Variation in Morphological and Quality Parameters in Garlic (Allium sativum L.) Bulb Influenced by Different Photoperiod, Temperature, Sowing and Harvesting Time

Photoperiod (light) and temperature as abiotic factors having significant impact on the garlic bulb morphology and quality. In various bulb plants including garlic, bulbing is affected by photoperiod, temperature, sowing date and the plant age. In this backdrop experiments were performed to understand the effect of different photoperiods (10 h/14 h, 12 h/12 h and 14 h/10 h (light/dark)), temperatures (25 °C/18 °C and 30 °C/20 °C (light/dark)), sowing dates (D0801: 1st August, D0901: 1st September and D1001: 1st October) and plant ages (A80, A60 and A40: 80, 60 and 40 days after planting) on garlic cultivars viz; G103, G024 and G2011-5. Parameters including morphological (plant height, fresh weight and pseudostem diameter), bulb attributes (diameter, weight, height and bulbing index), growth period and bulb quality related traits (total soluble solid (TSS), contents of soluble protein, soluble sugar, total sugar, glucose, sucrose, fructose, starch, total phenol and total flavonoid) were assayed. Longer photoperiod (14 h), higher temperature (30 °C), early sowing (D0801) and maximum plant age (A80) had maximum morphological and bulb quality related traits for cv. G103. These results showed that early sowing, maximum plant age, longer photoperiod and higher temperature are important for garlic bulb formation and quality. Moreover, the regulation of garlic bulb morphology and quality is achievable over the switch of sowing date, plant age, light and growth temperature.


Introduction
Garlic (Allium sativum L.) is the second most significant Allium crop. In addition to its regular use as a vegetable and condiment, it is known for its medicinal and nutraceutical properties. Growth of garlic mainly depends on the time of planting as the vegetative growth is stimulated under a short photoperiod and low temperature and bulb production is enhanced by a long photoperiod and high temperature. Sowing date and plant age significantly influence the various growth and developmental traits of garlic bulb [1][2][3]. Garlic is produced in most areas with mild winters and some rainfall, followed by dry summers [4,5]. Since all garlic cultivars are sterile, reproduction is generally vegetative with the cloves serving as propagation material [6]. Garlic bulb maturation is characterized by the In China, garlic is consumed as a spice and as a seasonal vegetable. Garlic bulbs are considered a high-value vegetable throughout Asia and the demand for bulbs is increasing. Though, variations in climate, geography, photoperiod and temperature significantly alter the bulb production and quality. In addition, the influence of photoperiod and temperature variations on the development of garlic bulbs is poorly understood. One of the most significant limitations of the research of photoperiod and temperature in garlic is the difficulty in controlling for plant age. A systematic understanding of the photoperiod and temperature environments required for garlic growth would improve our knowledge of the bulbing process, facilitate the production of bulbs in different environments and geographies, and enrich our understanding of growth regulation in other bulbous plant species. As a result, year-round production of garlic may be possible. Experiments have been designed to evaluate the effect of different sowing dates and plant ages in combination with photoperiod and temperature regimes on garlic bulb morphology and quality parameters.

Effect of Cultivar, Sowing Date, Plant Age, Photoperiod and Temperature Treatments on Garlic Morphological Traits
The effect of each factor on garlic morphological traits was analyzed (Tables 1 and 2). All of the studied factors, including cultivar (C), photoperiod (L), temperature (T), sowing date (D) and plant age (A) had a highly significant effect on plant height, fresh weight, pseudostem diameter, bulb characteristics (diameter, weight and height), bulbing index (BI) and growth period (Tables 1  and 2). The longer photoperiod (14 h) and higher test temperature (30 • C) presented a significant enhancing effect on the BI, plant height, maturity and bulb characteristics of garlic among the treatments (Tables 1 and 2). Cv. G024 had the highest BI, while cv. G103 produced the highest plant height, bulb characteristics and shortest growth period (Tables 1 and 2). Early sowing date (D 0801 ) and maximum plant age (A 80 ) had a significant effect on all of the studied indicators (Tables 1 and 2; Figures S1-S8).

Discussion
The results of our study showed that bulb characteristics and bulb quality related traits were higher at early sowing and maximum plant age (Tables 1-4; Figures S1-S18). The response of varied sowing dates on bulb development and physiology is different [34]. Long photoperiod and high temperature are suitable for healthy bulb growth. Rahman and Talukda [35] have also reported the similar results. Ali and El-Said [36] evaluated the effect of different planting times and varieties on garlic bulb yield and quality attributes and the results exhibited that early planting resulted in maximum bulbs. In the present study bulbing reduced constantly with late sowing (Tables 1 and 2; Figures S4-S7). The primary phenological stages in the garlic production are the number of days from sowing to emergence, emergence to bulb development, clove development, leafing, bulb initiation and maturity [37]. We observed similar results among the bulb characteristics. Sowing dates significantly influenced the growth and developmental traits of garlic (Tables 1 and 2; Figures S1-S8). Garlic bulb growth was affected by varied dates of planting. Upsurge in bulb growth was stimulated in early sowing (Tables 1  and 2; Figures S4-S7). The valuable effect on plant growth because of early sowing had been stated by Qaryouti and Kasarawi [38]. The garlic plant morphology and bulb quality traits have been observed in maximum quantity with early sowing in comparison with late sowing (Tables 1-4; Figures S1-S18). This results in maximum vegetative growth and bulbing, ultimately the plants have enlarged bulbs. Similar results have been reported by Das et al. [39] and Rahim et al. [40]. The response of different planting dates and plant age on bulb growth was very different. Early planting and maximum plant age constantly increased the bulb characteristics while late planting and minimum plant age decreased the bulb characteristics. The length and girth of bulb was noted at maximum with the first sowing date (Tables 1 and 2; Figures S1-S8). Early plantation also significantly increased the weight and size of bulb. Highest bulb weight might be due to adequate cool and dry climate, which probably increased the vegetative growth and size of bulb. Early plantation produced highest yield due to the large size production of bulb. Late planting reduced significantly the number of cloves and clove size. It might be due to the reason that the plant did not receive a long cool growth period, which was crucial for the development of the bulb as specified by Rahim [41]. On the basis of the above discussion we can assume that the early sowing, maximum plant age of garlic, long photoperiod and high temperature increased the bulbing potential.
According to Brewster [42], onion stops making leaf blades and transfers to the formation of bulb scales when bulb formation is initiated, and the development from leaf growth to bulb formation depends on both the photoperiod and temperature [43]. Moreover, high levels of photosynthetically active radiation combined with a long photoperiod increased both bulb development and final bulb size [44]. Bulb diameter and weight significantly enhanced with increasing photoperiod and temperature (Tables 1 and 2; Figures S1-S8), and a positive effect was observed on garlic bulb formation and quality. Hence, these results recommended that plant growth and bulb development increased as a result of a long photoperiod and high temperature. The bulb formation is strongly influenced by the choice of the cultivar, which correlates with the local weather and location [24]. So, one of the most important features to obtain high production is selecting the cultivar, which is appropriate for the climate and environment of the area. Garlic bulb development is induced by the day length and under an ideal growth temperature, which may vary among garlic cultivars [15]. Thus, to grow healthy garlic bulbs, we have investigated the environmental effects on the characteristics of different commercially used garlic cultivars to evaluate the bulbing performance and bulb quality.
The significant interaction between the photoperiod and temperature resulted in the identification of a critical photoperiod and temperature for bulbing ( Figures S1-S8). Photoperiod might affect the development of primary and secondary metabolites in plants. In Arabidopsis, the long photoperiod condition increases the contents of sucrose, vitamin C and other metabolites [45]. In our study, the phenolic compounds in the bulb of garlic increased at a long photoperiod and high temperature, but we did not observe this surge in the short photoperiod (Tables 3 and 4; Figures S17 and S18). Similarly, sugars in garlic bulbs, relatively lower in short day photoperiod, also revealed a positive response to the long-day photoperiod and high temperature (Figures S11-S18). Garlic bulb under medium-light and long-light conditions had higher soluble protein content than under short-light conditions (Tables 3  and 4; Figure S10). This is constant with the results in bulb characteristics (Tables 1 and 2; Figures S1-S8). Thus, lengthening the light duration and increasing the temperature can improve the quality of garlic bulb according to our investigation. Talbott and Zeiger [46] proposed that light eminence might affect the translocation of carbohydrates, which may affect starch break-down. During a red light the decline of starch is detected, whereas blue light increased the absorption of this polysaccharide in plants [47]. Du Toit et al. [48] stated that mature bulbs of Lachenalia could have diverse forms of starch and soluble sugars throughout the growth period. Several periods increased absorption of starch in bulbs of 'Rupert' thanin 'Ronina', both cultured under white and blue light. This might specify that the plants of 'Rupert' were in a dissimilar developing phase, near to the flowering time, in comparison to the plants from other treatments [49,50]. Ohyama et al. [51] have demonstrated that giving the growing point of mother and new bulblets of tulip cultured in the open field not only increase but also a different kind of carbohydrates might vary. Our investigation provided novel evidence on what way the photoperiod and temperature influence the alterations in primary and secondary metabolites in garlic bulb (Tables 3  and 4; Figures S9-S18).
In the present study it was observed that a longer photoperiod and higher temperature resulted in a higher amount of soluble protein, sugars and phenolic compounds could contribute to the nutritive quality traits as well as the defense mechanism against the environmental alterations. Increased levels of nutritional traits reflected that the bulbs grew at a longer photoperiod and higher temperatures owned not only developed phenolics but they also had higher quantities of protein and sugars (Tables 3  and 4; Figures S9-S18), which upsurged the general antioxidant properties of these bulbs. Present results could be used as a valuable tool for producing garlic bulbs with higher nutritive quality during the growing season in diverse topographical atmospheres. Increased growing temperature, produced fruit with the higher phenolic contents [52]. Similarly, in the present study, the total phenolics and total flavonoids as well as soluble protein and sugars of garlic cultivars increased due to an increase in growth temperature (Tables 3 and 4; Figures S9-S18). Previous studies have also linked increased phenol content with defense and plant growth protection [53,54].
Therefore, garlic bulbs produced at a longer photoperiod and higher temperatures have higher antioxidants, which is an optimistic nutritional enrichment. These possible health benefits of phenolic compounds depend on their interest and uptake, which in order are determined by their assembly plus their conjugation with added phenolics, consist of glycosylation/acylation, molecular size and solubility [55][56][57][58]. The phenolic compounds measured in the current investigation were similar to those detected in other studies [59,60]. As reported earlier [60], phenolic compounds in the garlic bulb, which contained mainly the total phenols and total flavonoids, are strongly affected by the plant genotype. Besides the genotypic dependence, the environmental variations also influence the quantity of phenolics [60]. Furthermore, Mpofu et al. [61] has reported that the environmental factors impart greater influence on the content of phenolics compared to genotypes. Though these researchers evaluated diverse genotypes in their study, the key likely reason could have been that these researchers collected their samples from diverse fields, where all the environmental factors including soil pH, temperature and rainfall were different. In comparison, currently we used controlled environments in which the only altering treatments were the photoperiod and temperature. The contents of phenolic compounds determined in the garlic cultivars in the current research were parallel to those detected in earlier investigations [62][63][64][65][66], establishing that the biosynthesis of phenolic compounds in plants and the garlic bulb was strongly affected by biotic and abiotic factors such as the photoperiod and temperature.
Afterward, the experimental cloves were planted in the field on 1 st August, 1 st September and 1 st October 2018 (these dates corresponded to the sowing date treatments of D 0801 , D 0901 and D 1001 , respectively) at 5 cm depth, 5 cm plant to plant spacing and 20 cm row to row spacing. The plot length and width were 1 m and 3 m, respectively. On 30 th January 2019, the experimental plants of garlic cultivars were transplanted into 15 cm × 15 cm pots and were cultured with organic substrate 'Jiahui' (20%-25% of organic matter, 8%-10% of humic acid, 6.5-6.8 of pH; Liaocheng, Shandong Province, China). Four uniform garlic plants were received by each pot, and for each replication, nine pots were used per treatment. Three replications were used for this trial. On 30 th January 2019, the experimental plants were conditioned for 48 h under 20 • C/18 • C (light/dark) and 80% RH to reduce transplanting shock, before treatment.
On 1 st February 2019, the experimental plants were exposed to different combinations of photoperiod (10 h/14 h, 12 h/12 h and 14 h/10 h (light/dark)) and temperature (25 • C/18 • C and 30 • C/20 • C (light/dark)) treatments in six separate growth chambers (Ningbo Jiangnan Instrument Factory, Zhejiang Province, China) with 70% RH (relative humidity) and 105 µmol m −2 s −1 PAR (photosynthetic active radiation). On 12 th March 2019, 40 days after photoperiod and temperature treatment, six plants from six pots from each treatment of each replication were sampled randomly. After measurement of morphological traits, the bulbs were rinsed with distilled water and dried with absorbent paper. The evenly mixed samples were then absorbed in liquid nitrogen for several seconds and stored at −80 • C in freezer until analysis of the physiological and nutritive quality traits. Three technical replicates were used for analysis.

Effect of Plant Age, Photoperiod and Temperature on Garlic Morphological and Physiological Traits (Trial 2)
The trial was a four-factor (plant age × cultivar × photoperiod × temperature) complete randomized experiment. Uniform cloves of three garlic cultivars (G103, G024 and G2011-5) were selected as the experimental material. Garlic cloves were panted in the field on 20 th September, 10 th October and 1 st November, 2018, these dates corresponded to the plant age treatments. The cultural practices were same as in trial 1. On 8 th December 2018, thirty-eight days after the last planting date, plants at different ages were transplanted in pots (same organic substrate was used as in trial 1).
On 8 th December 2018, the experimental plants were conditioned for 48 h under 20 • C/18 • C (light/dark) and 80% RH to decrease transplanting shock, before treatment. On 10 th December 2018, the experimental plants were exposed to different combinations of photoperiod (10 h/14 h, 12 h/12 h and 14 h/10 h (light/dark)) and temperature (25 • C/18 • C and 30 • C/ 20 • C (day/night)) treatments in six separate growth chambers (Ningbo Jiangnan Instrument Factory, Zhejiang Province, China) with 70% RH (relative humidity) and 105 µmol m −2 s −1 PAR (photosynthetic active radiation). The age of the plants at the time of photoperiod and temperature treatments was A 80 , 80 days after planting; A 60 , 60 days after planting; A 40 , 40 days after planting. Management practices were the same as that for the trial 1.
On 10 th March 2019, 90 days after the photoperiod and temperature treatment, six plants from six pots from each treatment of each replication were sampled randomly. After measurement of morphological traits, the bulbs were rinsed with distilled water and dried with absorbent paper. The evenly mixed samples were then absorbed in liquid nitrogen for numerous seconds and stored at -80 • C in a freezer until analysis of the physiological and nutritive quality indices. For analysis three technical replicates were used.

Assay of Garlic Morphological Indices
The garlic morphological traits were evaluated in the laboratory using a measuring tape (plant height), electronic balance (Changzhou, Jiangsu Province, China; fresh weight and bulb weight) and electronic Vernier caliper (Guanglu, Guilin Province, China; pseudostem diameter, bulb height and bulb diameter). The bulbing index (BI) was calculated as the ratio of the bulb diameter to pseudostem diameter. Bulbing was considered to start when BI = 2 [67]. Growth period (number of days) was calculated from the transplanting date (10 December and 01 February) in growth chambers to the harvest date.

Evaluation of Garlic Bulb Quality Related Traits
TSS (total soluble solid) content of the garlic bulb juice was measured by a digital refractometer (PAL-1, Minato-ku, Tokyo, Japan). The Coomassie Brilliant Blue (CBB) method was used to measure the soluble protein content [68]. Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 mL 95% ethanol and to this solution 100 mL 85% (w/v) phosphoric acid was added. The resultant solution was diluted to a final volume of 1 liter. Final concentrations in the reagent were 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol and 8.5% (w/v) phosphoric acid. The absorbance was taken at 595 nm and content of protein was calculated from standard curve.
Assessment of the soluble sugar content in garlic cloves was done according to Fei et al. [69]. The extracts of H 2 O were diluted with 80% methanol with a concentration of 10 mg/mL. One milliliter diluted extracts and 1.0 mL of 5% phenol were mixed and 5.0 mL H 2 SO 4 was added and mixed carefully. After cooling absorbance was noted at 485 nm and concentration of soluble sugar was determined from the standard curve of glucose solution (concentration range from 0 to 100 µg/mL). Results were expressed as % of glucose equivalents for g of garlic cloves. Total sugar content was assessed according to McCready et al. [70]. A 500 mg sample was extracted in ethanol. After centrifugation at 5000× g for 10 min supernatant was mixed with 15 mL hydrochloric acid (1N) and incubated in water bath for 20 min. Afterward, 1 mL of hydrolyzed extract was taken in a test tube and 4 mL of anthrone reagent was added. After 10 min absorbance was read at 620 nm and calculations were done from standard curve of glucose.
Glucose content was determined according to the method of Miller [71]. Samples were extracted in distilled water and supernatant was mixed with 1 mL of alkaline copper tartrate reagent and the resulting solution was incubated in a boiling water bath for 10 min. The test tubes were removed and cooled followed by an addition of 1 mL of arsenomolybdate reagent. The volume of each test tube was made up to 10 mL with distilled water. After 10 min an absorbance reading was taken at a wavelength of 620 nm. The amount of reducing sugar present in the sample was calculated from the standard curve of glucose. Sucrose content was examined according to Handel,[72]. After extraction the supernatant was mixed with 2 N NaOH and incubated in the water bath for 10 minutes. After cooling, 3.5 mL hydrochloric acid (30%), and 1 mL resorcinol (0.1%) were added, and resulting solution was mixed thoroughly. After incubating at 80°C in water bath for 30 min samples were cooled and read at 480 nm.
Measurement of fructose content was done according to Ashwell [73]. A 50 mg sample was extracted in 4 mL of 80% ethanol in a water bath at 80°C. Supernatant was collected and residue was again extracted in 2 mL of 80% ethanol. Ten milligrams of activated carbon was added to the supernatant to decolorize it. Two milliliters of 0.1% resorcinol and 1 mL of H 2 O were added and incubated at 80°C in a water bath for 10 min. After cooling at room temperature OD (optical density) was read at 480 nm and calculation was done using standard curve of fructose. Starch content was assessed according to McCready et al. [70]. Residue left after sugar estimation was dried and extracted in 3 mL of water at 100°C in a water bath for 20 min. Thereafter, 2 mL of 9.2 N perchloric acid was added and allowed to stand for 10 min followed by centrifugation at 4000× g for 15 min. After that 2.4 mL water and 1.6 mL of 9.2 N perchloric acid were added and allowed to stand for 10 min. Samples were again centrifuged and supernatants were mixed. To 0.4 mL extract was added 4 mL anthrone-sulfuric acid reagent and incubated at 90°C for 15 min. After cooling absorbance was recorded at 620 nm.
An assessment of total phenol content was done according to Singleton and Rossi [74]. One hundred milligrams of the sample was extracted in methanol and the extract was centrifuged at 3000× g for 10 min. One milliliter of supernatant was mixed with 1 mL FC (Folin phenol) reagent and 3 mL of 20% Na 2 CO 3 . After 30 min absorbance was taken at 765 nm wavelength. A standard curve of gallic acid was used for calculation. The method of Yong et al. [75] was used to measure total flavonoids content. After extracting a 100 mg sample in 30% ethanol and 5 mL of 5% sodium nitrite was added to the supernatant and allowed to stand for 5 min. Afterwards 10% aluminum nitrite solution was added followed by the addition of sodium hydroxide. The absorbance was taken at 510 nm and standard curve of rutin was used for calculation.

Statistical Analysis
The data were analyzed using analysis of variance (ANOVA) as a 3 × 3 × 3 × 2 (sowing date × cultivar × photoperiod × temperature) factorial design for trial 1 and 3 × 3 × 3 × 2 (plant age × cultivar × photoperiod × temperature) factorial design for trial 2. SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for analysis. Tukey HSD (Honest Significant Difference) tests were used for separations of mean among treatments at p < 0.05. Three replicates were used for both trials.

Conclusions
In conclusion, the contents of the primary and secondary metabolites were significantly inclined by photoperiod, temperature, sowing date and plant age. Bulb characteristics reduced from the earliest to the latest sowing dates with an increase in photoperiod and temperature. Though, not only bulb growth but also the quality of the garlic bulb was affected by the photoperiod, temperature, sowing dates and plant ages. Variation in the sowing date, plant age, light (photoperiod) and temperature affected the soluble protein, sugars and phenolic compounds that affected the bulb development. Present research establishes a key foundation for selection of management and growing conditions from sowing to photoperiod and temperature regimes for better yield of garlic in varied environments.