Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars.


Introduction
Production of transformants is an important technique for the analysis of gene expression and for the development of next-generation breeding methods. Shoot regeneration from calli and cultured cells is not only a crucial step in the production of transformants; it is also a useful regulatory functions of light remain unknown for the photoregulation of shoot regeneration in calli derived from immature barley embryos.
The present study examines endogenous hormone levels in calli derived from immature embryos cultured in different light conditions (16-h photoperiod and continuous darkness) for barley cultivars showing different responses to light conditions. Barley cultivars are classified as either photo-inhibition or photo-induction types. Those types respectively show higher shoot regeneration efficiency in continuous darkness and a 16-h photoperiod. In the photo-inhibition type, ABA is highly accumulated in calli. We investigate the expression of ABA biosynthesis gene and effects of exogenous ABA on shoot regeneration. In addition, the role of ABA for light regulation of shoot regeneration is discussed.

Plant materials and tissue culture
In a field of the Institute of Plant Science and Resources of Okayama University in 2009 and 2014, four barley cultivars were grown: Kanto Nijo-5 (KN5), K-3 (K3), Golden Promise (GP), and Lenins (LN). Husk-removed immature seeds were sterilized. Immature embryos (1-1.5 mm long) were excised aseptically. Modified MS medium with maltose (30 g l -1 ) was used as a basal medium [40]. The callus-induction medium was supplemented with 9 μM of 2,4-dichlorophenoxyacetic acid (2,4-D). ABA and fluridone were dissolved in ethanol at 10 mM concentration. These solutions were used as stock solutions. The callus-induction medium was supplemented with ABA (1 and 5 μM) or fluridone (0.1 and 0.5 μM). The regeneration medium was supplemented with 6 μM of IAA and 0.2 μM of zeatin. These media were solidified with 0.2% of Gelrite (Merck and Co. Inc.).
For callus induction, 14 immature embryos were placed on 20 ml of the callus-induction medium in a 90 × 15 mm Petri dish with the scutellum in contact with the medium. After four weeks of incubation, calli were transferred to the regeneration medium after removing shoots and roots derived from zygotic embryos. Shoot regeneration was recorded four weeks later. Petri dishes were kept at 25±2°C. Petri dishes were incubated in a 16-h photoperiod (16-h photo) or continuous darkness (Dark) during four weeks of callus induction. As a light source, white fluorescent lamps (FL40S; Toshiba Corp.) were used. The light intensity was 61.5 μmol m -2 s -1 . The light condition during shoot regeneration was a 16-h photoperiod. All experiments had three replications, each with three dishes. The shoot regeneration percentage was evaluated according to the percentage of embryos with calli regenerating shoots. Regeneration percentages of green and albino shoots were, respectively, the percentages of embryos with at least one green shoot and those with only albino shoots. Effects of light conditions during callus induction on shoot regeneration and endogenous hormone contents in calli were examined in plants grown in 2009. Treatments of ABA and fluridone, and gene expression analysis were examined in plants grown in 2014.

Quantification of endogenous hormones in calli
Extraction, purification, and quantification of indole acetic acid (IAA), isopentenyladenine (iP), trans-zeatin (tZ), Gibberellin A1 (GA1), GA4, ABA, jasmonic acid (JA), jasmonyl isoleucine (JA-Ile), and salicylic acid (SA) from calli were conducted as described by Tsukahara et al. [42]. Calli derived from immature embryos cultured for two weeks were used for quantification in four cultivars. Approximately 100 mg fresh weight (FW) of calli was used in each of three independent experiments. Endogenous hormone levels in calli cultured with fluridone were determined in KN5 and LN.

Statistical analysis
Differences in mean values were tested using Student's t-test or Duncan's multiple range test.

Light control of shoot regeneration in barley cultivars
Immature embryos were cultured in a 16-h photoperiod and continuous darkness during the callus induction. In KN5, shoot regeneration percentages were, respectively, 25.2±4.3% and 61.8±6.5% in a 16-h photoperiod and continuous darkness ( Table 1). The shoot regeneration percentage in a 16-h photoperiod was significantly lower than those in continuous darkness. Efficient shoot regeneration in continuous darkness was also observed in K3. However, shoot regeneration percentages of GP were, respectively, 79.3±4.8% and 54.7±2.9% in a 16-h photoperiod and continuous darkness. Shoot regeneration was inhibited by the culture in continuous darkness. Also, LN showed lower shoot regeneration percentage in continuous darkness. These cultivars were classified into photo-inhibition type (KN5 and K3) and photo-induction type (GP and LN) according to their response to light. No significant difference was found in albino shoot production between light conditions among all cultivars.

Endogenous hormone contents in calli
Endogenous hormone contents were determined in calli cultured in a 16-h photoperiod and continuous darkness. Contents of GA1, tZ, and JAIle were low in calli, irrespective of light conditions (Fig 1 and S1 Fig). Although contents of iP were also low, K3 showed significantly

Effects of exogenous ABA and fluridone on tissue culture traits
Germination of zygotic embryos and callus growth were inhibited by exogenous ABA in both light conditions (Fig 2). In KN5 and GP, shoot regeneration was inhibited in cultures with ABA in a 16-h photoperiod (Fig 3). Total regeneration percentages (green and albino) were reduced depending on the concentration of ABA in continuous darkness. Reduction of total regeneration percentages depending on the concentration of ABA was also observed in cultures of K3 and LN in both light conditions. In all cultivars, shoot regeneration was inhibited by exogenous ABA. No significant difference was found in the inhibitory effects of exogenous ABA on shoot regeneration between light conditions. An ABA biosynthesis inhibitor, fluridone, exhibited no marked effects on callus growth, whereas zygotic embryos germinated vigorously on the callus-induction medium with fluridone (Fig 2). Albino shoot production was enhanced strongly by the culture with fluridone in both light conditions. Total regeneration percentages of KN5 were increased at 0.1 μM of fluridone treatment and were decreased at 0.5 μM of treatment in a 16-h photoperiod (Fig 4). In continuous darkness, the total regeneration percentages decreased depending on the concentration of fluridone. In KN5, fluridone enhanced shoot regeneration at the lower concentration in a 16-h photoperiod and inhibited shoot regeneration in continuous darkness. In K3, total shoot regeneration percentages were increased depending on the concentration of fluridone in a 16-h photoperiod. In continuous darkness, although shoot regeneration percentages were decreased at 0.1 μM of fluridone treatment, a higher shoot regeneration percentage was

Endogenous hormone contents in calli cultured with fluridone
Endogenous hormone contents were examined in calli cultured with fluridone in KN5 and LN. Contents of tZ and iP were low in calli. The effects of fluridone were unclear (Fig 5). Fluridone had no effect on the endogenous level of SA. Moreover, GA1, JA and JAIle showed low contents in calli (data not shown). Endogenous contents of ABA were reduced strongly in cultures with fluridone in both light conditions. Furthermore, fluridone treatments decreased

Expressions of ABA catabolism and biosynthesis genes in calli
Expressions of HvABA8'OH-1 and HvNCED1, which were respectively associated with ABA catabolism and biosynthesis, were examined in calli. Expressions of HvABA8'OH-1 showed similar levels in a 16-h photoperiod and continuous darkness in KN5, GP, and LN. No significant effect of light conditions was detected in the regulation of HvABA8'OH-1 expression in these cultivars (Fig 6). However, HvABA8'OH-1 was highly expressed in continuous darkness in K3. HvABA8'OH-1 expression represented light dependency independent of photo-regulation type for shoot regeneration.
Expressions of HvNCED1 were significantly higher in a 16-h photoperiod than in continuous darkness in K3, GP, and LN (Fig 6). Expression levels differed among cultivars in a 16-h photoperiod. The photo-inhibition type showed higher expressions than the photo-induction type. Expression of HvNCED1 in calli was regulated by the light condition during callus induction.

Discussion
Shoot regeneration is regulated by light conditions that prevail during callus induction in immature barley embryo culture. The responsiveness to light differs among cultivars [40]. In a 16-h photoperiod, shoot regeneration of KN5 and K3 (photo-inhibition type) was inhibited. In contrast, shoot regenerations of GP and LN (photo-induction type) were enhanced in a 16-h photoperiod. These results support those of an earlier study [40]. For investigation of the regulatory mechanisms of light in immature barley embryo culture, endogenous hormone contents were examined in calli cultured under a 16-h photoperiod and continuous darkness. Higher accumulations of ABA were observed in a 16-h photoperiod in photo-inhibition type, although the levels of ABA were lower in continuous darkness. Exogenous ABA supplemented with the callus-induction medium inhibited callus growth and shoot regeneration. Furthermore, ABA biosynthesis inhibitor, fluridone, decreased endogenous ABA in calli. Photo-inhibition of shoot regeneration was reduced in KN5 and K3. These results indicate that the photo-inhibition of shoot regeneration is associated with the fluctuation of endogenous ABA contents in KN5 and K3.
The effects of ABA on tissue culture traits in several species have been investigated. Rajasekaran et al. [44] reported that exogenous application of ABA enhanced somatic embryogenesis and that it reduced the formation of non-embryogenic callus in Napier grass. Positive effects of ABA on shoot regeneration were also observed in calli derived from immature embryos of wheat [45,46], embryos and anthers of rice [6,47,48], cotyledons of rape [4,5], hypocotyls of carrot [2], and immature embryos of coconut [49]. However, ABA acts as a negative factor affecting shoot regeneration in calli derived from immature embryos of corn [50], leaf explants of alfalfa [51], immature seeds of Hevea [52], immature seeds of sunflower [53], and nodal segments of peach [54]. In the present study, ABA inhibits shoot regeneration in calli derived from immature barley embryos, whereas ABA promotes plant regeneration in barley anther culture [55]. Consequently, the effects of ABA on tissue culture traits differ depending on the species and explant source. In rice and cactus, callus growth was enhanced by low concentration of ABA; it was inhibited by high concentration [7,56]. Furthermore, somatic embryo formation and plant regeneration were enhanced by low concentration of ABA and were inhibited by high concentrations in sweet orange and melon [3,57]. ABA is necessary for shoot regeneration at a low concentration. A higher concentration of ABA might act as an inhibitor on tissue culture traits. However, Qureshi et al. [58] reported that ABA inhibited shoot regeneration in calli derived from wheat embryos in an early stage (10-14 days after pollination; DAP), whereas shoot regeneration was enhanced in calli derived from late stage embryos (21-25 DAP) through the inhibition of precocious germination. The developmental stage of seed affects sensitivity to ABA in wheat [59]. Recently, receptor like protein kinase 1 (RPK1) was identified as a QTL for the regulation of plant regeneration in Arabidopsis; more importantly, Light Inhibition of Shoot Regeneration Is Regulated by ABA in Barley  RPK1 is involved in the ABA signal transduction pathway [23]. Zhang et al. [24] reported that expressions of several miRNA are decreased in the embryogenic calli of Larix leptolepis and reported that the targets of these miRNA are positive regulators of ABA responses. These results indicate that ABA signals are involved in regulating shoot regeneration. Sensitivity to ABA might be associated with different responses of tissue culture traits.
In expression analysis, the expression of HvNCED1 was increased in a 16-h photoperiod. The NCED encodes 9-cis-epoxycarotenoid deoxygenase, which is a major enzyme for ABA biosynthesis, and acts at a key regulatory step in ABA biosynthesis. Thompson et al. [60] reported that the expression of NCED was increased in the light period and decreased in the dark period of a 12-h photoperiod in tomato. Moreover lower expressions were maintained in continuous darkness followed by a 12-h photoperiod. In tartary buckwheat, sprout of Hokkai T10 showed higher expressions of NCED in a 16-h photoperiod and lower expressions in continuous darkness [61]. HvNCED1 is also positively regulated by light in calli derived from immature barley embryos. In the photo-inhibition type, the increase of HvNCED1 expression activates ABA biosynthesis and induces high accumulation of endogenous ABA. Eventually, higher accumulation of ABA inhibits shoot regeneration. Rikiishi et al. [40] reported that blue light signals act in photo-inhibition of shoot regeneration in immature barley embryo culture. Expression of HvNCED1 might be regulated by blue light signals.
In this study, immature embryos were derived from plants grown in different years. Effects of light conditions during callus-induction on shoot regeneration (Table 1) and endogenous hormone contents in calli (Fig 1 and S1 Fig) Fig 5). However, endogenous ABA contents were higher in a 16-h photoperiod than in continuous darkness, as shown in Fig 2 (P<0.05) and Fig 5 (0.05<P<0.10). These results indicate that photo-inhibition type shows marked response to light conditions in distinct experiments. In this study, plants were grown in a field, not in a growth chamber. The quality of immature embryos is affected strongly by the vegetative conditions of the mother plants [33]. Different levels of shoot regeneration percentage and endogenous hormone content might be derived from the growth conditions of plants. However, effects of light conditions were not detected in photo-induction type in Fig 3 and Fig 4. Sensitivity to light conditions might be different between photo-inhibition and photo-induction types. Lower sensitivity induces unstable conditions of the photo-induction type.
In the photo-induction type, the reduction of endogenous ABA contents by fluridone showed no effects on the shoot regeneration, although exogenous ABA inhibited plant regeneration similarly to the photo-inhibition type. Because GP and LN had innately lower concentrations of ABA in both light conditions, photo-induction of shoot regeneration was independent of the endogenous ABA. Auxin-related and cytokinin-related processes are important for shoot regeneration and are highly conserved in plants [23]. In this study, endogenous IAA contents were reduced in continuous darkness in LN. In photo-induction type, shoot regeneration might be regulated by light through the auxin function.
Endogenous ABA content was reduced by the cultures with fluridone, independent of the light conditions. Fluridone reduces ABA contents and affects chloroplast development because fluridone inhibits the desaturation of phytoene at the early step of carotenoid biosynthesis [62][63][64]. Hoekstra et al. [65] reported that the production of albino shoot regeneration was enhanced strongly by fluridone in barley anther culture. Albino shoot regeneration was increased in calli cultured with fluridone in this study. However, shoot regeneration percentages were reduced in cultures with higher concentrations of fluridone. Nevertheless, higher concentrations had no greater effect on the reduction of endogenous ABA. Fluridone has inhibitory effects on shoot regeneration independent of ABA biosynthesis. Other inhibitors are expected to be necessary for the investigation of ABA effects on shoot regeneration. Han et al. [66] developed an inhibitor of NCED. Our results show that photo-inhibition of shoot regeneration is regulated by NCED expression. Endogenous ABA might be reduced by an inhibitor of NCED in avoiding albino shoot production. The effects of ABA on shoot regeneration can be evaluated precisely in immature barley embryo culture.
Shoot regeneration from de-differentiated tissues is an important step in the production of transgenic plants and for demonstrating totipotency. Rikiishi et al. [40] reported that the genes responsive to auxin and cytokinin showed different expression ratios depending on the light conditions in calli of KN5 and K3. This result indicates that auxin and cytokinin have an important role in the regulation of shoot regeneration in immature barley embryo culture. Results of this study show that ABA acts as an inhibitor of shoot regeneration in calli derived from immature embryos. This study also elucidates the role of ABA for the photo-regulation of shoot regeneration. Shoot regeneration is controlled by a regulatory network involving crosstalk among auxin, cytokinin, and ABA. Auxin and cytokinin have been investigated and understood in the function of regulating tissue culture traits. However, the physiological functions of ABA on tissue culture traits remain unclear. Endogenous contents of ABA and shoot regeneration capacity can be modified by simple light signals, suggesting that immature barley embryo culture is a useful tool for investigating the relation between ABA and tissue culture traits.