INTRODUCTION
Garlic (Allium sativum L.) is the second most important crops from the Allium group after onion which is used as a spice, and medicine in traditional and modern therapeutics (Bayan et al., 2014). The high demand for garlic in Indonesia has been fulfilled through imports from China. Indonesiagovernment’s efforts to reduce garlic import was by expanding the planting area and intensifying garlic cultivation. The availability of planting material in terms of the amount and quality is one of the priorities to support the programs. Garlic is propagated vegetatively using bulbs since the flowers of the commercial varieties are generally sterile (Dufoo-Hurtado et al., 2015).
Bulbs cannot be planted immediately as seeds after
being harvested because they are dormant even under favorable environmental conditions (Lopez- Bellido et al., 2016). The period of time required for garlic bulbs to break dormancy were varied among genotypes, but it is typically around 6 to 8 months.
Some studies had shown that garlic bulbs can remain dormant for even longer periods up to 22 weeks (Palupi et al., 2021).
Dormancy in garlic bulbs is controlled by a number of factors, including temperature, light, and plant hormones (Ai et al., 2023; Sasmitaloka et al., 2021). The bulbs are in a deeply dormant state after harvested (Lopez-Bellido et al., 2016). Dormancy in garlic bulbs is divided into three stages: true dormancy, post-dormancy, and sprouting which is indicating dormancy has ended (Sharma &
ARTICLE INFO Keywords:
Allium sativum Dormant clove Sensitivity index Untargeted metabolite Vernalization
Article History:
Received: May 17, 2023 Accepted: May 30, 2023
*) Corresponding author:
E-mail: *) [email protected];
ABSTRACT
The garlic seed bulbs cannot be planted immediately after harvest because has dormant period. Bulb dormancy can be broken by exposure of pre-planting bulbs to low temperatures. The research aimed to determine the sensitivity of different bolting types of garlic genotypes at low temperatures and the role of low temperatures on dormancy break. The experiment was conducted from March to June 2019 at the Universitas Brawijaya, using a nested design with three replications. Chinese hardneck and softneck, Sangga Sembalun and Tawangmangu Baru were the garlic accessions used in the study. The storage temperature treatments at 3 and 7°C; and room temperature at 27°C. Chinese softneck bulb had the highest sprouting and rooting after 3 and 7°C storage and was sensitive to low temperatures. The Chinese hardneck had the lowest sprouting and was highly insensitive to cold stress. Sangga Sembalun and Tawangmangu Baru had sprouted bulbs in between these Chinese genotypes, and they were insensitive and highly insensitive to low temperatures, respectively. Metabolites of 5-hydroxymethyl-2-furancarboxaldehyde, palmitic acid, diallyl trisulfide, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one played important roles in the bulb response to low temperature stress and directly or indirectly involved in the sprouting and rooting in dormant garlic cloves.
ISSN: 0126-0537Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia, Decree No: 30/E/KPT/2018
Cite this as: Kendarini, N., Aisyah, S. I., Maharijaya, A., & Sobir. (2023). The sensitivity of four garlic genotypes on low temperatures and the role on dormancy breaking. AGRIVITA Journal of Agricultural Science, 45(3), 483–498. http://doi.
org/10.17503/agrivita.v45i3.4177
The Sensitivity of Four Garlic Genotypes on Low Temperatures and the Role on Dormancy Breaking
Niken Kendarini1,2*), Syarifah Iis Aisyah3), Awang Maharijaya3), and Sobir3**)
1) Plant Breeding and Biotechnology Program, Graduate School, IPB University, Bogor, West Java, Indonesia
2) Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
3) Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Bogor, West Java, Indonesia
Lee, 2016; Lopez-Bellido et al., 2016). Exposure to low temperatures is one of the most important factors in breaking garlic dormancy (Ben-Michael et al., 2020). Vernalization is a process by which plants are exposed to cold temperatures in order to break dormancy. The vernalization temperature for breaking bulb dormancy ranges from 5–18°C (Madhu et al., 2019). Higher or lower temperatures will delay dormancy breaking, and the bulb’s dormancy disappears when shoots appear at the tip of the clove neck (Sharma & Lee, 2016; Petropoulos et al., 2016).
Genetic factors affect the rate of bulb dormancy breaking by low temperature treatment.
The duration to low temperature exposures for dormacy breaking was varied among garlic genotypes (Sharma et al., 2016). The different responses among genotypes are related to the sensitivity of the genotypes to the low temperature stress given during storage. Information about the sensitivity of varieties to low temperatures in the vernalization treatment to break dormancy can help growers select the right varieties at the right vernalization temperature and duration.
Low temperature is an abiotic stress that can have a significant impact on plant growth and development. It can affect plant morphology, physiology, and biochemistry (Yadav et al., 2020).
The response of plants to low temperature stress can be seen from the change of the metabolites they produce, which served as changes in metabolism (Genga et al., 2011). Secondary metabolites are biosynthetic compounds derived from primary metabolites, mainly produced as a response to abiotic and biotic environmental stresses.
Metabolomics have been used to study plant responses to abiotic stresses, some of which are studies of rice plant responses to Fe stress (Turhadi et al., 2019).
Some researchers have studied the variability of Indonesia’s local garlic genotypes.
Sandrakirana et al. (2020) stated differences in the quantitative characters of the garlic genotypes, suggesting there was variability among local garlic genotypes. However, Hardiyanto et al. (2008) using RAPD and isoenzyme analysis found the value of genetic relationships were 0.53-0.91 and 0.54-0.94, respectively. The vegetative propagation of garlic causes relatively narrow genetic variability but still has a quite high variation in certain characters.
Garlic can be differentiated based on the hardness
of its pseudostem into the hardneck type (A.
sativum ssp ophioscorodon) and softneck type (A.
sativum ssp sativum). The hardneck garlic has a hard pseudostem and is generally planted in areas with low temperatures, and the softneck type has a soft pseudostem and is cultivated in warmer area (Etoh & Simon, 2002). The variability of garlic can also be grouped based on the maturity time (growth cycle). Indonesian garlic varieties were harvested 99 to 137 days after planting (DAP) in Kota Batu, East Java (data unpublished). The garlic varieties were classified according to maturity time into the following categories: early (< 120 DAP), midseason (120-140 DAP), and late (>200 DAP) (da Cunha et al., 2014).
Previous studies in Indonesian garlic genotypes have shown that vernalization is commonly used to break garlic bulb dormancy.
Still, the response pattern, the visual index of dormancy, and the sensitivity of garlic genotypes with different maturity times and pseudostem types to vernalization treatment have yet to be thoroughly investigated. This study was conducted to determine the effect of storage temperature on the formation of shoots and roots in dormant garlic bulbs, the sensitivity of different garlic genotypes to vernalization treatment, and the metabolites formed in response to garlic bulbs when they underwent vernalization and played a role in breaking the dormancy of the bulbs.
MATERIALS AND METHODS
The research was conducted from March to June 2019 at the Plant Breeding Laboratory, Faculty of Agriculture, Universitas Brawijaya. The genetic material used in this study was garlic from Chinese:
Chinese hardneck (G1) and softneck (G2), and Indonesian garlic varieties: Sangga Sembalun (G3) and Tawangmangu Baru (G4). Sangga Sembalun was obtained from garlic growers in Sembalun, West Nusa Tenggara, and the Tawangmangu Baru (G4) came from Tawangmangu, Solo, Central Java.
The garlic bulbs of the Indonesian varieties were harvested eight weeks earlier and had been cured and dried. Bulbs before treatment were sorted by the cloves criteria with an internal shoot height inside the cloves <30% of the whole clove length by taking random samples from the bulb seed lot (Fig.
1A). According to Lopez-Bellido et al. (2016), garlic cloves with a shoot height less than 20-30% of the total clove length indicate that the cloves were still
dormant. The harvesting time of Chinese hardneck and softneck garlic was unknown because they were purchased from the local market in Malang, East Java, so the height of the internal shoots inside the cloves was used as a reference.
Bulbs were selected for relatively uniform size according to the morphology of each genotype, intact, solid, healthy, and not damaged. The morphology of each genotype was shown in Table 1. The experiment used a nested design with the garlic genotypes nested in the bulb storage temperature and was arranged in the randomized complete design with three replications.
Bulbs were stored at room temperature of 27°C as a control treatment (non-vernalization) and at 3±0.5°C and 7±0.5°C as low temperatures treatment (vernalization). The bulbs were put into vegetable plastic nets and stored in the dark for each treatment storage temperature. The vernalization bulbs were stored in the refrigerator with a relative humidity (RH) of 70-75%, and non- vernalization bulbs were stored on the shelves in the store room with an RH of 65-85%. Observations of garlic cloves were done every week during the six weeks of storage and the sample bulbs were taken randomly from the plastic nets.
Fig. 1. The morphology of garlic cloves before and after vernalization treatments: (A) internal height shoots in cloves <30% as cloves criteria before treatment, (B) shoots height in Sangga Sembalun cloves stored at T3 (3°C); T7 (7°C); T27 (27°C) after six weeks storage, (C) rooting stage of the Chinese softneck cloves stored T3 (3°C ); T7 (7°C ); T27 (27°C) after six weeks storage
The observed characters were bulb weight loss, number and percentage of sprouted cloves per bulb, and visual index of dormancy (VID). Cloves that sprout were cloves with a shoot length of >1 mm from the tip of the clove. Observation of VID was carried out by splitting the cloves longitudinally to determine the position of the shoots inside the cloves. The visual index of dormancy (VID) (Lopez-Bellido et al., 2016) was calculated by the formula (1):
……….(1) Observations on rooting characters were the number of roots per clove, the percentage of rooted cloves per bulb, and root length per clove. The observation of non-target metabolite compounds was analyzed using gas chromatography-mass spectrometry (GCMS). The treated bulbs were then extracted using ethanol maceration’s Vargas method (2016). The extracts of garlic bulbs were analyzed using the GC Agilent Technology model 7890A GC
system coupled with the Mass Spectrometer of Agilent Technology model 5975C.
The combined data were analyzed for analysis of variance (ANOVA) with the F test at a 5% level; if the variances were significantly different, the analysis was continued with the Least Significant Difference (LSD) test at a 5% level.
Character correlation was done by Pearson test.
Genotype sensitivity was analyzed using a stress susceptibility index (SSI) (Fisher & Maurer 1978), which was calculated by the formula (2):
…..…(2) Where: Ys = average specific genotype of abiotic stress (vernalization) conditions, Yp = average of an optimum condition genotype (non-vernalization), Ῡs = average of all abiotic stress (vernalization) genotypes, and Ῡp = average of all optimum condition (non-vernalization) genotypes.
Table 1. Genetic material and morphological characters of bulbs and cloves of four garlic genotypes used in the experiment
Characters Genotype
G1 G2 G3 G4
Bulb weight (g) 36.51b 42.84a 10.66d 17.59c
Cloves number per bulb 7.15d 12.77a 8.89c 10.20b
Clove weight (g) 5.88a 3.38b 1.21d 1.56c
Clove diameter (mm) 25.07a 16.11b 11.40b 12.16b
Clove length (mm) 29.62a 29.06a 18.12b 21.66b
Category of maturity time Late (>180 DAP) Late (>180 DAP) Early (<120 DAP) Mid (120-140 DAP) Bolting type Complete bolting Non-bolting Incomplete bolting* Incomplete bolting*
Pseudostem type Hardneck Softneck Hardneck Hardneck
Remarks: The numbers followed by the same letter in the same row are nonsignificant differences based on the Least Significant Different (LSD) test (α=5%); G1 = China hardneck, G2 = China softneck, G3 = Sangga Sembalun, G4 = Tawangmangu Baru; *data unpublished.
Table 2. Stress susceptibility index of four garlic genotypes to low temperature
G Characters
Average Criteria
PWL NSC PSC VID NR PRC RL
G1 0.53 0.58 0.60 -0.92 0.77 0.44 0.63 0.38 HI
G2 5.43 0.70 0.71 0.38 0.75 0.43 0.61 1.29 S
G3 1.56 0.84 0.85 -0.20 0.76 0.47 0.62 0.70 I
G4 -1.35 0.58 0.61 0.39 0.78 0.40 0.65 0.29 HI
Remarks: G1 = Chinese hardneck, G2 = Chinese softneck, G3 = Sangga Sembalun, G4 = Tawangmangu Baru; PWL = percentage of weight loss bulb, NSC = number of sprouted cloves, PSC = percentage of sprouted cloves, VID = visual index of dormancy, NR = number of root per clove, PRC = percentage of rooted cloves, RL = root length; HI = highly insensitive, I = insensitive, S = sensitive
Determination of the genotype sensitivity to low temperatures was a modification of the criteria according to Kumar et al. (2014). The genotype criteria were distinguished to highly insensitive (HI) genotype if SSI < 0.50, insensitive (I) genotype if 0.50 ≤ SSI < 0.75, moderate (M) genotype if 0.75
≤ SSI < 1.0 and sensitive (S) genotype if SSI ≥ 1.0.
Data on metabolite compounds was collected by making a genotype as a replication for the same treatment (vernalization and non-vernalization).
The data was then analyzed using principal cluster analysis (PCA). The software used for data analysis was Microsoft Excel 2010, SAS 9.4, and R Studio.
RESULTS AND DISCUSSION Sensitivity of Garlic Genotypes to Low Temperature (Vernalization Treatment)
Genotypes response to abiotic and biotic stresses are varied depending on the genetic makeup of each. Based on the observations, the bulbs of Chinese hardneck and softneck garlic genotypes had different sensitivity to low temperatures during vernalization treatment. Those were in the same category based on the maturity time, the late-season group (Table 1). The Indonesian varieties, Sangga Sembalun and Tawangmangu Baru, had slightly different sensitivities to low temperatures. Sangga Sembalun was categorized as an early-season, and Tawangmangu Baru was a mid-season group based on the maturity time. However, Tawangmangu Baru and Chinese hardneck had the same sensitivities to low temperatures. The differences in the maturity time of garlic genotypes did not give the same pattern to the sensitivities to cold stress. It was suspected that the pseudostem hardness type might influence garlic bulbs’ sensitivity to low temperatures.
Chinese softneck was a non-bolting type and sensitive to low bulb storage temperatures (Table 2). Softneck garlic (A. sativum ssp sativum) is usually grown in warmer areas than in temperate climates (Etoh & Simon, 2002). According to Yeshi et al. (2022), plants that grew in warmer subtropical climates will be more sensitive to low temperature stress than plants from temperate climates. Battla &
Benech-Arnold (2015) stated that seed dormancy in summer species or warm-season plants would end faster when treated with low temperatures. Those statements were in line with the characteristic of Chinese softneck, which was sensitive to low storage temperature and had cloves with the highest sprouting and rooting compared to other
genotypes (Table 3 and Table 4). The linear regression equation showed that the Chinese softneck garlic has faster growth of sprout genotype than other garlic genotypes (Table 5). These results implied that Chinese softneck which was non- bolting types garlic would require a relatively short period and a higher temperature in the vernalization treatment than other garlic genotypes to break the bulb dormancy. Generally, the sensitivity index can be used to select genotypes that are tolerant to environmental stress (Zasari et al., 2020). That statement showed that the Chinese softneck most likely has a low tolerance to cold temperature stress.
Chinese hardneck which is complete bolting type garlic was highly insensitive to low temperatures (Table 2). The linear regression equation showed that the Chinese hardneck has a slower shoot elongation rate than other genotype bulbs stored at the same low temperatures (Table 5). Hardneck garlic (A. sativum ssp ophioscorodon) is usually grown in areas with high latitudes or temperate climates with low temperatures (Etoh and Simon 2002). This could be the cause of Chinese hardneck garlic being a highly insensitive genotype to low temperature based on the results of this study. The Chinese hardneck garlic had a sprout growth rate slower than other garlic genotypes from the equation of linear regression and the lowest sprouting cloves (Table 3 and Table 5). It was indicated that Chinese hardneck needed longer duration or lower temperature in vernalization treatments to break bulb dormancy than other garlic genotypes. According to Markovic et al. (2020), plants from areas with low temperatures generally require a longer period of low temperatures to break their dormancy.
Tawangmangu Baru was a highly insensitive genotype to low temperatures, similar to the Chinese hardneck, while Sangga Sembalun was insensitive.
The similarity in the sensitivity of Tawangmangu Baru and Chinese hardneck was strengthened by the results of Hardiyanto et al. (2008), who grouped Tawangmangu Baru and Tiongkok (which assumed Chinese hardneck) garlic into the same cluster based on isozyme and RAPD analysis, while Sangga Sembalun and Honan (which assumed Chinese softneck) were in the same cluster.
Tawangmangu Baru and Sangga Sembalun were incomplete bolting types, so those varieties were the hardneck groups (data unpublished).
Sangga Sembalun and Tawangmangu Baru are Indonesian garlic varieties and have been planted in the center of garlic cultivation areas around Indonesia, which have a tropical climate with warmer temperatures than subtropical ones. The research result of Hirata et al. (2016) showed that 44.4% of garlic from Southeast Asia were incomplete bolting. Indonesian local garlic was assumed to have originally come from hardneck garlic from temperate climates, which then endured mutation, biotic-abiotic stresses, and the selection and eventually spread and adapted well to tropical climates in the center of garlic cultivation areas. Although those varieties were in the same group, Tawangmangu Baru could produce bulblets, flower stalks (scape), and spathe, while Sangga Sembalun only had bulblets within the pseudostem (data unpublished). These
differences might lead to differences in sensitivity to low temperatures. The differences between Tawangmangu Baru and Sangga Sembalun not only showed in the qualitative (including bolting characters) and quantitative characters (data unpublished) but also in the genetics based on molecular and isozyme analysis, which showed that those varieties were located in different clusters on the dendogram of the RAPD and isozyme analysis (Hardiyanto et al., 2008). These results confirmed that Tawangmangu Baru and Sangga Sembalun were phenotypically and genetically different. The difference in sensitivity to low temperature indicated Tawangmangu Baru needed more time to break the dormancy than Sangga Sembalun at the same vernalization temperature.
Table 3. The genotype and storage temperature on the percentage of weight loss of bulb and sprouting characters of four garlic genotypes
Genotype Temperature (°C)
3 7 27
Percentage of weight loss/bulb (%)
G1 5.44b 7.75b 6.60b
G2 10.49a 15.35a 11.35a
G3 2.51b 5.57b 4.49b
G4 3.45b 6.35b 4.27b
Number of sprouted cloves/bulb
G1 0.03d 0.06c 0.00a
G2 5.44a 6.75a 0.08a
G3 4.39b 4.86b 0.03a
G4 2.64c 4.94b 0.00a
Percentage of sprouted cloves/bulb (%)
G1 0.33d 0.78b 0.00a
G2 42.00b 49.56a 0.67a
G3 50.44a 54.67a 0.33a
G4 25.11c 47.78a 0.00a
Visual index of dormancy (%)
G1 77.55b 84.82c 66.76b
G2 107.13a 113.97b 102.91a
G3 107.60a 130.92a 102.75a
G4 105.33a 120.10ab 89.02a
Remarks: Numbers followed by the same letter in the same column are nonsignificant different by the Least Significant Different (LSD) test (α=5%); G1 = Chinese hardneck, G2 = Chinese softneck, G3 = Sangga Sembalun, G4 = Tawangmangu Baru
The Role of the Low Temperature to Break Bulb Dormancy in Garlic
The Chinese softneck as a sensitive genotype to low temperature was the genotype with the highest reduction in bulb weight among garlic genotypes at all storage temperatures (Table 3). The garlic genotypes responded differently to storage temperature on bulb quality (El-Sanousy et al., 2017).
The Chinese softneck had large bulbs and cloves and many cloves per bulb (Table 1). The size of bulbs and cloves of the Chinese softneck affected the rate of bulb weight loss. Large bulbs and cloves had a wider surface area, resulting in higher transpiration, causing a higher decrease in bulb water content, contributing to a decrease in bulb weight. This
experiment results agreed with Abubakar, Maduako,
& Ahmed (2019), smaller onion bulbs had a slower loss weight rate than medium and jumbo-size bulbs.
A different pattern was shown by the Chinese hardneck, which had larger bulb and clove sizes and fewer clove numbers than the Chinese softneck, but bulb weight loss was low (Table 1 and Table 3). The difference between Chinese hardneck and softneck was the sprouting rate of clove per bulb. Chinese softneck had a higher number and percentage of sprouted cloves than Chinese hardneck. Sprouting in cloves causes the rate of respiration in garlic bulbs to increase. Abubakar, Ahmed, & Mashigira (2019) stated that a high respiration rate tended to cause more rapid weight loss.
Table 4. Root growth characteristics of four garlic genotypes under different low temperature treatments
Genotype Temperature (°C)
3 7 27
Root number/clove
G1 1.94b 3.06a 0.00a
G2 5.00a 3.08a 0.00a
G3 0.00c 2.22ab 0.00a
G4 1.11b 1.67b 0.00a
Percentage of rooted cloves/bulb (%)
G1 2.59b 3.89a 0.00a
G2 4.26a 5.37a 0.00a
G3 0.00c 3.33a 0.00a
G4 1.85b 4.07a 0.00a
Root length/clove (cm)
G1 1.17b 4.74b 0.00a
G2 3.32a 9.32a 0.00a
G3 0.00b 2.52c 0.00a
G4 0.99b 2.44c 0.00a
Remarks: Numbers followed by the same letter in the same column are not significantly different by the Least Significant Different (LSD) test (α=5%). G1= Chinese hardneck; G2= Chinese softneck; G3= Sangga Sembalun; G4=
Tawangmangu Baru
Table 5. Linear regression on the character of the growth rate of shoot height on cloves of four garlic genotypes in the different storage temperatures
Storage temperature
(°C) Genotype
G1 G2 G3 G4
3 Y= 10.09x + 0.26
R2= 0.96 Y= 24.62x – 18.57
R2= 0.91 Y= 9.91x + 27.33
R2= 0.95 Y= 9.45x + 19.93 R2= 0.98
7 Y= 9.26x + 5.79
R2= 0.90 Y= 26.04x – 21.55
R2= 0.97 Y= 13.08x + 6.15
R2= 0.92 Y= 14.39x + 17.87 R2= 0.96
Remarks: G1 = Chinese hardneck; G2 = Chinese softneck; G3 = Sangga Sembalun; G4 = Tawangmangu Baru
Chinese softneck was the sensitive to low temperature genotype and had the highest number of sprouted cloves at all low storage temperatures, and Chinese hardneck the highly insensitive genotype was the lowest (Table 2 and Table 3).
According to Akan et al. (2022), softneck and hardneck garlic ecotypes had different responses to storage in sprouting cloves. Because the Chinese softneck was sensitive to low temperatures it was indicated as the most responsive genotype to vernalization than other genotypes at the same storage duration. Chinese softneck had a higher number of sprouted cloves per bulb but had a lower percentage of sprouted cloves than Sangga Sembalun at 3°C. This was because the Chinese softneck had more cloves than Sangga Sembalun (Table 1). So, the average percentage of cloves sprouting per bulb of Chinese softneck was less than Sangga Sembalun. At 27°C, the number and percentage of cloves sprouted in all genotypes was very low and not significantly different. At high temperatures, the enzymes associated with growth are inactive, and the increase of ABA inhibitor and the decrease of endogenous cytokinin and gibberellin would inhibit germination(Petropoulos et al., 2016; Wu et al., 2016).
The visual index of dormancy (VID) value can indicate shoot growth inside the cloves. The low temperature increased the VID value, as indicated by the increase in internal shoot height, which was higher than at room temperature. At 3°C the Sangga Sembalun had the highest VID value but was not significantly different from Chinese softneck and Tawangmangu Baru. At 7°C Sangga Sembalun was not significantly different from Tawangmangu Baru (Table 3). The highest VID value at 27 °C was shown by the Chinese softneck but not significantly different from Sangga Sembalun and Tawangmangu Baru. Chinese hardneck had the smallest VID value in all storage temperatures. In this experiment, shoot height for all genotypes tested at 27 °C was increased in VID value of more than 60%. The RH of the control treatment (room temperature) ranged from 65-80%, which affects the increase in the VID value. Abubakar, Ahmed, & Mashigira (2019) stated that fluctuations in temperature and high RH can trigger the germination of garlic cloves.
The rooting of garlic cloves showed a slightly different response pattern from the sprouting.
Chinese softneck had the highest number of roots
per clove, the percentage of rooted cloves per bulb at 3 °C, and the longest root length at 3 and 7 °C (Table 4). Sangga Sembalun showed no root growth at 3°C. Under the storage treatment of the Chinese softneck had the highest number of roots per clove but was not significantly different from the Chinese hardneck and Sangga Sembalun. The percentage of rooted cloves was not significantly different in all garlic genotypes tested at 7°C. Bulbs stored at 27°C showed no root growth in all garlic genotypes.
According to Islam et al., (2019), optimal root growth in garlic cloves occurs at more than 90% humidity.
In this experiment, the RH in the refrigerator ranged from 70-75%, 65-85% at the room temperature.
The Pearson correlation showed a significant positive correlation between the percentage of weight loss of bulbs and the number of sprouting cloves, so the higher the percentage of bulb weight loss, the higher the number of sprouting cloves (Table 6). This was consistent with the condition of Chinese softneck garlic which had lots of sprouts on the clove/bulb and the highest percentage of bulb weight loss (Table 3). A decrease in bulb weight during storage can indicate internal shoots growing, which causes the increase of respiration (Vázquez- Barrios et al., 2006; Petropoulos et al., 2016).
The number of roots had a negative and significant correlation with the VID value, which meant that the fewer the roots, the higher the shoots.
This pattern was demonstrated by the Chinese hardneck, which grows roots before sprouting.
Sangga Sembalun had shoot growth faster at 3 and 7°C, but root growth could not be observed at 3°C. In comparison, the Chinese softneck grew shoots and roots on its cloves at all vernalization temperatures. The difference in these patterns was caused by genotypes, each of which has a different genetic makeup. According to Liu et al. (2020), some genotypes would grow roots earlier than sprouting, while others grew shoots faster than roots. Chinese softneck was sensitive to low temperatures and not only the genotype with the highest sprouting but also the highest rooting evidence in cloves.
The VID value had a positive and significant correlation with clove diameter, which means the larger the clove diameter, the higher the VID value.
Desta et al. (2021) explained that large bulbs germinate faster due to the more excellent supply of carbohydrates in the cloves. The pattern was suited to Chinese softneck garlic. However, this
pattern could not be applied to Chinese hardneck which has large clove diameters but the lowest VID values for each temperature treatment. Chinese hardneck was a highly insensitive genotype to low temperature. These inferred that the genotype needed a longer duration in vernalization than other
genotypes with different sensitivities to break the dormancy of the bulb. Lower temperatures was required for the bulbs to stimulate shoot growth.
Hardneck garlic is generally cultivated in temperate climates with lower temperatures than in subtropical climates (Etoh & Simon 2002).
Table 6. Correlation between sprout and root characters, and morphology of bulb and clove of four garlic genotypes in the different storage temperatures
PWL Morphology Sprout Root
CW NC DC LC NSC PSC VID NR RL
NSC 0.99* -0.74ns -0.96ns 0.94ns -0.89ns 1.00ns
PSC 0.91ns 0.95ns 0.99ns -0.71ns 0.99ns 0.91* 1.00ns
VID -0.96ns -0.46ns -0.80ns 0.99* -0.68ns 0.94ns -0.71ns 1.00ns
PRC 0.98ns 0.86ns 0.99* -0.85ns 0.97ns -0.98ns 0.98ns -0.87ns 1.00ns
NR 0.53ns 0.97ns 0.76ns 0.99* -0.99ns -0.54ns 0.84ns -0.22* 0.70ns 1.00ns RL 0.98ns 0.86ns 0.99ns -0.85ns 0.97ns -0.98ns 0.98ns -0.85ns 0.99* 0.70ns Remarks: * = significant, ns = non significant correlation based on Pearson correlations (α=5%); PWL = percentage of weight loss per bulb, CW = clove weight, NC= number of cloves per bulb, DC = diameter of clove, LC = length of clove, NSC = number of sprouted cloves, PSC = percentage of sprouted cloves, VID = Visual index of dormancy, RN = root number per clove, PRC = percentage of rooted cloves, RL = root length
Table 7. Metabolites detected in garlic bulbs, loading factor values of the principal component (PC) of vernalized and non-vernalized bulbs
Metabolite NV V Principal component (PC)
PC1 PC2 PC3 PC4
2,3-dihydro-3,5-dihydroxy-6-methyl-4(h)-pyran-4-one √ √ -0.01 0.14 -0.25 0.58
5-hydroxymethyl-2-furancarboxaldehyde √ √ -0.78 -0.37 -0.20 0.25
Palmitic acid methyl ester √ √ 0.13 -0.15 0.16 0.29
Palmitic acid √ √ 0.29 -0.10 0.60 0.29
Methyl (9z,12z)-9,12-octadecadienoate √ √ 0.03 0.25 -0.03 0.39
Diallyl trisulfide √ √ 0.40 -0.64 -0.50 0.08
8,11-octadecadienoic acid, methyl ester √ √ 0.00 -0.03 -0.03 -0.10
Linoleic acid √ √ 0.12 0.30 -0.12 0.49
Tricosane √ √ 0.01 0.02 -0.01 -0.03
Oleic acid amide C18H35NO √ √ 0.14 -0.05 -0.05 0.05
Z-12-pentacosene - √ 0.01 0.00 -0.03 -0.02
Pentacosane - √ 0.00 0.01 -0.02 -0.01
Docosene - √ 0.01 0.00 -0.03 -0.03
10-heneicosene (c,t) √ √ -0.11 0.23 -0.05 0.16
Cyclotetracosane √ √ -0.02 0.01 -0.05 -0.07
(23s)-ethylcholest-5-en-3-?-ol - √ 0.03 0.01 -0.06 -0.07
Acetamide, n-(1-dimethylaminomethylene-2-oxopropyl)- - √ 0.01 0.01 -0.02 -0.04
Linolelaidic acid, methyl ester - √ 0.19 0.11 0.02 -0.20
Linolenic acid, methyl ester √ √ -0.01 -0.38 0.49 0.04
Table 7. (continued)
Metabolite NV V Principal component (PC)
PC1 PC2 PC3 PC4
Gamma.-sitosterol - √ 0.01 0.00 0.04 0.00
(9z)-9-tricosene - √ 0.02 0.02 0.10 0.01
3,4-dihydro-2-naphthalinmethanol √ √ 0.10 -0.10 0.23 0.23
Peri-xanthenoxanthene-4,10-dione, 2,8-bis(1-methylethyl)- - √ 0.01 0.00 0.04 0.00
3-vinyl-3,6-dihydro-1,2-dithiine # √ √ 0.08 -0.08 0.12 0.27
Alpha.-tolualdehyde - √ 0.01 0.01 0.10 0.00
Nonadecene √ √ -0.11 0.05 0.03 0.21
Cyclohexene 4-(4-Ethylcyclohexyl)-1-Pentyl- - √ 0.01 0.01 0.00 -0.02
Stearic Acid - √ 0.02 0.01 -0.02 0.00
Eicosanol - √ 0.00 0.01 0.00 -0.01
Dimethyl Trisulfide - √ -0.01 0.01 0.00 0.02
E Z-1,312-Nonadecatriene - √ -0.01 0.02 0.00 0.02
Oleic Acid Methyl Ester - √ 0.01 0.01 -0.01 -0.01
2-Propanamine N- (1-Methylethyl)- - √ 0.01 0.00 -0.02 0.03
Diallyl tetrasulfide - √ 0.01 0.00 0.00 0.02
7-Heptadecene, 1-chloro- - √ 0.05 0.04 0.14 -0.06
Stearic Acid Methyl Ester - √ 0.01 0.01 0.00 -0.02
13-Methyloxacyclotetradecane-2,1,1-Dione - √ 0.01 0.01 0.00 -0.02
Cyclopentadecane - √ 0.01 0.02 0.00 -0.04
3-deoxy-d-mannonic acid - √ 0.01 0.01 -0.01 0.00
Alpha.-tocopherol - √ 0.01 0.01 0.00 -0.02
3-vinyl-3,4-dihydro-1,2-dithiine √ √ 0.00 -0.08 -0.02 -0.31
1,2-dithiolane √ - -0.01 -0.05 -0.01 -0.12
11-octadecenoic acid, methyl ester √ √ -0.01 -0.03 0.02 -0.01
Stigmastan-3,5-diene √ - -0.01 -0.06 0.04 0.04
14-.beta.-h-pregna √ - -0.02 0.00 -0.01 -0.03
13-tetradecenyl acetate √ √ -0.02 0.01 -0.01 -0.01
2-furancarboxaldehyde, 5-methyl- - √ 0.04 -0.01 -0.07 0.04
Heptacosane, 1-chloro- - √ 0.00 0.00 -0.01 -0.01
8-methyl-6-nonenamide - √ 0.01 0.00 -0.02 -0.02
Diallyl disulfide - √ 0.05 0.03 -0.08 -0.07
2-propanamine, n- (1-methylethyl)- - √ 0.02 0.00 -0.03 0.00
Oleic acid, methyl ester - √ 0.01 0.01 0.00 -0.02
4-pyrimidinol, 5-methoxy- - √ 0.05 -0.01 -0.12 0.16
(1rs,4rs)-5,5-ethylenedioxy-7-oxabicyclo[2.2.1]hept-2-ene - √ 0.01 0.00 -0.01 0.03
N-nonadecane - √ 0.01 0.01 0.00 -0.03
Z-14-nonacosane - √ 0.01 0.01 0.00 -0.02
9-hexacosene - √ 0.00 0.02 0.00 -0.01
Table 7. (continued)
Metabolite NV V Principal component (PC)
PC1 PC2 PC3 PC4
Eicosane - √ 0.01 0.00 0.00 -0.01
Z-11-tetradecenoic acid - √ 0.01 0.01 -0.01 -0.02
Methyl 9-cis,11-trans-octadecadienoate - √ 0.00 0.00 0.00 -0.01
2-methyl-3-(methylthio)furan - √ 0.01 0.01 -0.01 0.00
Henicosane - √ -0.01 0.01 0.00 0.01
Octadecane - √ 0.01 0.01 0.00 -0.03
Eigenvalue 15.38 4.83 1.60 1.00 Variance (%) 64.06 20.13 6.66 4.18 Cumulative variance (%) 64.06 84.19 90.85 95.03 Remarks: Ö = detected, - = not detected, numbers in bold and highlight have a loading factor value of >0.5
a
b
0 5 10 15 20 25 30 35 40 45
Non-vernalization Vernalization
Relative concentration (%) A
b
a
0 2 4 6 8 10 12
Non-vernalization Vernalization
Relative concentration
B
b
a
02 46 108 1214 1618 2022 24
Non-vernalization Vernalization
Relative concentration (%)
C
b a
02 4 68 1012 1416 18
Non-vernalization Vernalization
Relative concentration (%)
D
Fig. 2. Relative concentration alteration of the metabolites in the vernalized and non-vernalized garlic bulbs. (A) the metabolite 5-hydroxymethyl-2-furancarboxaldehyde, (B) palmitic acid, (C) diallyl trisulfide, and (D) 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one
The results of this study indicated that the number of roots had a positive and significant correlation with clove diameter. This result followed the opinion of Lopez-Bellido et al. (2016) that the roots were grown in outside and inside the circle of the clove disc. These resulted the larger the diameter of the clove would produce the more surface area for the roots to grow. Root length had a positive and significant correlation with the percentage of rooted cloves, which indicated that after the cloves grow roots, the roots will continue to grow elongated.
Based on the results of the GCMS analysis, 63 non-target metabolite compounds were detected and identified from all observed bulbs (Table 7).
Metabolites detected in vernalized garlic bulbs were 95.24% (60 metabolites), while only 36.67% (22 metabolites) were detected in nonvernalized bulbs.
In the vernalization treatment, low temperature is a form of abiotic stress that can affect the physiology and biochemistry of plants. Low temperature is abiotic stress affecting plants’ morphology, physiology, and biochemistry (Yadav et al., 2020).
Plants will respond to abiotic stress by changing the expression patterns of genes that encode proteins that control the biosynthesis of metabolites needed for defense against these conditions (Ljubej et al., 2021). The many metabolites detected in vernalized garlic bulbs indicated that the metabolism occurring in the bulbs was more complex. According to Arbona et al. (2013), more metabolite compounds were formed as a response of plants to environmental stress caused by a more complicated metabolism in these plants.
Based on the PCA results from the data of metabolites, it was detected that there were four PCs (PC1-PC4) that had an eigenvalue >1.0 (Table 7). The metabolites with a loading factor value of more than 0.5 for each PC1-PC4 were 5-hydroxymethyl-2-furancarboxaldehyde, palmitic acid, diallyl trisulfide, and 2,3-dihydro-3,5-dihydroxy- 6-methyl-4(H)-pyran-4-one (Table 7). PC with an eigenvalue of less than 1.0 has less diversity, so the influence on the contribution of cumulative diversity is low, while characters with a loading factor of more than 0.5 contribute to maximum diversity (Girden 2001; Woolford 2015). Therefore, the metabolites 5-hydroxymethyl-2-furancarboxaldehyde, palmitic acid, diallyl trisulfide, and 2,3-dihydro-3,5-dihydroxy- 6-methyl-4(H)-pyran-4-one contributed to the varies of the response of garlic bulbs to low temperatures by vernalization.
The 5-hydroxymethyl-2-furancarboxaldehyde is a derivative of furan containing both aldehyde and alcohol functional groups, which are formed from the simple sugar molecules glucose and fructose under hydrothermal acid conditions (Kowalski et al., 2013; Yang et al., 2019). The relative concentration of 5-hydroxymethyl-2-furancarboxaldehyde tended to be higher in non-vernalized bulbs or stored at room temperature of 27 °C (Fig. 2A).
The lower concentration of 5-hydroxymethyl-2- furancarboxaldehyde in vernalized garlic bulbs indirectly indicates that glucose and fructose as essential substrates of 5-hydroxymethyl-2- furancarboxaldehyde had not been changed to 5-hydroxymethyl-2-furancarboxaldehyde. Glucose and fructose concentrations increase in leaves during low temperature stress, which functions as osmoprotectants that stabilize proteins and membranes and maintain cell osmotic pressure (Orzechowski et al., 2021). Glucose and fructose produced from hydrolyzed carbohydrates at low temperatures were also used as a source of energy for plant growth, and a source of carbon for seedling growth during the transition to autotrophy (Ohanenye et al., 2019; Wang et al., 2021). This was indicated also in this research that the vernalized bulbs have more shoots and roots than non-vernalized bulbs (Table 2).
Palmitic acid was a metabolite compound with a loading factor value of more than 0.5 (Table 7). The results showed that vernalized garlic bulbs had an increased relative concentration of palmitic acid compared to non-vernalized garlic bulbs (Fig.
2B). This result was the same as the research result of Hu et al. (2017) which showed that the palmitic acid content increased when Bermuda grass was exposed to low temperature stress. Cold temperature stress causes a decrease in plasma membrane fluidity due to the change from the liquid- crystalline phase to the gel phase. The response of plants to maintain membrane stability was to increase unsaturated fatty acids (linolenic acid and linoleic acid) and saturated fatty acids (palmitic acid) in their membranes (Petrov et al., 2016; Hu, et al., 2017). The increased concentration of palmitic acid was assumed to be related to the sprouting and rooting of garlic bulbs/cloves. Palmitic acid supports forming of new membranes in cells. Seed germination was associated with the production of numerous new membranes incorporating polar lipids that require more palmitic acid (Zhukov, 2015).
The diallyl trisulfide was detected and acted as an important metabolite in the metabolism of garlic bulbs during vernalization treatment based on the result of PCA analysis (Table 7). The results of this study indicated that diallyl trisulfide increased in garlic bulbs stored at low temperatures (vernalized) (Fig. 2C). At low temperature stress, the accumulation of reactive oxygen species (ROS) will increase, which can cause cell damage (Zhang et al., 2016). Diallyl trisulfide, which has the ability as an antioxidant can suppress the accumulation of ROS, is an antifungal, anti-inflammatory, antitumor, antidiabetic, anticancer, chemopreventive agent (Zakarova et al., 2014; Lee et al., 2015). The research of Marques et al. (2021) resulted that garlic extracts were able to induce budburst in bud dormancy in Kiwi vines in the two seasons. Diallyl trisulfide is the major organosulfides in garlic oil besides diallyl disulfide. Diallyl trisulfide (DAT) reacts rapidly with glutathione (GSH) to release hydrogen sulfide (H2S) through thiol−disulfide exchange followed by allyl perthiol reduction by GSH, and diallyl disulfide is not a rapid H2S donor (Zakarova et al., 2014). Hydrogen sulfide (H2S) is regulating essential plant processes such as seed germination, root growth, flowering, crosstalk with plant hormones, and other functions (Zhou et al., 2021). From those statements, diallyl trisulfide was also involved in breaking dormant cloves via H2S to support sprouting and rooting in garlic cloves.
The metabolite 2,3-dihydro-3,5-dihydroxy- 6-methyl-4(H)-pyran-4-one had a function as an antioxidant (Sachdev et al., 2021). This research showed a relative concentration of the metabolite 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4- one was higher in vernalized bulbs (Fig. 2D). The accumulation of ROS increases when low temperature stress occurs and can be reduced by antioxidants to prevent cell damage due to further ROS actions.
Garlic sprouting is also associated with increases in antioxidant activity (Zakarova et al., 2014).
CONCLUSION
Low temperatures above 0 to 10°C can break dormancy by accelerating the growth of shoots and roots in dormant garlic bulbs. Genotypes responded differently to the vernalization treatment, indicating the differences in genetic makeup, and it was also shown in their sensitivity to low temperatures. The Chinese softneck (non-bolting) was the sensitive genotype to low temperatures and had the highest
sprouting and rooting values in dormant clove. The Chinese hardneck (complete bolting) was the highly insensitive genotype to low temperatures and had the lowest sprouting in cloves. Tawangmangu Baru and Sangga Sembalun were incomplete bolting types (hardneck group) that were highly insensitive and insensitive genotypes to low temperatures, respectively, and had the sprouting and rooting in the cloves between Chinese hardneck and softneck. The highly insensitive genotypes to low temperatures needed more time to break bulb dormancy in the vernalization treatment than the sensitive genotypes. The morphology of clove and bulbs correlated with the sprouting and rooting of garlic. Vernalization of bulbs causes alteration in the numbers and composition of the metabolites produced by garlic bulbs. Metabolites that function as energy providers, membrane stabilizers, and antioxidants tend to increase in vernalized bulb to overcome cold stress and were associated with supporting shoot and root growth in garlic cloves.
ACKNOWLEDGEMENTS
This work was supported by the Indonesia Endowment Fund for Education of the Minister of Finance through the scheme of the scholarship program.
REFERENCES
Abubakar, M. S., Maduako, J. N., & Ahmed, M. (2019).
Effects of storage duration and bulb sizes on physiological losses of Agrifound light red onion bulbs (Allium cepa L.). Agricultural Science and Technology, 11(1), 90–97.
Abubakar, M.S., Ahmed, M., & Mashigira, S.A. (2019).
Effects of storage duration and bulb size on physiological losses of white creole onion (Allium cepa L.) bulbs stored in improved naturally ventilated storage structure. African Journal of Agriculture Technology and Environment, 8(2), 14-23. https://ajate.com.ng/wp-content/
uploads/2023/02/2-Abubakar-AJATE-Vol-8- No-2.pdf
Ai, P., Xue J., Zhu, Y., Tan, W., Wu, Y., Wang, Y., Li, Z., Shi, Z., Kang, D., Zhang, H., Jiang, L., & Wang, Z. (2023). Comparative analysis of two kinds of garlic seedings: qualities and transcriptional landscape. BMC Genomics, 24:87. https://doi.
org/10.1186/s12864-023-09183-x
Akan, S., Karakan, F. Y., & Horzum, Ö. (2022).
Differential response of softneck and hardneck garlic ecotypes to quality attributes for long-
term storage. Emirates Journal of Food and Agriculture, 34(4), 346–357. https://doi.
org/10.9755/ejfa.2022.v34.i4.2845
Arbona, V., Manzi, M., de Ollas, C., & Gómez-Cadenas, A. (2013). Metabolomics as a tool to investigate abiotic stress tolerance in plants. International Journal of Molecular Sciences, 14(3), 4885–
4911. https://doi.org/10.3390/ijms14034885 Batlla, D., & Benech-Arnold, R. L. (2015). A framework
for the interpretation of temperature effects on dormancy and germination in seed populations showing dormancy. Seed Science Research, 25(2), 147–158. https://doi.org/10.1017/
S0960258514000452
Bayan, L., Koulivand, P. H., & Gorji, A. (2014). Garlic: a review of potential therapeutic effects. Avicenna Journal of Phytomedicine, 4(1), 1–14. https://doi.
org/10.22038/ajp.2014.1741
Ben-Michael, T. E., Rozenblat, L., Faigenboim, A., Shemesh‐Mayer, E., Forer, I., Peters, R., Klein, J. D., Rabinowitch, H. D., & Goldstein, R. K.
(2020). From embryo to adult: Low temperatures affect phase transitions of Allium sativum L. From germination to flowering. Agronomy, 10(11).
https://doi.org/10.3390/agronomy10111651 da Cunha, C. P., Resende, F. V., Zucchi, M. I., &
Pinheiro, J. B. (2014). SSR-based genetic diversity and structure of garlic accessions from Brazil. Genetica, 142(5), 419–431. https://doi.
org/10.1007/s10709-014-9786-1
Desta, B., Tena, N., & Amare, G. (2021). Growth and bulb yield of garlic as influenced by clove size.
Hindawi The Scientific World Journal, 7351873, 7. https://doi.org/10.1155/2021/7351873
Dufoo-Hurtado, M. D., Huerta-Ocampo, J., Barrera- Pacheco, A., Barba de la Rosa, A. P., &
Mercado-Silva, E. M. (2015). Low temperature conditioning of garlic (Allium sativum L.) “seed”
cloves induces alterations in sprouts proteome.
Frontiers in Plant Science, 6, 1–15. https://doi.
org/10.3389/fpls.2015.00332
El-Sanousy, M.A., Haridy, A.G.H, Abd-El-Aal, S.A., &
Badawy, A.S. (2017). Quantity and quality losses in some garlic (Allium sativum L.) cultivars under different storage methods. Assiut Journal of Agriculture Science, 48(5), 300-307. Retrieved from https://search.emarefa.net/en/detail/BIM- 886319-quantity-and-quality-losses-in-some- garlic-allium-sativum-l.
Etoh T., & Simon, P.W. (2002). Diversity, fertility and seed production of garlic. In H.D.
Rabinowitch, & L. Currah (Eds.), Allium Crop Scence: Recent Advances. (pp. 101-117).
Wallingford, UK. CAB International. https://doi.
org/10.1079/9780851995106.0101
Fischer, R. A., & Maurer, R. (1978). Drought resistance in spring wheat cultivars. I. Grain yield responses.
Australian Journal of Agricultural Research, 29(5), 897–912. https://doi.org/10.1071/
AR9780897
Genga, A., Mattana, M., Coraggio, I., Locatelli, F., Piffanelli, P., & Consonni, R. (2011). Plant Metabolomics: A Characterisation of Plant Responses to Abiotic Stresses. Abiotic Stress in Plants - Mechanisms and Adaptations, iii. https://
doi.org/10.5772/23844
Girden ER. (2001). Evaluating research articles from start to finish. Thousand Oaks, Calif., Sage Publications. California. Retrieved from https://
eric.ed.gov/?id=ED409805
Hardiyanto, Devy, N.F., & Martasari, C. (2008).
Identifikasi kekerabatan genetik klon-klon bawang putih Indonesia menggunakan isozim dan RAPD. Jurnal Hortikultura, 18(4), 385-394.
https://repository.pertanian.go.id/server/api/
core/bitstreams/577c3469-d66f-4bba-8d49- 6ca80ad00822/content
Hirata, S., Abdelrahman, M., Yamauchi, N., & Shigyo, M. (2016). Diversity evaluation based on morphological, physiological and isozyme variation in genetic resources of garlic (Allium sativum L.) collected worldwide. Genes and Genetic Systems, 161–173. https://doi.
org/10.1266/ggs.15-00004
Hu, Z., Amombo, E., Gitau, M. M., Bi, A., Zhu, H., Zhang, L., Chen, L., & Fu, J. (2017). Changes of antioxidant defense system and fatty acid composition in Bermudagrass under chilling stress. Journal of the American Society for Horticultural Science, 142(2), 101–109. https://
doi.org/10.21273/JASHS03999-16
Islam, M. N., Wang, A., Pedersen, J. S., Sørensen, J.
N., Körner, O., & Edelenbos, M. (2019). Online measurement of temperature and relative humidity as marker tools for quality changes in onion bulbs during storage. PLoS ONE, 14(1), 1–19. https://doi.org/10.1371/journal.
pone.0210577
Kowalski, S., Lukasiewicz, M., Duda-Chodak, A., & Ziȩc, G. (2013). 5-hydroxymethyl-2-furfural (HMF) -heat-induced formation, occurrence in food and
