LaCl 3 treatment improves the Agrobacterium -mediated immature embryo 1 genetic transformation efficiency in maize

1 Agrobacterium -mediated genetic transformation of immature embryo plays an 2 important auxiliary role in the study of gene function and molecular breeding in maize. 3 However, the relatively low genetic transformation efficiency is still the bottleneck of 4 the application of this method, especially in commercial scale production application. 5 In this study, we found that pretreatment of immature embryos with LaCl 3 , a Ca 2+ 6 channel blocker, could improve the infection efficiency of Agrobacterium tumefaciens , 7 increase the proportion of resistant calluses, obtain more positive regenerated plantlets, 8 and finally improve the transformation efficiency in maize. This optimization 9 provides a new direction for improving the efficiency of plant genetic transformation 10 mediated by Agrobacterium tumefaciens .

Maize, a monocotyledon food crop, is also a feed crop and an energy crop. It is 2 the world's most widely grown crop and one of the most productive food crops in the 3 world. About one-third of the world's population depends on corn as a staple food. 4 Maize is a C 4 plant, which is also a model plant for studying the photosynthesis 5 (Mookkan, et al. 2017). Due to time and labor consuming and the existence of 6 interspecific reproductive barrier, which prevents target traits from being introduced 7 into recipient plants, so the utilization of excellent germplasm resources is limited to a 8 certain extent by conventional breeding (Ahmar, et al. 2020). However, transgenic 9 technology has greatly promoted the process of obtaining various resistance candidate 10 genes and new varieties based on gene function research. Even so, the efficient, short 11 cycle and stable genetic transformation system of maize is still a hindrance. 12 Transgenic technology is a powerful method to cultivate high yield, high quality, 13 resistant to biological and abiotic stress varieties of crop. During the development of 14 maize transgenic technology, scientists have invented many transformation methods, 15 such as electroporation (Fromm, et al. 1986), particles bombardment (Klein,et al. 16 1988), polyethylene glycol (PEG) treatment of protoplasts (GOLOVKIN, et al. 1993), 17 silicon carbide fibers (Kaeppler, et al. 1994) and Agrobacterium-mediated 18 transformation (Ishida, et al. 1996). Among these transgenic transformation methods, 19 Agrobacterium-mediated transformation not only has a clear mechanism, simple 20 operation, low cost, but also has stable inheritance of exogenous genes and low copy 21 number (Liu, et al. 2017). Due to its many advantages, Agrobacterium-mediated 22 transformation is the most widely used genetic transformation method, especially in 23 commercial production (Chen, et al. 1998;Hiei, et al. 1997). 24 Since 1987, Grimsly et al. first used Agrobacterium to infect maize, and proved 25 that this method could transform maize. Subsequently, Ishida et al. established 26 relatively stable Agrobacterium-mediated genetic transformation system used maize 27 immature embryos as the explant for the first time in 1996. There have been many 28 studies on Agrobacterium-mediated optimization of immature maize embryo genetic 29 transformation system. Among them, many factors such as the vector, the genotype of    production still has some problems. Although Agrobacterium-mediated genetic 5 transformation efficiency of immature embryo in maize has been greatly improved 6 through continuous system optimization, and this method has also been widely used 7 in the commercialization of maize breeding, the low transformation efficiency is still 8 an urgent bottleneck to overcome in the application of maize genes function study and 9 molecular breeding.

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It is well known that Agrobacterium tumefaciens, as a naturally occurring 11 gram-negative bacterium, contains tumor induce (Ti) plasmid, which contains T-DNA 12 that can be integrated into recipient plant genomes after being horizontally transferred 13 into plant cells. Hence, Ti plasmid is modified to transform the target genes into the 14 plant genome, so as to achieve the transformation of the target genes in the recipient 15 species, therefore, known as "the smallest genetic transformation engineer in nature"   tumefaciens. 33 In this study, we developed an efficient optimize system that used the Ca 2+        36 37 The red fluorescence distribution of immature embryos and resistant calluses 38 6 were observed with a multifunctional zoom microscope (Nikon AZ100) at 510 nm 1 -560 nm. The fluorescence signal value in the pictures was calculated by self-written 2 script. The significance of the data was analyzed by t-test.  After all positive regeneration plantlets (T 0 ) were obtained, the editing efficiency 22 was calculated as: the editing efficiency (%) = number of edited regeneration plantlets 23 / number of positive regeneration plantlets × 100. 24 The significance of the data was analyzed by t-test.  To test the hypothesis mentioned above, we simultaneously pretreated the 1 immature embryos of ND101 with infection medium containing different 2 concentrations of LaCl 3 , which is a calcium channel blocker, at 45℃ for 5 minutes. 3 Meanwhile, the infection medium without LaCl 3 was used as the control. The intact 4 immature embryos isolated from tassels which 9 to 12 days after pollination as 5 explants, and used 25 immature embryos for each treatment and kept the embryos in 6 the infection medium for no more than 60 minutes. After the pretreatment, the 7 immature embryos were infected by Agrobacterium tumefaciens EHA105 harboring 8 the binary vector with RFP reporter gene ( fig. S1). Then they were transferred to 9 co-culture medium for co-cultivation respectively. pretreatment were significantly higher than the control group and those of other 15 concentrations ( Fig. 1 A and B); in addition, the infection efficiency of 10 mM LaCl 3 16 pretreatment was the highest (Fig. 1C). After that, we observed the callus induced 17 after 14 days under the screening condition and calculated the rate of callus, and 18 pre-differentiated callus clumps cultured for 12 days were also followed ( Fig. 1A and   19 D). The results indicated that pretreatment with 10 mM LaCl 3 had the best 20 performance in the state of pre-differentiation, had no effect on callus formation and 21 significantly improved the infection efficiency mediated by Agrobacterium.   Fig. 2A), and the proportion of resistant callus was consistent with this (Fig. 2B). The 37 results suggested that pretreatment with LaCl 3 of immature embryos improved the  culturing for 20 days on regeneration medium, and it was obvious that the 14 experimental group pretreated with 10 mM LaCl 3 was better than the control group 15 (Fig. 3A). Furthermore, we counted the regeneration frequency that the proportion of 16 tissues differentiated with elongated shoots are in the total calluses. The results 17 showed that after LaCl 3 treatment, the regeneration frequency increased from 13.2% 18 to 27.2%, more than twice as high as in the control group (Fig. 3B). Subsequently, we 19 identified the bar positive T 0 plantlets by PCR and calculated the transformation 20 efficiency. The results revealed that the transformation efficiency increased from 8.40% 21 to 17.60% after LaCl 3 pretreatment. (Fig. 3C and D). Moreover, the number of 22 positive T 0 plantlets transplanted into the nutrition bowl of LaCl 3 pretreatment was 23 twice that of the control group, which was basically consistent with the above 24 conclusion (Fig. 3E). Our results indicated that pretreatment with LaCl 3 of immature 25 embryos improved Agrobacterium-mediated transformation efficiency in maize.

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To address whether LaCl 3 pretreatment of immature embryos had effects on the 27 morphology and fertility of regenerated plants, we followed the growth, development

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Base on the above, we established an optimized system to improve the efficiency 32 of Agrobacterium mediated genetic transformation by pretreating immature embryo 33 with LaCl 3 in maize (Fig. 4).

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The optimized protocol was validated by CRISPR/Cas9 system 36 To verify the validity of the optimized protocol, we constructed 6 CRISPR/Cas9 37 vectors targeting four maize genes, including four single-editing-target vectors and 38 two double-editing-target vectors. After that, we transformed the five of the vectors 1 into maize inbred line ND101 using the optimized protocol, while the other one 2 transformed using the non-optimized protocol as the control, and estimated the 3 regeneration frequency, transformation efficiency and editing efficiency of the 4 different transformation protocol respectively. The results suggested that the 5 regeneration frequency increased from 11.09% to 20.52%-25.21% after optimization, 6 and the average regeneration frequency was 23.03%. Consistently, the transformation 7 efficiency increased from 7.17% to 11.98%-12.95% after optimization, and the 8 average transformation efficiency was 13.25%. The regeneration frequency and 9 transformation efficiency were both doubled compared with control (Table 1) (Fig. S3A and B). Based on the above results, it is further confirmed that 18 LaCl 3 pretreatment improves the genetic transformation efficiency of immature 19 embryos mediated by Agrobacterium and has no effect on editing efficiency. frequency and transformation efficiency. We transformed 6 CRISPR/Cas9 vectors for 28 batch system verification, which revealed that the method protocol is indeed effective 29 in improving the regeneration frequency, transformation efficiency, and also the 30 delivery efficiency of editing vectors, and that had no effect on editing efficiency.

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Hence, in the present investigation, inhibition of Ca 2+ signal transduction triggered by 32 Agrobacterium infection in explants improves the efficiency transformation.  by Agrobacterium is improved. 16 In the LaCl 3 concentration test experiment, we found that high concentration of  S4A). Besides, we pretreated the immature embryos of 25 ND88 in the same way, which is one recalcitrant maize inbred line. Infection 26 efficiency was nearly doubled from 46.88% to 100%, and confocal results also 27 showed that the fluorescence quantity of RFP transient expression in immature 28 embryos after 2 days of co-culture was significantly higher than that in the control 29 group ( fig. S4B and C). The results further confirmed the feasibility that inhibition of 30 Ca 2+ signal transduction in explants could improve the infection efficiency and 31 transformation efficiency. 32 In summary, this optimized protocol provides a new idea for improving the 33 genetic transformation efficiency of maize. Meanwhile, perhaps in the near future, by 34 further deepening the optimization system, we may overcome the genotype dependent 35 obstacles of the Agrobacterium-mediated genetic transformation during the operation, 36 and also provide more opportunities for further basic research on crop gene function 37 and molecular breeding.   soil. In the above statistical analysis, error bars represent means ± SEMs. Statistical 7 differences were analyzed by student's t-test, n=10.