Reinvigoration of diploid strawberry (Fragaria vesca) during adventitious shoot regeneration

Diploid strawberry (Fragaria vesca ‘Baiguo’) is a model plant for studying functional genomics in Rosaceae. Adventitious shoot regeneration is essential for functional genomics by Agrobacterium tumefaciens-mediated transformation. An efficient shoot regeneration method using diploid strawberry leaf explants was conducted on 1/2MS + 1/2B5 medium that contained 2.0 mg L−1 TDZ over 14 days of dark culture; this induced the maximum percentage of shoot regeneration (96.44 ± 1.60%) and the highest number of shoots per explant (23.46 ± 2.14) after 11 weeks of culture. The explants considerably enlarged after 12 days; then, turned greenish brown after 30 days, yellowish brown after 36 days, and completely brown and necrotic after 48 days. Large numbers of adventitious shoots were produced from 48 to 66 days, and the shoots elongated from 66 to 78 days; this represents a critical period of reinvigoration, which included 30 days for leaf explant chlorosis, 36 days for adventitious shoot appearance, and 48 days for generation of numerous shoots. During the reinvigoration process, higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days. Our results indicate that these genes may regulate reinvigoration of shoot regeneration.

Diploid strawberry (Fragaria vesca), also known as the woodland strawberry, has a small genome (2n = 2× = 14, 240 Mbp), easy vegetative propagation, and a small, herbaceous stature 1 . These advantages make diploid strawberry more suitable as an attractive model than the commercial octoploid strawberry (Fragaria × ananassa) (2n = 8× = 56) and other plants of Rosaceae for functional genomics research 2 . Adventitious shoot regeneration is essential for subsequent functional genomics research by Agrobacterium tumefaciens-mediated transformation and is affected by internal and external influences.
Notably, we found that regenerating competence recovered in the nearly brownish diploid strawberry explants, and shoots were regenerated from 30 to 48 days of culture. This may represent a selective process in which cells with low competence in response to the plant growth regulator treatment are killed, whereas a few competent cells from specific leaf tissue, potentially parenchymatic cells, start to divide and differentiate into a new adventitious shoot over time 11 . Similarly, "reinvigoration" of plants has been pursued by methods such as grafting and 6-benzyladenine metabolism. The term reinvigoration refers to the reversion of senescence 12,13 and was used to describe the reversion of senescence that occurs when leaf explants are cultured in the regeneration medium 14 . This senescence is a general response that occurs in all strawberry explants, cultivated or wild, and it is reflected by chlorosis of the explant prior to regeneration 8,15 . Reinvigoration is critical for efficient shoot regeneration in diploid strawberry. However, less is known about molecular aspects of leaf explant reinvigoration events. Molecular mechanisms underlying shoot regeneration have been extensively studied, and considerable evidence has demonstrated important roles of plant growth regulators, such as cytokinins 16 , auxin 17 , and gibberellin 18 , in regulating shoot regeneration. Consequently, the genes involved in hormonal control may affect reinvigoration of leaf explants during shoot regeneration.
In this study, we reported a reinvigoration phenomenon during adventitious shoot regeneration of diploid strawberry, analysed the detailed changes over a long period (at least 11 weeks), and carried out a detailed transcript analysis of related genes during reinvigoration. The relationships between reinvigoration of adventitious shoot regeneration and transcript level variations were also revealed.

Results and Discussion
Reinvigoration during adventitious shoot regeneration of diploid strawberry F. vesca 'Baiguo' leaf explants. The morphogenic responses of somatic tissue have been studied in vitro. There are several lines of F. vesca and F. ananassa that have been tested for in vitro regeneration and showing a different behaviour 19 . Several blackberry and raspberry lines have been tested for in vitro regeneration and showed different morphogenic responses to plant growth regulator combinations 20 . Our results showed that adventitious shoot regeneration of diploid strawberry F. vesca 'Baiguo' differed from that of octoploid strawberry 'Honeoye' . The octoploid strawberry leaf explants were green during regeneration (Fig. 1A). The explants showed elongation and enlargement after 6 days of culture. The shoot organogenesis from very small callus was formed from the cuts of explants after 15 days culture by histological observations 8 . A large callus formed after 18 days of culture. Relatively small shoots were observed that were sporadically dispersed within the callus on day 21 of culture, and clustered shoots were observed on day 27. The number of adventitious shoots and their growth increased over time, with most shoots being longer on day 33 of culture 8 . During whole shoot regeneration of octoploid strawberry, the leaf explants remained green and invigorated, and adventitious shoots typically appeared after 33 days.
Compared with octoploid strawberry 'Honeoye' , adventitious shoots regenerated more in browning leaf explants of F. vesca 'Baiguo' (Fig. 1B). After 12 days of culture, the F. vesca 'Baiguo' explants considerably enlarged. After 30 days of culture, the explants turned greenish brown. After 36 days of culture, the explants turned yellowish brown, produced a yellow-green callus, and sporadically formed adventitious shoots around the wound edges. After 48 days of culture, the explants turned completely brown, but many shoots appeared. From 48 to 66 days of culture, adventitious shoots nearly doubled in number and elongated. After 66 days of culture, adventitious shoots were even more elongated. Therefore, the critical periods occurred when there was reinvigoration of completely brown explants that resulted in shoot regeneration, which included 30 days for chlorotic leaf explants, 36 days for adventitious shoot appearance, and 48 days for generation of several shoots.
Dark treatment effects on reinvigoration during adventitious shoot regeneration from the leaf explant of F. vesca. Subsequently, to study the effect of dark treatment on reinvigoration, various periods of initial dark culture (from 0 to 28 days) were applied. Although dark treatments did not influence the reinvigoration outcomes, the duration of dark culture did (Tables 1 and S1). Both the control explants (0 days, no initial dark culture treatment) and explants initially kept under dark culture conditions regenerated adventitious shoots. The adventitious shoot regeneration rate (from 90.67 ± 7.34% to 96.44 ± 1.60%) and number of shoots per explant (from 19.62 ± 1.88 to 23.46 ± 2.14) after the initial 7, 14, and 21 days of dark culture were not different from that in the control, but were significantly higher than that during the initial 28 days of dark culture (63.56 ± 10.45% and 7.86 ± 1.52 for adventitious shoot regeneration rate and number of shoots per explant, respectively) (p < 0.05) (Tables 1 and S1). On the other hand, dark treatments did not influence the reinvigoration process, the duration of dark culture did ( Fig. 2A). However, dark treatments only influenced reinvigoration for the first 7 days. The other dark treatments are lower than 0 day in adventitious shoot regeneration rate between 42-60 days after culture ( Fig. 2A). Furthermore, chlorosis of leaf explants after the initial 7, 14, and 21 days of dark culture from 30 to 48 days was less obvious than in the control, but more obvious than that in the initial 28 days of dark culture (Figs 2B and S2); this might be attributed to light having negative interactions with hormones that induces oxidative stress which kills cells 21 , dark incubation enhancing the accumulation of endogenous IAA 22 , and dark treatments leading to the reduction of photosynthetic capacity and a reduced efficiency of the chloroplasts 8,10,23 . We also found that, when the regenerated shoots were inoculated on 1/2MS + 1/2B 5 medium, the leaves of shoots turned yellow after 66 days; then, there was reinvigoration, which facilitated more continuous shoot growth and vigorous shoot generation after 66 days of culture (Fig. 2C). The results showed that dark treatments did not affect the leaf explant reinvigoration, but the duration of dark culture did. The best dark culture condition for reinvigoration (14 days) was then used to study the molecular aspects of reinvigoration.   www.nature.com/scientificreports www.nature.com/scientificreports/ began to enlarge or produce shoot primordia (Fig. 3A,B). This is consistent with zeatin concentration variation in in vitro-cultivated strawberry (F.ananassa cv. 'Honeoye') leaf explants; the zeatin concentrations peaked at 5.98 ng g −1 Fw during meristemoid formation and reached 7.69 ng g −1 Fw during shoot primordia formation 8 . www.nature.com/scientificreports www.nature.com/scientificreports/ Furthermore, the ZOG and Ciszog transcript levels were the highest at 72 days of culture, during which senescence was delayed and adventitious shoots very elongated. This could be explained by the two-step adventitious shoot regeneration process that was first observed in in vitro-derived lingonberry leaves 25 . www.nature.com/scientificreports www.nature.com/scientificreports/ Cytokinin hydroxylase (CKH) is involved in trans-zeatin biosynthesis, and contributes to tissue and organ formation 26 . The CKH transcript level significantly increased from 0 to 24 days of culture, which was during leaf explant shoot primordia formation. The CKH transcript level was higher from 30 to 48 days of culture, during which reinvigoration occurred and leaf explants began to produce multiple shoots (Fig. 3A,B).
Cytokinin oxidase/dehydrogenase (CKX) is involved in cytokinin degradation and plays important roles in plant meristem activity and morphogenesis, including causing changes in root growth and xylem differentiation 27 , and has opposite regulatory functions in root and shoot meristems 28,29 . The transcript levels of some CKX genes (CKX2, CKX3, CKX5, CKX6, and CKX7) increased during the initial days of culture (from 0 to 6 days), which was when there was leaf explant enlargement (Fig. 3A,B). The CKX2, CKX3, and CKX7 transcript levels significantly increased from 30 to 48 days of culture, during which leaf explants were reinvigorated and many shoots appeared. However, the CKX2 and CKX3 levels were lower at 12 and 18 days than at 6, 24, 30, and 36 days. Additionally, the CKX7 level was substantially lower at 30 and 36 days compared with 12, 42, and 48 days. These peaks were consistent with the findings of zeatin concentration variation in in vitro-cultivated strawberry leaf explants 8 .
Noticeable increases in the transcript levels of cytokinin biosynthesis-related genes were associated with the critical periods, including the first 6 days, during which there was explant enlargement, and 30 to 48 days of culture, during which there was diploid strawberry explant reinvigoration. The results obtained are in consistent with the initial phase of leaf senescence due to cytokinin degradation by CKX.
YUCCA (YUC) catalyses hydroxylation of the tryptamine amino group, which is a rate-limiting step in auxin biosynthesis 30,31 . The YUC genes play essential roles in auxin biosynthesis and plant development, including floral organ and vascular tissue formation in Arabidopsis 32 , crown root development control in rice 33 , and soybean growth and development alteration 34 . The YUC1, YUC2, YUC3, YUC5, YUC7, YUC8, YUC9, YUC10, and YUC11 transcript levels significantly increased from 0 to 6 days of culture, which was when there was leaf explant enlargement. This can be explained by dark incubation having a positive effect on auxin-related gene stimulation, and thus endogenous auxin production and eventually leaf explant enlargement 22,35 . The transcript level of YUC6 is higher in 0 day than in 6 days may be ascribed to wounding first triggers short term wound signaling that lasts from seconds to hours, then the wound signal spreads very rapidly from the wound site to mesophyll cells and activates YUC expression in mesophyll cells 36 . The YUC2, YUC5, YUC6, YUC9, and YUC10 transcript levels significantly increased during reinvigoration (Fig. 3A,B). These noticeable increases in YUC gene transcript levels during the critical periods (including 30, 36, and 48 days of culture) indicate that they have roles in diploid strawberry reinvigoration.
Gibberellin 2-beta-dioxygenase (GA2ox) is involved in GA inactivation, which is associated with Glycine max development 37 , might play critical roles in cell division 38 . The GA2ox transcript level was substantially higher from 30 to 48 days of culture, which was when explant reinvigoration was observed (Fig. 3A,B). GA20ox might modify cell growth and development associated with explant reinvigoration. The GA20ox transcript level was much higher before 48 days of culture, which indicates that it is related to explant reinvigoration (Fig. 3A,B). The increased GA2ox levels prior to shoot regeneration, concomitant with a reduction in GA20oxydase (involved in GA synthesis), might indicate that a reduction in GAs preceded adventitious shoot formation.
Briefly, we report an efficient protocol that used 1/2MS + 1/2B 5 medium supplemented with 2.0 mg L −1 TDZ, 20 g L −1 sucrose, and 6 g L −1 agar (pH 5.875) for in vitro shoot regeneration of diploid strawberry. During adventitious shoot regeneration, the obvious reinvigoration of nearly brownish diploid strawberry explants aroused our attention. The transcript level changes of shoot regeneration-related genes may provide a possible explanation of such reinvigoration. Plant hormone synthesis-related genes play essential roles in plant development. Higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days of culture during leaf explant reinvigoration. Thus, we conclude that efficient recovery of nearly brownish diploid strawberry explants is closely associated with hormone synthesis-related genes. www.nature.com/scientificreports www.nature.com/scientificreports/ at dark and 50 at 16-h light photoperiod) tested for shoot induction were analysed by preparing three subsequent repetitions of three 100 mL glass flasks (90 mm in diameter with 40 mL of medium added), each of which contained 25 segmented leaf explants. The adventitious shoot regeneration rate (%) and number of regenerated shoots per explant were calculated for each glass flask. The morphogenic responses of segmented leaf explants were observed and photographed every 6 days. The adventitious shoot regeneration rates were also recorded. There were three repetitions of each dark duration treatment (225 leaf explants for each dark treatment), and the experiment was repeated three times (repetition1: from 12, Feb. to 30, Apr., 2015; repetition 2: from 12, Jun. to 30, Aug., 2015; repetition 3: from 18, Mar. to 5, Jun., 2016).

Methods
Finally, the best regeneration condition was determined (initial 0, 7, 14, 21, or 28 days of dark culture) and applied to prepare leaf explants for transcript-level analysis of related genes at 0, 6, 12, 18, 24, 30, 36, 42, Table 2. The sequences of the oligonucleotide primers for qRT-PCR analysis were used in this study. Note: F, forward primer; R, reverse primer.