The gene TaCB1 overcomes genotype dependency in wheat genetic transformation

Genotype dependency is the most important factor in wheat genetic transformation, which further limits 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 e�ciency. In the present study, a wheat gene TaCB1 in the WUSCHEL family was identi�ed to dramatically increase the transformation e�ciencies of many wheat varieties without genotype dependency after its over-expression. The expression of TaCB1 in wheat calli did not prohibit shoot differentiation and root development. The application of TaCB1 can lighten the requirement to wheat immature embryo for plant regeneration. Transgenic wheat plants can be clearly recognized by the visible phenotype of wide �ag leaves. The promise function of TaCB1 on improving transformation e�ciency was also tested in T. monococcum, triticale, rye, barley, and maize.


Abstract
Genotype dependency is the most important factor in wheat genetic transformation, which further limits 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 e ciency.In the present study, a wheat gene TaCB1 in the WUSCHEL family was identi ed to dramatically increase the transformation e ciencies of many wheat varieties without genotype dependency after its over-expression.The expression of TaCB1 in wheat calli did not prohibit shoot differentiation and root development.The application of TaCB1 can lighten the requirement to wheat immature embryo for plant regeneration.Transgenic wheat plants can be clearly recognized by the visible phenotype of wide ag leaves.The promise function of TaCB1 on improving transformation e ciency was also tested in T. monococcum, triticale, rye, barley, and maize.

Main
The cereal crops, such as common wheat (Triticum aestivum L.) and its relative species, maize (Zea mays L.), barley (Horderum vulgare L.), and Indica rice (Oryza sativa L. ssp.indica), are not easy to be transformed and their transformation e ciencies are usually low.Recently, a big progress on wheat transformation mediated by Agrobacterium tumefaciens called as PureWheat technique has been made by Japan Tobacco Inc using modi ed tissue culture media and transformant selection regime, by which the transformation e ciency for a model wheat genotype Fielder is up to 50% 1 .Richardson et al. 2 employed this technique and obtained a transformation e ciency around 45% for a few Australian wheat cultivars.Moreover, 15 commercial Chinese hexaploid wheat cultivars were transformed with the e ciency from 2.9% to 22.7% using of the PureWheat technique 3 .However, PureWheat still has limitation in transforming many commercialized wheat cultivars which show strong genotypedependency, being low e ciencies or recalcitrant to transformation even using PureWheat 2,3 .Therefore, overcoming genotype-dependence in transformation is still needed for wheat improvement.
Although different tissue culture media and physical treatment displayed some effects on the improvement of the genetic transformation e ciency of cereal crops, it is well known that the genotype restriction is still the key limited factor in the transformation manipulation.Many researches have been conducted on the identi cation of plant genes related to in vitro regeneration, and a few candidate genes affecting somatic embryogenesis have been characterized such as somatic embryogenesis receptor kinase (SERK 4 ), LEAFY COTYLEDON1(LEC1) (LEC1 5 ), LEAFY COTYLEDON2(LEC2 6 ), NiR 7 , BABY BOOM (BBM 8 ), WUSCHEL (WUS 9,10 ).Especially, co-overexpression of BBM and WUS2 genes led to high transformation frequencies in several previously transformation-recalcitrant maize inbred lines 11 .Moreover, maize mature embryos and leaf tissues were all used to generate transgenic plants with available e ciencies assisting by the two morphogenic regulators of BBM and WUS2.In addition, the coexpression of BBM and WUS2 obviously enhanced the transformation e ciencies of some recalcitrant genotypes of sorghum [Sorghum bicolor (L.) Moench], Indica rice (Oryza sativa L. ssp.Indica), and sugarcane (Saccharum o cinarum L.).However, over-expression of WUS2 and BBM in the cereal crops led to many negative effects such as callus necrosis, di cult differentiation of shoots and roots, decreased fertility of transgenic plants, aberrant, stunted, and twisted phenotypes 11 .Most recently, the expression of GRF4-GIF1 chimera was found to achieve a transformation e ciency of 60% in teraploid wheat (Desert King and Kronos) and hexaploid wheat (Fielder), even 9-19% in two non-transformable hexaploid wheat (Hahn and Cadenza), using the PureWheat protocol 12 .In this paper we report that overexpression of a wheat gene TaCB1 related to e cient plant regeneration in the WUS family dramatically improve the transformation frequency of wheat as well as a few other cereal plants with less genotype-dependence and no obvious negative effects on callus differentiation and plant phenotype.
The WUS gene is an important regulator for somatic embryogenesis in Arabidopsis 13 .Based on the Arabidopsis WUS sequence, two wheat homologous genes (TaCB1 and TaCB2) were obtained.Gene TaCB1 was more closely related to Arabidopsis WOX5 and WOX7 genes containing WUS related homeobox domain (Extended data Fig. 1) according to the description on the classi cation of AtWUS protein structure by Graaff et al. 14 , which belongs to WOX5 type in the WUS gene family and speci cally is expressed in roots tips 15 .While, TaCB2 gene was more closely related to AtWUS gene (Extended data Fig. 1) and can be known as TaWUS exactly.Further, six homologous TaCB1 genes were obtained from a common wheat line CB037 (AABBDD, 2n=42), and three from a T. monococcum accession CItrl3961 (AA, 2n=14), and an Ae.speltoides (SS, 2n=14) accession PI554241 (Supplementary Table 1), respectively.Thus, we speculated that TaCB1 has at least nine copies in common wheat.Three homologous TaCB2 genes (TaWUS-A, TaWUS-B and TaWUS-D) were also obtained from CB037 (Supplementary Table 1).
The genomic sequences of TaCB1, TaCB2-A (TaWUS-A), TaCB2-B (TaWUS-B) and TaWUS-D isolated from CB037 were manipulated under the control of maize ZmUbi promoter and Nos terminator and then linked onto an expression vector pWMB111, respectively, which only contains the Bar gene in the T-DNA region (Fig. 1a).When the construct pWMB111-TaCB1 was introduced into two wheat genotypes (Fielder and CB037) by Agrobacteriummediated approach, the transformation e ciencies were nearly 100%, but the transformation e ciencies using the control vector pWMB111 were around 50% (Fig. 1a).While the transformation e ciencies using the constructs TaWUS-A, TaWUS-B and TaWUS-D were lower than that using pWMB111 because many regeneration shoots in the transformation experiments did not produce roots and were excluded in the transgenic plants.We observed that TaCB2 greatly promoted shoot production among which TaWUS-D was most effective.Therefore, to further verify the function of TaCB1 and TaCB2 in regeneration, vectors pWMB111, pWMB111-TaCB1 and pWMB111-TaWUS-D were transformed into several widely commercialized wheat cultivars, such as Zhongmai895, Jimai22, Jing411, Lunxuan987 and Zhengmai9023.Results showed that TaWUS-D could improve the transformation e ciencies of the cultivars except Zhengmai9023.While the e ciencies of all the varieties were greatly improved by TaCB1, nearly reaching 100% in Fielder, CB037 and Zhongmai 895 (Fig. 1b, 1d).These results indicate that TaCB1 is the best choice in the transformation e ciency improvement.
In the past ve years, 29 hexaploid wheat varieties in total including some special recalcitrant varieties were successfully transformed with various target genes by application of TaCB1.Without using TaCB1 in the transformation, Ningchun4 didn't regenerate green shoots, Jimai22 and Kenong199 showed only a few green shoots, and Fielder showed less than 10 green shoots on each immature embryo.Using TaCB1, all varieties displayed dozens of green shoots on each immature embryo (Fig. 2).Jimai22 is the most widely cultivated variety in China with 2 million hectares each year, and its immature embryos derived calli are very poor in quality.However, the quality of the Jimai22 calli overexpressing TaCB1 was dramatically improved (Fig. 2), and the transformation e ciency of this cultivar was increased from 5.8% to 55.4% (Fig. 1c and Supplementary Table 2).Similar improved results were also achieved in other wheat cultivars with the assistance of TaCB1 for obtaining transgenic plants (Extended data Fig. 2 and Supplementary Table 2).As shown in Fig. 1d and Supplementary Table 2, the application of TaCB1 increased the transformation e ciencies of CB037, Fielder and Kenong199, which are regarded as easily transformed wheat varieties with a transformation e ciency of 20-45% under the using of a common vector, to 96.2%, 94.5% and 75.7% from 39.9%, 45% and 17.7%, respectively.With the assistance of TaCB1, the transformation e ciencies of poorly transformed varieties Zhongmai895, Sumai3 and Jing411 (transformation e ciency is less than 8% using a common vector) were 82.7%, 57.4% and 17.5%, respectively.The transformation e ciencies of non-transformable varieties Bs366, Ningchun4, Aikang58, Xinong979, and Sunstate were 83.5%, 29.3%, 21.8%, 16.7%, and 9.1% using TaCB1, respectively.Moreover, transgenic plants were successfully generated from Zhengmai6694, Zhengmai9170, Zhongmai175, Luohanmai (a special landrace genotype) and Cang6005 using TaCB1 with an e ciency of 6.1-97.8%.During the past a few years, 2322 immature embryos of Fielder were transformed with a common vector, and 3290 immature embryos of Fielder and 5459 immature embryos of Jimai22 were transformed with a TaCB1 containing vector.The immature embryos used for transformation experiment from most varieties exceeded 1,000 in amount (Supplementary Table 2).Parts of the transgenic plants were con rmed by the Southern blotting, PCR and QuickStix Kit (Extended data Fig. 3).The above data demonstrated that the application of TaCB1 greatly improves the transformation e ciency with less genotypedependency in wheat.
Theoretically, Agrobacterium-mediated plant transformation depends on infection e ciency of bacteria and the ability of host cell regeneration.Obviously, the application of TaCB1 can enhance the ability of the host tissues for callus induction and regeneration.In this case, whether a target gene could transfer into wheat cells will limit the nal transformation e ciency, and high transformation e ciency could be achieved if the expression cassette can be well delivered into the target cells by the bacteria with a high e ciency.The sensitivity of different wheat genotypes to Agrobacterium varied in a large extent.Interestingly, despite genotypes differ in their transformation e ciency, the e ciencies were less related to their regeneration abilities even if TaCB1 was applied.According to our evaluation in a previous study 16 , the order of regeneration capacity for the widely cultivated Chinese commercial wheat cultivars was Zhongmai895, Ningchun4, Aikang58, Xinong979, and Jimai22.While the transformation e ciency of Jimai22 was 55.4% by virtue of TaCB1, being higher than Aikang58, Xinong979 and Ningchun4 in this study.To explain this phenomenon, two maize genes associated with anthocyanin biosysthesis ZmR and ZmC1 17 were transformed into three types of wheat genotypes with different transformation e ciency: high group (Fielder and Zhongmai895), medium group (Jimai22), and low group (Ningchun4, Aikang58, and Sunstate).The order of transient expression e ciency from high to low was Fielder, Zhongmai895, Jimai22, Ningchun4, Aikang58, and Sunstate (Extended data Fig. 4), which was consistent with the order of transformation e ciency using TaCB1.Therefore, we speculated that the transient transformation e ciencies might be more important than the regeneration ability on the premise of using TaCB1.In short, the application of TaCB1 basically could overcome genotype limitation in wheat genetic transformation when TaCB1 can be delivered into the immature embryos of the wheat genotypes by Agrobacterium infection.
The TaCB1 gene can make wheat transformation more e cient and stable.High temperature during growth period of mother plants, especially at the grain lling stage, negatively affected production and differentiation of embryonic calluses derived from the immature embryos, and nally led to transformation failure 18,19 .In the PureWheat standard methods, the physiological status of wheat immature embryos is also addressed to determine plant regeneration ability and transformation e ciency.When wheat mother plants are subjected to biotic or abiotic stress, the physiological state of the immature embryos will be deteriorated.Usually, these wheat immature embryos cannot generate embryonic callus.There was no good quality callus produced from the immature embryos of CB037 and Kenong199 plants with poor growing status in normal transformation experiments (Fig. 3a); while in the transformation with the assistance of TaCB1, the immature embryos from CB037 plants produced brown and limited grown calluses, from which some green shoots were still generated (Fig. 3b, c).Although calli of Kenong199 produced many roots (generally in our experiences, the quality of the calluses producing roots is very poor, which is normally di cult to generate shoots), but they also can generate many shoots by virtue of using TaCB1.Therefore, the application of TaCB1 can lighten the requirements to wheat immature embryo status in Agrobacterium-mediated genetic transformation.Moreover, under the presence of TaCB1 transgenic wheat plants were also obtained with an e ciency of 54.0% using the previously published media 18 Gene TaCB1 driven by the maize ubiquitin promoter was also tested in species in the tribe Triticeae.A T. monococcum line PI428182 and a tetraploid wheat line Dwar ng Polish wheat were transformed for generating transgenic plants under the assistance of TaCB1, and their transformation e ciencies were 94.5 and 15.1% (Table1), respectively.The transformation frequency of a barley cultivar Vlamingh was increased to 78.6% under the premise of using a vector containing TaCB1 from 10.1% employing a common vector.A few commercial barley genotypes including Supi3, Zhepi8, Baudin and Buloke, which were all recalcitrant to transformation, gave transformation frequencies of 24.7%, 20.3%, 17.6%, and 12.2%, respectively, on the premise of using TaCB1 (Table1).Additionally, TaCB1 also played a positive function in the transformation of Secale cereale L. and triticale (×Triticosecale Wittmack) with the transformation frequencies of 7.8% for Lanzhou Heimai and 53.3% for Linfen45, respectively (Table1).
To test the function of TaCB1 in more genotypes, TaCB1 was transferred into Japan Tobacco Inc via patent collaboration treaty (PCT).The transformation e ciency of Fielder was increased to 100% from 60% in control; the transformation e ciency of another wheat line Norin61 was increased to 24.3% from 0 in control; while the transformation e ciency of Chinese Spring wheat also enhanced to 29.8% was tested in maize, and the transformation e ciencies of two inbred lines B73 and A188 were from 16.4% in the control (Table2).The function of TaCB1 e increased to 23.2% and 38.1% from 0 and 8.1% in the control, respectively (Table2).
Compared to other reported regeneration related genes BBM, WUS2, and GRF4-GIF1, TaCB1 has certain advantages in the genetic transformation of wheat.Firstly, application of TaCB1 can signi cantly increase the transformation e ciency to high level.The transformation e ciency of some wheat varieties like Fielder, CB037, Zhoumai18, and Zhengmai6694 could be as high as 100% in a small scale and 90% in a large scale in many repeated experiments during last ve years (Supplementary Table 2).Moreover, one callus could generate dozens of green shoots with three replications (Fig. 2d), and most shoots produced could develop into normal plants with healthy roots (Extended data Fig. 4).Therefore, the transformation e ciency is calculated based on the immature embryos used for transformation, and only one shoot is considered for each immature embryo even though more shoots might be generated from a single embryo.If all the generated shoots are considered, the transformation e ciency will be far more than 100%.We found that wheat TaCB2, which is the homologous gene of maize WUS2 (Extended data Fig. 1), showed a much lower function on the promotion of transformation e ciency than wheat TaCB1 (Fig. 1a  and 1b).Secondly, TaCB1 can be singly used to increase transformation e ciency while the other reported genes needed to be co-expressed together in maize, sorghum and wheat transformation 11,12,20 .Moreover, the two genes of WUS2 and BBM needs to be eliminated from the transformed calli during the transformation process using Cre/LoxP system driven by dehydration inducible promoter because of their negative effects on callus differentiation and root development 11,20 .The TaCB1 gene is not necessary to be removed during the transformation as it has no negative effects.Lastly, TaCB1 can be used as a marker to distinguish transgenic plants.Although overexpression of TaCB1 had no negative effect on shoot differentiation and root development, it has some phenotypes in transgenic wheat plants including wide, short ag leaves and thick stems (Fig. 3d and Supplementary Table2).Therefore, the TaCB1 transgenic wheat plants can be easily recognized by its phenotype.
The newly identi ed TaCB1 in this study as a promising regenerative gene has a great potential application in genetic transformation and genome editing for cereal crops.We have incorporated TaCB1 onto the vectors containing double T-DNA region and Cas9 expression cassette, on which TaCB1 was linked with the Bar selection marker and Cas9, and TaCB1 can be removed together with Bar and Cas9 in the progenies of transgenic or edited plants.Although the application of TaCB1 cannot directly contribute to the frequency improvement of generating marker-free or Cas9 cleavage plants, it can greatly help to obtain more candidate plants for identifying marker-free or mutants.Similarly, the application of TaCB1 can help to reduce workload for identifying marker-free transgenic and Cas9-free edited plants by selecting the TaCB1 phenotype in the segregation population.
In summary, a total of 31 hexaploid wheat cultivars were transformed by using TaCB1, and the genotype dependency in transformation was overcome.In addition, overexpression of TaCB1 also signi cantly increased the transformation e ciencies of T. monococcum, triticale, rye (Secale cereale, RR, 2n=14), barley, and maize.The application of TaCB1 can make the genetic transformation and genome editing of wheat and other crops more e cient and economical by improving plant regeneration, reducing the requirement for embryo quality, and identifying transgenic or edited plants by visible phenotypes.

Plant and cultivation conditions
Wheat cultivars Zhengmai7698, Zhengmai1342, Zhengmai1860, and Zhengmai6694 were kindly provided by Prof. Weigang Xu at Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou, Henan province; Zhongmai895 and Zhongmai175 by Prof. Yong Zhang at Institute of Crop Sciences, Chinese Academy of Agricultural Science (ICS-CAAS), Beijing; Luohanmai by Prof. Ming Hao at Sichuan Agricultural University, Chengdu, Sichuan province; Cang6005 and Ji5265 provided by Prof. Xiulin Guo and Hui Li at Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, Hebei province, respectively.Fielder and Chinese Spring were obtained from Yokohama City University.Norin61 was obtained from Kyoto University.Wheat cultivars CB037, Ningchun4 and Kenong199 and a rye cultivar Lanzhou Heimai were maintained in our laboratory.A tetraploid (Triticum polonicum, AABB, 2n=28) line Dwar ng Polish wheat was provided by Prof. Minyi Wang at Sichuan Agricultural University, Chengdu, Sichuan province.Other wheat genotypes, including winter wheat cultivars Jimai22, Aikang58, Xinong979, and Jing411, were requested from the National Germplasm Bank at ICS-CAAS.All the barley cultivars Vlamingh, Supi3, Zhepi8, Baudin, and Buloke were kindly provided by Prof. Yanhao Xu at Yangtze University, Jingzhou, Hubei province.A T. monococcum (AA, 2n=14) accession PI428182 was kindly provided by Prof. Yueming Yan at Capital Normal University, Beijing; and triticale (AABBRR, 2n=42) cultivars ZS3297, ZS1257, ZS3224 and Linfen45 by Prof. Zengyuan Wang at ICS-CAAS and Prof. Fangpu Han at the Institute of Genetics and Developmental Biology of Chinese Academy of Sciences, Beijing, respectively.Maize inbred lines A188 and B73 were obtained from National Agriculture and Food Research Organization in Japan.

Cloning of TaCB1
The amino acid coding sequence of Arabidopsis WUS (AJ012310) registered in NCBI was used as a query for searching the homologous gene of wheat via tblastn, in which the organism was set as Triticum aestivum, resulting in only one sequence FN564431.1.Based on this sequence, a pair of primers of TaCB1F (GTGTCAATGGAGGCGCTGAGCG) and TaCB1R (ATGCGTGCGTGCGACGTTGATT) were designed to amplify TaCB1 from the genomic DNA of wheat line CB037.
It was found that TaCB1 is not the original WUS, so the protein sequence of AtWUS was used as a query for tblastn in IWGSC, and three contigs were obtained.According the contig sequences, a speci c primer pairs was designed to amplify the sequence, designated TaCB2.The identi cation and application of TaCB1 has been granted for Chinese invention patent (ZL201710422896.6)and applied for PCT with Japan Tobacco Inc (PCT/CN2018/090239).

Vector construction
The primers CB1SmaF: AAACCCGGGATGGAGGCGCTGAGCGG and CB1KpnR: AAAGGTACCTTAGACCAGATACCGAT were used to perform PCR ampli cation using pMD-18T-TaCB1 as a template with high delity enzyme KOD.Then, the PCR product and the pWMB003 vector (containing the ZmUbi promoter and the Nos terminator) were digested with KpnI and SmaI to obtain a 773 bp product and a 4535 bp vector backbone.Next, the target PCR product and the vector backbone were ligated to generate an intermediate expression vector pWMB003-TaCB1.The vectors pWMB003-TaCB1 and pWMB111 (containing a Bar expression cassette controlled by the Ubi promoter) were digested with HindIII to produce a 3033 bp TaCB1 expression frame and a 10,170 bp vector backbone, respectively.Finally, the two enzyme-digested products were ligated to generate the target expression vector pWMB111-TaCB1 for the transformation of wheat, T. monococcum, rye, triticale, and barley.The TaCB1 expression cassette was inserted into the vector pLC41 containing a Bar expression frame controlled by the 35S promoter and the Nos terminator for wheat varieties Chinese Spring and Norin61, and maize transformation.The expression vector containing TaCB2 was also constructed by the same method.The pWMB202 vector containing two anthocyanin biosynthesis related genes ZmR and ZmC1 17 were used to detect the transient transformation e ciency in different wheat genotypes.

Plant transformation
Wheat transformation.Spikes of wheat were harvested at 14 d post anthesis (DPA).Immature wheat grains were carefully collected.In the aseptic conditions, grains were surface-sterilized with 70% ethanol for 1 min, 5% sodium hypochlorite (NaClO) for 15 min, and rinsed 5 times with sterile water.Fresh immature embryos of wheat were isolated and transformed by Agrobacterium-mediated transformation to obtain transgenic plants following the protocol described by Ishida et al. 1 with slight modi cations.In brief, immature wheat embryos were incubated with Agrobacterium strain C58C1 harbouring vectors for 5 min in WLS-inf medium at room temperature, and cocultivated for 2 d on WLS-AS medium with scutellum facing upwards at 25 °C in darkness.After co-cultivation, embryonic axes were removed with a scalpel and the scutella were transferred onto plates containing WLS-Res medium for delay culture for 5 d under the same condition.Afterwards, tissues were cultured on WLS-P5 medium for callus induction.Two weeks later, calli were placed on WLS-P10 medium for 3 weeks in darkness.Embryogenic calli were then differentiated on LSZ-P5 medium at 25°C with 100 μmol/m 2 /s light.Regenerated shoots were transferred into cups lled with LSF-P5 medium for elongation and root formation.Plantlets with well-developed root systems were transplanted into pots and cultivated in growth chambers.The transformation of T. monococcum, rye, and triticale were performed by the same methods aforementioned for wheat.Barley transformation.Barley transformation was performed following the previously published protocols 21 with a slight modi cation.Immature embryos of barley were isolated after sterilization of the immature grains by the same methods as wheat, subsequently incubated with Agrobacterium for 10 min, and co-cultivated for 2 d on CM medium.Then, embryo axes were removed and the scutella were cultured on the rst selection medium.After 2 weeks, tissues were transferred onto the second selection medium.Three 3 weeks later, embryonic calli were cultured on DM medium at 25 °C with 100 μmol/m 2 /s light for differentiation.Shoots were timely moved into a plastic box containing RT medium.Lastly, plants were transplanted into pots lled with soil.Maize transformation.Maize transformation was performed following the previously published protocols 22 .Between 8 and 15 DPA, spikes containing immature embryos were harvested.Immature embryos were isolated and infected by Agrobacterium for 5 min.Then, embryos were transferred onto LS-AS solid medium with the scutellum face up, and incubated in darkness at 25°C for 7 d.After the co-cultivation steps, embryos were transferred onto the rst selection medium for 10 d, and then moved to the second selection medium and the third selection medium for 21 d, respectively.Next, embryonic calli were transferred onto LSZ medium for shoot differentiation.
The regenerated shoots were transferred into cups lled with LSF medium.Finally, plants with developed roots were transplanted into pots containing appropriate soil.

Detection of transgenic
Quickstix detection.plants were detected for the Bar gene by the QuickStix Kit (EnviroLogix) according to the manufacturer's instructions, which is used to detect the protein encoded by the Bar gene.PCR analysis.Genomic DNA was extracted from the leaves of transgenic plants using a NuClean PlantGen DNA kit (CWBIO, Taizhou, China).The primer pair 5'-ACCATCGTCAACCACTACATCG-3' and 5'-GCTGCCAGAAACCACGTCATG-3' was used to detect the Bar gene in the transgenic plant samples by PCR ampli cation with a 429-bp fragment.Southern blotting analysis.Total genomic DNA was extracted followed a standard CTAB method 23 , and 10 μg DNA from each sample was digested with HindШ.Then, the digested DNA samples were fractionated on a 0.8% agarose gel and transferred onto a nylon Hybond-N + membrane (Roche, Mannheim, Germany) with a membrane transfer instrument (Model 785, Hercules, California, Bio-Rad).The PCR products of Bar gene (429-bp) were labeled with Digoxigenin and used as probes to hybridize with the digested DNA on the membrane.The hybridization and detection steps were performed according to the instructions for the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Mannheim, Germany).The transformation e ciencies of Fielder and CB037 using the vectors containing TaCB1 and TaCB2 genes as well as the common vector pWMB111.c: The transformation e ciencies of Zhongmai895, Jimai22, Zhengmai9023, Lunxuan987 and Jing411 on the premise of using the vectors containing TaCB1 and TaWUS-D genes as well as the common vector pWMB111.d: The transformation e ciencies of 29 wheat varieties using the TaCB1 containing vector and the common vector.0: the transformation e ciency is 0%; and *: no data.

Figure 1 Effects
Figure 1