Growth and Acclimation of In Vitro-Propagated M9 Apple Rootstock Plantlets under Various Visible Light Spectrums

This study aimed to explore the suitable light quality condition for ex vitro acclimation of M9 apple plantlets. Light quality treatments were set as followed; monochromatic LEDs (red (R), green (G), blue (B)) and polychromatic LEDs (R:B = 7:3, 8:2 and 9:1; R:G:B = 6:1:3, 7:1:2 and 8:1:1). Plant height of R, R9B1, and R8G1B1 treatments were significantly higher than the other treatments. The number of leaves and SPAD value of B were significantly higher than the other treatments. Root fresh weights of R9B1 and R7G1B2 treatments showed an increase of at least 1.7-times compared to R, G and R8B2. R8G1B1 accumulated higher starch contents than the other treatments. Photosynthetic rate of R9B1 and R8B2 were significantly higher than the other treatments. In terms of stomatal conductance and transpiration rate, treatments with high blue ratio such as B, R7B3 had higher values. Rubisco concentration was high in R and B among monochromatic treatments. In conclusion, red light was effective to increase photosynthetic rate and biomass and blue light increased chlorophyll content and stomatal conductance. Therefore, for R9B1 and R8G1B1, a mixture of high ratio of red light with a little blue light would be proper for the acclimation of in vitro-propagated apple rootstock M9 plantlets to an ex vitro environment.


Introduction
Plant tissue culture is an effective method for mass production of virus-free plantlets [1]. However, typical environmental characteristics of in vitro such as high relative humidity, low light intensity, and artificial supply of sugar and growth regulator through culture medium are major factors that are associated with low survival rates in transplanting virus-free plantlets to ex vitro [2]. Thus, acclimation process for a certain period is required for a successful survival in ex vitro conditions [3]. Acclimation refers to the manipulation of ex vitro environment to ensure that plantlets have resistance to harsher conditions compared to in vitro conditions without fatal growth impediment [4]. Factors affecting acclimation include light, humidity, and carbohydrate concentration of culture medium [5].
Among them, light is a crucial factor affecting photomorphogenesis and photosynthesis of plants, which have a significant influence on the acclimation of the in vitro-propagated plantlets. For example, in previous studies related to acclimation of in vitro-propagated plantlets, the effect of light intensities on photosynthetic capacity and the activity of enzymatic antioxidants have been examined in various crops [6][7][8][9] but the studies showing the effect of light quality are limited. In general, red light induces the accumulation of biomass and stem elongation [10], while blue light induces changes in   100  100  0  0  100  0  100  0  100  0  0  100  R9B1  100  90  0  10  R8B2  100  80  0  20  R7B3  100  70  0  30  R8G1B1  100  81  10  9  R7G1B2  100  70  9  21  R6G1B3 100 59 10 31 z Fraction of red, green, and blue wavelengths in terms of photosynthetic photon flux density (PPFD).

Growth Characteristics
The growth characteristics of in vitro-propagated apple plantlets acclimated under different light quality treatments were compared. Plant height, stem diameter, shoot and root fresh weights, SPAD value (chlorophyll content index) and survival rate were measured at 6 weeks after transplantation. Plant height and stem diameter on the basal and 4th leaf from apical meristem were measured using a ruler and a digital Vernier caliper (NA530-300S; Bluebird, Seoul, Korea). Fresh weight of shoot and root were measured with an electronic scale (SI-234; Denver Instrument, Denver, CO, USA). The SPAD value of the 3rd or 4th leaf from an apical meristem was measured by a portable chlorophyll meter (SPAD-502; Konica Minolta, Tokyo, Japan).

Photosynthetic Rate
Photosynthetic rate, stomatal conductance, and transpiration rate of apple plantlets were measured using a portable photosynthesis device (LI-6400; Li-Cor, Lincoln, NE, USA) equipped with a clear chamber bottom (2 × 3 cm) in a standard leaf chamber (LI-6400-40; Li-Cor, Lincoln, NE, USA) at 5 weeks after transplantation. The measurement was performed from 9:00 to 13:00, two hours after the light was on. The measurement conditions were set similarly to the cultivation environment; air flow rate 400 µmol·s −1 , CO 2 concentration 500 µmol·mol −1 , relative humidity 70% and block temperature 25 • C. The measurement was conducted under each lighting source at the light intensity of 100 µmol·m −2 ·s −1 .

Rubisco Concentration
Fresh leaf sample of 0.5 g was lyophilized with liquid nitrogen and stored at −70 • C until analysis. The sample was grinded using a mortar and extracted with 5 mL of 25 mM PBS (pH 7.4) solution. The extracted sample was stored at 4 • C in a 2 mL microtube for 2 h. After then, centrifugation was carried out at 12,000× g and 2 • C for 20 min and supernatant of the sample (1.5 mL) was used for analysis. All reagents were prepared at room temperature before analysis. The quantitative determination of Rubisco concentration was performed using a Rubisco ELISA Kit (MBS779145; MyBioSource, San Diego, CA, USA) and the optical density of final reaction solution was measured at 450 nm by a multi-mode microplate reader (Synergy HTX; BioTek, Winooski, VT, USA).

Starch Content
Freeze-dried shoot of apple plantlets was powdered using a Tube Mill control (IKA, Wilmington, NC, USA) and stored at 4 • C until analysis. A powdery sample (0.1 g) was added to a 15 mL conical tube, mixed with 80% ethanol of 10 mL, and then vortexed. After this, centrifugation was performed at 4 • C for 10 min at 3250× g at the centrifuge (5810R; Eppendorf, Hamburg, Germany). The pellet was stored at −80 • C until analysis.
The starch content of the pellet was analyzed using the modified dinitrosalicylic acid method [33]. The pellet was dissolved in distilled water of 2 mL and then autoclaved at 121 • C for 30 min. The solution was mixed with 0.2 M Na-acetate (pH 5.5) buffer, 1 mL 30 U amyloglucosidase (Sigma-Aldrich) and 1 mL 10 U β-amylase (Sigma-Aldrich) and centrifuged at 13,000× g for 10 min. The supernatant (50 µL) of the centrifuged sample was mixed with 0.5 mL of DNS (dinitrosalicylic acid reagent) and reacted in boiling water at 100 • C for 5 min. After completely cooling, 0.9 mL of distilled water and 0.1 mL of each reaction solution were mixed and measured by absorbance at 525 nm using a spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan). The starch content of each sample was shown as mg glucose (Sigma-Aldrich) per dry weights of in vitro-propagated apple plantlets.

Statistical Analysis
Each measurement parameter had five replicates per light quality treatment except for photosynthetic rate and Rubisco concentration. Four replicates were used for photosynthesis parameters and Rubisco analysis. All measured data were analyzed using the SAS program (SAS 9.4; SAS Institute, Cary, NC, USA). One-way analysis of variance (ANOVA) was performed and significant comparison among treatments means were conducted by Duncan's Multiple Range Test (DMRT).

Growth Characteristics
The plant height of apple plantlets was significantly higher values for R and R9B1 with a high ratio of red light that the others at 6 weeks after transplantation ( Figure 2). These treatments significantly increased about 1.3 times more than B which had the lowest value.

Growth Characteristics
The plant height of apple plantlets was significantly higher values for R and R9B1 with a high ratio of red light that the others at 6 weeks after transplantation ( Figure 2). These treatments significantly increased about 1.3 times more than B which had the lowest value. In terms of stem diameter, the top indicated the part connected with the scion part and the basal part was designated at the bottom. Stem diameters at top and bottom were measured at 6 weeks after transplantation ( Figure 3). Stem diameter by light quality did not show a significant difference in both parts. However, changes of stem diameter at the top tended to be similar to those of plant height. For example, R in monochromatic treatment and R9B1 and R8G1B1 in combination treatment, which were all high ratio of red light, recorded high values ( Figure 3A). In terms of stem diameter, the top indicated the part connected with the scion part and the basal part was designated at the bottom. Stem diameters at top and bottom were measured at 6 weeks after transplantation ( Figure 3). Stem diameter by light quality did not show a significant difference in both parts. However, changes of stem diameter at the top tended to be similar to those of plant height. For example, R in monochromatic treatment and R9B1 and R8G1B1 in combination treatment, which were all high ratio of red light, recorded high values ( Figure 3A). The number of leaves was significantly higher in B treatment at 6 weeks after transplantation ( Figure 4A). The total leaf area did not show significant difference, but R and R9B1 treatment showed high values ( Figure 4B). B treatment induced the highest SPAD value and the other treatments except B showed no significant difference value at 6 weeks after transplantation ( Figure 4C). The number of leaves was significantly higher in B treatment at 6 weeks after transplantation ( Figure 4A). The total leaf area did not show significant difference, but R and R9B1 treatment showed high values ( Figure 4B). B treatment induced the highest SPAD value and the other treatments except B showed no significant difference value at 6 weeks after transplantation ( Figure 4C).
Although no significant difference between the treatments was observed in the shoot fresh weight, there was a significant difference in the shoot dry weight at p < 0.01. R, R9B1 and R8G1B1 showed high values, and R9B1 had a significantly 1.4-fold higher value than R8B2 ( Figure 5A,C). Significant differences were also shown in root fresh and R9B1 and R7G1B2 treatments had at least 1.7 times higher values compared to R, G, and R8B2. The root dry weight also showed a similar tendency with root fresh weight and the highest values were observed in R9B1 and R7G1B2 ( Figure 5B,D). Additionally, the survival rate of apple seedlings was recorded as from 83% to 96% regardless of light treatments ( Figure 6).  Although no significant difference between the treatments was observed in the shoot fresh weight, there was a significant difference in the shoot dry weight at p < 0.01. R, R9B1 and R8G1B1 showed high values, and R9B1 had a significantly 1.4-fold higher value than R8B2 (Figures 5A,C). Significant differences were also shown in root fresh and R9B1 and R7G1B2 treatments had at least 1.7 times higher values compared to R, G, and R8B2. The root dry weight also showed a similar tendency  with root fresh weight and the highest values were observed in R9B1 and R7G1B2 (Figures 5B,D). Additionally, the survival rate of apple seedlings was recorded as from 83% to 96% regardless of light treatments ( Figure 6).

Photosynthetic Parameters
The photosynthetic rate was significantly lower in G and B treatments, and R9B1 and R8B2 treatments were significantly higher than the others except for R7G1B2 at 5 weeks after with root fresh weight and the highest values were observed in R9B1 and R7G1B2 (Figures 5B,D). Additionally, the survival rate of apple seedlings was recorded as from 83% to 96% regardless of light treatments ( Figure 6).

Photosynthetic Parameters
The photosynthetic rate was significantly lower in G and B treatments, and R9B1 and R8B2 treatments were significantly higher than the others except for R7G1B2 at 5 weeks after

Photosynthetic Parameters
The photosynthetic rate was significantly lower in G and B treatments, and R9B1 and R8B2 treatments were significantly higher than the others except for R7G1B2 at 5 weeks after transplantation ( Figure 7A). Stomatal conductance and transpiration rate for R7B3 and B showed significantly higher value compared to the other treatments ( Figure 7B,C). The change of intercellular CO 2 concentration also showed a similar trend with the stomatal conductance (data not shown). transplantation ( Figure 7A). Stomatal conductance and transpiration rate for R7B3 and B showed significantly higher value compared to the other treatments ( Figures 7B,C). The change of intercellular CO2 concentration also showed a similar trend with the stomatal conductance (data not shown). Rubisco concentration showed high values in R and B among monochromatic treatments, which were significantly higher than those for RB and all RGB mixed treatments ( Figure 7D).

Starch Content
Starch content was higher in R among monochromatic group and in R9B1 among RB combination treatments and R8G1B1 among RGB combination treatment had significantly higher starch content than those of G, B, R8B2, R7B3, R7G1B2, and R6G1B3 treatments at 6 weeks after transplantation ( Figure 8).  Rubisco concentration showed high values in R and B among monochromatic treatments, which were significantly higher than those for RB and all RGB mixed treatments ( Figure 7D).

Starch Content
Starch content was higher in R among monochromatic group and in R9B1 among RB combination treatments and R8G1B1 among RGB combination treatment had significantly higher starch content than those of G, B, R8B2, R7B3, R7G1B2, and R6G1B3 treatments at 6 weeks after transplantation ( Figure 8). transplantation ( Figure 7A). Stomatal conductance and transpiration rate for R7B3 and B showed significantly higher value compared to the other treatments ( Figures 7B,C). The change of intercellular CO2 concentration also showed a similar trend with the stomatal conductance (data not shown). Rubisco concentration showed high values in R and B among monochromatic treatments, which were significantly higher than those for RB and all RGB mixed treatments ( Figure 7D).

Starch Content
Starch content was higher in R among monochromatic group and in R9B1 among RB combination treatments and R8G1B1 among RGB combination treatment had significantly higher starch content than those of G, B, R8B2, R7B3, R7G1B2, and R6G1B3 treatments at 6 weeks after transplantation ( Figure 8).

Discussion
The plant's characteristics controlled by the plant photoreceptors were plant height, leaf angle, shape, and size [34]. Our results showed that plant height of in vitro-propagated apple plantlets showed significantly higher values at the R, R9B1 and R8G1B1 treatments at 6 weeks after transplantation and B had significantly the lowest value ( Figure 2). Blue light absorbed by phototropin in the hypocotyl cell makes the cryptochrome formation signal more efficient and inhibited the hypocotyl elongation of Arabidopsis [35]. In addition, inhibitory effects on plant growth by blue light (400-500 nm) were reported in other plant species such as pea, Arabidopsis, lettuce and soybean [36][37][38] and similar responses were shown in the in vitro-propagated apple plantlets. The top part of the stem diameter also showed a similar tendency to the plant height ( Figure 3A), suggesting that a high ratio of R light promoted secondary growth after the primary growth of cells and tissues derived from the apical meristem. Our results were consistent with the results of Li et al. [39], who reported that stem diameter of tomato seedling was significantly higher under the 75% red light treatment in red and blue combination treatment. The M9 seedlings used in this study are one of the apple cultivars used as dwarfing rootstocks. Rootstock is the base and root portion of grafted plants. A scion (shoot), the flowering and/or fruiting part of the plant, is grafted onto a rootstock. Therefore, the stem diameter can be an important factor when the scion is grafted onto a rootstock. The observed positive effects of thickest stem diameter rootstocks were rootstock's vigorous root system which absorb water and nutrients more efficiently [40]. It was reported that grafted trees with thicker stem diameter produced significantly more fruits [41]. Thus, in this study R8B1 and R8G1B1 with high stem diameter could have a positive impact on growth and fruit after transplanting to field. In addition, Shin et al. [42] observed that leaf elongation and expansion of in vitro-cultured Doritaenopsis plants could be promoted by red light, which supports our result that the vigorous leaf growth was observed in R and R9B1 ( Figure 4B).
SPAD value (an indirect index of chlorophyll content) of blue light was significantly higher at 6 weeks after transplantation than the others ( Figure 4C). Our results are supported by previous studies where blue light had positive effects on chlorophyll formation as well as stomatal opening in other plant species [42][43][44]. The fast increase of leaf area and stem diameter was occurred in R and R9B1 treatments which also had high shoot fresh weights ( Figure 5A). The expansive leaves intercept more light, which can remarkably increase biomass even under low light intensity. These results are in agreement with those found by Li and Kubota [45]; the biomass of baby leaf lettuces significantly increased, presumably due to enhanced light interception by enlarged leaf area under low light intensity. Meanwhile, relatively low shoot dry weight was observed in treatments with a high ratio of blue since blue light inhibited the growth of fresh and dry weight of leaves [38,45,46] ( Figure 5A,C). Liu et al. [47] reported that red light induces root elongation by promoting polar IAA migration from apical meristem to root. Therefore, the values of R9B1 and R7G1B2 with a relatively high ratio of red light-induced vigorous root growth in this study ( Figure 5B,D). The vigorous root growth of apple seedlings could contribute to successful transplanting to field due to its essential physiological function such as water and mineral uptake.
Blue light is important for chlorophyll synthesis and chloroplast development and red light is a major light for the development of photosynthetic apparatus [48], which means that each monochromatic light has its own role in plant development and photosynthesis. However, it has a relatively limited effect over mixed light. The photosynthetic rate showed the highest value of R9B1 in red and blue combination treatment, and the monochromatic treatments showed a lower photosynthetic rate compared to other treatments at 5 weeks after transplantation. Our results were in agreement with Matsuda et al. [49], who reported that the photosynthetic capacity of spinach leaves grown in red and blue ratio of 9:1 was higher than that of spinach cultivated in monochromatic light of red. In our study, improved carbon assimilation rate by the radiation of high ratio of red light treatment contributed to significant increases in growth parameters such as plant height, stem diameter and leaf area. Meanwhile, monochromatic green light showed the lowest photosynthetic rate ( Figure 7A). This was consistent with the tomato study by Wu et al. [50], in which the photosynthetic rate of Solanum Lycopersicum seedlings under the green light was significantly reduced. The green light may be effective to plant growth in addition to red and blue lights because green light can penetrate into the plant canopy and the leaves of the lower canopy can use the transmitted green light in fixing CO 2 [51,52]. Although the photosynthetic rate was low in the monochromatic green light, the polychromatic LEDs (R:G:B) showed a high level of photosynthetic rate. The dry weights of the shoot and root were higher under all RGB treatments than the monochromatic green light ( Figure 5C,D). Since blue light plays a role in stomata opening, blue light showed significant high stomatal conductance and transpiration rate in monochromatic light treatments. In addition, Shimazaki et al. [53] reported that the addition of blue light to red light-based light quality activates signaling that brings fast stomata openings which supported our result of high stomatal conductance and transpiration rate at the treatment of R7B3. Photosynthetic rates showed a different trend with stomatal conductance and transpiration rate, indicating that even though the in vitro-propagated apple plantlets had less stomata opening at 5 weeks after transplantation, it did not significantly affect photosynthetic rate due to abundant CO 2 . Rubisco, a key enzyme in Calvin cycle, begins carbon dioxide assimilation action through carboxylation of RuBP. Rubisco was significantly higher in R and B than the others at 5 weeks after transplantation ( Figure 7D). In a previous study, proteins synthesized in red light were as active as those synthesized in blue light but a higher Rubisco concentration per unit leaf area was observed under blue light [54]. This implies that blue light was more effective in Rubisco protein synthesis than red light. However, our results of Rubisco concentration showed no correlation with the results of the photosynthetic rate which were consistent with the result of Ernstsen et al. [55], who reported that light quality had a small or no effect on Rubisco induction.
Light quality can regulate carbohydrate metabolism of higher plants [56]. In most plants, starch and sucrose are the major storage form of carbohydrates and the principal form in which carbon is transported through the plants [39]. The starch content was showed a higher value in monochromatic treatment of R, red and blue combination treatment of R9B1 and red, blue and green combination treatment of R8G1B1 at 6 weeks after transplantation (Figure 8). Maas [57] and Li et al. [39] reported that red light increased starch accumulation in rose plants and cotton plantlets. The increment in starch content by a high ratio of red light treatment might be due to the increase in carbon through possible inductions in the photosynthetic rate [39]. The photosynthetic rate also showed a similar trend to that of starch content (Figures 7A and 8). In this study, there was a significant increase in photosynthetic rate and starch content under the combination of R and B LED lights, especially R9B1, compare with the other treatments. Previous studies suggested that the spectral energy distribution of red and blue wavelength coincided with the absorption spectrum of chlorophyll, and therefore photosynthetic rate was stimulated [58]. Therefore, this combination light might be effective for the accumulation of soluble carbohydrates in the apple plantlet. Therefore, our results suggest that the increase in shoot biomass of the apple plantlets may be caused by an accumulation in starch content by a red light.

Conclusions
Plant tissue culture is an effective method for producing virus-free plantlets. However, due to the environmental characteristics of in vitro, the manipulation of the acclimation environment is crucial to increase the survival rate of the plantlets in indoor places such as plant factories with artificial lighting. In particular, the light environment such as light quality and light intensity is an essential factor for the successful acclimation of the in vitro-propagated plantlets. In this study, we demonstrated that a high ratio of red light was effective to increase photosynthetic rate and biomass and blue light was an adequate light source to increase chlorophyll content and stomatal conductance of virus-free apple plantlets. Therefore, our study suggested that R9B1 or R8G1B1 would be a proper lighting condition for successful acclimation of in vitro-propagated apple rootstock M9 plantlets to ex vitro conditions. Our trial for the acclimation of virus-free apple plantlets showed an applicability of environmental control technology in closed-type plant production systems to fruit seedlings, a new target crop group. Moreover, this technology can be applied to vertical farming in urban areas in the near future.