genotypes of germplasm in the crop species can be transformed at a high frequency. Emerging technologies in genome editing



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also need efficient protocols to introduce macromolecules into plant cells and to regenerate plants from the edited cells. Clearly, both transformation and genome editing approaches require a very similar set of techniques. In dicotyledonous species, the gene transfer method of choice since the very early stage has been a transformation mediated by the soil bacterium Agrobacterium tumefaciens. This bacterium can transfer its DNA (T-DNA or transfer DNA) to plant cells to incite plant tumors called crown galls (Ream, 1989). By the mid-1980s, the methods to generate transgenic plants without causing tumors were developed for tobacco, petunia, and other dicotyledons (Fraley et al., 1986). However, it was generally believed that A. tumefaciens could not transform monocotyledons because these plants are outside the host range of crown gall disease (De Cleene and De Ley, 1976). Therefore, until the mid-1990s, DNA was delivered to monocotyledonous cells mainly by direct methods. For example, DNA was taken up by protoplasts, which are naked cells created by enzymatic degradation of cell walls, by electric pulses, or by polyethylene glycol treatments. Most commonly, plant tissues were bombarded with metal particles coated with DNA. Because A. tumefaciens can transfer a small number of copies of relatively large DNA segments with defined ends to plant chromosomes with few rearrangements (Hooykaas and Schilperoort, 1992); delivery of DNA mediated by A. tumefaciens was also highly desirable for cereals. Although many studies in the 1980s indicated that DNA could be transferred by A. tumefaciens to cereal cells, attempts similar to the methods used to generate transgenic plants in tobacco, petunia, and other dicotyledons, such as the cocultivation of leaf segments with A. tumefaciens, all failed or provided controversial results. Then, transgenic plants were obtained from calli induced from mature embryos of rice (Hiei et al., 1994) and from immature embryos of maize (Ishida et al., 1996) after the tissues were cocultivated with A. tumefaciens. A key success factor in these studies was the use of cells that were dedifferentiated or undifferentiated, divided or about to divide, and capable of regenerating plants. Mature and immature embryos turned out to be good sources of such cells. These precedents inspired studies in other monocotyledonous plants, and quite a few cereal and grass species can now be transformed efficiently (Hiei et al., 2014).

5.3 WHEAT Wheat is the No. 1 crop in the world in many ways, including the global acreage for production and the amount of the grain internationally

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traded. Two major species, bread wheat Triticum aestivum L. and durum wheat Triticum durum Desf., are grown worldwide to provide materials for a vast variety of foods and beverages, including breads, noodles, and cakes. Despite the global importance of the crop, progress in productivity improvement has been slow compared to maize and rice, possibly for two major reasons: complex genome structure and recalcitrance in tissue culture. T. aestivum and T. durum are hexaploid and tetraploid species, respectively, and T. aestivum has a genome that is 40 times and five times the size of rice and maize, respectively. Although the term “genome” was coined from the studies of the ploidy variation in wheat, the complexity has been a major hurdle in the study of wheat genomics. Fortunately, rapidly advancing sequencing capabilities have been lowering the hurdle, and a “draft sequence” of wheat genome was produced in 2014 (IWGSC,The International Wheat Genome Sequencing Consortium, 2014). The hurdle in tissue culture has also been lowered after years of effort. The first transgenic wheat was created by particle bombardment in 1992 (Vasil et al., 1992). Soon after efficient protocols of transformation mediated by A. tumefaciens were developed in rice and maize, cultivar Bobwhite of wheat was transformed by A. tumefaciens (Cheng et al., 1997), and several reports followed. As with other cereals, the selected starting tissue were immature embryos. Progress made thereafter has been somewhat slower, and the frequency of transformation reported was mostly less than 5% of the inoculated tissue pieces (Wan and Layton, 2006; Wu et al., 2008; Risacher et al., 2009; He et al., 2010; Binka et al., 2012). Ishida et al. (2015) optimized the protocol to obtain a frequency of transformation as high as 90% for cultivar Fielder, and other research groups successfully confirmed that the protocol was efficient, and the results were reproducible (Richardson et al., 2014;Wang et al., 2016a).The testing of various genes in wheat by this protocol is now possible for the improvement of agronomical characteristics (Ashikawa et al., 2014). Ishida et al. (2015) noticed that the list of key factors in wheat transformation, including the choice of genotype, quality and stage of immature embryos, media composition, strain of A. tumefaciens, pretreatment of embryos, and handling of tissues, was not much different from those studied previously. However, the optimal ranges of many of these factors were much narrower in wheat than in rice and maize, suggesting that the narrow windows were a key reason for the slow progress. These factors are discussed in detail in the following sections.



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5.3.1  Wheat Tissues Immature embryos were cocultivated with A. tumefaciens following the protocol of Ishida et al. (2015).They emphasized that the quality of the starting tissues was critically important and the embryos must be collected from healthy plants of the wheat cultivar Fielder grown in a well-conditioned greenhouse. The developmental stage of the embryo is also important, and the size of the embryos is a good indicator of the stage. Ishida et al. (2015) found that immature embryos that are between 2.0 and 2.5 mm in length, which is bigger than the embryos used in the preceding reports, were optimal for transformation. There are similarities and differences in the transformation protocols among cereals. For example, before the cocultivation, immature embryos are pretreated with centrifugation and are heated in rice (Hiei and Komari, 2008) and maize (Ishida et al., 2007). Although the embryos are centrifuged at 20,000 ×g in the protocol of Ishida et al. (2015), heating was not effective and thus not employed in wheat. After the cocultivation step, the embryo axis was removed from the protocol of Ishida et al. (2015). Removal of the axis prior to cocultivation or the process without the removal resulted in poor transformation. Such a step was not found in the transformation protocols of any other cereals. Immature embryos appear to be the best starting tissues with respect to efficiency of transformation in cereals in the recent literature. However, there is a criticism of dependency on immature embryos, as the year-round supply of immature tissues is a tedious, labor intensive, delicate, and comparatively expensive process that requires maintaining donor plants under controlled conditions and avoiding stresses to the plants as much as possible (Parmar et al., 2015; Tamas-Nyitrai et al., 2012). On the other hand, the transformation of alternative tissues, such as calli induced from mature or immature embryos and maintained in vitro, is generally inefficient and faces high genotypic hurdles.The development of efficient methods for diverse genotypes is likely another tedious, labor intensive, delicate, and comparatively expensive process. Therefore, our recommendation is the collaboration between elite teams with suitable facilities and expert staff focusing on transformation technology for immature embryos and users of the technology.

5.3.2  Tissue Culture The composition of media for wheat transformation, in terms of types and concentrations and of inorganic salts, vitamins, amino acids, sugars, and

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g­ elling agents in the media, is generally not much different from those for other major cereals and does not vary much among the reports of wheat transformation. On the other hand, the modification of phytohormones in the media has been attempted more extensively. Habib et al. (2014) cultured immature embryos of 26 cultivars, including 12 commercially grown cultivars, on media with a wide variation in hormone composition. They were able to induce calli and regenerate plants in all the genotypes tested. They then focused on cultivar Sehar-2006, which demonstrated a good response and successfully obtained transgenic plants from 2.45% of the embryos; they were able to obtain transgenic plants from 65% of the bombarded callus pieces. Khokhar et al. (2016) found callus induction from immature embryos of Pakistani wheat cultivars was better on a medium with 2 ppm of 2,4-dichlorophenoxyacetic acid (2,4-D) than on media with 4 or 6 ppm of 2,4-D. They also observed that callus induction was improved by the addition of 300 ppm of casein to the medium. Sabetta et al. (2016) found callus induction in durum wheat was better on a medium with 3,6-dichloro-­2methoxybenzoic acid (Dicamba) than on a medium with 2,4-D. The addition of certain minerals could also make a difference. For example, Delporte et al. (2014) observed the efficient regeneration of plants from calli induced from immature embryos and bombarded with a gene for antifreezing protein and a BASTA resistance (bar) gene by adding Cu2+ to the media either for initial callus induction or for regeneration culture. In protocols of transformation mediated by A. tumefaciens, optimization of the inoculation step, such as the adjustment of acetosyringone concentration (Mitić et al., 2014; Manfroi et al., 2015)—a phenolic compound used to induce the virulence genes of the bacteria—in the media for cocultivation of plant tissues and the bacteria, seems to be important. In a unique method developed by Risacher et al. (2009), an inoculum was injected into the immature spikelets in vivo before the embryos were isolated and cultured in vitro.

5.3.3  Selection Markers Similar to the transformation protocols of other cereals, the choice of selection marker genes and selective agents is critical in wheat transformation, and the bar gene and a hygromycin resistance gene were often used to protect transformed cells from selection pressure by herbicides or antibiotics (Ishida et al., 2015). A phosphomannose isomerase gene was also a preferred marker to make transformed wheat cells grow on a medium with mannose as the sole carbon source (Gadaleta et  al., 2006). Kanamycin, a selective



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agent most frequently added to media for the transformation of dicotyledons, was not very effective in wheat transformation (Jhinjer et al., 2017). Once transformants are selected, the selection marker genes are not only unnecessary but also undesired in the light of public concern regarding transgenic wheat. Methods to remove selection marker genes were well established in other plants (Komari et al., 1996) and tested successfully in wheat (Wang et al., 2016b). If the transformation process is efficient enough, transformants could be obtained without using selection markers. Wheat tissues may be cultured without selective agents after A. tumefaciens infection (Moghaieb et al., 2014; Richardson et al., 2014; Wang et al., 2016a) or bombardment (Rooke et al., 1999, Gadaleta et al., 2008; Qin et al., 2014), and cells with transgenes may be screened by molecular techniques, such as polymerase chain reactions.

5.3.4  Strains of A. tumefaciens The choice of strains and vectors is another key factor in transformation mediated by A. tumefaciens. In wheat, the preferred strains were EHA105 and AGL1, which were derivatives of the highly virulent strain A281 (Ishida et al., 2015). Vectors that carried the virulence genes from A281 were also used successfully in wheat (Mitić et al., 2014) and in durum wheat (Wang et al., 2016b).

5.3.5  Genotype of Wheat A large difference among genotypes in the efficiency of transformation is commonly recognized in many plant species. Because tissue culture processes are usually a core component of transformation protocols, transformation amenability (TFA) is often related to the tissue culture response of genotypes. Therefore, tests of tissue culture without gene transfer steps are highly recommended for the examination of transformation protocols in any genotypes. A general rule is that the better the tissue culture response, the higher the transformation frequency. Cultivar Bobwhite has been a popular genotype in wheat tissue culture and was naturally the first cultivar that was transformed by particle bombardment (Vasil et al., 1992) and by A. tumefaciens (Cheng et al., 1997). Ishida et al. (2015) found that cultivar Fielder, another genotype with a good tissue culture response, exhibited a higher frequency of transformation. While leading cultivars of maize and rice may be widely grown globally in various production areas, wheat cultivars are quite regional. Each of the wheat cultivation regions tends to have its own regional cultivars adapted

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to the climate, soil, and other conditions. Therefore, development of transformation technology for a wide range of cultivars is especially important in wheat. Haliloglu et  al. (2013) examined the tissue culture response of 14 Turkish winter wheat cultivars and observed a wide variation in callus induction from immature embryos. While calli were formed from 99.5% of the Bobwhite embryos—a control variety—the highest frequency recorded among the regionals was 45% in cultivar Surak. Once calli were induced, plant regeneration was possible in all of the cultivars tested. The top four cultivars were then successfully transformed by A. tumefaciens. Delporte et al. (2014) also detected a large difference in the frequency of regeneration of plants from cells cultured from mature embryos of four regional cultivars in Europe. As mentioned above, Habib et al. (2014) reported a varietal difference among 26 Pakistani cultivars and transformation of a cultivar showing a good response. Sabetta et  al. (2016) and Wang et  al. (2016b) optimized transformation protocols for durum wheat cultivars. The presence of a wide variety of genotypes does not mean that the protocol is different for each genotype. Fortunately, the protocol reported by Ishida et al. (2015) was reasonably good for six Australian, two Mexican, two durum cultivars (Richardson et al., 2014) and for 15 Chinese cultivars (Wang et al., 2016a), although efficiency of transformation varied considerably among the cultivars in these studies. The highest frequency of independent transformants per immature embryos infected with A. tumefaciens in the study of Wang et al. (2016a) was 37.7% of cultivar CB037, which was comparable to the control cultivar Fielder.The frequencies recorded by Richardson et al. (2014) varied between 1.5% and 51%. Also in transformation by particle bombardment, one protocol was good for more than 30 elite cultivars from various areas (Sparks and Jones, 2014) and another was good for six Australian cultivars (Ismagul et al., 2014a).

5.4 BARLEY Barley (Hordeum vulgare L.) is the fourth cereal in farming acreage and a major staple crop in relatively dry regions in the world because of its remarkable drought tolerance. The importance of this crop for humans is as great as the three major cereal crops—maize, wheat, and rice—as food and feedstock and for the production of beer and syrup; however, the amount of production in the world is far below the level of the three major crops. In addition, knowledge gained from studies on barley, which has a simple diploid genome, might be useful for studies on wheat with a complex hexaploid genome.



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The first transgenic barley plants were created through the particle bombardment of immature embryos, calli induced from immature embryos, and embryos derived from microspores of cultivar Golden Promise (Wan and Lemaux, 1994). Transformation of Golden Promise mediated by A. tumefaciens was reported soon after the success in rice and maize transformation (Tingay et  al., 1997). Initially, the frequency of transformants was not very high, between 0.3% and 7.9% of the tissue pieces treated, but recent studies have reported the frequency of around 25% or higher in the immature embryos infected with A. tumefaciens (Hensel et al., 2008; Bartlett et al., 2008; Harwood, 2014). The highest frequency in the study by Hensel et al. (2008) was 86.7%. Then, we found that Golden Promise could be transformed much more efficiently by modifying the protocol of Hensel et al. (2008); on average, 2.7 independent transgenic plants per immature embryo infected with A. tumefaciens were obtained. The details of the modification (published in a patent document, EP2599382A1) are discussed below. Therefore, transformation methods for barley are well established by now and could be employed in basic and applied studies quite efficiently. The transformation process in barley may still be less efficient than in rice but more efficient than in wheat, maize, and other cereals.

5.4.1  Barley Tissues and Culture Techniques Immature embryos and calli induced from immature embryos were transformed in many studies. Naturally, the list of the critical factors for high efficiency transformation is similar to that of wheat. The embryos must be collected from healthy plants, not damaged by diseases or insects, and grown in a controlled environment (Bartlett et al., 2008; Harwood, 2016). The developmental stage of the embryos must be exact; the optimal size of the embryos was approximately 1.5 mm in length in the study by Harwood (2014). Unlike other major cereals, the removal of the embryo axis immediately following embryo preparation, and incubation of the embryos with the scutellum side down during the coculture with A. tumefaciens were recommended (Harwood, 2014, Ismagul et al., 2014b). Because it is relatively easy to prepare microspores compared with other cereals, transformation of microspores or calli induced from microspores was attempted in barley (Ji et al., 2013). In addition, ovules soon after pollination (Holme et  al., 2012), mature embryos (Uçarli et  al., 2015), calli induced from mature embryos (Gürel et al., 2015), immature inflorescences, and shoot base segments (Pasternak et al., 1999) were examined as starting

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tissues. However, the efficiency of transformation was generally lower in these tissues than that of immature embryos. Various additives to media have been worth testing. The addition of 2+ Cu to culture media was effective in a number of reports (Bartlett et al., 2008; Ji et al., 2013; Harwood, 2014). Gürel et al. (2015) added cellulose and lectin to the inoculum suspension of A. tumefaciens and observed an elevated transformation efficiency. In our modification of the protocol of Hensel et  al. (2008), 5 μM of 2,3,5-triiodobenzoic acid (TIBA) was added to the medium for cocultivation of A. tumefaciens and immature embryos of cultivar Golden Promise.The TIBA is classified as an “antiauxin” compound and is thus somewhat inhibitory to the growth of the cells. However, this addition elevated the efficiency of transformation for an unknown reason. Further modification was then made: the embryos were heated at 43°C for 5 min prior to cocultivation and placed with the scutellum side up on the cocultivation medium.The duration of the cocultivation was limited to 1 day, and the embryos were centrifuged at 20,000 ×g for 10 min after cocultivation. The embryos were then cut into small pieces before the selection culture. As mentioned above, 2.7 independent transgenic barley plants were obtained per embryo after the modification.

5.4.2  Selection Markers A kanamycin resistance gene was used in the earlier studies (Tingay et al., 1997).Then, the hygromycin resistance gene was preferentially used in later studies (Kapusi et al., 2013; Harwood, 2014; Ismagul et al., 2014b) because hygromycin could select the transformants effectively (Kumlehn et  al., 2014). The bar gene was not used as frequently in barley transformation as in other cereals, possibly because barley cells tend to escape from the pressure of the selective agents for the marker (Harwood, 2014). Holme et al. (2012) attempted the transformation of ovules soon after pollination without using selection markers. Thus, the ovules infected with A. tumefaciens were cultured without selection pressure. The frequency of transformation was low (less than 0.8%), but transformants obtained in four barley cultivars were poor in tissue culture response. Holme et al. (2012) appealed that the time required for transformation from the infection to potting in this method was short (less than 10 week), while the transformation of immature embryos typically took longer than 10 week. The cotransfer of a β-glucuronidase (gus) gene and the hygromycin resistance gene from separate T-DNAs was tested in barley, and selection marker-free transgenic plants were later segregated (Kapusi et al., 2013).



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5.4.3  Strains of A. tumefaciens In barley, the preferred strains were derivatives of A281, such as AGL0 and AGL1. The vectors that carried the virulence genes from A281 were also used successfully in barley (Harwood, 2014).

5.4.4  Genotype of Barley Again, in barley, only limited genotypes could be transformed. Most of the studies of barley transformation utilized cultivar Golden Promise or, less frequently, cultivar Igri. These barley cultivars exhibited a relatively good tissue culture response. Golden Promise is the genotype that can be transformed most efficiently. As mentioned above, an efficient protocol reported by Hensel et al. (2008) was further modified by us so that 2.7 independent transgenic plants per immature embryo of this genotype were obtained. Efficiency of transformation of other cultivars is generally low. Hensel et al. (2008) tested nine other cultivars and found that three of them could not be transformed. The frequency for the other cultivars was somewhat lower than Golden Promise and ranged between 0.5% and 12%. Hisano and Sato (2016) and Hisano et al. (2017) recognized an issue under these circumstances that genetic complementation analysis employing transformation techniques is virtually restricted to alleles nonfunctional in Golden Promise or Igri. Their hunt for quantitative trait loci (QTLs) for good transformation response from Golden Promise is discussed below.

5.5  OTHER SMALL GRAIN CEREALS Compared to wheat and barley, few reports on tissue culture and transformation technology for oat, rye, and triticale have been published to date. The limitation is likely related to the small acreage of the production of these crops and, consequently, to the low level of funding for such research.

5.5.1 Oat Oat (Avena sativa L.) is a crop adapted to cool wet climates (e.g., Northern Europe) and the sixth largest cereal in farming acreage. Although oatmeal and rolled oats are traditionally popular foods, the current production of oat is primarily as a feed crop. The first transgenic oat plants were created from calli induced from immature embryos by particle bombardment (Somers et al., 1992). A number of studies then followed to optimize factors like selection marker genes and starting tissues for the method (Torbert et al., 1995, 1998; Gless et al., 1998;

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Zhang et al., 1999; Kaeppler et al., 2000; Kuai et al., 2001). The transformation of oat mediated by A. tumefaciens was reported by Gasparis et al. (2008). Vectors that carried the virulence genes from strain A281 were also used successfully in this study, and the frequency of transformation (12.3% of immature embryos of cultivar Bajka infected with A. tumefaciens strain) was reasonably high. Therefore, a basic technical platform was well established, but we could not find any reports on further developments since then.

5.5.2 Rye Rye (Secale cereale L.) is a crop remarkably tolerant to cool climate and less-fertile soil conditions. Rye is a main cereal crop in Central and Northern Europe, where wheat cannot be grown. Rye bread and alcoholic beverages, such as beer, whisky, and vodka, are major products from rye for human consumption, and rye is also very important feed for livestock. The first rye transformants were created by bombarding calli induced from immature embryos with a bar gene and a gus gene (Castillo et al., 1994). Although Castillo et al. (1994) selected transformed cells in tissue culture by phosphinothricin (PPT), Popelka et al. (2003) tested a similar method but cultured the bombarded calli and regenerated plants without any selective agents. They then sprayed the regenerants with BASTA, whose active ingredient was PPT, to select transformants.They claimed that transformants with a single copy of the transgene was effectively obtained by this method because the ratio of such plants was 40%. Popelka and Altpeter (2003) also reported transformation of rye mediated by A. tumefaciens. Immature embryos of rye were precultured for 5 days and infected with strain AGL0. Paromomycin-tolerant transformants were obtained from up to 4% of the embryos. However, even in a recent review (Singh and Prasad, 2016), it was observed that only a few reports on rye genetic transformation are available. Still, because studies on tissue culture response to rye genotypes have been continued (Targonska et al., 2013) and regeneration from cells cultured from leaf-base segments derived from mature seeds was recently established (Haliloglu and Aydin, 2016), more efficient and convenient transformation technology will likely be developed in the near future.

5.5.3 Triticale Unlike the other cereals discussed in this chapter, which have been key staples throughout the history of human civilization, triticale (Triticosecale) is a recent crop. It is an intergeneric hybrid between wheat (T. aestivum or T. durum) and rye, and the superior characteristics of the parents—for example, grain quality, yield potential of wheat, and stress tolerance and disease



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r­esistance of rye—were intended to be combined. Major regions for triticale cultivation include Poland, Hungary, Belarus, Germany, France, China, and Australia. Livestock may be fed with the grain or with the foliage directly or as silage.The grain is also processed for bread, pasta, cereal products, and cookies for human consumption. The first transgenic triticale plants were obtained by bombarding scutellum tissues from embryos with a bar gene and a gus gene (Zimny et al., 1995). Transformation of triticale mediated by A. tumefaciens was first reported in 2005 by Nadolska-Orczyk et al. Like other cereal species, vectors that carried the virulence genes from A281 were higher in frequency of transformation than other combinations of strains and vectors (NadolskaOrczyk et  al., 2005; Hensel et  al., 2012). Precultured immature embryos were the starting tissues in these studies. Both kanamycin and hygromycin could be used as selective agents. The highest frequency of transformation in these studies was 16% (Nadolska-Orczyk et al., 2005). A unique method was tested in triticale (Ziemienowicz et  al., 2012). A complex of DNA carrying a gus gene, virD2 protein of A. tumefaciens, and RecA protein was introduced into microspores being mediated by Tat2 cell-penetrating peptide. The gus gene was integrated into plant chromosomes and expressed in the cells and plants derived from the microspores.

5.6  FUTURE TASKS IN TRANSFORMATION OF SMALL GRAIN CEREALS 5.6.1  Means to Overcome Genotype Differences The issue that only limited genotypes may exhibit a good tissue culture response and be transformed efficiently is rather serious in any cereal species. Theoretically, the issue could be approached from two directions: (1) the development of methods good for recalcitrant genotypes, and (2) genetic modification of recalcitrant genotypes. In either approach, a universal “genotype-­independent” solution is highly preferable but could likely be more difficult to find. Thus, improvement in a genotype-by-­ genotype manner is more realistic and equally useful. The former protocol-oriented approach has already been discussed in the section of each crop. Hopefully, the sustained efforts in various genotypes could continuously improve transformation technologies. Studies like the one by Seldimirova et al. (2016) regarding phytohormone distribution in calli, regenerating tissues, and shoot and leaf primordia may aid optimization of media composition.

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The latter genetic approach has been attempted in many plant species. For example, in wheat, recombinant inbred lines were examined for the frequency of regeneration of plants from calli to map three major QTLs (Khan, 2015). However, QTL mapping related to tissue culture and transformation is not generally straightforward since assays of large mapping populations by tissue culture and transformation protocols demand a lot of labor. Further tedious effort is needed to transfer the identified QTLs to genotypes of interest by breeding, possibly with the assistance of genetic markers. Again, only reliable assays in each step is the actual experiments of tissue culture and transformation using a sufficient number of plant lines. Furthermore, the effects of the QTLs may not be the same in a different genetic background and/or may appear to be “diluted” in later breeding generations. Consequently, trials to dissect the genetic difference and to exploit genes related to good responses in tissue culture and transformation have achieved limited success. Recently, Hisano and Sato (2016) and Hisano et  al. (2017) started a unique attempt. They crossed Golden Promise and recalcitrant cultivars of barley and examined the transformation of the immature embryos from the F1 plants. The embryos are in the F2 generation and, hence, in a segregating population. The embryos with the genes to provide a good response have a higher chance of being transformed, and they identified three “TFA” loci. Similarly, Wang et al. (2016a) examined the transformation of immature embryos from F1 hybrids between Fielder and Chinese cultivars of wheat. In this way, a segregating population of plant tissues were cultured/transformed so that QTLs for a good response are enriched in regenerated plants by relatively small-scale experiments. Therefore, this kind of approach presents a solid potential toward the genetic improvement of small grain cereals to demonstrate better response in tissue culture and transformation. Factors involved in cell division and plant development may be exploited to make plant cells demonstrate better responses in tissue culture and transformation. Lowe et al. (2016) were able to transform genotypes of maize with poor tissue culture response by expressing maize transcription factors (Baby Boom involved in embryogenesis and Wuschel2 involved in the maintenance of shoot apical meristems) in starting tissues. Such factors may be effective in small grain cereals. The search for useful factors had also begun in wheat. Delporte et al. (2013) examined genes expressed in the dedifferentiation of cells from wheat embryos and plant regeneration process.



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She et al. (2013) studied genes differentially expressed in genotypes of good or poor tissue culture responses in the presence or absence of 2,4-D and suggested that a catalase gene, TaCAT1, may be involved in the tissue culture response. Zhou et al. (2013) cultured immature embryos from Chinese commercial cultivars with or without A. tumefaciens and found genes specifically expressed in cells infected with the bacterium. The use of known and newly found genes, which may improve the response in tissue culture and transformation, would likely contribute greatly to the development of efficient transformation protocols in small grain cereals.

5.6.2  Gene Editing The finding, modification, development, and exploitation of nucleases that can specifically cut genomic DNA at desired locations in vivo provided excellent opportunities to directly edit genes in diverse organisms. Imprecise repair of the cut sites may easily knock out the genes, and the specific digestion may efficiently allow integration of foreign DNA segments to modify the genes in a desired manner. Therefore, gene editing is a very powerful technology to improve crops, including small grain cereals. Many of the modifications by gene editing are identical to those that could occur through natural mutations and conventional breeding, and varieties developed by gene editing could be considered as nontransgenic crops. Because the high cost of deregulation of genetically modified crops and lack of public acceptance of such crops have been serious problems in the commercialization of biotechnology varieties—especially in crops for direct human consumption and in small-acreage crops like small grain cereals—gene editing would likely provide highly attractive options for improving varieties of small grain cereals. A Chinese group has been presenting the results of highly efficient gene editing in wheat (Liang et al., 2016; Zhang et al., 2016). Lawrenson et al. (2015) and Kapusi et al. (2017) reported gene editing in barley. These studies suggested that the nucleases and related technologies demonstrated first in other crops may work efficiently in wheat, barley, and probably in other small grain cereals. If so, key issues in successful gene editing include how efficiently proteins and nucleic acids are introduced into target cells and how efficiently plants are regenerated from the edited cells. Clearly, the key issues for gene editing and genetic transformation largely overlap, and nearly all of the discussion points of transformation technology in this chapter are relevant to gene editing.

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5.6.3  Development of Useful Traits and Commercialization of Biotechnology Crops Although genes for high productivity, stress tolerance, insect resistance, and herbicide tolerance have been investigated in various crop species, few reports of such studies have been published in small grain cereals. This is possibly because genetically modified varieties in wheat and other small grain cereals have not been commercialized to date. A rare example was a study by Zhang et al. (2014), which demonstrated an elevated level of photosynthesis in wheat by the expression of maize genes for pyruvate phosphate dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPC). On the other hand, studies concerning disease resistance in wheat have been quite popular. Wheat plants transformed with genes for transcription factors from a Triticum species were higher in the expression of six defense genes and resistant to Gaeumannomyces graminis (Liu et  al., 2013). Wheat plants transformed with a defensin gene was rust resistant (Kaur et al., 2017). The expression of a gene for UDP-glucosyltransferase provided resistance against Fusarium graminearum (Li et al., 2015) but reduced the efficiency of transformation mediated by A. tumefaciens (Zhou et al., 2017). Continued efforts to study disease resistance are very important. In wheat, a new strain of rust fungus might become a major threat in the near future. The strain, UG99, was first reported in Brazil in 1985 and caused serious damages in Bangladesh in 2016 and in India in 2017, arousing the concern over an imminent global outbreak (personal communication). Therefore, the development of technology to counter the spread of UG99 is an urgent task. The efforts are critically important not only for wheat, as rust disease caused by Puccinia fungi is also a major problem in other cereals. The necessity of genetic engineering to cope with such a severe issue may be understood by the public. For the commercialization of transgenic crops, it is important to create what are known as “quality events,” which are typically characterized as being single-copy transformants without any nontarget foreign DNA pieces, and to analyze the sites of DNA integration in detail. Certain preliminary studies have begun in wheat and barley. In particle bombardment in wheat, more single-copy plants were obtained by treating the DNA with a phosphatase (Tassy et  al., 2014). Chromosomal locations of b­ ombarded segments in wheat were examined by fluorescence in situ hybridization (FISH), showing that foreign DNAs tended to be integrated close to the end regions of chromosomes more frequently (Han et  al., 2015).



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Integrated chromosomes were identified for three single-copy transgenic wheat plants by flow cytometry (Cápal et al., 2016). The transfer of the socalled vector backbone sequences in durum wheat was suppressed by vectors with two or more repeats of border signal sequences of T-DNA in transformation mediated by A. tumefaciens (Wang et al., 2016b). Junctions of foreign DNAs were sequenced in barley plants created by transformation mediated by A. tumefaciens (Bartlett et al., 2014). Although 33% of the plants lacked the right border and 14% of the plants showed certain rearrangements of the transferred DNA segments, both continued efforts in wheat and barley and initiation of similar studies in other small grain cereals are very important.

5.7 CONCLUSION Wheat, barley, oat, rye, and triticale were all transformed first by p­ article bombardment in the 1990s and later by the methods mediated by A. tumefaciens (Table  5.1). Immature embryos were the best starting tissues, A. tumefaciens strains derived from strain A281 and vectors carrying virulence genes from A281 provided higher efficiency of transformation, and the hygromycin resistance gene and the bar gene were the choices for selection markers. Many more studies concerning transformation techniques in wheat and barley were published than those in the other cereals, and, consequently, wheat and barley may be transformed much more efficiently than the other crops. Even in wheat and barley, the development of highly efficient protocols was a recent event, and thus wide application of the techniques to various basic and commercial studies remains a future task. The costs for deregulation and limited public acceptance are delaying development and commercialization of transgenic varieties. Therefore, the improvement of these crops through the emerging technology of gene editing, which requires a set of techniques mostly shared by transformation, will be an attractive option. A major obstacle is the fact that limited genotypes are amenable to tissue culture and transformation. Both the improvement of the protocols and exploitation of genes related to good response in tissue culture and transformation have been attempted and must be further pursued.

ACKNOWLEDGMENTS The authors thank Naoki Takemori and Kumiko Donovan for their support and helpful discussions.

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Wheat Barley

Embryogenic callus Immature embryo Immature embryo Immature embryo

Oat

Leaf base segment Immature embryo

Rye

Embryogenic callus Precultured immature embryo Immature embryo Precultured immature embryo

Triticale

Bar/BASTA (1–40) Bar/PPT (5–10) Bar/bialaphos (5) Hygromycin phosphotransferase / hygromycin (30–100) PPT acetyl transferase/PPT (2–4) Neomycin phosphotransferase II / kanamycin (50) Bar/PPT (5–30) Neomycin phosphotransferase II / paromomycin (30–50) Bar/PPT (2–6) Neomycin phosphotransferase II / kanamycin (50)

Independent events/explant

Reference

Biolistic A. tumefaciens Biolistic A. tumefaciens

0.005 0.70 0.073 2.73

Vasil et al. (1992) Ishida et al. (2015) Wan and Lemaux (1994) EP2599382A1

Biolistic A. tumefaciens

0.05 0.123

Gless et al. (1998) Gasparis et al. (2008)

Biolistic A. tumefaciens

0.004 0.04

Biolistic A. tumefaciens

0.054 0.16

Castillo et al. (1994) Popelka and Altpeter (2003) Zimny et al. (1995) Nadolska-Orczyk et al. (2005)

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Table 5.1  Overview of reported frequencies of transformation in small grain cereals Selection marker/selective agent Delivery Crop Explant (mg/L) method



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