Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos.

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., 'Nipponbare') regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The natural mutation rate of 1.2 × 10-8 calculated using the variants in the seed-propagated plants was consistent with the values reported previously. The ratio of single nucleotide variants (SNVs) among the variants in the seed-propagated plants was 91.1%, which is higher than 56.1% previously reported, and not significantly different from those in the regenerants. Overall, the ratio of transitions to transversions of SNVs was lower in the regenerants as shown previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

High-throughput phenotypic screening of random genomic fragments in transgenic rice identified novel drought tolerance genes.

KEY MESSAGE: Novel drought tolerance genes were identified by screening thousands of random genomic fragments from grass species in transgenic rice. Identification of agronomically important genes is a critical step for crop breeding through biotechnology. Multiple approaches have been employed to identify new gene targets, including comprehensive screening platforms for gene discovery such as the over-expression of libraries of cDNA clones. In this study, random genomic fragments from plants were introduced into rice and screened for drought tolerance in a high-throughput manner with the aim of finding novel genetic elements not exclusively limited to coding sequences. To illustrate the power of this approach, genomic libraries were constructed from four grass species, and screening a total of 50,825 transgenic rice lines for drought tolerance resulted in the identification of 12 reproducibly efficacious fragments. Of the twelve, two were from the mitochondrial genome of signal grass and ten were from the nuclear genome of buffalo grass. Subsequent sequencing and analyses revealed that the ten fragments from buffalo grass carried a similar genetic element with no significant homology to any previously characterized gene. The deduced protein sequence was rich in acidic amino acid residues in the C-terminal half, and two of the glutamic acid residues in the C-terminal half were shown to play an important role in drought tolerance. The results demonstrate that an open-ended screening approach using random genomic fragments could discover trait genes distinct from gene discovery based on known pathways or biased toward coding sequence over-expression.

The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation.

Although great progress has been achieved regarding wheat genetic transformation technology in the past decade1-3, genotype dependency, the most impactful factor in wheat genetic transformation, currently limits the capacity for wheat improvement by transgenic integration and genome-editing approaches. The application of regeneration-related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency4-11. In the present study, we found that overexpression of the wheat gene TaWOX5 from the WUSCHEL family dramatically increases transformation efficiency with less genotype dependency than other methods. The expression of TaWOX5 in wheat calli prohibited neither shoot differentiation nor root development. Moreover, successfully transformed transgenic wheat plants can clearly be recognized based on a visible botanic phenotype, relatively wider flag leaves. Application of TaWOX5 improved wheat immature embryo transformation and regeneration. The use of TaWOX5 in improvement of transformation efficiency also showed promising results in Triticum monococcum, triticale, rye, barley and maize.

Tissue culture protocols for gene transfer and editing in maize (Zea mays L.).

Efficient methods for gene transfer to maize were developed in the 1990s, first mediated by particle bombardment and then by Agrobacterium tumefaciens. Both methods can efficiently create high-quality events. Genetically modified varieties were commercialized in 1996 and are now planted in more than 90% of the US corn field. Tissue culture protocols for both methods have been well developed and widely employed. Thus, various factors, including handling before gene delivery, techniques to protect cells during gene delivery, and culture media, have been well optimized for various genotypes. Typical protocols for both methods are herein presented to show major outputs from the studies conducted since the early 1990s. As the bombardment protocols tended to be optimized specifically for limited genotypes, the one for B104, a new public inbred with favorable agronomic characteristics, is shown. The Agrobacterium protocol is suitable for various inbred lines, including B104. These protocols are also useful starting points in the optimization of tissue culture for gene editing. The rate-limiting step in both transformation and gene editing is in tissue culture and plant regeneration from modified cells in elite germplasm. Despite the prolonged efforts, large varietal differences in tissue culture responses remain a serious issue in maize. Recently, protocols using morphogenic regulator genes, such as Bbm and Wus2, have been developed that show a strong potential of efficiently transforming recalcitrant varieties.

Binary vectors and super-binary vectors.

A binary vector is a standard tool in the transformation of higher plants mediated by Agrobacterium tumefaciens. It is composed of the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, selectable marker genes, reporter genes, and other accessory elements that can improve the efficiency of and/or give further capability to the system. A super-binary vector carries additional virulence genes from a Ti plasmid, and exhibits very high frequency of transformation, which is valuable for recalcitrant plants such as cereals. A number of useful vectors are widely circulated. Whereas vectors with compatible selectable markers and convenient cloning sites are usually the top criteria when inserting gene fragments shorter than 15 kb, the capability of maintaining a large DNA piece is more important for consideration when introducing DNA fragments larger than 15 kb. Because no vector is perfect for every project, it is recommended that modification or construction of vectors should be made according to the objective of the experiments. Existing vectors serve as good sources of components.

Rice, indica (Oryza sativa L.).

Indica varieties, which are generally recalcitrant to tissue culture and transformation, occupy 80 % of rice cultivation area in the world. Therefore, transformation method for indica rice must be improved greatly so that global rice production would take full advantage of cutting-edge biotechnology. An efficient protocol for indica transformation mediated by Agrobacterium tumefaciens is hereby described. Immature embryos collected from plants in a greenhouse are cocultivated with A. tumefaciens after pretreatment with heat and centrifuging. The protocol was successfully tested in many elite indica cultivars such as IR8, IR24, IR58025B, IR64, IR72, Suweon 258, and Nanjing 11, yielding between 5 and 15 of independent transgenic plants per immature embryo. The use of immature embryos is recommended because gene transfer to them could be much more efficient and much less genotype dependent than gene transfer to callus.

Wheat (Triticum aestivum L.) transformation using immature embryos.

Wheat may now be transformed very efficiently by Agrobacterium tumefaciens. Under the protocol hereby described, immature embryos of healthy plants of wheat cultivar Fielder grown in a well-conditioned greenhouse were pretreated with centrifuging and cocultivated with A. tumefaciens. Transgenic wheat plants were obtained routinely from between 40 and 90 % of the immature embryos, thus infected in our tests. All regenerants were normal in morphology and fully fertile. About half of the transformed plants carried single copy of the transgene, which are inherited by the progeny in a Mendelian fashion.

Progress of cereal transformation technology mediated by Agrobacterium tumefaciens.

Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.

Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed.

Here, we provide comprehensive, highly efficient protocols for Agrobacterium tumefaciens-mediated transformation of a wide range of rice genotypes. Methods that use either immature embryos (japonica and indica rice) or calli (japonica cultivars and the indica cultivar, Kasalath) as a starting material for inoculation with Agrobacterium are described. Immature embryos are pretreated with heat and centrifugal force, which significantly enhances the efficiency of gene transfer, and then infected with Agrobacterium. Callus is induced from mature seeds and infected. Transformed cells proliferated from these tissues are selected on the basis of hygromycin resistance, and transgenic plants are eventually regenerated. A single immature japonica or Kasalath embryo will produce between 10 and 18 independent transgenic plants; for other non-Kasalath indica varieties, the number of transgenic plants expected will be between 5 and 13. For japonica and Kasalath, transformants should be obtained from between 50 and 90% of calli. From inoculation with Agrobacterium to transplanting to soil will take 55 d for japonica and Kasalath, and 74 d for indica other than Kasalath using the immature embryo method, and 50 d for japonica and Kasalath using the callus method.

Agrobacterium-mediated transformation of maize.

Maize may be transformed very efficiently using Agrobacterium tumefaciens-mediated methods. The most critical factor in the transformation protocol is the co-cultivation of healthy immature embryos of the correct developmental stage with A. tumefaciens; the embryos should be collected only from vigorous plants grown in well-conditioned glasshouses. With the protocol described here, approximately 50% of immature embryos from the inbred line A188 and 15% from inbred lines A634, H99 and W117 will produce transformants. About half of the transformed plants are expected to carry one or two copies of the transgenes, which are inherited by the progeny in a mendelian fashion. More than 90% of transformants are expected to be normal in morphology. The protocol takes about 3 months from the start of co-cultivation to the planting of transformants into pots.

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.).

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.