Red and Blue Light-Emitting Diodes Significantly Improve in vitro Tuberization of Potato (Solanum tuberosum L.)

Solanum tuberosum ‘Kufri Jyoti’ is widely cultivated in India. For the experiment tubers were provided by the Central Potato Research Institute (CPRI, Shimla, India). The characteristics of cultivar include the early time to maturity, medium-size tubers, and upright plant type. The experiments were done at Potato Phenomics Laboratory established at Department of Biotechnology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar, Madhya Pradesh, India (23.8388°N, 78.7378°E). The potato tubers collected were washed in running water, dried, and sprouted under dark conditions at 37 °C with 80% relative humidity. The sprouted plantlets (4–5 cm in height) were then transferred to the medium-size pots (25 cm) filled with coco-peat and sand (3 : 1 ratio). The plants were maintained in the plant growth chambers (Matrix Eco India, size 1.5 m × 1.2 m × 2 m, providing a total growth area of 4.2 m² with 40-cm high clearance) at 22 ± 2 °C temperature 16/8 h photoperiod exposed to the fluorescent light and required humidity (65 ± 10%). The plants were watered with tap water as per requirements.

The stem taken from 4- to 6-week-old plants were cut into pieces (1.5 cm) containing nodal part, washed with detergent (Triton-X100), and disinfected with 0.1% sodium hypochlorite followed by 70% ethanol and then washed thrice with autoclaved water, and dried under the laminar flow. Single nodal explants were aseptically inoculated into the Murashige and Skoog (1962) medium (MS) containing 3% sucrose and 0.8% agar adjusted to pH 5.8. The cultures were incubated at 22 ± 2 °C with 60% relative humidity in the plant growth chamber fitted with different LED lights. Each treatment contained 60 nodal explants, and a total of 180 explants were taken upon repeating the experiment thrice.

The LED light source was procured from the supplier (Sagar, Madhya Pradesh, India) with the corresponding red (650 nm) and blue (460 nm) wavelength. The LED source comprised stripes (5 × 100 cm) containing 40 LEDs, which created a light-emitting set with 100 μmol·m⁻²·s⁻¹ total photosynthetic photon flux density (PPFD). The nodal tissues were maintained under the exposure of red (R) and blue (B) LED such as 100% R (R₁₀₀), 100% B (B₁₀₀), and their combinations of RB such as 30% R and 70% B (R₃₀B₇₀), 50% R and 50% B (R₅₀B₅₀), and 70% R and 30% B (R₇₀B₃₀) LED spectra. The nodal tissues maintained under W₁₀₀ LED without supplementation of red and blue lights were used as a control, while the nodal tissues maintained under continuous dark were used as a positive control. The photoperiod, photosynthetic photon flux density (PPFD), day/night temperature, and relative humidity in the plant growth chamber were set as 16/8 h (day/night), 210 μmol·m⁻²·s⁻¹, 22 ± 2 °C, 65 ± 10%, respectively.

The percentage of in vitro tuberization was calculated according to the number of stolons (arising on the nodal cuttings) converting into tubers divided by the total number of the inoculated single-node cuttings. The stolons and further developed tuber samples were collected individually upon each LED light treatment. Observations on the tuber induction period, number of stolons converting to initial tubers, the total number of tubers, tuber weight, and overall yield were taken upon 30 days of tuber induction.

The soluble sugar content in tubers was extracted as described (Ciha & Brun 1978) and quantified using the phenol-sulfuric acid method (Dubois et al. 1956). Concentrated sulfuric acid hydrolyzes the glycosidic linkages to neutral sugars, which then partially dehydrated via elimination of three molecules of water to form furfural or furfural derivatives. The colored compounds developed by the condensation of furfural or furfural derivatives with phenol were estimated using the spectrophotometer (Shimadzu 1800, Japan) at 490 nm. Various concentrations of D-glucose (10–100 μg·ml⁻¹) were used as standards for the estimation of total carbohydrates. The tuber starch was hydrolyzed into simple sugars using perchloric acid, and the quantity of simple sugars was estimated by the an-throne method (Sadasivam & Manickam 1992). The D-glucose solution (50–500 μg·ml⁻¹) was used as a standard for the estimation of starch.

Extraction of the soluble proteins was done using the phosphate buffer following the standard protocol (Sambrook et al. 1989), and quantification of the protein was done using the Bradford re-agent (Bradford 1976). Solution of bovine serum albumin (1–10 μg·ml⁻¹) was used as a standard for the estimation of total proteins.

The calcium-dependent protein kinases (StCDPK), calmodulin proteins (StCaM1), and the resulting metabolites of the lipoxygenase (StLOX) pathway have been reported as the major signaling pathway genes regulating the potato tuberization. Total RNA was extracted from tuberizing tissues by using TRIzol reagent kit (Invitrogen, USA) following the manufacturer's instructions.

The SuperScript^™ First-Strand cDNA synthesis kit (Invitrogen, USA) was used taking 1.0 μg RNA. A standard protocol was followed for the cDNA synthesis using Oligo-dT primers. The mixture was incubated at 42 °C for 50 min to complete the reverse transcription reaction (Sambrook et al. 1989). The cDNA was used as a template for the quantitative gene expression analysis. Real-time quantitative amplification (q-PCR) of the major tuberization pathway genes, such as CaM1, StCDPK, and LOX, was carried out in a real-time PCR system (Applied Biosystems, USA) using the gene-specific primers. The RT-PCR reaction was performed using the SYBR-green real-time PCR kit (Invitrogen, USA). The actin (NCBI accession No. XM_015308091.1) gene-specific primers were used as an internal gene amplification control where the proportional threshold (Ct) values were normalized to the actin gene to achieve the comparative expression. Different primer sequences used for the qPCR analysis are shown in Table 1.

The in vitro grown stolons and tuber tissues developed from the single nodal explants upon the treatment with various LED lights were collected simultaneously depending on their stage of formation. The proteins were extracted following the standard protocol (Sambrook et al. 1989). The LOX activity was quantified using the extracted proteins following the procedure as reported (Gökmen et al. 2002). A standard substrate solution was prepared via mixing 2.0 ml of potassium phosphate buffer (0.1 M; pH 6.0), 2.0 μl of Tween-20, and 2.0 μl of pure linoleic acid (Sigma, USA). The gentle stream of air was passed through the solution for aeration, and the reaction was initiated by adding 100 μl of crude enzyme extract to the solution. The blank consisted of only substrate solution (2.0 ml) added with distilled water (100 μl). The synthesis of hydroperoxides due to the presence of a conjugated hydroperoxydiene moiety was monitored using the spectrophotometer (Shimadzu 1800, Japan) at 234 nm. One unit of LOX activity was defined as an increase in absorbance at min per mg of protein under assay conditions.

The data were reported as the mean of three replicates (± standard error, SE). Data were analyzed by ANOVA and Tukey HSD test with the statistical package SPSS 15.0 unless otherwise stated.

Exposure of R and B LED light spectra and their various combinations altered the in vitro tuberization process on the nodal tissues grown on the MS media (Fig. 1). An early stolons formation was observed on the nodal explants maintained under R₁₀₀ (7–8 days) and R₃₀B₇₀ (8–9 days) light spectra compared with other light treatments and the controls. Observations further revealed that the stolons developed under the exposure of R₃₀B₇₀ combination of LED light showed early tuber formation (within 12–14 days) and enhanced number of stolons converting into tubers (143.67 ± 5.37), with improved tuber size (4.10 ± 0.45 mm), tuber weight, and tuber yield (83.48 ± 3.50 g) in comparison with those under the exposure of other light spectra (Table 2). Interestingly, delayed tuber induction was also observed with the nodal tissues maintained without light and under W₁₀₀.

Figure 1

A slight increase in the total soluble sugar content was observed with the microtubers developed under the exposure of the R₃₀B₇₀ (8.1 ± 0.42 mg·g⁻¹ of tuber FW) and R₁₀₀ (7.9 ± 0.71 mg·g⁻¹ of tuber FW) compared with those developed under W₁₀₀ and other light combination (Fig. 2A). The highest starch and protein contents in the microtuber tissues were found under the B₁₀₀ and the R₃₀B₇₀. The R₃₀B₇₀ combination of LED light spectra induced maximum content of starch (174.1 ± 5.76 mg·g⁻¹ tuber FW) and the protein (24.2 ± 1.65 mg·g⁻¹ tubers FW). The lowest content of starch was quantified in the microtubers grown under the W₁₀₀ light (Fig. 2B–C).

Figure 2

The qPCR analysis of major tuberization-specific genes, such as calmodulin (StCaM1), calcium-dependent protein kinase (StCDPK), and the lipoxygenase (LOX), indicated a significantly enhanced expression in the stolon tissues developed under the exposure of R₁₀₀ LED (2.65-, 2.84-, and 3.21-folds, respectively) followed by the R₃₀B₇₀ combination of LED light (2.39-, 2.56-, and 2.95-folds, respectively) as compared with the control (Fig. 3A). The expressions of StCaM1, StCDPK, and LOX in the initial tuber tissues developed under the exposure of R₃₀B₇₀ and B₁₀₀ LED light were significantly enhanced in comparison with the other light combinations and the control W₁₀₀ light as well as the dark conditions. Exposure to R₃₀B₇₀ LED light enhanced the expression of the StCaM1, StCDPK, and LOX genes by 1.93-, 2.01-, and 2.20-folds, respectively, in comparison with the control light conditions as shown in Figure 3B. In addition, an enhanced expression of these genes was also observed in the developed tuber tissues grown under R₃₀B₇₀ LED light combination in comparison with those grown under dark and W₁₀₀. A noteworthy reduction in the expression of all the genes was observed in the developed tubers grown under all kinds of light spectra (Fig. 3C).

Figure 3

Spectrophotometric analysis depicted an enhanced LOX enzyme activity in stolons and initial and developed tuber tissues grown under the exposure of B₁₀₀ and R₃₀B₇₀ light combinations. The R₃₀B₇₀ LED spectrum induced the LOX activity by 2.20-fold in stolon (Fig. 4A), 2.06-fold in initial tubers (Fig. 4B), and 1.74-fold in developed tubers (Fig. 4C), respectively, in comparison with the W₁₀₀ LED light. The LOX activity was also enhanced in the stolons, initial tubers, and developed tuber tissues under the B₁₀₀ LED light as compared with the control light condition. The increase in the LOX enzyme activity in the stolons and tuber tissues also coincides with the enhanced LOX gene expression as was evident in the qPCR analysis as represented in Figure 3.

Figure 4

Various spectra of light impact photosynthesis and morphogenesis and hence alter plant growth and developmental process (Seabrook 2005; Li et al. 2018). The results exhibited that the degree of in vitro tuberization on the nodal explants varied with exposure to different LED lights; therefore, it was tempting to find out the pathway reliant upon the light involved in tuberization. The one important reason might be the difference in the photosynthetic capacity, hormone content, and efficient translocation of carbohydrates from the source (MS media in the case) under different spectra of LED light which resulted in varied tuberization. The light-mediated regulation of the endogenous plant hormones, including GA, abscisic acid (ABA), and indole acetic acid (IAA), has already been reported (Aksenova et al. 1989; Fixen et al. 2012; Matsuo et al. 2019). The GA has been recognized as an important plant hormone regulating the photoperiodic-mediated tuberization in the potato and reported to induce stolon formation via the longitudinal cell expansion by the transverse orientation of microtubules and microfibrils to the cell axis (Aksenova et al. 1989). Thus, the plant hormone GA plays an essential role in stolon formation and elongation during tuber formation; however, its higher concentration prevents the conversion of stolons to tubers (Fixen et al. 2012). A recent report also exhibited an increase in bioactive GA content in the plant grown under the R LED compared with those grown under other light conditions (Matsuo et al. 2019). However, the addition of the B LED to R light increased the transcript level of the GA inactivation gene, thus balancing endogenous GA contents in the plants. The GA level decreases when stolon tips started to swell to form the initial tuber (Sarkar 2008). In this study, an early stolon induction was observed in the case of the nodal explants growing under the R₁₀₀ LED light; however, a lesser number of initial tubers with lower mass per tuber were also observed (Table 2). This might be due to continuous biosynthesis of the endogenous GA content under the R₁₀₀ monochromatic light, which induced the stolons formation, but the efficiency of conversion of stolons to the tuber was redundant. In addition, it was observed that the nodal explants growing under the B₁₀₀ LED light showed delayed stolon initiation and tuber formation but increased the number of tubers with the greater mass per tuber. This might be due to reduction in the GA content essential for the conversion of the stolons to tubers. However, insufficient biosynthesis of bioactive GA content under B₁₀₀ LED light might have delayed the stolon initiation. The combined spectra of R₃₀B₇₀ LED efficiently provided the required wavelength of light, resulting in biosynthesis of balanced hormone GA and IBA for early stolons formation, efficient conversion of the stolons into the tuber, and translocation of the carbohydrates (from either media or the nodal explants) for early tuberization with enhanced tuber mass and yield.

The light quality has been recognized as an essential factor regulating the protein and carbohydrate metabolism in plants and the transportation, as well as accumulation of soluble carbohydrates such as starch which ultimately determine the growth of tuber tissues of potato (Malone et al. 2006; Asghari-Zakaria et al. 2009; Kang et al. 2013). The blue light has been reported to induce sugar and starch contents in potato tuberizing under the in vitro conditions on the growth media (Li et al. 2020). A similar result was also observed in this study. An increase in starch content in micro-tubers grown under B₁₀₀ LED spectra represented the accumulation of starch content in the tuber tissues, which was also associated with the increase in the tuber size and mass. Similar results of enhanced biosynthesis of the starch and protein in the tissues under the B dominant light conditions validated our study (Chen et al. 2020; Li et al. 2017). However, the soluble sugar content in microtubers developed under R₁₀₀ LED light remained higher which indicated a reduction in transportation and accumulation of sucrose as starch in the tissues. Quantification of the starch and soluble protein content enhanced in the tuber tissues developed under the exposure of R₃₀B₇₀ combination of LED light indicated better translocation of the available carbohydrates from the growth medium leading to storage of starch in the tuber.

The change in the concentration of cytosolic Ca²⁺ is one of the primary events in the transduction of different signals leading to variation in metabolic processes. The concentration of free cytosolic Ca²⁺ in plant cells elevates via various extrinsic and intrinsic stimuli. The calcium-binding proteins are present in the cell initiate downstream signaling events leading to alteration in gene expression resulting in variation in the morphological and physiological parameters (Tuteja & Mahajan 2007; Khan et al. 2014). In case of potato tuberization, the change in cytosolic Ca²⁺ triggers a cascade of phosphorylation of tuber-inducing proteins including the calcium-binding modulator proteins (calmodulin, StCaM1) and calcium-dependent protein kinases (StCDPK1) (Jena et al. 1989; Raíces et al. 2001; Upadhyaya et al. 2016). These proteins have been recognized as a key mediator in sucrose-signaling pathways during tuber induction and development (MacIntosh et al. 1996; Raíces et al. 2003). The blue and red-light irradiations have been reported to enhance the cytosolic Ca²⁺ through the Ca²⁺ channels via the action of phototropin 1 and two proteins (Babourina et al. 2002; Harada et al. 2003; Stoelzle et al. 2003). In this study, an early tuber induction, increase in tuber number, and yield under the R₃₀B₇₀ could also be attributed to enhanced expression of the calcium-dependent StCaM1 and StCDPK gene as evident in the qPCR expression analysis (Fig. 3). Therefore, change in concentrations of cytosolic Ca²⁺ due to the combined RB LED spectrum could be a major factor in enhanced expression of the calcium-signaling pathway genes leading to early tuber induction and tuber yield as evident in this study.

Variation in cytosolic Ca²⁺ also resulted in the StCDPK-mediated activation of the NADPH oxidase and cellular oxidative burst, which ultimately induced an intracellular signaling pathway resulting in the generation of reactive oxygen species (ROS) that damage membrane lipids and proteins (Grant et al. 2000; Kobayashi et al. 2006). In addition to ROS, the enhanced LOX enzyme which activity was highly correlated with the cellular redox state also catalyzed the peroxidation of damaged membrane lipid components, particularly unsaturated fatty acids, which ultimately participated in the biosynthesis of the tuber-inducing essential plant hormones (Koda 1997; Maccarrone et al. 2000). The enhanced expression of the LOX gene was also corroborated with induced tuber formation in potatoes as reported earlier (Nam et al. 2005). An enhanced expression of the LOX gene in the stolons and the developing tubers growing under the R₃₀B₇₀ light spectrum might be due to the cytosolic change in the Ca²⁺ ions leading to enhanced ROS-mediated LOX cascade products inducing the in vitro tuberization. Products of the LOX cascade, such as jasmonic acid (JA) and tuberonic acid (TA), have been reported to play multiple roles as growth regulators, as well as signal molecules to initiate potato tuberization by antagonizing the effects of GA (Pinsky et al. 1971; Jackson & Willmitzer 1994; Smith 1994). Moreover, the LOX enzyme activity strikes exclusively at the tip of stolons, developing tubers, which ultimately reduces at the maturing stage of the tuber formation (Kolomiets et al. 2001; Pelacho & Mingo-Castel 1991). An enhanced LOX enzyme activity and gene expression in the stolons tissues and initial tubers were also observed in this study (Fig. 4A–B). Therefore, the R₃₀B₇₀ combination of LED spectrum control the expression of the LOX genes in the tissues, resulting in an efficient in vitro tuberization even without the addition of external enzymes or sucrose in the growth medium. The report on LOX cascade-regulated tuber formation via direct interaction with light and low temperature suggested an important downstream signal molecule in photoperiod-controlled signaling pathways (Nam et al. 2005; 2008). Taken together, an association of change in the LOX activity profile and tuber formation under the in vitro condition exposed to the LED light irradiation could not be ignored. A line diagram describing the possible pathways involved in light-mediated induction of potato tuberization is presented in Figure 5.

Figure 5