Micropropagation of Prunus scoparia, a Suitable Rootstock for Almond under Drought Conditions

ABSTRACT

Prunus scoparia is a wild deciduous shrub, usually living on dry calcareous soils of the rocky mountains and has been used as a grafting rootstock for domesticated almonds to provide drought resistance. In the current study, micropropagation ability of P. scoparia was investigated using cytokinin and auxin. Uniform nodal shoot pieces (3–5 cm in length) of seedlings were used as explants. The explants were disinfected with 10% sodium hypochlorite solution. For adventitious shoot induction and proliferation, Murashige and Skoog (MS) media containing 7.00 g/l agar and 30.00 g/l sucrose containing five concentrations of benzyl adenine (BA) (0.00, 0.50, 1.00, 2.00, and 4.00 mg/1) and also containing six concentrations of Thidiazuron (TDZ) (0.00, 0.50, 1.00, 2.00, 5.00, and 7.00 mg/1) were compared. For rooting, in vitro shoots (2–3 cm) were transferred into ½ MS medium supplemented with 30 g/l sucrose, 7.50 g/l agar, and different concentrations of IBA (0.00, 0.25, 0.50, and 1.00 mg/l) and NAA (0.00, 0.25, 0.50, and 1.00 mg/l). Based on the results obtained for shoot proliferation, only 2.00 and 4.00 mg/l BA and 2.00 mg/l TDZ concentrations generated shoots, while other treatments did not show shoot proliferation. Among the three treatments that generated shoots, the best results for shoot number, leaf number, and leaf color quality were observed in media containing 2.00 mg/l TDZ. Based on the results obtained for rooting, the effect of IBA concentrations on the rooting percentage, root number, and root length was significant. Among IBA concentrations, only 0.50 mg/l IBA induced rooting, while there was no rooting in the media containing other IBA concentrations. None of the NAA concentrations showed rooting. In conclusion, MS culture medium supplemented with 2.00 mg/l TDZ and ½ MS culture medium supplemented with 0.50 mg/l IBA are suggested for in vitro shoot proliferation and rooting of P. scoparia, respectively. The results presented herein could be used for in vitro selection and micropropagation of P. scoparia.

KEYWORDS:

• Wild species
• auxin
• tissue culture
• shoot proliferation
• rooting

Introduction

The genus Prunus covers some of the most important temperate stone fruit species. In 2014, world annual production of stone fruit (almond, apricot, cherry, nectarine, peach, and plum) exceeded 45.30 million metric tons (FAO, 2014). Because of their economic importance, Prunus species have been the subject of numerous studies including breeding (Scorza et al., 1985; Webster et al., 2000), in vitro organogenesis, and somatic embryogenesis (Canli and Tian, 2008; Cheong and Pooler, 2004; Liu and Pijut, 2008) and molecular genetics (Zhebentyayeva et al., 2008). Historically, much attention has been given to the development of superior cultivars and dwarfing rootstocks. Dwarfing rootstocks that are disease and pest resistant, tolerant to environmental stresses, and provide precocious bearing have been the objectives of many rootstock development programs. Despite all breeding attempts to develop a “perfect” rootstock, some commonly used rootstocks have undesired traits, and still, there is a need to develop new rootstocks and/or cultivars. For instance, some dwarfing cherry rootstocks, i.e., Gisela-5 and Maxima-14, are susceptible to crown rot Phytophthora spp. (Exadaktylou and Thomidis, 2005), and some peach–almond hybrid rootstocks are difficult to root if not micro-propagated (Felipe, 1995).

Almond (Prunus dulcis Mill.) is a species of genus Prunus, subgenus Amygdalus (Rosaceae) which is commercially grown worldwide (Madam et al., 2011). The domesticated almond is thought to have originated in the arid mountainous regions of Central Asia (Ladizinsky, 1999). In this area, some wild almond species are also found growing throughout Southwest and Central Asia. Iran as an almond origin is extremely rich in wild almond species (Ghahreman and Attar, 1999). These wild species provide an enlarged pool of available germplasm and suitable characteristics such as late bloom and self-fertility and resistance to drought, salinity, and low winter temperatures (Denisov, 1980). Moreover, wild almond species demonstrate a greater resistance to abiotic and biotic stresses, and so represent valuable germplasm sources for breeding (Gradziel et al., 2001). On the other hand, some of these species can be used directly as rootstocks for almond, usually in non-irrigated conditions. In several parts of Iran, grafting of the cultivated almonds on wild almonds has a 50–60-year-old record. Prunus scoparia, P. elaeagnifolia, and P. eburnean, naturally distributed in many regions of Iran, have been used in arid and semi-arid areas to control soil erosion and watersheds (Khadivi-Khub and Anjam, 2016). Among them, P. scoparia is distributed in different natural regions of Iran. It is a wild deciduous shrub or small tree, usually living on dry calcareous soils of the rocky mountains and has been used as a grafting rootstock for domesticated almonds to provide drought resistance (Khadivi-Khub and Anjam, 2016). Furthermore, it is well adapted to diverse conditions of its growing region, providing an extensive germplasm resource for domestication and improvement and can have the potential of being a dwarfing rootstock for almond, and also improving other Prunus species as a gene donor (Khadivi-Khub and Anjam, 2016).

Micropropagation has many advantages for clonal propagation of fruit trees (Stushnoff and Fear, 1985) and is important for regeneration following transformation (Ainsley et al., 2000) and cryopreservation (Channuntapipat et al., 2000). The majority of the cultivated and some of the wild Prunus species have been cultured in vitro. Adventitious shoot regeneration from various explant sources has been reported for P. armaniaca, P. avium, P. domestica, P. cerasus, P. dulcis, P. mumeand, and P. serotina (Lane and Cossio, 1986; Liu and Pijut, 2008; Matt and Jehle, 2005; Miguel et al., 1996; Ning et al., 2007; Nowak and Miczynski, 1997; Petri and Scorza, 2010; Song and Sink, 2005).

Khadivi-Khub and Anjam (2016) reported that P. scoparia reduced plant height and increased fruit yield of almond scion under drought stress and rocky calcareous soils and can be used as a dwarfing rootstock for almond. To realize the potential uses of a particular species, it is necessary to develop effective regeneration, propagation, and conservation strategies. Thus, this species can be produced in large scale clonally for nurseries and farmers. Therefore, an optimized protocol is needed for micropropagation to the clonal propagation of this species. However, we found no report about in vitro adventitious shoot regeneration, and/or micropropagation of P. scoparia. Thus, in the present study, we investigated micropropagation ability of P. scoparia using cytokinin and auxin types. To the best of our knowledge, this is the first study reporting in vitro regeneration (and possibly vegetative propagation) of P. scoparia.

Materials and methods

Plant material

The seeds of P. scoparia were collected from Fadak Park in Arak region from Markazi provinces in Iran. The region is placed at 34°05ʹ45” N latitude, 49°45ʹ22”E longitude, and 1730-m height above sea level and has an annual average temperature of 13.80 °C and an annual precipitation of 320.20 mm. Seeds were washed under tap water and cleaned using a metal strainer and cultured for producing the young seedlings. Uniform nodal shoot pieces (3–5 cm in length) of seedlings were used as explants.

The explants were surface disinfected with 70.00% ethanol for 30 s. After several treatments to obtain the best method, the explants were then disinfected with 10% sodium hypochlorite solution (1.00% active chlorine) for 10 min and rinsed three times with sterile distilled water.

Shoot proliferation

The MS media (Murashige and Skoog, 1962) containing five concentrations of benzyl adenine (BA) (0.00, 0.50, 1.00, 2.00, and 4.00 mg/1) and also the media containing six concentrations of Thidiazuron (TDZ) (0.00, 0.50, 1.00, 2.00, 5.00, and 7.00 mg/1) were compared for shoot proliferation.

All the explants were cultured in MS medium and transferred to growth room with a light intensity of 3500 lux, photoperiod of 16/8 h light/dark, and a constant temperature of 25 ± 1°C. Each treatment included four replications and each replicate included five explants. Leaf number, shoot number, and leaf color quality were recorded. Leaf color quality was scored as follows: 1- one to two shoots and one to three leaves with green-yellow color, 3- two to three shoots and three to five leaves with light green, and 5- three to five shoots and five to ten leaves with normal green.

Rooting

First, the proliferated shoots were elongated on MS medium supplemented with 1.00 mg/l GA₃. After 2 weeks, in vitro shoots (2–3 cm) were transferred to ½ MS medium supplemented with 30 g/l sucrose, 7.50 g/l agar, and different concentrations of IBA (0.00, 0.25, 0.50, and 1.00 mg/l) and different concentrations of NAA (0.00, 0.25, 0.50, and 1.00 mg/l). All the treatments were maintained in the dark for 10 days and then were transferred to photoperiod of 16/8 h light/dark. Each treatment included three replications and each replicate included three shoots. Rooting percentage (%), root number, and root length (cm) were recorded after 20 days.

Statistical analysis

The experiments were carried out based on completely randomized design (CRD). Statistical analysis of the data was carried out using SAS software and obtained means were compared using Duncan’s Multiple Range Test (p ≤ 0.05).

Results and discussion

Shoot proliferation

Analysis of variance showed that the effect of cytokinin treatments on leaf number, shoot number, and leaf color was significant (Table 1). Among BA concentrations, only 2.00 and 4.00 mg/l treatments generated shoot, while there were no shoots in the media containing other BA concentrations (Table 2). Furthermore, among TDZ concentrations, only 2.00 mg/l treatment generated shoots, while there were no shoots in the media containing other TDZ concentrations. Among the three treatments that generated shoots, the highest shoot number (2.33) was observed in the media containing 2.00 mg/l TDZ which showed significant differences with BA treatments. As shown in Table 2, the number of leaves on shoots produced on media containing 2.00 mg/l TDZ (5.60) was significantly higher than that of generated by other treatments. Shoots produced on media containing 2.00 mg/l BA had significantly higher leaf number (2.13) than on media containing 4.00 mg/l BA (0.36). In addition, leaf color quality in media containing 2.00 mg/l TDZ (2.79) was significantly better than other treatments. The use of TDZ for in vitro adventitious shoot induction and proliferation has also been reported by other studies, such as those with almond (Ainsley et al., 2000; Arab and Shekafandeh, 2016). Furthermore, media containing 2.00 mg/l BA showed better leaf color quality than media containing 4.00 mg/l BA and there was a significant difference between them.

Table 1. Analysis of variance for the effect of BA and TDZ treatments on shoot proliferation of the P. scoparia.

		Mean square
S.O.V.	Degree of freedom	Shoot number	Leaf number	Leaf color quality
Cytokinin	10	0.437**	1.441**	0.681**
Error	44	0.041	0.095	0.062
C.V. (%)	-	24.90	32.68	28.11

**: Significant at 0.01 probability.

Table 2. The effect of BA and TDZ treatments on shoot proliferation of the P. scoparia.

Cytokinin treatment	Shoot number	Leaf number	Leaf color quality
TDZ (mg/l)
0.00	0.00^b	0.00^c	0.00^c
0.50	0.00^b	0.00^c	0.00^c
1.00	0.00^b	0.00^c	0.00^c
2.00	2.33^a	5.60^a	2.79^a
5.00	0.00^b	0.00^c	0.00^c
7.00	0.00^b	0.00^c	0.00^c
BA (mg/l)
0.00	0.00^b	0.00^c	0.00^c
0.50	0.00^b	0.00^c	0.00^c
1.00	0.00^b	0.00^c	0.00^c
2.00	0.70^b	2.13^b	1.73^b
4.00	0.60^b	0.36^c	0.60^c

In columns, means followed by the same letters are not significantly different with Duncan test at 0.05 level of probability.

Plant growth regulators have a great influence on shoot regeneration. In Prunus regeneration studies, TDZ and BA are the cytokinins used most, while kinetin and isopentenyl adenine (2iP) have been used less (Ruzic and Vujovic, 2008). Results about the effectiveness of PGR on the regeneration of Prunus are contradictory. Often TDZ has been reported more effective than BA (Bhagwat and Lane, 2004; Canli and Tian, 2008; Espinosa et al., 2006; Matt and Jehle, 2005; Perez-Tornero et al., 2000), while in some reports, BA has been found more effective than TDZ (Nas et al., 2010; Ruzic and Vujovic, 2008; Tang et al., 2002). These contradictory results could be ascribed to the use of different species, explant types, and also possible effects of genotypes (Nas et al., 2010).

The TDZ concentrations more than 2.00 mg/l (5.00 and 7.00 mg/l) did not show shoot induction and proliferation and agreed with findings of Nas et al. (2010) in P. microcarpa subsp. tortusa. The TDZ is known to promote shoot regeneration within a narrow concentration range, but at high concentrations, it is usually inhibitory (Ning et al., 2007). Thus, it is suggested that in general, 2.00 mg/l TDZ concentration could be appropriate for adventitious shoot induction and proliferation of P. scoparia. Cytokinins are essential factors for breaking the apex dormancy and inducing axillary shoot proliferation that influence the success of in vitro multiplication (Silva et al., 2003).

Rooting

Based on the results obtained, the effect of IBA concentrations was significant on the rooting percentage (%), root number and root length (Table 3). Among IBA concentrations, only 0.50 mg/l concentration induced rooting, while there was no rooting in the media containing other IBA concentrations. None of the NAA concentrations showed rooting (data not shown) and, thus they were removed from the analysis. Rooting percentage, root number, and root length in media containing 0.50 mg/l IBA was 35.00 %, 1.50 no., and 2.50 cm, respectively (Table 4, Figure 1). Thus, it is suggested that 0.50 mg/l IBA concentration could be appropriate for rooting of adventitious shoots of P. scoparia. Also, Sadeghi et al. (2015) reported that ½ MS medium with 0.50 mg/l IBA was found as the best medium for in vitro rooting of Prunus empyrean proliferated shoots as it produced higher root number, root length, and rooting percentage. Furthermore, use of auxins for in vitro rooting stage has also been reported by other researchers such as a study on GF677 that the highest rooting percentage was found in media containing 0.50 mg/l IBA (Vaez-Livari and Salehi-Soghadi, 2005). It has also been reported that IBA induced lateral rooting better than indole acetic acid (IAA) (Riov, 1993; Spethmann and Hamzah, 1988; De Klerk et al., 1999; Ludwig-Muller, 2000). Furthermore, IBA is more stable and less sensitive to auxin degrading enzymes (Riov, 1993). It has been reported that rooting is low in most of the species related to almond (Ainsley et al., 2001). In the present work, the rooting percentage was moderate which was agreed with findings of Choudhary et al. (2015) in almond.

Table 3. Analysis of variance for the effect of IBA treatments on rooting of the P. scoparia.

		Mean square
S.O.V.	Degree of freedom	Rooting percentage	Root number	Root length
IBA	3	11.81*	0.164*	0.375**
Error	4	0.61	0.012	0.008
C.V. (%)	-	33.83	9.82	7.78

*: Significant at 0.05 probability; **: Significant at 0.01 probability.

Table 4. The effect of IBA treatments on rooting of the P. scoparia.

IBA treatment (mg/l)	Rooting percentage (%)	Root number	Root length (cm)
0.00	0.00^b	0.00^b	0.00^b
0.25	0.00^b	0.00^b	0.00^b
0.50	35.00^a	1.50^a	2.50^a
1.00	0.00^b	0.00^b	0.00^b

In columns, means followed by the same letters are not significantly different with Duncan test at 0.05 level of probability.

Figure 1. Shooting and rooting of P. scoparia. (A) Shoot induction, (B) shoot growth 2 week after induction, (C) shoot growth after subculture in MS medium supplemented with 1.00 mg/l GA₃, (D) rooting.

Conclusion

Shrubs of P. scoparia show great tolerance to abiotic stresses such as drought, salinity, low soil fertility, and low winter temperatures. Therefore, P. scoparia may represent an important genetic resource to be used in breeding programs to generate new almond cultivars and/or directly as a rootstock for almond which is more adapted to climate change. Finally, MS culture medium supplemented with 2.00 mg/l TDZ and ½ MS culture medium supplemented with 0.50 mg/l IBA are suggested for in vitro proliferation and rooting of P. scoparia, respectively. The results presented herein could be used for in vitro selection and micropropagation of P. scoparia.