Establishment of A Rapid and Stable Infected System by Agrobacterium-Mediated Transformation of Germination Seeds in Diploid Strawberry.

Highlight: Using the CHLH (the H subunit of magnesium chelatase magnesium chelatase) as a reporter gene, we first have successfully established Agrobacterium -mediated transformation of germinating seeds (AMTGS) in diploid 20 strawberry ( Fragaria vesca ), providing a useful tool for identification of gene function. Abstract Strawberry ( Fragaria ) is regarded as a model plant for both Rosaceae and non-climacteric fruit ripening. Although much 4 progress has been made in identification of gene function using stable and transient genetic transformation systems in 5 strawberry, the limitation is, more or less, are present. To this end, development of a rapid, efficient, and stable 6 transformation system is required for strawberry research and breeding. Here, using diploid Hawaii-4 ( Fragaria vesca ) seeds and a reporter gene of CHLH (the H subunit of magnesium 9 chelatase magnesium chelatase) key to chlorophyll synthesis, we first develop a rapid, efficient, and stable infected 10 system by the Agrobacterium -mediated seed infection to silence the reporter gene, reaching an infection frequency with 11 28.3% through a series of optimization elements, including seed full imbibition and initial germination, shaking infection 12 for 24 h, dark cultivation on MS medium for 3 d at 24 ℃ , light culture on MS-Tim medium for 1 week at 24 ℃ , and 13 vector construction tagged with fluorescence label. Taken together, radicle-emergence germination seeds, appropriate 14 Agrobacterium concentration and infection time are critical for successful infection, finally obtaining the infected 15 kanamycin-resistant seedlings of T1 generation by infected wild seeds within 1 month and T2 generation-infected plants 16 within 4 months.


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The mature and immature embryo-generated calli are also used to develop infected plants in rice, maize, wheat and 10 barley [21][22][23]27]. Notably, non-tissue culture-based methods, such as the floral dip infection, is widely and efficiently 11 used in Arabidopsis as a model system [24][25][26], and the Agrobacterium-mediated infection of germinating seeds 12 (AMTGS) of Arabidopsis is also early reported [31]. In recent years, the AMTGS of kenaf (Hibiscus cannabinus) seeds   that it is tend to usage of the diploid for scientific studies rather than the octaploid, and the results gained from the diploid 23 are appropriate for the octaploid application. Thereby, in the present study, we recruit the diploid and integrate these 24 reported methods, apart from the GFP and GUS genes (Fig. 2), we also used the reporter gene CHLH to construct the 25 recombination pK7GWIWG2 (II) RR-FveCHLH vector carried with the two reporter genes, DsRed and CHLH, which 26 are used as early selection. On the basis of DsRed fluorescence, it is easy to isolate early potential infected seedlings, and 27 further confirming to be positive infected plants by kanamycin selection (Fig. 3 and 4). The first selection by DsRed 28 fluorescence may bypass a large quantity of seeds used for kanamycin selection, not only saving more time and labor, but also and making it easier to obtain infected plants. One month after the infection, through the reporter gene CHLH, we 1 may observe the leaves of infected plants with a loss-of-green phenotype (Fig. 5). In all, through the two reporter genes, 2 the Agrobacterium-mediated infection of strawberry seeds was first established successfully.

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Determination of seed imbibition extent and optimum kanamycin screening concentration 7 To establish rapid and stable infected system in strawberry, the Agrobacterium-mediated infection of germinating seeds 8 in diploid strawberry was first studied based on the previous reports [31,32]. We first find that there was no significant 9 difference in the germination rate of strawberry seeds under different light culture conditions, including light culture, 10 dark culture, and light culture after dark culture; also the three treatments had a few, half, and 90 % of seed germination 11 respectively with 4-d, 5-d and 6-d imbibition (Fig. 1a).

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To confirm optimal kanamycin concentration for screening, the sterilized strawberry seeds were cultured in MS 13 selection medium with kanamycin concentration at 0, 25, 50, 75, 100 mg/L for 15 days. The results showed that as the 14 concentration of kanamycin was 0 mg/L, 95 % of the seeds germinated, the plants grew normally and the leaf color was 15 dark green. Compared with no selection pressure, when the concentration of kanamycin increased by 25 mg/L, 91.7 % of 16 the seeds germinated, except for the roots with slightly-slow growth, the germinated seedlings continued to growth as the 17 same as the wild-type; when the concentration of kanamycin was 50 mg/L, 41.7 % of the seeds germinated, but the first 18 leaf of the germinated seedlings could not fully unfold; when the selection pressure was increased to 75 mg/L, only 20 % 19 of the seeds could germinate and the color of the two cotyledons of the seedlings were yellow and white; when the 20 selection pressure increased to 100 mg/L, 5 % of the plants germinated and died of browning (Fig. 1b, c). Taken together, 21 75 mg/L of kanamycin in MS-selection medium was selected as an antibiotic selection concentration.

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Optimization of infection parameters 24 After 3-d culture in the selection medium, the GFP could be observed in the successfully-infected plants (Fig. 2a, b).

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Similarly, the unsuccessfully-infected seedlings had no GUS expression (Fig. 2c, e), while the successfully-infected 26 seedlings showed blue in the roots and true leaves with GUS expression (Fig. 2d, f). Base on the observation of both GFP  The CHLH encoding the H subunit of magnesium chelatase magnesium chelatase involved in chlorophyll biosynthesis is 7 chosen as a reporter gene, which is used to early assess effectiveness in the seed infection system, due to its silencing 8 phenotypes with yellow/white leaves easy to be observed [33-36].

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Based on the optimal infection conditions obtained above, we infected germinating seeds at the stage of radicle 10 emergence of diploid strawberry 'Hawaii-4' using Agrobacterium strain GV3101 strain carrying the pK7GWIWG2 (II)

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RR -FveCHLH vector. After 3-day infection under dark culture, the seeds were transferred to MS-selection medium.

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After 3 days again, the seeds were screened by DsRed fluorescence. Then after 2 weeks, the fluorescent seedlings were 13 transferred to quartz sand and watered using nutrient solution containing 75 % kanamycin. The whole processes for 14 infection of germination strawberry seeds are shown in Fig. 3. We found that the DsRed + fluorescence observed in

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T2 seedlings of FveCHLH-RNAi plants retains kanamycin-resistance 1 T1 seeds of FveCHLH-RNAi infected plants were spread on the quartz sand soaked in the nutrient solution containing 75 2 mg/L kanamycin. In the presence of kanamycin, the seeds sensitive to kanamycin cannot germinate or the cotyledons 3 turn yellow 10 days after germination, and the true leaves could not develop. T2-resistant seedlings could germinate on 4 quartz sand with kanamycin and continued to grow normally (Fig. 6a). The Kanamycin resistant bands and DsRed bands 5 were detected in T2 generations (Fig. 6b). These results demonstrate that the infected plants can obtain by seed infection 6 and the relative genetic characteristics may be integrated into the T1 progeny. To this end, the rapid and stable infected 7 system is successfully established by the Agrobacterium-mediated infection of germinating seed.    higher Agrobacterium concentration causes harm to explant differentiation including browning even death, and a lower 7 concentration is not enough Agrobacterium for T-DNA integration of explants with lower infection efficiency. In the 1 present study, we find that the germination seeds were more tolerant to Agrobacterium tumefaciens, and OD 600 of 1.5 is 2 an optimal concentration for highest infection efficiency (Fig. 2). Finally, the appropriate concentration of kanamycin is

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which is further confirmed to be positive infected plants by kanamycin selection (Fig. 3 and 4). The first selection by

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DsRed fluorescence may bypass a large quantity of seeds used for kanamycin selection, not only saving more time and 20 labor, but also making it easier to obtain infected plants. One month after the infection, through the reporter gene CHLH,

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we may observe the leaves of infected plants with a loss-of-green phenotype (Fig. 5).

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To further confirm the Agrobacterium-mediated infection of the germination seeds, we also carried out kanamycin 23 resistance and PCR analysis in T2 seedlings (Fig. 6): (a) when strawberry seedlings were selected on medium containing 24 75 mg/L kanamycin, the true leaves of kanamycin-resistant T2 seedlings can develop normally, whereas the growth of 25 wild-type seedlings and kanamycin-sensitive T2 seedlings was partially inhibited and true leaves failed to develop; (b) 26 through PCR test, the integration of DsRed gene and Kanamycin resistance gene into the genome of kanamycin-resistant 27 T2 seedlings have been confirmed, whereas in wild-type seedlings and kanamycin-sensitive T2 seedlings, corresponding 28 gene integration has not been detected. By analysis of the infected characteristics of transformed progeny (T1 and T2 29 generation), we provide a line of evidence to demonstrate that the Agrobacterium-mediated infection of strawberry seeds was first established successfully. Based on the early report [41], we also speculate that transgenic epidermal cell division 1 (L1 layer) is likely as a result of phenotype 1, and the transgenic anticlinal cell divisions (L2 layer) is likely as a result of 2 phenotype 2, to some extents, the phenotype 1 is easy to generate T2, the chimeric phenotypes are generated.

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The seed culture condition in light incubator is as a 16 h photoperiod with a light intensity of 30,000 LX 29 (250μmol·m-2·s-1) at 24 ℃. Each treatment was inoculated with 20 seeds (three repeats) for germination.     reporter gene were used for early screening and statistics. After 2 weeks, the seedlings with fluorescence were 2 transplanted into quartz sand and covered with cling film for three days to prevent water loss, and watered with the 3 kanamycin-added nutrient solution to allow the seedlings to grow under appropriate selection pressure at condition with a 4 greenhouse at 24 ℃, light/dark 16/8 h.

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To determine factors related to infection efficiency, we performed a series of experiments, including OD values 6 (optical density at 600 nm, OD 600 =0.5, 1, 1.5, and 2), infection time (12 h, 24 h, and 36 h), and seed germination status (1 7 represents the seed that has not yet exposed the radicle; 2 represents the seed that has just sprouted the radicle; 3 8 represents that the seed has two cotyledons,). The optimal infection parameters were determined by investigation of GFP 9 using stereo fluorescence microscope (ZEISS-Axiocam 506 color) and GUS Histochemical Stain Kit (Solarbio, G3060).

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Kanamycin resistance test of T1 seeds 4 A number of seeds were obtained from a single inbred T1 generation, sprinkled on quartz sand, and irrigated with 5 kanamycin (75 mg/L). After 10 days of culture, the sensitivity of the plants to kanamycin resistance was observed, and 6 DNA molecules were analyzed for the resistant seedlings.                  efficiency. h Effect of seed germination status on infection efficiency (1 represents the seed that has not yet exposed the 6 radicle; 2 represents seed infected at the stage of radicle emergence; 3 represents that the seed has two cotyledons). i       c and e No GUS blue in the un-infected seedlings. d and f GUS blue in successfully-infected seedlings. c and d Infection seedlings after 3-d culture in MS-selection medium. e and f Infected seedlings after 2 weeks culture. g Effect of OD values on infection e ciency. h Effect of seed germination status on infection e ciency (1 represents the seed that has not yet exposed the radicle; 2 represents seed infected at the stage of radicle emergence; 3 represents that the seed has two cotyledons). i Effect of infection time on infection e ciency. Each treatment was treated with 20 seeds. The error bars represent the standard error (n=3). Different letters indicate statistically signi cant differences at P < 0.05 as determined by Duncan's test. Bars = 0.2 cm.    Line1 is T2-sensitiveseedlings, line2 is positive control, and line3-5 is T2-resistant seedlings.