Strawberry is a model plant for both Rosaceae and non-climacteric fruit ripening. Although much progress has been made underlying the molecular mechanism of strawberry fruit ripening using transiently-genetic fruit transformation and stably-genetic transformation systems, in which some limits are existed [1–14, 16, 37]. Notably, it is previously reported that the Agrobacterium-mediated seed transformation may facilitate the entry of Agrobacterium tumefaciens into the seeds to integrating a target gene into the embryo cells [31–32], similar to the pollen-tube mediated genetic transformation method widely used in Arabidopsis thaliana [24, 30]. Reference of the seed transformation method [31–32], we first establish the Agrobacterium-mediated transformation of the germination seeds in diploid strawberry by a series of optimization conditions.
First, some factors key to transformation efficiency were confirmed, including seed imbibition time, suspension Agrobacterium concentrations, Agrobacterium infection time, kanamycin concentration in selection of transgenic plants. (1) Full imbibition is requisite for seed transformation. It is early reported that in Arabidopsis seed transformation, 12-h imbibition has the highest transformation efficiency, less than 9 h showing no transformants . In strawberry, we demonstrate that seed imbibition to reach both testa rupture and, radicle emerging is vital for transformation efficiency (Fig. 2). In addition, Agrobacterium concentrations and infection time are also critical to transformation efficiency . We also confirm that 24-h infection time has maximum transformation efficiency, in consistent with the previous reports in seed transformation [31, 32]. Generally, in the conventional leaf disk method, Agrobacterium with concentration at 0.3–0.5 of OD600 has high vigor and is used as an optimal concentration for infection , in that a higher Agrobacterium concentration causes harm to explant differentiation including browning even death, and a lower concentration is not enough Agrobacterium for T-DNA integration of explants with lower transformation efficiency. In the present study, we find that the germination seeds were more tolerant to Agrobacterium tumefaciens, and OD600 of 1.5 is an optimal concentration for highest transformation efficiency (Fig. 2). Finally, the appropriate concentration of kanamycin is also pivotal for selection of transgenic positive plants. In Arabidopsis seed transformation, T1 and T2 generation positive seedlings could be selected on medium containing 100 mg/L kanamycin . In Kenaf seed transformation, F0 and F1 plants were successfully obtained on medium containing 50 mg/L kanamycin . In the present study, we find that strawberry seedlings were selected on medium containing 75 mg/L kanamycin, in which positive transgenic plants could developed true leaves, whereas not in the control (Fig. 2). Taken together, we confirm high efficiency transformation parameters in the Agrobacterium-mediated transformation of the germination seeds of diploid strawberry, including radicle initially-emerging seeds, 1.5-OD600 Agrobacterium, 24-h infection, and 75 mg/L kanamycin in transgenic plant selection.
In fact, how to early select positive transgenic seedlings is more important in genetic transformation. It is previously demonstrated that the reporter gene is a good strategy for early transformation selection, such as neomycin phosphotransferase gene (NPTⅡ), green fluorescent protein (GFP), red fluorescent protein (DsRed), and β-glucuronidase (GUS) genes [31, 40]. In the present study, apart from the GFP and GUS genes (Fig. 2), we also used the reporter gene encoding magnesium chelatase H subunit (CHLH) functioned in chlorophyll biosynthesis, in that when its expression is downregulated, resulting in yellow or white leaf phenotype and thus using as a good report gene [33–36]. We construct the recombination pK7GWIWG2 (II) RR-FvCHLH vector with two reporter genes, DsRed and CHLH, which used as early selection. On the basis of DsRed fluorescence, it is easy to pick out early potential transgenic seedlings, which is further confirmed to be positive transgenic plants by kanamycin selection (Figs. 3 and 4). The first selection by DsRed 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 transgenic plants. One month after the transformation, through the reporter gene CHLH, we may observe the leaves of transgenic plants with a loss-of-green phenotype (Fig. 5). In all, through the two reporter genes, the Agrobacterium-mediated transformation of strawberry seeds was successfully established.
To further confirm the Agrobacterium-mediated transformation of the germination seeds, we also carried out kanamycin resistance and PCR analysis in T2 seedlings (Fig. 6): (a) when strawberry seedlings were selected on medium containing 75 mg/L kanamycin, the true leaves of kanamycin-resistant T2 seedlings can develop normally, whereas the growth of wild-type seedlings and kanamycin-sensitive T2 seedlings was partially inhibited and true leaves failed to develop; (b) through PCR test, the integration of DsRed gene and Kanamycin resistance gene into the genome of kanamycin-resistant T2 seedlings have been confirmed, whereas in wild-type seedlings and kanamycin-sensitive T2 seedlings, corresponding gene integration has not been detected. By analysis of the transgenic characteristics of transformed progeny (T1 and T2 generation), we provide strong evidence to confirm that the Agrobacterium-mediated transformation of strawberry seeds was successfully established.
In summary, we have first established a fast and efficient protocol for Agrobacterium-mediated transformation of strawberry seeds: (1) seed imbibition time (just to the point of radicle emergence) is a key step; (2) Agrobacterium concentration and the infection time are also critical for successful transformation; (3) the selection of kanamycin-resistant seedling takes 1 month, T2 generation transgenic plants are obtained within 4 months; (4) the transformation efficiency is 10%. Thus, this method can greatly shorten the experimental cycle and simplify the operation processes. Give that strawberry is a model plant for both Rosaceae and non-climacteric fruit ripening, the Agrobacterium-mediated transformation of strawberry seeds is to be widely used and will facilitate fruit development research and breeding.