Effects of multiple factors on hyperhydricity of Allium sativum L.

Scientia Horticulturae 217 (2017) 285–296

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Effects of multiple factors on hyperhydricity of Allium sativum L. Min Liu, Fangling Jiang, Xiangyu Kong, Jie Tian, Zexiu Wu, Zhen Wu ∗ College of Horticulture, Nanjing Agricultural University/ Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing, 210095, PR China

a r t i c l e

i n f o

Article history: Received 23 November 2016 Received in revised form 1 February 2017 Accepted 4 February 2017 Keywords: Garlic Hyperhydricity Plantlets in vitro Multiple factors Shoot proliferation

a b s t r a c t Hyperhydricity is a common physiological disorder during plant in vitro culture and seriously affected regeneration and micropropagation of plants. Garlic is very susceptible to hyperhydricity. However, effects of multiple factors on hyperhydricity of garlic remain unclear. To clear the regularity of occurrence of hyperhydricity and to obtain a high-efficiency regeneration system of garlic, we systematically investigated effects of explants, media components, culture conditions, and exogenous additives on hyperhydricity. Our results showed that shoots were more easily hyperhydric than plantlets. Shoots induced by inflorescences showed a higher hyperhydric rate and proliferation coefficient than those induced by bulbs. Genotype, physiological age, and explant size affected hyperhydricity of shoots in initial culture, not that of plantlets in subculture. Younger inflorescence and smaller explant were more easily hyperhydric. Dose-dependent manners of cytokinins and gelling agents involved in hyperhydricity were found. Hyperhydricity was aggravated at increased cytokinin concentrations and was alleviated by increased gelling agent and sucrose concentrations, ventilation, and illumination intensity. Media with pH higher than 6.0 and lower than 5.8 resulted in more hyperhydricity. Shoots and plantlets were much more likely to be hyperhydric in MS medium than that in B5 medium. Hyperhydricity was relieved by 50 ␮M salicylic acid, 250 ␮M ascorbic acid, 10 ␮M spermidine, and 50 ␮M hydrogen peroxide, but aggravated by high concentrations of hydrogen peroxide and spermidine. Mannitol had no effect on hyperhydricity, whereas polyethylene glycol 6000 induced it. Positive correlations of shoot proliferation and hyperhydricity were found under different treatments of cytokinins, gelling agents, and explants which included genotype, organ type, physiology age and size. A regeneration system of garlic with high proliferation coefficient and low hyperhydric rate was established based on the results above. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Hyperhydricity, previously known as vitrification, is a common morphological, anatomical, and physiological disorder during plant in vitro culture (Kevers et al., 2004). It was first reported more than 50 years ago by Phillips and Mathews (1964) in shoot tip cultures of carnation. At present, more than 200 species have been found to be hyperhydric, and about 150 of them are easily but significantly hyperhydric (Bakir et al., 2016; Chakrabarty et al., 2006;

Abbreviation: Ag+ , silver ion; AsA, ascorbic acid; B5, gamborg; BA, benzyladenine; H2 O2 , hydrogen peroxide; ICM, initial culture medium; MA, mannitol; MS, murashige and Skoog’s; NAA, ␣-naphthaleneacetic acid; NH4 + , ammonium nitrogen; NO3 − , nitrate nitrogen; PA, polyamine; PC, proliferation coefficient; PEG 6000, polyethylene glycol 6000; pH R, plantlet hyperhydric rate; pH I, plantlet hyperhydric index; SA, salicylic acid; SCM, subculture medium; SHR, shoots hyperhydric rate; SHI, shoots hyperhydric index; Spd, spermidine; KT, kinetin. ∗ Corresponding author. E-mail address: [email protected] (Z. Wu). http://dx.doi.org/10.1016/j.scienta.2017.02.010 0304-4238/© 2017 Elsevier B.V. All rights reserved.

Mayor et al., 2003; Wu et al., 2009; Zhou, 1995). The common feature of hyperhydricity in these species is excessive hydration of tissues (Fernandez-Garcia et al., 2008), which results in its their difficulty in differentiation, multiplication, rooting, and survival after transplant (Picoli et al., 2001). In addition, hyperhydricity significantly affects applications of in vitro culture in industrial production and scientific research. For about 60% of plants, hyperhydricity was reported to result in great loss (Tabart et al., 2015; Tian et al., 2016; van den Dries et al., 2013; Wu et al., 2009). Germplasm resources conservation of endangered species became more difficult because of hyperhydricity (Pence et al., 2014). Hyperhydricity of transgenic plants caused waste of previous experimental efforts (vanAltvorst et al., 1996). Water, macronutrients, micronutrients, plant growth regulators, vitamins, and sugar in the growth media supply 95% of nutrients and energy for explants (Razdan, 2003). Different microclimates have been created in culture vessels according to required conditions (Lai et al., 2005). The specificity of explants, media, and microenvironments results in complexity of impact factor of

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hyperhydricity. Impact factors of hyperhydricity can be classified into four aspects: 1) explant, including genotype, physiological age, organ type, and size (Fei and Weathers, 2015; Mayor et al., 2003; Tsay et al., 2006; Vasudevan and Van Staden, 2011); 2) media components, such as basal medium, plant growth regulators, and gelling agents (Ivanova and van Staden, 2008; Vasudevan and Van Staden, 2011); 3) culture conditions, such as illumination intensity and ventilation condition (Ivanova and Van Staden, 2010; Saez et al., 2012; Tsay et al., 2006); and 4) exogenous additives, such as SA, PEG 6000, H2 O2 , and Ag+ (Hassannejad et al., 2012; Sen and Alikamanoglu, 2013; Tian et al., 2015; Vinoth and Ravindhran, 2015). Complex impact factors of hyperhydricity directly results in the difficulty of prevention and control hyperhydricity. Corresponding control technologies were proposed based on impact factors above, such as the following: 1) suitable selected genotype or organ as explants (Tsay et al., 2006; Vasudevan and Van Staden, 2011); 2) adjusted media components, such as reduced concentration of NH4 + and cytokinins, increased concentration of calcium, iron, magnesium (Ivanova and Van Staden, 2009; Machado et al., 2014; Vasudevan and Van Staden, 2011; Yadav et al., 2003); 3) improved culture microenvironment, such as increased ventilation (Ivanova and Van Staden, 2010; Perez-Tornero et al., 2001); and 4) added exogenous additives, such as Ag+ , SA, PA, phloroglucinol or polysaccharide-producing bacterial Pseudomonas spp. (Hassannejad et al., 2012; Tabart et al., 2015; Teixeira da Silva et al., 2013; Ueno et al., 1998; Vinoth and Ravindhran, 2015). Using one or several preventative methods together described above usually cannot prevent hyperhydricity ideally. Besides, many methods are specific to a limited number of plant species and cannot be applied to others. Furthermore, different plant species need distinct media components and culture conditions, and adjustment ranges of media components vary with species. Therefore, regularity of occurrence of hyperhydricity in specific plant species should be studied individually and comprehensively. Garlic (Allium sativum L.), an annual or biennial herb belonging to the Amaryllidaceae family, is a worldwide vegetable, a medicine, and a condiment with considerable edible and medicinal value. Garlic preparations are popular across the world because of their antibacterial, anticancer, and anti-cardiovascular benefits (Li et al., 2016; Ried, 2016; Varshney and Budoff, 2016). Garlic was asexually propagated for centuries. Accumulation of virus due to vegetative propagation throughout the year cause considerable garlic degeneration (Ramírez-Malagón et al., 2006). Micropropagation is the most effective and economical method to producing large quantities of genetically uniform, pathogen-free garlic plants within a short time. However, even though first in vitro system of garlic has been established for 40 years (Kehr and Schaeffer, 1976), it has not yet been applied to industrial production. High frequency of hyperhydricity is a major concern (Liu et al., 2015). During in vitro culture of garlic, hyperhydricity easily burst on a large scale, sometimes reaching rates of up to 100%. Hyperhydric garlic plantlets is difficult to reverse, causing high losses in terms of labor, materials, and financial resources. To study the effects of impact factors on hyperhydricity of garlic systematically, we investigated the effects of explant (genotype, organ type, physiological age, and explant size), media components (cytokinins, gelling agents, sucrose, and basal medium), culture conditions (media pH, illumination intensity, and ventilation condition), and exogenous additives (AsA, SA, Spd, H2 O2 , MA, and PEG 6000) on hyperhydricity of shoots and plantlets in vitro. Relationships between shoot proliferation coefficient and hyperhydricity were discussed. Besides, a high-efficiency regeneration system of in vitro garlic micropropagation was proposed. These experiments contribute to our understanding of effects of multiple factors on hyperhydricity, and provide a reference for prevention of hyperhydricity in other plant species.

2. Materials and methods 2.1. Plant materials Four varieties of garlic (A. sativum L. cultivars Ershuizao, Xuzhoubai, Cangshan, and Zhengyuezao) bulbs were germinated and grown in a greenhouse at the Horticultural College of Nanjing Agricultural University, Nanjing, China. Inflorescences were harvested 200–220 days after planting and bulbs after 280 days. 2.2. Garlic shoots and plantlets induced by inflorescences or bulbs 2.2.1. Inflorescence-induced pathway Inflorescences with a rachis of 5 cm were harvested 210 days after planting, immersed in saturated detergent water for 20 min, and rinsed with running water for 30 min. Clean inflorescences were placed on a clean bench, immersed in 70% ethyl alcohol and washed with sterile water. Subsequently, inflorescences were immersed in 2% sodium hypochlorite for 12 min and washed five times with sterile water. After drying on the bench, the bracts and degraded primordial flowers were removed. The inflorescences were then cut lengthwise into four pieces and cultured in an initial culture medium (ICM, B5 medium + 8.8 ␮M BA + 0.54 ␮M NAA + 0.65% agar + 3% sucrose, pH = 5.8). After 20 days, normal shoots were selected and subcultured in a subculture medium (SCM, B5 medium + 4.4 ␮M BA+ 0.54 ␮M NAA+ 0.65% agar + 3% sucrose, pH = 5.8). 2.2.2. Bulb-induced pathway Same-size bulbs of four varieties were selected and kept at 4 ◦ C for 10 days to break dormancy. Induction of shoots and plantlets by bulbs was performed following the published protocol (Tian et al., 2015), using ICM as described above to induce shoots. After 20 days, normal shoots were selected and subcultured in SCM. All in vitro-grown explants were grown at 25 ± 1 ◦ C with a 12-h photoperiod per day. All explants except for different illumination intensities treatments were grown under cool white fluorescent light at 100 ␮mol m−2 s−1 (Philips, China). pH of all treatments except for different pH treatments is 5.8. pH values were adjusted by a Phs-3C (Leici, Shanghai, China). 2.3. Experimental design and treatments 2.3.1. Effects of subculture time on hyperhydricity Inflorescences of Ershuizao after 210 days planting were selected for inducing shoots, and other treatments were the same as described under Section 2.2. Normal plantlets were selected and subcultured in SCM media three times every 20 days. 2.3.2. Effects of explants on hyperhydricity 2.3.2.1. Explants of different genotype and organ type. Inflorescences and bulbs of Ershuizao, Zhengyuezao, Cangshan, and Xuzhoubai were selected as explants for inducing shoots. After 20 days, normal shoots of each treatment were selected and subcultured in SCM. 2.3.2.2. Explants of different physiological age and size. Ershuizao inflorescences whose physiological ages were 200, 210, and 220 days were cut lengthwise into one (1 mm3 ), two (0.5 mm3 ), three (0.33 mm3 ), and four (0.25 mm3 ) pieces as explants and cultured in ICM. After 20 days, normal shoots of each treatment were selected and subcultured in SCM. All other processes and conditions were the same as described in Section 2.2. 2.3.3. Effects of media components on hyperhydricity 2.3.3.1. Different basal medium. Ershuizao inflorescences were cultured in media containing MS or B5 medium, and other substances

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were the same as those in ICM. After 20 days, normal shoots of two treatments were selected and subcultured in the media above with a different basal medium. 2.3.3.2. Different sucrose concentrations. Ershuizao inflorescences were cultured in media with sucrose (15, 30, or 45 g L−1 ), and other substances were the same as those in ICM. After 20 days, normal shoots of each treatment were selected and subcultured in the media above with different sucrose concentrations. 2.3.3.3. Different cytokinin levels. Ershuizao inflorescences were cultured in media with different KT (0.46, 0.92, 1.85, 3.71, and 7.43 ␮M) or BA (2.2, 4.4, 8.8, 17.6, and 35.2 ␮M) levels, and other substances were the same as those in ICM. After 20 days, normal shoots of each treatment were selected and subcultured in the media above with different cytokinin varieties and concentrations. 2.3.3.4. Different gelling agents. Ershuizao inflorescences were cultured in media with agar (Solarbio, Beijing, China; 0.35, 0.50, 0.65,

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2.4. Data collection and analysis Treatments were performed in triplicate. Ten bottles per replicate were cultured, with four explants per bottle. The PC of every explant; SHR and shoot hyperhydric level were investigated on the 20th day of initial cultivation. The PHR and plantlet hyperhydric level were investigated on the 20th day of subculture. The hyperhydric level of every shoot and plantlet was scored and recorded using a rating scale of 0–4, as the grading standard of Tian et al. (2015). Normal shoots and plantlets without hyperhydric ones was calculated. SHI and PHI were calculated to evaluated the severity of hyperhydric symptom. Related indexes were calculated as follows: Proliferation coefficient of every inflorescence/bulb (PC) = shoots of every explant (shoot length longer than 0.3 cm) × section of every inflorescence/bulb; Shoots/plantlets hyperhydric index (SHI/PHI) =

Hyperhydric shoots/plantlets × 100 Totalshoots/plantlets

 Shoots/plantlets hyperhydric index(SHI/PHI) =

[(The numberof hyperhydric shootorplantlets of this index) × (Hyperhydric index)]

0.80, or 0.95 g L−1 ) or GELRITE (Yuhan, Shanghai, China; 0.20, 0.35, 0.50, 0.65, or 0.80 g L−1 ), and other substances were the same as those in ICM. After 20 days, normal shoots of each treatment were selected and subcultured in the media above with different gelling agents and concentrations. All other processes and conditions were the same as described in Section 2.2. 2.3.4. Effects of culture conditions on hyperhydricity 2.3.4.1. Different media pH. Ershuizao inflorescences were cultured in media with pH values of 5.4, 5.6, 5.8, 6.0, or 6.4, and other substances were the same as those in ICM. After20 days, normal shoots of each treatment were selected and subcultured in the media above with different media pH values. 2.3.4.2. Different ventilation conditions. Ershuizao inflorescences were cultured in media with a ventilation cap (TQPG-76, Hualian, Xuzhou, China) or a sealed cap (MFPG, Hualian, Xuzhou, China); other substances were the same as those in ICM. After 20 days, normal shoots of each treatment were selected and subcultured in the media above with different ventilation conditions. 2.3.4.3. Different illumination intensities. Ershuizao inflorescences were cultured in media with different illumination intensities (50, 100, 200, or 300 ␮mol m−2 s−1 ) in the chamber (RDN-560E-4, Dongnan Instrument Co, Ltd, Ningbo, China), and other substances were the same as those in ICM. After 20 days, normal shoots of each treatment were selected and subcultured in the medium above with different illumination intensities. All other processes and conditions were the same as described in Section 2.2. 2.3.5. Effects of exogenous additives on hyperhydricity Normal Ershuizao shoots induced by the inflorescence pathway were selected and subcultured in SCM with filter-sterilized H2 O2, SA, Spd, AsA, and MA (0, 10, 50, 250, or 1250 ␮M) or PEG 6000 (0, 10, 20, 40, and 60 g L−1 ). All other processes and conditions were the same as described in Section 2.2.

Total number of shoots investigated

× 100

N1, N2, N3, and N4 represented the numbers of hyperhydric shoots or plantlets whose hyperhydric index was one, two, three, and four, separately. Normal shoot number (NS) = PC × (1-SHR) Normal plantlet number (NP) = NS × (1-PHR) Data were compared by analysis of variance (ANOVA) and correlation analysis using SPSS 22.0 (IBM SPSS Statistics; IBM Corporation, Somers, NY), and differences between the means were considered significant at P < 0.05 by the least significant difference (LSD) test. Figures were drawn using Microsoft Excel 2016 and SigmaPlot 13.0 (Systat Software, Inc., San Jose, CA). 3. Results and discussion 3.1. Normal and hyperhydric shoots and plantlets of garlic The first clear symptoms of hyperhydricity in garlic shoots were observed on the 10th day of initial culture. Shoots of one explant were either completely hyperhydric or altogether normal (Fig. 1a and b). Normal plantlets developed more leaves than shoots (Fig. 1a and c). Hyperhydric shoots were succulent, less green, and vitreous (Fig. 1b), and hyperhydric plantlets showed a translucent, intumescent, curled, and brittle appearance (Fig. 1d). This morphology disorders resulted in the difficulty in recovering, proliferation, and rooting (Kevers et al., 2004). Also, most of the hyperhydric garlic shoots were necrotic during subculture or died during transplant to the field. Hyperhydricity was mainly occurred in shoots and plantlets. Hyperhydric protocorm-like-body was also found in orchid plants, such as Doritaenopsis (Zhou, 1995). 3.2. Shoots were much more easily hyperhydric than plantlets of garlic More than half of the garlic shoots were hyperhydric during initial culture. Also, the first two cultures lost lots of shoots and plantlets due to hyperhydricity (Supplementary Fig. 1), which indicates it is a serious problem in garlic. Plantlets of three subcultures had a lower hyperhydric rate and index than shoots which induced by initial culture (Supplementary Fig. 1). This can be explained by the fact that plantlets developed more roots and leaves (Fig. 1a and c) and had a better ability to maintain normal. Different from increased hyperhydricity with adding culture times of Paeonia suffruticosa (Bouza et al., 1994), we found that hyperhydricity reduced

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Fig. 1. Normal and hyperhydric shoots and plantlets induced by Ershuizao inflorescence (a) normal garlic shoots, (b) hyperhydric garlic shoots, (c) normal garlic plantlets, (d) hyperhydric garlic plantlets.

with increased subculture times (Supplementary Fig. 1). These could be explained by culture time. Shoots of Paeonia suffruticosa were subcultured every 5 weeks (Bouza et al., 1994) which was 15 days longer than our study. In fact, prolonged exposure of plantlets in media induced more hyperhydric shoots and plantlets (data not shown). 3.3. Effects of explants on shoot proliferation and hyperhydricity of garlic 3.3.1. Effects of genotype and organ type Even shoots induced by inflorescences were much more easily hyperhydric; they had a drastically higher PC compared to those induced by bulbs (Table 1). For example, the PC of Ershuizao shoots induced by inflorescences was about 6-fold and SHR 1.85-fold more compared to those of shoots induced by bulbs (Table 1). Therefore, the inflorescence-induced pathway is more efficient than the bulb-induced pathway. In addition, shoots induced directly from inflorescences take about 10–18 days, which is about one-fifth to one-third of the callus pathway duration (Luciani et al., 2006). In terms of saving time and cost, the inflorescence pathway is very efficient and economical to produce large genetically uniform plants within a short time for industrial garlic production. Therefore, prevention of hyperhydricity in shoots and plantlets induced by inflorescences was much more meaningful. Shoots of Ershuizao and Zhengyuezao induced by inflorescence had a higher SHR and SHI than that of Xuzhoubai and Cangshan (Table 1). It is well known that hyperhydricity is a physiological disorder (Muneer et al., 2016). Effects of genotype on hyperhydricity of garlic shoots may cause by the variety character. Garlic of asexual reproduction can be divided into two groups based on their commodity organs: varieties that are harvested for their inflorescences and those for their bulbs. Ershuizao and Zhengyuezao have a good reproductive and bolting ability whose inflorescences are mainly harvested for commodity, while Xuzhoubai and Cangshan are poor in terms of bolting and mainly harvested for bulbs. The differences of hyperhydricity among genotypes induced by inflorescences disappeared during subculture (Table 1), suggesting that environmental differences rather than genetic differences are the determinants of hyperhydricity of plantlets during subculture. Besides, shoots induce by inflorescence of normal plantlets’ offspring were found hyperhydric (data not shown), which proved that non-hyperhydric ability cannot be inherited. Bulbs of four genotypes exhibited no difference in hyperhydricity of shoots and plantlets (Table 1). Hyperhydricity of different varieties were suggested to be related to bolting ability. Interestingly, the trend of PC was the same as hyperhydricity (Table 1), which indicates bolting ability may affect PC, too.

3.3.2. Effects of explant physiological age and size Hyperhydricity of shoots decreased with increasing physiological age of inflorescences (Table 2). More ease of hyperhydricity of younger inflorescences could be expected due to they were more susceptible to in vitro conditions. But physiological age had no effect on hyperhydricity during subculture (Table 2). PC showed the same trend with SHR. This fact may due to the developmental degree of floral organs. Garlic floral organs can develop into small aerial bulbs with increasing physiological time in the field, which limits the differentiation of adventitious shoots in in vitro culture (Kamenetsky and Rabinowitch, 2001). The SHR and SHI increased with the decrease of the explant size. The SHR induced by 0.50-mm3 - and 0.33-mm3 -sized explants had no significant difference, but SHI induced by the 0.33 mm3 explants was much higher than that induced by the 0.50 mm3 ones. This fact shows that hyperhydricity of the former was more pronounced and that the increasing SHI was due to the aggravated symptoms rather than an increasing number of hyperhydric shoots. No significant differences were found in hyperhydricity of plantlets of different physiological age and size during subculture (Table 2). PC of every inflorescence was calculated based on the PC of every explant of different size. Under the same physiological age, cutting explants into two or three pieces result in higher PC. This could mainly because smaller-sized explants had more wound areas and contact area with the media, helping explants acquire more nutrients and plant growth regulators. However, the PC of the smallest size (0.25 mm3 ) was surprisingly less than those of 0.50-mm3 - and 0.33-mm3 -sized ones (Table 2). It is possible that some buds were destroyed by cutting. A significant interaction between the physiological age and size on the PC, SHR and SHI was found (Table 2). Concurrently, physiology age had a much greater impact than explant size on proliferation and hyperhydricity of shoots. We recommend selecting explants of 210 days and cutting them into two or three pieces to acquire highest numbers of normal shoots and plantlets. 3.4. Effect of media components on hyperhydricity of garlic 3.4.1. Effects of basal medium SHR and SHI of MS media was much higher than those of B5 media (Fig. 2a). The PHRs of two treatments had no significant difference, whereas the PHI in MS media was notably higher than that in B5 media (Fig. 2b), which indicates that symptoms of plantlets were aggravated in MS medium. Similarly, Thomas et al. (2000) found that watermelon cultured in B5 showed better growth and less hyperhydricity than in MS medium (Thomas et al., 2000). It had been proposed that the ratio of NH4 + and NO3 − in media may be the cause (Ivanova and Van Staden, 2009). B5 is a medium with high

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Table 1 Effects of genotype and organ type on proliferation and hyperhydricity during in vitro cultivation of garlic. Organ type

Genotype

Shoot hyperhydric rate (%)

Shoot hyperhydric index (%)

Plantlet hyperhydric rate (%)

Plantlet hyperhydric index (%)

Proliferation coefficient

Normal shoots

Normal plantlets

Inflorescence

Ershuizao Zhengyuezao Cangshan Xuzhoubai

52.50 ± 1.44a 51.67 ± 1.67a 36.67 ± 2.20b 35.00 ± 1.44b

36.58 ± 0.74a 35.92 ± 1.30a 27.58 ± 0.93b 27.33 ± 0.87b

37.50 ± 1.44a 35.83 ± 2.20a 36.67 ± 1.67a 34.17 ± 0.83a

21.00 ± 0.72a 20.63 ± 1.08a 20.63 ± 0.51a 21.13 ± 1.23a

40.20 ± 2.12a 37.60 ± 2.11a 18.20 ± 1.54b 20.26 ± 1.43b

19.10 18.17 11.53 13.39

11.93 11.81 7.49 9.04

Ershuizao Zhengyuezao Cangshan Xuzhoubai Organ type effect Genotype effect

28.33 ± 0.83c 27.50 ± 1.44c 26.67 ± 1.67c 26.67 ± 0.83c F-** F-*

11.33 ± 0.30c 11.42 ± 0.51c 11.17 ± 0.68c 10.25 ± 0.43c F-** F-**

18.33 ± 2.20b 18.33 ± 0.83b 19.17 ± 2.20b 16.67 ± 1.67b F-NS F-NS

9.38 ± 0.07b 8.38 ± 0.36b 8.25 ± 0.29b 9.13 ± 0.22b F-NS F-NS

6.70 ± 0.56c 6.10 ± 0.60c 8.10 ± 0.50c 8.60 ± 0.69c F-** F-*

4.80 4.42 5.94 6.31 – –

3.72 3.65 4.75 5.05 – –

Bulb

Note: Data are the mean ± SD. The different letters mean significant difference. NS: not significant. *and * indicate significance at P < 0.05 and P < 0.01, respectively. Table 2 Effects of physiological age and size of explant on shoot proliferation and hyperhydricity during in vitro cultivation of garlic. Physiological age (day).

Size (mm3 ).

Shoot hyperhydric rate (%).

Shoot hyperhydric index (%).

Plantlet hyperhydric rate (%).

Plantlet hyperhydric index (%).

Proliferation coefficient.

Normal shoots.

Normal plantlets.

0.200

1.00. 0.50 0.33 0.25

63.33 ± 0.83d. 66.67 ± 1.67 cd 74.17 ± 2.20b 82.50 ± 1.44a

37.75 ± 0.06d. 40.33 ± 0.30c 48.50 ± 0.90b 56.67 ± 1.16a

30.00 ± 1.44a. 30.83 ± 0.83a 33.33 ± 0.83a 34.17 ± 0.83a

20.25 ± 0.63a. 20.58 ± 0.44a 20.80 ± 0.68a 19.75 ± 0.76a

38.20 ± 1.69b 46.90 ± 1.02a 48.10 ± 1.38a 41.90 ± 1.27b

14.01 15.63 12.43 7.33

9.80 10.81 8.28 4.83

210

1.00 0.50 0.33 0.25

50.00 ± 1.44ef 53.33 ± 0.83e 52.50 ± 1.44e 69.17 ± 2.20c

31.25 ± 0.52f 33.75 ± 0.14e 38.33 ± 0.33 cd 47.42 ± 0.58b

30.83 ± 0.83a 31.67 ± 0.83a 31.67 ± 1.67a 32.50 ± 1.44a

20.52 ± 0.58a 19.75 ± 0.87a 19.83 ± 0.36a 19.42 ± 0.93a

27.40 ± 0.69 cd 40.20 ± 1.74b 39.10 ± 1.43b 31.00 ± 1.13c

13.70 18.76 18.57 9.56

9.48 12.82 12.69 6.45

36.00 ± 0.76h 43.33 ± 0.83g 46.67 ± 1.67fg 53.33 ± 2.20e F-** F-* F-*

27.00 ± 1.01g 30.83 ± 0.44f 33.83 ± 0.73e 37.92 ± 0.55d F-** F-** F-**

28.33 ± 4.41a 29.17 ± 3.00a 32.50 ± 1.44a 33.33 ± 1.67a F-NS F-NS F-NS

21.08 ± 0.22a 19.83 ± 0.22a 20.25 ± 0.29a 20.42 ± 0.55a F-NS F-NS F-NS

19.60 ± 1.38e 25.80 ± 1.15d 27.30 ± 1.05 cd 20.80 ± 1.34e F-** F-* F-NS

12.54 14.62 14.56 9.71 – – –

8.99 10.36 9.83 6.47 – – –

1.00 0.50 0.33 0.25 Physiological age effect Explant size effect Physiological age × size 220

Note: Data are the mean ± standard error. The different letters mean significant difference. NS: not significant. ** and * indicate significance at P < 0.05 and P < 0.01, respectively.

concentration of NO3 − , and MS is a medium with high NH4 + which represents a great growth-limiting stress to plants (Sarasketa et al., 2016). Nielsen and other basal mediums were selected to observe hyperhydricity, but the PC of shoots was seriously decreased (data not shown). Therefore, B5 medium was recommend to use in garlic in vitro culture.

3.4.2. Effects of sucrose Three percent sucrose is the common concentration for most plant species using during in vitro culture. Increasing sucrose concentrations from 30 g L−1 to 45 g L−1 resulted in a significant decrease of hyperhydricity in shoots and plantlets (Fig. 2c and d), which agreed with data in Petunia (Zimmerman and Cobb, 1989). Sucrose plays two important roles in providing a carbon source and maintaining osmotic pressure of media during in vitro culture. We hypothesized that alleviation of hyperhydricity under high concentration was caused by osmotic potential and available water change. For shoots, hyperhydricity was unchanged under low sucrose concentrations. However, alleviation of hyperhydricity was found in broccoli shoots (Yu et al., 2011). For plantlets, low concentrations of sucrose promoted hyperhydricity in garlic (Fig. 2d). A similar trend was found in Artemisia annua plantlets (Fei and Weathers, 2015). We thought nutrition deficiency of plantlets under low concentrations of sucrose may be one cause of increasing hyperhydricity of plantlets.

3.4.3. Effects of cytokinins An upward trend of hyperhydricity of shoots and plantlets with increasing concentrations of KT and BA was observed (Table 3). The highest hyperhydricity rate was 94.17% under 0.34 ␮M KT during initial culture. Similarly, a dose-dependent manner of cytokinins involved in hyperhydricity was found in many other species (Ivanova and Van Staden, 2011; Vasudevan and Van Staden, 2011). Recent evidence suggested that cytokinins could induce ethylene biosynthesis (Zd’arska et al., 2013). We suggested ethylene accumulation under high concentrations of cytokinins could possibly be related to hyperhydricity. Besides having a great effect on hyperhydricity, cytokinins also play an essential role in breaking the apex dormancy and in shoots proliferation (Kadota and Niimi, 2003; Werner et al., 2001). The PC of every inflorescence increased with adding concentrations of KT and BA and peaked when KT concentration reached 1.85 ␮M and BA reached 8.8 ␮M or greater (Table 3). The lowest concentration of KT to achieve the best proliferation was lower than that of BA, indicating KT was more effective than BA in shoots proliferation. To prevent hyperhydricity and obtain more normal shoots, 4.4 ␮M BA was suggested for use in initial culture and 2.2 ␮M BA in subculture. 3.4.4. Effects of gelling agents A dramatic increase of hyperhydricity under low gelling agent concentration was found (Table 4). Low concentrations of agar would syneresis during in vitro culture (Ghashghaie et al., 1991). The lower the gelling agent concentration, the more severe the syneresis. In addition, chelators excreted by plants may dissolve

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Fig. 2. Effects of basal media and sucrose concentration on hyperhydricity during in vitro cultivation of garlic. (a) SHI and SHR under different basal media, (b) PHI and PHR under different basal media, (c) SHI and SHR under different sucrose, (d) PHI and PHR under different sucrose.

Table 3 Effects of cytokinin variety and concentration on shoot proliferation and hyperhydricity during in vitro cultivation of garlic. Cytokinin

Concentration (␮M)

Shoot hyperhydric rate (%)

Shoot hyperhydric index (%)

Plantlet hyperhydric rate (%)

Plantlet hyperhydric index (%)

Proliferation coefficient

Normal shoots

Normal plantlets

KT

0.46 0.92 1.85 3.71 7.43

47.50 ± 1.44de 50.00 ± 2.89d 74.17 ± 0.83c 89.17 ± 3.00ab 94.17 ± 2.20a

23.83 ± 1.88h 28.67 ± 1.06g 47.08 ± 0.44d 57.50 ± 2.89b 65.25 ± 0.88a

24.17 ± 0.83e 35.00 ± 2.89d 44.17 ± 0.83c 52.50 ± 1.44b 65.83 ± 2.20a

10.50 ± 0.88f 22.17 ± 0.51d 26.67 ± 0.68c 30.50 ± 0.38b 36.92 ± 0.30a

26.70 ± 1.12c 32.20 ± 1.76b 39.20 ± 1.79a 40.30 ± 1.17a 40.50 ± 1.71a

14.02 16.10 10.13 4.37 2.36

10.63 10.47 5.65 2.07 0.81

BA

2.2 4.4 8.8 17.6 35.2

40.00 ± 2.50e 48.33 ± 3.33d 55.83 ± 0.83d 76.67 ± 4.41c 85.00 ± 2.89b

19.83 ± 0.55i 30.50 ± 0.52g 34.58 ± 0.51f 43.00 ± 0.88e 52.00 ± 1.01c

22.50 ± 1.44e 25.83 ± 2.20e 33.33 ± 1.67d 50.00 ± 2.89bc 55.83 ± 3.63b

10.58 ± 0.58f 12.83 ± 0.30e 21.92 ± 0.60d 27.25 ± 1.26c 30.17 ± 1.20b

24.60 ± 1.01c 31.90 ± 1.48b 39.10 ± 1.22a 39.60 ± 1.92a 41.90 ± 1.40a

14.76 16.48 17.27 9.24 6.29

11.44 12.22 11.51 4.62 2.78

Note: Data are the mean ± SD. The different letters mean significant difference.

the GELRITE (van den Dries et al., 2013). So, we considered that gelling agents of low concentration increased the availability of water and moisture of the culture vessel, allowed a high uptake of water, ultimately resulting in hyperhydricity. It has been reported that hyperhydricity can be alleviated by increasing concentration of gelling agent (Casanova et al., 2008). Similar results also were found in our study. When concentrations of agar and GELRITE increased to 0.65% and 0.50% separately or above, the SHR did not change and the SHI decreased (Table 4), indicating that hyperhydric symptoms were relieved under higher concentration of gelling agents

(Table 4). The amount of GELRITE used to solidify media was lower than that of agar (Franck et al., 2004). Therefore, under the same concentration of gelling agents, agar media had more available water. This could explain why hyperhydricity under agar media was more severe than that under GELRITE media of the same concentration. The PC decreased notably under high concentrations of gelling agents. This could because high gel strength affects the absorption of nutrients and plant growth regulators (Franck et al., 2004). We recommend using 0.65% agar or 0.50% GELRITE in garlic in vitro culture.

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Table 4 Effects of gelling agent variety and concentration on shoot proliferation and hyperhydricity during in vitro cultivation of garlic. Gelling agents

Concentration (%)

Shoot hyperhydric rate (%)

Shoot hyperhydric index (%)

Plantlet hyperhydric rate (%)

Plantlet hyperhydric index (%)

Proliferation coefficient

Normal shoots

Normal plantlets

Agar

0.35 0.50 0.65 0.80 0.95

75.83 ± 2.20a 62.50 ± 1.44b 53.33 ± 0.83c 45.00 ± 2.89c 46.67 ± 3.33c

42.83 ± 0.79b 39.08 ± 0.65c 36.08 ± 0.74 d 31.25 ± 0.14ef 30.92 ± 0.30fg

50.83 ± 2.20a 42.50 ± 1.44b 35.83 ± 2.20c 32.50 ± 1.44 cd 31.67 ± 1.67 cd

33.00 ± 0.43b 26.83 ± 0.46c 20.83 ± 0.30d 19.00 ± 0.66e 18.75 ± 0.14e

42.20 ± 1.71a 40.90 ± 1.05ab 39.10 ± 1.59ab 32.50 ± 1.20c 30.90 ± 0.84c

10.20 15.34 18.25 17.88 16.48

5.01 8.82 11.71 12.07 11.26

GELRITE

0.20 0.35 0.50 0.65 0.80

75.00 ± 3.82a 54.17 ± 4.17bc 45.83 ± 2.20c 49.17 ± 3.00c 45.00 ± 2.89c

46.17 ± 0.67a 37.67 ± 1.17 cd 33.08 ± 0.51e 31.17 ± 0.46ef 29.00 ± 0.52g

51.67 ± 0.83a 44.17 ± 2.20b 32.50 ± 1.44 cd 29.17 ± 0.83d 30.00 ± 1.44d

35.83 ± 0.55a 26.25 ± 0.29c 20.00 ± 0.63de 19.17 ± 0.22e 18.67 ± 0.96e

40.70 ± 1.58ab 37.80 ± 1.48b 30.60 ± 1.27c 28.80 ± 1.53 cd 25.80 ± 1.19d

10.18 17.33 16.58 14.64 14.19

4.92 9.67 11.19 10.37 9.93

Note: Data are the mean ± SD. The different letters mean significant difference.

3.5. Effects of culture conditions on hyperhydricity of garlic 3.5.1. Effects of media pH The lowest levels of hyperhydricity were observed in media whose pH was 5.8 and 6.0, followed by 6.4, 5.6, and 5.4, in an increasing order (Fig. 3). The common pH for most species during in vitro culture is 5.8. No significant differences in four parameters were found between pH 5.8 and 6.0 (Fig. 3). A pH below 5.8 had a much more serious impact on hyperhydricity than a pH value above 6.0. Almost all shoots were hyperhydric in media whose pH was 5.4 (hyperhydric rate was 97.5%). This could be related to the fact that lower pH values affect the solidity of agar more readily. The lower the pH, the more difficult it is for the media to solidify and the more water becomes available. In addition, studies have shown that low pH levels are associated with inhibited cation uptake (Pasqua et al., 2002). Less cation uptake also may affect hyperhydricity. Media pH values of 5.8–6.0 are recommended during garlic in vitro cultivation. 3.5.2. Effects of ventilation Hyperhydricity of garlic shoots and plantlets in media with ventilation caps were reduced compared to those with airtight caps (Fig. 4a and b). Lai et al. (2005) found that hyperhydricity in shoot cultures of Scrophularia yoshimurae could be prevented by sufficient gas exchange during cultivation (Lai et al., 2005). Some researchers thought that hermitically sealed culture vessels were the main cause of hyperhydricity (Casanova et al., 2008; Majada et al., 2000). To keep the environment sterile, plantlets are required to be kept in a relatively limited space, which results in lack of air; impeded exchange of CO2 and O2 ; and accumulation of harmful substances, such as ethylene (Ivanova and Van Staden, 2010). When the humidity is prevented by ventilation or by bottom cooling, hyperhydricity considerably decreases (Park et al., 2004; Saher et al., 2005b). In our study, caps with filter membrane and ventilation tube below increased gas exchange between the culture vessel and outer environment, reduced harmful air, and decreased moisture to some extent. Increasing the ventilation is a good way to prevent hyperhydricity, but too much also can affect growth of plantlets. Thomas et al. (2000) found that more aeration (2.5 cm diameter) led to serious media dehydration and growth limitation (Thomas et al., 2000). 3.5.3. Effects of illumination intensity Hyperhydricity parameter of shoots and plantlets maintained a downward trend with illumination intensity (Fig. 4c and d). This could be related to increasing of transpiration and water consumption with raising illumination intensity. Even no difference in hyperhydric rate was found between 300 and 200 ␮mol m−2 s−1 ; the hyperhydric index at 300 ␮mol m−2 s−1 was lower than that at

200 ␮mol m−2 s−1 (Fig. 4c and d), which indicates that the hyperhydricity symptom was reduced by higher illumination intensity. However, we found high illumination intensity produced a pale green color in the shoots and plantlets, which is same as that found by Perez-Tornero et al. (2001). We recommended to use 200 ␮mol m−2 s−1 light to induce more normal plantlets. 3.6. Effects of exogenous additives on hyperhydricity of garlic SA is an signal in response to various stresses in plants (Loake and Grant, 2007); its function in alleviated hyperhydricity was reported in oregano (Andarwulan and Shetty, 1999); our finding was in accordance with this finding. The PHR and PHI under 50 ␮M SA were significantly lower than those of other SA treatments, and no differences were found among control, 10, 250, and 1250 ␮M SA treatments (Fig. 5a), which indicating concentration of SA is important to alleviate hyperhydricity in garlic. Recently, Hassannejad et al. (2012) found that 5 ␮M SA improved hyperhydricity reversion in Thymus daenensis. Therefore, SA is also a potential substance to control and reverse hyperhydricity for other species. Spd played a dual role in hyperhydricity of garlic: low concentrations of Spd (10 and 50 ␮M) significantly reduced hyperhydricity, and high concentrations of Spd (250 and 1250 ␮M) increased hyperhydricity (Fig. 5b). The PHI under 10 ␮M Spd was significantly lower than that under 50 ␮M Spd, indicating the former concentration had a better alleviating effect than the latter one. More hyperhydric shoots were found in 1250 ␮M Spd compared to 250 ␮M (Fig. 5b). Recent evidence showed that PA particularly may be involved in the reversion of hyperhydricity, and its contents were reduced in hyperhydric shoots (Hassannejad et al., 2012). Tabart et al. (2015) found Spd reduced the percentage of hyperhydricity in apple. Researchers suggested that free Spd supplementation could help facilitate osmotic stress tolerance (Liu et al., 2004), maintenance of polyamine levels necessary for cell osmoregulation, antioxidant protection, cell wall cross-linking, and plant growth regulation (Tabart et al., 2015). However, high concentrations Spd caused the damage to the plantlets and result in more hyperhydricity. In previous work, we observed that H2 O2 could induce hyperhydricity (Tian et al., 2015). Similarly, when H2 O2 concentration was 250 ␮M or higher, the PHR and PHI increased with increasing H2 O2 concentrations. Interestingly, we found 50 ␮M H2 O2 reduced hyperhydricity significantly in this study. When H2 O2 concentration was 50 ␮M, PHR and PHI values were 26.67% and 13.75% respectively, which are 25% less than those of controls (Fig. 5c). However, 10 ␮M H2 O2 had no significant effect on alleviating hyperhydricity. Hyperhydricity in relation to oxidative stress has been studied in Dianthus caryophyllus (Saher et al., 2005a), Mammillaria gracilis (Balen et al., 2009), and Euphorbia millii (Dewir

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Fig. 3. Effects of pH value on hyperhydricity during in vitro cultivation of garlic. (a) SHI and SHR under different pH value, (b) pH I and pH R under different pH value.

et al., 2006). Moreover, Tian et al. (2015) found that apoplastic oxidation is a key factor of hyperhydricity. H2 O2 , a type reactive oxygen species, also may play a signaling role in plant stress responses (Foyer and Noctor, 2005). The alleviation in hyperhydricity could be related to the signaling role of H2 O2 . AsA significantly reduced the hyperhydric index. PHR under 250 ␮M AsA were significantly lower compared to controls and other AsA treatments (Fig. 5d). As an important antioxidant substance (Valpuesta and Botella, 2004), the lessening of hyperhydricity by AsA could be related to the alleviation of oxidative stress. The PHI and PHR were unchanged under increasing MA treatments (Fig. 5e). However, Fei and Weathers (2015) found that adding MA could reduce hyperhydricity in Artemisia annua at low sucrose concentrations. MA reduced hyperhydricity probably by increasing osmotic potential, as observed in other species (Kadota et al., 2001; Yadav et al., 2003). PEG 6000 significantly aggravated hyperhydricity (Fig. 5f), which is in accordance with the findings of Sen and Alikamanoglu (2013) in sugar beet. The PHR reached its highest values when concentrations were 40 and 60 g L−1 . PEG 6000 is often used to decrease water potential and increase osmotic pressure in media (Chazen et al., 1995; Sen and Alikamanoglu, 2013). PEG can decrease O2 movement by increasing solution viscosity, making water less available to the cultured plants (Verslues et al., 1998). However, the reduction of available water did not stop the increase of hyperhydricity. Oxidative stress was thought to involved in hyperhydricity induction by PEG 6000, according to Sen and Alikamanoglu (2013).

3.7. Positive correlation between shoot proliferation and hyperhydricity From an industrial perspective, increasing PC and decreasing hyperhydricity are the main goals of shoot induction of in vitro cultivation. Impact factors, such as sucrose concentration, only affected hyperhydricity and not proliferation of garlic. It is not hard to selected the best treatment for micropropagation based on hyperhydricity. However, genotype, physiological age, explant size, gelling agents and cytokinins not only have a significant impact on proliferation coefficient but also affect hyperhydricity seriously. A significant positive correlation between proliferation coefficient and hyperhydricity of shoots was found under these conditions (Supplementary Tab 1). The highest correlation was 0.990 under different genotype and organ type. Increasing the PC and decreasing hyperhydric rate at the same time seem to be a contradiction which also existed in other species during shoot induction (Kadota and Niimi, 2003). Balance of PC at a high level and hyperhydricity at a low level is essential for reducing costs and saving time in terms of scientific study and industrial production. However, no method that could increase proliferation and reduce hyperhydricity was found until now.

3.8. Impact factors of hyperhydricity Hyperhydricity is considered a physiological disorder that can be induced by different stress conditions (Bakir et al., 2016), and its impact factors are very complex. Effects of 17 factors

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Fig. 4. Effects of veneration condition and illumination intensity concentration on hyperhydricity during in vitro cultivation of garlic (a) SHI and SHR under different veneration condition, (b) SHI and SHR under different veneration condition, (c) SHI and SHR under different illumination intensity, (d) SHI and SHR under different illumination intensity.

on hyperhydricity and proliferation were investigated in our study. Considering that studying all factors in one experiment is unrealistic, we divided them into four varieties (explant, media components, culture conditions, and exogenous additives) and studied them separately. Optimizing one condition did not always result in a satisfactory result to overcome hyperhydricity (Supplementary Fig. 2a). For example, impact factors of hyperhydricity in Aloe polyphylla were studied for several years to optimize its regeneration system (Ivanova et al., 2006; Ivanova and Staden, 2009; Ivanova and van Staden, 2008, 2009, 2010, 2011). Except for factors above, Ag+ and phloroglucinol also were reported to be potential substances in the control of hyperhydricity (Bernard et al., 2015; Gao et al., 2017; Teixeira da Silva et al., 2013). The hyperhydricity of shoots and plantlets does not always show the same trend. For example, explant effects on hyperhydricity, which include genotype, organ type, explant size, and physiological age, are only limited to initial culture but disappear during subculture (Supplementary Fig. 2). Shoots and plantlets under different media compositions and culture conditions basically showed the same trend in hyperhydricity (Supplementary Fig. 2). SHR and PHR had a great degree of dispersion under different treatments (Supplementary Fig. 2a), which indicated adjusting the media components or endogenous additives in the media to suitable concentrations is very important. Low concentrations of H2 O2 had no effect on hyperhydricity, and high concentrations aggra-

vated hyperhydricity. Only under the suitable concentrations of H2 O2 could hyperhydricity be relieved. In fact, some factors could affect another factor. For example, the media pH affected the solidification of gelling agents. BA caused a reduction of polyamines in plantlets. Some factors possibly could be related to the same pathway. For example, both basal media and pH affect the root uptake of nitrogen (Pasqua et al., 2002). Oxidative stress was found under SA, Spd, H2 O2 , and PEG 6000 treatments (Hassannejad et al., 2012; Sen and Alikamanoglu, 2013; Tabart et al., 2015; Tian et al., 2015). Given the limited information available, further research is needed to explore the mechanisms.

3.9. Optimized regeneration system of garlic Based on the results above, we optimized regeneration system of garlic. Inflorescences of Ershuizao were harvested 210 days after planting, cut lengthwise into two or three pieces, and cultured in media (B5 medium + 4.4 ␮M BA + 0.54 ␮M NAA + 0.65% agar + 1.5% sucrose, pH = 5.8). After 20 days’ culture, normal shoots were selected and subcultured in media (B5 medium + 2.2 ␮M BA + 0.54 NAA ␮M + 0.65% agar + 3% sucrose, pH = 5.8). In vitrogrown explants were grown in vessels with ventilation caps under 200 ␮mol m−2 s−1 (Philips, China). By using this method, we successfully reduced SHR and PHR to 0% (Supplementary Tab 2), and the regenerated plantlets with well-developed roots and shoots

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Fig. 5. Effects of exogenous additives on hyperhydricity during in vitro cultivation of garlic. (a) PHI and PHR under different SA, (b) PHI and PHR under different Spd, (c) PHI and PHR under different H2 O2 , (d) PHI and PHR under different AsA, (e) PHI and PHR under different MA, (f) PHI and PHR under different PEG 6000.

were successfully transferred to a greenhouse with a survival rate of 95%. 4. Conclusion Hyperhydricity of garlic was induced by multiple factors. Explant, media compositions, culture conditions and endogenous additives all had impacts on hyperhydricity. Shoots and plantlets induced by garlic inflorescences were much more susceptible to hyperhydricity than those induced by bulbs. Shoots induced by Ershuizao and Zhengyuezao inflorescences were much more likely

to be hyperhydric than that induced by Xuzhoubai and Cangshan. Hyperhydricity aggravated with the decrease of physiological age and explant size. Hyperhydricity of shoots and plantlets aggravated with increased cytokinin concentrations and decreased gelling agents. KT had a more significant effect on hyperhydricity than BA of the same concentration. Shoots and plantlets in the MS medium were more easily hyperhydric than those in B5 basal medium. When pH values were higher than 6.0 and lower than 5.8, hyperhydricity increased. Improvement of illumination intensity and ventilation condition alleviated hyperhydricity. Suitable concentrations of AsA, Spd, SA, and H2 O2 relieved hyperhydricity. However, high concen-

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trations of H2 O2 and Spd aggravated hyperhydricity. MA had not impact on hyperhydricity and PEG6000 induced hyperhydricity in plantlets. This study is the first comprehensive study concerning impact factors on hyperhydricity in garlic. Alleviation effects of AsA, Spd, SA, and H2 O2 on hyperhydricity was first reported in this study. We hope this study will provide useful information for studying the mechanisms of hyperhydricity as well as for prevention and control technologies of hyperhydricity in other plants species. Acknowledgements We acknowledge financial support of the National Natural Science Foundation of China (NSFC, Grant No. 31372056), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and China Scholarship Council (CSC, No.[2015]3022). The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017. 02.010. References Andarwulan, N., Shetty, K., 1999. Influence of acetyl salicylic acid in combination with fish protein hydrolysates on hyperhydricity reduction and phenolic synthesis in oregano (Origanum vulgare) tissue cultures. J. Food Biochem. 23, 619–635. Bakir, Y., Eldem, V., Zararsiz, G., Unver, T., 2016. Global Transcriptome Analysis Reveals Differences in Gene Expression Patterns Between Nonhyperhydric and Hyperhydric Peach Leaves Plant Genome-Us 9. Balen, B., Tkalec, M., Pavokovic, D., Pevalek-Kozlina, B., Krsnik-Rasol, M., 2009. Growth conditions in in vitro culture can induce oxidative stress in Mammillaria gracilis Tissues. J. Plant Growth Regul. 28, 36–45. Bernard, F., Moghadam, N.N., Mirzajani, F., 2015. The effect of colloidal silver nanoparticles on the level of lignification and hyperhydricity syndrome in Thymus daenensis vitro shoots: a possible involvement of bonded polyamines. In Vitro Cell. Dev.-Plant 51, 546–553. Bouza, L., Jacques, M., Miginiac, E., 1994. In vitro propagation of Paeonia suffruticosa Andr. cv. ‘Mme de Vatry’: developmental effects of exogenous hormones during the multiplication phase. Sci. Hortic.-Amsterdam 57, 241–251. Casanova, E., Moysset, L., Trillas, M.I., 2008. Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots. Biol. Plant. 52, 1–8. Chakrabarty, D., Park, S.Y., Ali, M.B., Shin, K.S., Paek, K.Y., 2006. Hyperhydricity in apple: ultrastuctural and physiological aspects. Tree Physiol. 26, 377–388. Chazen, O., Hartung, W., Neumann, P.M., 1995. The different effects of peg-6000 and nacl on leaf development are associated with differential inhibition of root water transport. Plant Cell Environ. 18, 727–735. Dewir, Y.H., Chakrabarty, D., Ali, M.B., Hahn, E.J., Paek, K.Y., 2006. Lipid peroxidation and antioxidant enzyme activities of Euphorbia milii hyperhydric shoots. Environ. Exp. Bot. 58, 93–99. Fei, L.W., Weathers, P., 2015. From leaf explants to rooted plantlets in a mist reactor. In Vitro Cell. Dev.-Plant 51, 669–681. Fernandez-Garcia, N., Piqueras, A., Olmos, E., 2008. Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation. Environ. Exp. Bot. 62, 168–175. Foyer, C.H., Noctor, G., 2005. Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17, 1866–1875. Franck, T., Kevers, C., Gaspar, T., Dommes, J., Deby, C., Greimers, R., Serteyn, D., Deby-Dupont, G., 2004. Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response. Plant Physiol. Biochem. 42, 519–527. Gao, H., Xia, X., An, L., Xin, X., Liang, Y., 2017. Reversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO 3 and its associated mechanism during in vitro culture. Plant Sci. 254, 1–11. Ghashghaie, J., Brenckmann, F., Saugier, B., 1991. Effects of agar concentration on water status and growth of rose plants cultured in vitro. Physiol. Plant. 82, 73–78. Hassannejad, S., Bernard, F., Mirzajani, F., Gholami, M., 2012. SA improvement of hyperhydricity reversion in Thymus daenensis shoots culture may be associated with polyamines changes. Plant Physiol. Biochem. 51, 40–46. Ivanova, M., Staden, J., 2009. Natural ventilation effectively reduces hyperhydricity in shoot cultures of Aloe polyphylla Schönland ex Pillans. Plant Growth Regul. 60, 143–150.

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