Liu Cong, Tang Dongying, Zhou Zhengkun, Zeng Hui,Hu Xiaochun, Tan Yanning, Qin Peng, Deng Yong, Wu Jicai,Wang Yan, Yang Yuanzhu, Yuan Dingyang, Liu Xuanming, Lin Jianzhong

Letter

Efficient Transformation ofRice Mediated byand Generation ofTransgenic Genic Male-Sterile Rice with High Nitrogen Use Efficiency

Liu Cong1, #, Tang Dongying1, #, Zhou Zhengkun1, #, Zeng Hui1, #,Hu Xiaochun2, Tan Yanning3, Qin Peng2, Deng Yong1, Wu Jicai1,Wang Yan1, Yang Yuanzhu2, Yuan Dingyang3, Liu Xuanming1, Lin Jianzhong1

(Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, College of Biology, Hunan University, Changsha 410082, China; Academy of Seed Industry of Hunan Yahua, Changsha 410001, China; State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Hunan Academy of Agricultural Sciences, Changsha 410125, China; These authors contributed equally to this work)

Although somerice varieties have already been transformed by, there is still great demand of developing transformation system for economically importantvarieties. Here, a fast and efficient method of- mediated transformation forrice was developed, and antransgenic genic male-sterile (GMS)rice 628S was generated using this method. The regenerated transgenic plantlets can be obtained within three months, and the transformation efficiency achieved about 17%.transgenic 628S exhibited an enhanced capacity of ammonium assimilation and more effective panicles under low nitrogen conditions. Moreover, the hybrid rice lines generated bytransgenic 628S showed 8%–27% yield advantage than those by the non-transgenic 628S. Taken together, these results provide an efficient transformation method forrice and an opportunity to improve nitrogen use efficiency (NUE) and the heterosis of hybrid rice.

Rice production is critical since it is the second largest cereal crop providing staple food for more than one third of world’s population. To meet the growing demand of rice, genetic engineering is becoming increasingly important, and a timesaving genetic transformation system with a convenient transgenic-positive screening method is urgently needed. Though several protocols forrice transformation have been developed, they often take more than three months to get the regeneration seedlings from explants, and the transformation efficiency ofrice is extremely lower than that ofrice (Lin and Zhang, 2005). Here, a fast and efficient transformation method forrice was established, and then an NADP(H)-dependent glutamate dehydrogenase [NADP(H)-GDH] genefromwas introduced into a GMSrice variety Xiangling 628S (hereafter called 628S) using this method.

was inserted into a modified plant binary vector (Fig. S1-A), and the pre-cultured seeds with embryogenic calli were directly used for-mediated transformation (Fig. S1-B). As the transformation vector contained a red fluorescence protein gene () and a hygromycin resistant gene () (Fig. S1-A), both red fluorescence and hygromycin-resistant screens were conducted to select the positive transformation, which greatly increased the positive rate and accelerated the transformation process (Fig. S1-C). Additionally, the optimized culture media significantly improved the growth quality and differentiation rate ofrice calli (Fig. S1-D to -G). Consequently, the regenerated transgenicrice plantlets with a transformation efficiency of 17% were obtained within 3 months, which was markedly shorter than otherreported methods with 3–6 months (Lin and Zhang, 2005). Meanwhile, another GDH gene, derived from, was also successfully introduced into thevarieties Zhu1S and Hua 819 by using this method, and the transformation efficiencies were 19% and 15%, respectively (data not shown). Subsequently, PCR and red fluorescence analysis found thathad been integrated into the genomic DNA and successfully expressed in transgenic rice lines (Fig. 1-A and Fig. S2). Semi-quantitative PCR analysis and Western blotting further found that the transgenic linesandexhibited the highest expression levels of(Fig. 1-B and -C).Notablely,was dominantly expressed in the roots of transgenic rice due to its expression drived by a root-specific expression promoter(Fig. S1-A) from tobacco () (Fig. 1-D). Taken together, a timesaving and high efficient method forrice transformation was successfully developed, andtransgenic 628S lines were generated.

GDH catalyzes reversible deamination of-glutamate to 2-oxoglutarate (2-OG) in the presence of NAD(P)H as a cofactor, and plays a complementary role for ammonium assimilation in higher plants. Whereas, NADP(H)-GDHs from micro-organisms were recently reported to significantly enhance NUE in rice as their higher affinity for NH4+(Yan et al, 2020). The affinity of NcGDH for NH4+was reported to be significantly higher than that of rice endogenous GDHs (Wang and Tian, 2001), implying that NcGDH can be used for improving the NUE in rice. Thus, the effects of NcGDH on rice nitrogen assimilation were subsequentlyinvestigated. NADP(H)-GDH activityanalysisshowed that NcGDH significantly improved both the aminating and deaminating activities of transgenic lines (Fig. 1-E). Furthermore, the hydroponics experiments showed that, compared to the non-transgenic plants,transgenic lines had a stronger nitrogen assimilation capacity with higher nitrogen content at low nitrogen conditions (Fig. 1-F). The field trial indicated that the effective panicles per plant oftransgenic lines were markedly higher than those of non-transgenic lines under low nitrogen fertility (0, 37.5 kg/hm2), but with no obviousdifference under high nitrogen fertility (112.5 and 187.5 kg/hm2) (Fig. 1-G and -H). These results demonstrated that heterologous expression ofimproved the growth and effective panicles in rice, especially at low nitrogen fertility.

Fig. 1. Identification and phenotypic analysis oftransgenic 628S.

A, Red fluorescence emitted from germinated seeds oftransgenic 628S (T1). The yellow and white arrows indicate germinated seeds with and without red fluorescence, respectively. B, Semi-quantitative PCR analysis ofexpression level in differenttransgenic lines and control plants (CK). C, Western blotting analysis oftransgenic lines and CK. D, Expression pattern analysis ofin transgenic lines by qRT-PCR. E, NADP(H)-GDH activities in the roots of CK andtransgenic lines. F, Nitrogen contents of CK andtransgenic lines. G, Phenotypes of CK andtransgenic lines grown in the field under different nitrogen fertilizer conditions. Urea was used as nitrogen fertilizer. H, Effective panicles per plant of CK andtransgenic lines under different nitrogen fertilizer (urea) conditions. I, Phenotypic comparison of hybrid rice generated by CK andtransgenic lines grown under low nitrogen conditions at the maturity stage. J and K, Effective panicles per plant (J) and grain yield per plant (K) of different hybrid rice lines. 628S,,andwere used as the female parents; H268 and H611 were used as the male parents. In B, C and E–H, CK represents the negative control (non-transgenic 628S) plants.was used as an internal reference in B–D; Scale bars in G and I are 10 cm. Data in D–F, H, J and K are presented as Mean ± SD (= 3). * and **,≤ 0.05 and≤ 0.01 by the Student’stest, respectively

It is well-known that a distant hybridization can significantly improve the heterosis of offspring, but it’s hard to overcome cross-incompatibility (Yuan, 1997). Genetic engineering is a powerful tool to take the advantageous genes from distantly related species, and to break the boundaries of species and expand the gene pool of crops. As 628S is a GMSrice variety and widely used for the hybrid rice production, the effects of NcGDH on agronomic traits of the hybrid rice generated by 628S were also investigated. Two eliterice varieties H268 and H611 were crossed withtransgenic 628S lines for generation of the heterozygous first filial generation (F1). Then, the hybrid rice lines (F1) were grown in a field supplied with 37.5 kg/hm2nitrogen fertility (in the form of urea) and the yield-component traits were determined after harvest. As expected, the hybrid rice lines generated bytransgenic 628S showed an obvious growth advantage and an enhanced hybrid vigor with more panicles and grain yields under low nitrogen condition (37.5 kg/hm2) (Fig. 1-I to -K). Notablely, compared to the non-transgenic lines, the grain yields of hybrid rice lines fromtransgenic 628S were increased by 8%–27% (Fig. S3). These results further indicated thatis a promising candidate gene to improve NUE and to increase the heterosis of hybrid rice at low nitrogen condition.

In conclusion, a timesaving and efficient method for-mediated transformation ofrice was developed, and antransgenic GMSrice 628S with higher NUE was generated.

ACKNOWLEDGEMENTs

This study was supported by the National Science Foundation of China (Grant Nos. 31571635 and 31871595), Hunan Provincial Important Science and Technology Specific Projects (Grant No. 2018NK1010), Natural Science Foundation of Hunan Province, China (Grant No. 2020JJ4004), Public Subject of State Key Laboratory of Hybrid Rice (Hunan Hybrid Rice Research Center) (Grant No. 2019KF02), and China Postdoctoral Science Foundation (Grant No. 2020M682561). We thank Dr. Liu Hong at Fujian Agriculture and Forestry University for providing the fungusand for the assistance with cloning.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

File S1. Methods.

Fig. S1. Schematic diagram of plant expression vector and transformation procedure.

Fig. S2. Identification oftransgenic 628S.

Fig. S3. Grain yield of different hybrid rice lines.

Table S1. Primers used in the study.

Table S2. Medium components used forrice transformation.

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Lin Jianzhong (jianzhlin@hnu.edu.cn); Liu Xuanming (xml05@hnu.edu.cn)

23 December 2020;

9 July 2021

Copyright ? 2021, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2021.09.002