Micropropagation of Arid Zone Fruit Tree, Pomegranate, cvs. ‘Malase Yazdi’ and ‘Shirine Shahvar’

ABSTRACT

An optimum culture medium for micropropagation of pomegranate, cvs. ‘Malase Yazdi’ and ‘Shirine Shahvar’ is defined and protocols have been developed for in vitro shoot proliferation and rooting of the cultivars. Axillary buds of young shoots were used as explants. The shoots were multiplied in vitro on Murashige and Skoog (MS) supplemented with 0, 1.5, 2, and 2.5 mg.l⁻¹ of N6-benzyladenine (BA). The micropropagated/cloned shoots were rooted on MS and half strength (1/2) of MS containing indole-3-butyric acid (IBA) at three concentrations (0, 0.5, and 1 mg.l⁻¹) and 0.5 mg.l⁻¹ naphthalene acetic acid (NAA). The cultivar ‘Malase Yazdi’ multiplied better than ‘Shirine Shahvar’. In both cultivars, the maximum shoot number with maximum shoot height was produced on medium with 2 mg.l⁻¹ BA. Control medium free from plant growth regulator also supported shoot multiplication. The cultured shoots exhibited the greatest number of leaves on a medium containing 2.5 mg.l⁻¹ BA. The cloned shoots of ‘Malase Yazdi’ rooted the best on half-strength MS medium with 0.5 mg.l⁻¹ IBA while shoots of ‘Shirine Shahvar’, rooted on 1 mg.l⁻¹ IBA and 0.5 mg.l⁻¹ NAA in half MS medium. This method can be used to quickly propagate and conservation of these two pomegranate cultivars for future prospects.

KEYWORDS:

• In vitro
• proliferation
• Punica granatum L.
• rooting
• tissue culture

Introduction

Pomegranate belongs to distinct Punicaceae family consisting of one genus (Punica). There were two species in this genus, P. granatum and P. protopunica (El-Agamy et al., 2010). It has been cultivated from many years ago in tropical and subtropical regions. Majorly, it is grown for using of its delicious fruits. Today, the medicinal properties of pomegranate increase the demand for this plant. Except its fruits, which have bioactive compounds such as anthocyanins having antioxidative properties, leaves, bark of young shoots and roots, flowers, seed oil, and fruit peel are usable in traditional medicine. Pomegranate seed oil has effective in inhibition of skin and breast cancers (Deepika and Kanwar, 2010; Dhinesh and Ramasamy, 2016; Poyrazoglu et al., 2002; Shaygannia et al., 2016).

Pomegranates is considered to be native of Iran, the Baluchistan region of South Pakistan to the Himalayas in northern India and Afghanistan. This plant is grown extensively in the driest regions of Southeast Asia, Malaysia, the tropical Africa of East India, and India. Pomegranates can be grown in variety of soils, ranging from acidic sandy loam to alkaline calcareous soils and even have the adaptability to drought. Typically, pomegranate was propagated with air layering and rooted-hardwood and softwood cuttings, which are cumbersome and time consuming. Using rootstocks in pomegranate propagation is not common. Apart from that, seed culture induces heterogeneity. These problems require the development of rapid and efficient protocols for the in vitro propagation of elite genotypes (Chauhan and Kanwar, 2012; Deepika and Kanwar, 2008; Holland et al., 2009; Naik et al., 1999; Ram Chandra and Dhinesh, 2010).

In recent years, the bacterial blight and wilt diseases causing enormous losses of the crop and propagation through conventional methods does not ensure diseases-free healthy plantlets (Teixeira Da Silva et al., 2013). In the large-scale propagation, the use of micropropagation techniques for over the past decades has increased significantly. The tissue culture has many benefits over the old propagation techniques, including uniform quality, seasonal independence, and rapid mass production of disease-free material and in vitro culture is the only mass propagation technique of healthy plants over a short period of time (Chauhan and Kanwar, 2012; Pal et al., 2014). In vitro propagation has been attempted in different pomegranate growing countries to propagate similar type and virus-free plants and there are some reports on tissue culture of pomegranate over the last few years (Kalalbandi et al., 2014; Kumar et al., 2017; Patil et al., 2011) and different samples such as shoot tips (Kalalbandi et al., 2014), nodal explants (Murkute et al., 2004), and cotyledons (Kanwar et al., 2010) were used. However, a more favorable method is the using of the axillary buds that is obtained from the nodal segments, as it ensures true to type of plants (Kalalbandi et al., 2014; Patil et al., 2011; Usharani et al., 2014). Propagation through lateral buds is free of disease, has very high rate of regeneration and better multiplication (Sujatha et al., 2005). Therefore, different results of different pomegranate cultivars have been reported and only one report on pomegranate micropropagation cv. ‘Malase Yazdi’ (Valizadeh Kaji et al., 2013b) has been reported, the aim of this study was to investigate the effect of different concentrations of BA on proliferation stage of explants tissue culture and effect of MS medium concentration and type of auxins (IBA and NAA) on rooting stage of explants. Then, the major purpose of this research was the development of a micropropagation protocol via proliferation of axillary shoots for the most popular cultivars under cultivation in Iran, ‘Malase Yazdi’ and ‘Shirine Shahvar’. To our knowledge, this is the first report on micropropagation of Iranian pomegranate cv. ‘Shirine Shahvar’.

Material and methods

Explant preparation and surface sterilization

The microcuttings of two pomegranate cvs. ‘Malase Yazdi’ and ‘Shirine Shahvar’, obtained from current season shoots of 10-year-old commercial pomegranate orchard in Yazd (Iran) were used. The experiments were carried out in the Tissue Culture Laboratory of Ardakan University. The leaves of shoots were trimmed, and explants were completely washed with running tap water for 15 min. The cut shoots were treated with 0.05% citric acid solution for 3 min and washed with sterilized distilled water. The stems were soaked in a solution contains citric acid (0.05%) and 0.1% mercury chloride (HgCl₂) for 3 min and for three times washed with sterilized distilled water. Axillary buds (1–1.5 cm) were removed from the shoots and the single node (1 cm) of shoots were detached in the laminar air flow hood and put in aseptic solutions for inception of in vitro tissue culture (Figure 1a).

Figure 1. Explants of pomegranate cv. ‘Malase Yazdi’. (a) Multiple shoot formations on MS medium with 2.0 mg.l⁻¹ BA in ‘Malase Yazdi’ (b) and ‘Shirine Shahvar’ (c). Rooted microcuttings in half MS medium with 0.5 mg. l⁻¹ IBA after 1 month of culture (d).

Establishment medium and culture condition

Aseptic microcuttings were cultured on establishment medium comprised of MS (Murashige and Skoog, 1962) medium without any growth regulators. Sucrose was added at 3.0% and before adding 0.8% (w/v) agar, the pH of the medium was adjusted to 5.8. Media was sterilled in autoclave at 121°C for 20 min. The medium was aliquoted to jars (60 × 100 mm) containing 25 ml each and left to be air-cooled for media solidification. Single-node explants of ‘Malase Yazdi’ and ‘Shirine Shahvar’ were inoculated in medium and kept in a culture room at 24 ± 3°C temperature in the light/dark (16/8) photoperiod at 2000–2500 lux for initiation of explant growth.

Proliferation

After 6 weeks of cultivation, the stems that were obtained from establishment medium, cut into samples with two nodes (1.5 cm) and placed on proliferation medium for producing more axillary shoots. Microshoots were subcultured on full-strength MS medium comprised of BA (0, 1.5, 2, and 2.5 mg.l⁻¹) and 3.0% sucrose and 0.8% agar. Two samples from each cultivar were placed on the four proliferation media and four replicates were used in each treatment. All explants were subcultured every week. After 6 weeks in culture, the number of stems and leaves per explant and the mean length of stem were evaluated.

Rooting and acclimatization

Shoots of both cultivars with 15–25 mm long developed in vitro proliferation stage, were excised into small cuttings and cultured in full- and half-strength MS media containing 0, 0.5, and 1 mg.l⁻¹ of IBA or 0.5 mg.l⁻¹ NAA for inducing rooting. Each treatment had four jars and there were two mirocuttings per jar for each cultivar. All explants were subcultured every week. The root length and number of root were determined at 4 weeks after incubation.

Rooted microcuttings were separated from the rooting medium and gently washed under fresh water to eliminate the medium from the roots, then the rooted microcuttings were transmitted to plastic pots containing autoclaved sand, vermicompost, and cocopeat in a 1:1:1 ratio. The transparent plastic bags were used as cover for keeping moisture. Pots were kept in a tissue culture laboratory and watered at intervals of 3–4 days for 7 days. After 7 days, pores were created on the cover and the plastic cover was removed after 6 days. Plants were then kept in the laboratory for 20 days and then transferred to direct sunlight condition for 14 days. The rate of plantlets survival was registered 20 day after transfer.

In vitro culture conditions

All cultures were done in a laminar air flow hood under similar conditions as described for establishment stage. All cultures used for proliferation and rooting stages were kept at 24 ± 3°C in light/dark (16/8) at 2000–2500 lux.

Experimental design and analysis

This research was performed on a completely randomized design (CRD) with four replicates per treatment. After 4–6 weeks, incubation observations on aseptic culture were recorded. Data were analyzed by analysis of variance (ANOVA) with means separated by Duncan’s multiple range test using SAS (v. 9.1) calculated the significance of differences.

Results

Optimization of shoot proliferation

The axillary buds began to grow in 2 weeks and cultured on MS medium after about 6 weeks that their long reached to 1.5 cm. Among the four BA treatments, medium containing 2 mg.l⁻¹ BA produced the most number of shoots in both cvs. ‘Malase Yazdi’ (3.7) and ‘Shirine Shahvar’ (3.5) and significant differences were observed between MS medium containing 2 mg.l⁻¹ BA and control treatment (without any plant growth regulator), but no significant difference was observed between other BA concentrations (Figure 1b,c). ‘Malase Yazdi’ had more leaves in comparison with ‘Shirine Shahvar’ and 2.5 mg.l⁻¹ BA produced the best number of leaves (13.5) in ‘Malase Yazdi’ and the same concentration of BA along with 2 mg.l⁻¹ BA produced more leaves in ‘Shirine Shahvar’. These two higher levels of BA produced significantly more leaves than the other treatments. The highest shoot length of both ‘Malase Yazdi’ (2.8 cm) and ‘Shirine Shahvar’ (2.6 cm) observed in control medium (Table 1).

Table 1. Effects of BA treatments on shoot proliferation of ‘Malase Yazdi’ and ‘Shirine Shahvar’.

	Treatments	Shoot number	Length of shoots (cm)	Leaf number
Cultivar (C)	‘Malase Yazdi’	2.6 a	1.98 a	8.6 a
	‘Shirine Shahvar’	2.6 a	2.16 a	6.5 b
BA (mg.l^−1)	BA1 (0)	1.18 c	2.7 a	3.37 c
	BA2 (1.5)	2.83 b	2.4 ab	6.7 b
	BA3 (2)	3.6 a	1.96 b	9.3 a
	BA4 (2.5)	2.9 ab	1.15 c	10.8 a
Cultivar × BA	‘Malase Yazdi’ × BA1	1.25 b	2.8 a	3.25 d
	‘Malase Yazdi’ × BA2	2.75 a	2.5 ab	7.5 c
	‘Malase Yazdi’ × BA3	3.7 a	2.12 abc	10.5 b
	‘Malase Yazdi’ × BA4	2.7 a	1.25 cd	13.5 a
	‘Shirine Shahvar’ × BA1	1.2 b	2.6 ab	3.5 d
	‘Shirine Shahvar’ × BA2	2.9 a	2.47 ab	6 c
	‘Shirine Shahvar’ × BA3	3.5 a	1.8 bcd	8.25 bc
	‘Shirine Shahvar’ × BA4	3.2 a	1.05 d	8.25 bc
Significance
Cultivar		NS	NS	**
BA		**	**	**
BA× Cultivar		*	*	**

NS: not significant

*P ≤ 0.05, **P ≤ 0.01 significant at 5% and 1% level, respectively.

Mean values with similar letters under different treatments have not significantly different from each other at P ≤ 0.05.

In vitro induction of adventitious roots

Among the various treatment half MS medium produced more root number and root length than full-MS medium. According to our finding, 0.5 mg.l⁻¹ IBA in half MS medium has been found to be most effective for in vitro rooting of ‘Malase Yazdi’ and the highest number of roots (3.5), and root length (3.8 cm) occurred in this medium. Root number produced in medium containing 0.5 mg.l⁻¹ IBA was significantly greater than control and NAA media (Figure 1). The rest of the auxin combinations induced significantly the shortest root length (Table 2).

Table 2. Effect of auxin treatments and medium concentration on rooting stage of ‘Malase Yazdi’ and ‘Shirine Shahvar’.

		Treatments	Root number	Root length (cm)
Cultivar (C)		‘Malase Yazdi’	2.6 a	1.98 a
		‘Shirine Shahvar’	2.6 a	2.16 a
Medium concentration		MS	2.03 a	1.9 b
		½ MS	2.15 a	2.4 a
Auxin (mg.l^−1)		Control (0)	0.06 c	2.5 a
		IBA1 (0.5)	2.8 a	2.6 a
		IBA2 (1)	2.6 ab	0.09 b
		NAA (0.5)	2.31 b	2.6 a
Cultivar × auxin		‘Malase Yazdi’ × control	0.5 c	2.4 ab
		‘Malase Yazdi’× 0.5 IBA	3.1 a	3.1 a
		‘Malase Yazdi’ × 1 IBA	2.5 ab	0.6 c
		‘Malase Yazdi’ × 0.5 NAA	2.1 b	2.52 ab
		‘Shirine Shahvar’ × control	0.75 c	2.67 ab
		‘Shirine Shahvar’ × 0.5 IBA	2.5 ab	2.02 b
		‘Shirine Shahvar’ × 1 IBA	2.75 ab	1.81 c
		‘Shirine Shahvar’ × 0.5 NAA	2.5 ab	2.7 ab
Medium concentration × auxin		MS × control	0.75 b	2.3 b
		MS × 0.5 IBA	2.75 a	2.02 b
		MS × 1 IBA	2.3 a	0.93 c
		MS × 0.5 NAA	2.25 a	2.5 ab
		½ MS × control	0.5 b	2.8 ab
		½ MS × 0.5 IBA	2.87 a	3.1 a
		½ MS × 1 IBA	2.87 a	0.9 c
		½ MS × 0.5 NAA	2.3 a	2.7 ab
Cultivar× medium concentration × auxin	‘Malase Yazdi’	MS × control	0.75 d	2.12 bc
		MS× 0.5 IBA	2.75 abc	2.5 bc
		MS× 1 IBA	2.5 abc	1 de
		MS× 0.5 NAA	2 c	2.42 bc
		½ MS × control	0.25 d	2.7 d
		½ MS × 0.5 IBA	3.5 a	3.8 a
		½ MS × 1 IBA	2.5 abc	0.37 e
		½ MS × 0.5 NAA	2.25 bc	2.62 b
	‘Shirine Shahvar’	MS × control	0.75 d	2.47 bc
		MS× 0.5 IBA	2.75 abc	1.55 dc
		MS× 1 IBA	2.25 bc	0.87 de
		MS× 0.5 NAA	2.5 abc	2.6 b
		½ MS × control	0.75 d	2.87 b
		½ MS × 0.5 IBA	2.25 bc	2.5 bc
		½ MS × 1 IBA	3.25 ab	1.5 dc
		½ MS × 0.5 NAA	2.5 abc	2.92b
Significance
Cultivar			NS	*
Medium concentration			*	**
Auxin			**	**
Cultivar × auxin			*	**
Medium concentration ×auxin			*	*
Cultivar × medium concentration × auxin			*	*

NS: not significant

*P ≤ 0.05, **P ≤ 0.01 significant at 5% and 1% level, respectively.

Mean values with similar letters under different treatments have not significantly different from each other at P ≤ 0.05.

‘Shirine Shahvar’ produced more roots (3.25) and longest roots (2.92 cm) on half MS medium with comprising of IBA (1 mg.l⁻¹) and NAA (0.5 mg.l⁻¹), respectively. Furthermore, there was no significant difference in root numbers of explants cultured in auxin treatments, but there was significant difference between root numbers of plantlets cultured in auxin treatments and medium avoided of NAA or IBA (Table 2).

Acclimatization

The plantlets were acclimated in plastic cups with sand, vermicompost, and cocopeat. The survival rate of plantlets was 60% and 80% for ‘Malase Yazdi’ and ‘Shirine Shahvar’, respectively. After 20 days of transfer to direct sunlight condition and remained constant thereafter and good growth during this period showed successful establishment of micropropagated plantlets.

Discussion

In this study, axillary buds from new growth were used as explants. While an explant gets older, the regeneration capacity decreases. In the same study, in banana, using young suckers as explants was effective during the establishment stage (Strosse et al., 2006). Similarly, Yildiz (2012) reported differences in regeneration capacity of seedlings in different ages that used as microcutting in flax (Linum usitatissimum L.).

In pomegranate, HgCl₂ and NaOCl are the most widely employed surface sterilants. In this research, HgCl₂ was effective in surface sterilization as Kalalbandi et al. (2014) reported by using 0.1% HgCl₂ for 10 min, minimum microbial contamination and the highest survival (90.58%) was observed. According to Naik et al. (1999) and Murkute et al. (2004), a common disinfectant in sterilization of pomegranate explants was 0.1% mercuric chloride.

Explant browning is an important problem in initiation stage of sterile cultures, because phenolic compounds and secondary metabolites were leaked from the cutting surface of explants (Krishna and Singh, 2007). Pomegranate that has the high amount of phenolic substances, then browning is a serious problem, especially in adult explants (Naik and Chand, 2011). The different approaches to overcome the harmful effects of browning are culture of young explants, or using new growth of branches, or using solutions containing antioxidants (Wang et al., 1994). One of the antioxidant’s role is preventing the oxidation of molecules. Free radicals are produced in oxidation reactions and can cause cell damage or death (Kumar et al., 2017). The addition of antioxidant in the medium at establishment stage is the best treatment for browning control (Hassan and Zayed, 2018). Singh and Patel (2016) mentioned that using antioxidants in medium or immersing explants in solution of antioxidant are the most commonly used methods to decrease browning in micropropagation of Punica granatum. The browning was increased by increasing explant size, and in microcuttings 1.5 cm long, reduction of browning was observed. In the same case, we used explants at the same size in this research and browning of explants was not observed, maybe it was because of using young segments and also citric acid at establishing stage.

Proliferation of ‘Malase Yazdi’ was slightly better than ‘Shirine Shahvar’. ‘Malase Yazdi’ showed a better result in leaf production but rooting rate of both cultivars did not have significant differences (Table 2). Valizadeh Kaji et al. (2013a) reported the same results and observed a little better result in proliferation stage of ‘Malase Saveh’ compared to ‘Yousef Khani’.

MS medium is the most common medium for micropropagation of pomegranate genotype, as used in this research. Also some reports showed using WPM medium (Chauhan and Kanwar, 2012). At proliferation stage with increasing BA concentration, leaves and shoots number increased significantly compared to control medium, then cytokinin is effective in proliferation stage of pomegranate explants. Two high level of cytokinins (2 and 2.5 mg.l⁻¹) produced less shoot length compared to control. As this research showed, addition of 2 mg.l⁻¹ BA caused improving shoot number (3.5 shoots per explant). Similarly, 2 mg.l⁻¹ BA increased shoot number (Golozan and Shekafandeh, 2010). BA has a more effective role in proliferation than other cytokinins. Deepika and Kanwar (2010) reported that BA showed better results in the callus induction of different explants of pomegranate. Singh and Patel (2014) showed the best result at multiplication stage in medium containing 1.0 mg.l⁻¹ BA + 1.0 mg.l⁻¹ kinetin. In one study, the MS medium with 2 mg.l⁻¹ BAP induced maximum shoot number per explant while MS medium + 2.5 mg.l⁻¹ BAP increased the length of shoot and number of leaves compared to other treatments (Kalalbandi et al., 2014). Researches on other plants such as blackberry (Jafari Najaf-Abadi and Hamidoghli, 2009), almond (Savita et al., 2010), and citrus (Baiea, 2009) showed the same results. According to some reports, MS medium with low concentration of plant growth regulator or without it is effective in increasing shoot length (Sivanesan et al., 2011).

Auxin addition to medium significantly produced more roots compared to medium without any auxin, but root length was not influenced by adding auxin to rooting medium. In this study, half MS medium was more effective in increasing root length. The maximum rooting with a higher root number per microshoots found on half MS basal medium in pomegranate (Singh et al., 2010). Also, half-strength WPM medium enhanced induction of root and in rooting of some plant species, medium with low concentrations of salt was effective (Rai et al., 2010; Thimmappaiah et al., 2007). In rooting stage of shoot explant of P. granatum cv. ‘Ganesh’ half MS medium containing NAA (0.1–0.5 mg.l⁻¹) induced rooting (Naik and Chand, 2011; Naik et al., 2000). Similarly, Singh et al. (2010) concluded the use of half-strength MS medium is more suitable for rooting of pomegranate explants and the best number of roots found on this medium. In the same case, half MS medium was effective in increasing the root number and length of almond (Isikalan et al., 2008). IBA is the best auxin for rooting of many plants (Hartman and Kester, 1990). Naik et al. (1999) reported that in micropropagation of pomegranate cv. ‘Ganesh’, the best rooting was achieved in half MS medium containing 1 mg.l⁻¹ IBA. Siwach and Gill (2011) obtained a similar result in Prunus rootstock regeneration and 0.5 mg.l⁻¹ NAA in MS medium, increased root numbers. Related results were obtained in this research without auxins and addition of IBA and NAA to the rooting medium of ‘Malase Yazdi’ and ‘Shirine Shahvar’ was effective.

Conclusion

Due to the nutritional and medicinal value of pomegranate, its drought tolerance, and following the increasing demand for the production and maintenance of this precious plant, it is very important to optimize its tissue culture. These finding could be a useful tool to development of confident micropropagation protocol for pomegranate and can be used for preservation of germplasm and production of disease free plant material.