Effects of Boron and Salinity on Red Raspberry in Vitro

ABSTRACT

Raspberry (Rubus idaeus L.) explants were cultured in vitro under two boron concentrations (0.1 and 0.5 mM) and three levels of NaCl salinity (0, 5, and 10 mM) on an MS medium. The high boron and salt treatment (0.5 mM B and 10 mM NaCl) diminished mean shoot length and fresh and dry weights of the explants; however, fresh-to-dry weight ratio was positively affected. When B increased in the medium, leaf chlorophyll content and fluorescence were reduced under saline conditions. The absorption of boron, chloride, and sodium was enhanced by increased concentrations of boron and NaCl in the medium. Moreover, B enhanced the uptake and accumulation of chloride and sodium in the explants, but the reverse was not true. Finally, the relative growth rate and relative performance of the explants, under the combined effect of B and NaCl, showed a rapid decline at high boron and salt treatment.

KEYWORDS:

• Boron addition
• growth
• ion content
• Rubus idaeus (L.)
• sodium chloride

INTRODUCTION

Boron is an essential element implicated in cell wall synthesis and structure, membrane stucture and function, metabolism of phenolic acids and auxins, and even in nitrogen metabolism (Ruiz et al., 1998). Plant requirements for boron differ widely among species, and the concentration range between deficient and toxic levels is narrower for this micronutrient than for any other (Bastías et al., 2004). High concentration of boron (B) may occur naturally in the soil or in the ground water or be added to the soil from fertilizers or irrigation water (Nable et al., 1997). Also, boron is often found in high concentrations in association with saline soils and saline water, and this kind of stress is frequently associated with arid and semiarid environments (Läuchli, 2002; Stemberg et al., 2001). Moreover, because boron is removed more slowly than salinity, it may be still present at excessive levels even after salt leaching (Alpaslam and Gunes, 2001). There is currently limited information on the response of plants to the combination of B toxicity and salinity, and there is no consensus about the mutual relations between salinity stress and B toxicity (Yerminahu et al., 2003). Bingham et al. (1987) reported that wheat plants respond independently of soil salinity levels and, according to Holloway and Alston (1992), excess external B and salinity interact to limit growth, yield, and yield components of wheat. A similar tendency was found by Alpaslam and Gunes (2001) in tomato. In contrast to this, an antagonistic effect between salinity and B toxicity was observed in 12 different kinds of plants (Ferreyra et al., 1997).

The increasing interest in the cultivation of red raspberry in warmer climates and in the mild southern parts of Europe (Neocleous et al., 2005a, 2005b; Oliveira et al., 1996, 1999), where the water salinity is one of the major limiting factors in agricultural production, requires a better understanding about the combined effect of salt with nutrients that have key role in plant development, such as boron. There are only a few studies carried out in comprehensive understanding of the principal physiological mechanisms of salt damage in red raspberries in vivo (Neocleous and Vasilakakis, 2007) and in vitro (Neocleous and Minas, 2007). Since no information was found on the the combined effect of salt and boron concentrations on red raspberry, the aim of this work was an effort to investigate the plant response and adaptation mechanisms in vitro and gather useful information to be used in open field cultivation.

MATERIALS AND METHODS

Apical shoots of red raspberry (Rubus idaeus cv. Autumn Bliss) 20 mm long were collected from actively growing plants in a greenhouse and they were used as explants. Explants were rinsed under running tap water followed by continuous agitation for 15 min in 15% commercial bleach (0.78% sodium hypochlorite) plus 0.1% (v/v) Tween 20 and rinsed in sterile distilled water. Each explant was transferred into a 330-mL fully transparent polycarbonate jar containing 60 mL of culture medium and placed on the upper surface of a solid MS (Murashige and Skoog, 1962) modified medium. The medium contained MS basic salts and vitamins, supplemented with 3% sucrose, 4.5 mg L⁻¹ BAP, 0.009 mg L⁻¹ IBA, and 55.7 mg L⁻¹ ascorbic acid and was solidified with 2.5 g L⁻¹ phytagel. Two B concentrations were used, the original concentration in MS (0.1 mM) and a 5 × concentration (0.5 mM) combined with three NaCl concentrations (0, 5, and 10 mM). The pH of the medium was adjusted at 5.6 before autoclaving at 1.5 atm and 120°C for 20 min. Cultures were incubated in a growth room at 23°C and 16 h photoperiod (2000 lux) for 20 days before evaluation for boron and salt concentrations effects.

The length, fresh, and dry weights (after drying at 68°C) of the micro shoots were measured and the ratio fresh:dry weight was calculated. Leaf chlorophyll fluorescence was estimated using chlorophyll fluorometer OS-30p (Opti-Sciences, Hudson). The OS-30p (Opti-Science, Hudson, NH, USA) measures chlorophyll fluorescence parameters F ₀ and F_m where F ₀ is the initial fluorescence, F_m is the maximum fluorescence, and the variable fluorescence F_v is calculated as: F_v = F_m − F ₀. To estimate the leaf chlorophyll content, three leaf disks of 2.5 cm² area were weighed and ground in 10 cm³ of N,N dimethylformamide. After 48 h storage in the dark at 5°C, the absorbance of the supernatant was measured at 647 and 664 nm with a Campesc M350 UV-Vis spectrophotometer (Campesc Ltd, Cambridge, U.K.) and chlorophyll content—a, b, and a + b—was computed according to Moran (1982).

To determine the mineral composition, plantlets were harvested and rinsed with distilled water. After drying, chloride concentration in the micro shoots was determined by titration against silver nitrate (Richards, 1954) and sodium by flame photometer Sherwood model 420, (Sherwood Scientific Ltd, Cambridge, U.K.). Boron was determined by the carmine method according to Hatcher and Wilcox (1950). The relative growth rate (RGR) was calculated as: RGR = (lnW ₂ − lnW ₁)/(t ₂ − t ₁) (Blackman, 1919), where W ₁ and W ₂ are plant dry weights at times t ₁ and t ₂. Finally, to compare the relative performance (RP) between treatments, the following equation was used to estimate the comparative changes dry weight: RP = [(Performance under stress)/(Performance under control conditions)] × 100.

Each treatment included 25 replicates (jars) randomly assigned. Differences between means were evaluated using Duncan's multiple range test at P ≤ 0.05.

RESULTS

B and NaCl Effect on Visual Symptoms

No visual symptoms were observed on micro shoots grown in culture media supplemented with various concentrations of B and NaCl in the present study.

B and NaCl Effect on Growth Parameters

As shown in Table 1, the presence of B and NaCl in the growing medium considerably affected the length and fresh and dry weights of the micro shoots. Particularly, the reduced growth occurred when explants were grown on culture medium containing 0.5 mM B and 10 mM NaCl. In contrast, the same concentrations increased the fresh-to–dry weight ratio (Table 1).

TABLE 1. Effect of B and NaCl concentration of the culture medium on the length, fresh and dry weights, and fresh-to–dry weight ratio per explant (Rubus idaeus L. ‘Autumn Bliss’)

B (mM)	NaCl (mM)	Shoot length (mm)	Fresh weight (g)	Dry weight (g)	Fresh/dry weight
0.1	0	35.4a	0.92a	0.15a	6.2a
	5	35.1a	0.91a	0.15a	6.3a
	10	34.6a	0.90a	0.14a	6.4a
0.5	0	34.8a	0.90a	0.14a	6.4a
	5	31.5a	0.87ab	0.13b	6.6ab
	10	25.0b	0.80b	0.11b	7.4b
Mean separation within columns followed by different letters are significantly different according to Duncan's multiple range test (P ≤ 0.05).

B and NaCl Effect on Chlorophyll Content and Chlorophyll Fluorescence

In comparison with the medium containing 0.1 mM B and 0 mM NaCl, the reductions in chlorophyll content and fluorescence were statistically significant when B concentration in the medium was 0.5 mM and NaCl 5 and 10 mM (Table 2). The maximum reduction was observed at 0.5 mM B and 10 mM NaCl and amounted to 30% and 9% for chlorophyll content and fluorescence, respectively (Table 2).

TABLE 2. Effect of B and NaCl concentration of the culture medium on the total chlorophyll (a + b) and chlorophyll fluorescence of raspberry explants (Rubus idaeus L. ‘Autumn Bliss’)

B (mM)	NaCl (mM)	Total chlorophyll (a + b) (mg g^−1 f.w.)	Fluorescence (Fv/Fm) ^z
0.1	0	2.29a	0.75a
	5	2.11ab	0.73ab
	10	2.07ab	0.72ab
0.5	0	2.12ab	0.73ab
	5	1.95b	0.71b
	10	1.60c	0.68c
^zFv/Fm is the variable fluorescence over the maximum fluorescence.
Mean separation within columns followed by different letters are significantly different according to Duncan's multiple range test (P ≤ 0.05)

B and NaCl Effect on Ion Content

By increasing the B concentration from 0.1 mM to 0.5 mM in the growing medium the B concentration of the explants increased by 2.5× as a mean for the various NaCl treatments (Table 3). Under saline conditions, increasing B concentration in the medium increased the chloride and sodium content of the explants by 1.6× and 3×, respectively (Table 3). In addition, by increasing NaCl from 0 to 10 mM, chloride and sodium contents of the explants showed an increase at low B concentration only at 10 mM, although at high B concentrations the increase was obvious at 5 and 10 mM NaCl (Table 3).

TABLE 3. Effect of B and NaCl concentration of the culture medium on B, Cl, and Na concentrations of raspberry explants (Rubus idaeus L. ‘Autumn Bliss’)

B (mM)	NaCl (mM)	B (μg g^−1 d.w.)	Cl (% d.w.)	Na (% d.w.)
0.1	0	87b	0.99c	0.10d
	5	76b	1.03c	0.10d
	10	82b	1.56b	0.37c
0.5	0	210a	1.49b	0.40c
	5	211a	2.13a	0.65b
	10	195a	2.13a	0.69b
Mean separation within columns followed by different letters are significantly different according to Duncan's multiple range test (P ≤ 0.05).

B and NaCl Effect on Relative Growth Rate and Relative Performance

The relative growth rate and the relative performance of the explants showed similar patterns for the various boron and salinity treatments (Fig. 1). Particularly, both showed a rapid decline when B and NaCl concentrations were highest in the medium (Fig. 1).

FIGURE 1. The effect of B and NaCl concentration of the culture medium on the a) relative growth rate and b) relative performance of raspberry grown in vitro (Rubus idaeus L. ‘Autumn Bliss’). Bars represent ± SE values.

DISCUSSION

The raspberry explants did not show any visual toxicity symptoms. It thus appears that either shoots grown in vitro and intact plants differ in their sensitivity, like in kiwifruit shoot cultures (T.E. Sotiropoulos and Dimassi, 2004), or the experimental period was too short for the symptoms to become visible.

In the present study, the explants responded to high B and NaCl concentrations in the growing medium by exhibiting decreased growth. Reduced growth in response to salinity and boron has been reported and in other plants in vivo (Bastías et al., 2004; Laüchli, 2002; Sternberg et al., 2001) and in vitro (Sotiropoulos and Dimassi, 2004). Additionally, interactive effects of excess boron and salinity on growth and yield have been reported previously in wheat (Grieve and Poss, 2000; Holloway and Alston, 1992), tomato and cucumber (Alpaslan and Gunes, 2001). Restriction of growth is mainly caused by salt-induced osmotic effects, ion toxicity, and mineral perturbations (Hasegawa et al., 2000). In the present study, B and NaCl application did not have a negative effect on the fresh-to-dry weight ratio. This may indicate that the restriction of growth in raspberry explants was not impaired with water limitations due to osmotic effects (Neocleous and Vasilakis, 2007). Possibly, ion effects on the cytoplasm or chloroplast functions and the energy required to regulate inorganic ions may limit the potential for plant growth (Stavarek and Reins, 1984; Zhu, 2001).

The leaf chlorophyll content and fluorescence was not significantly affected by NaCl concentration when B concentration was low. However, at high B concentrations in the medium the leaf chlorophyll content and fluorescence were reduced in the presence of 5–10 mM NaCl. From these results, it could be suggested that the application of boron enhanced the negative effect of salinity on photochemical parameters and subsequent photosynthetic processes in our experiment (Bounfour et al., 2002). Similarly, in vitro chlorophyll reduction has been reported as a result of B toxicity in apple cultures (T. Sotiropoulos et al., 2004). Free radical–induced oxidative stress has been associated with several cellular toxic processes, including oxidation damage to chlorophylls (Cobb, 1992) and Cl ions at toxic levels can cause blockage in the electron transport within photosystem II (Feirabend et al., 1992). As a consequence, the combined effect of B and NaCl on raspberries in vitro possibly induces a toxic effect in photochemical parameters associated with limited metabolic energy for plant growth.

High boron and saline conditions in the growing medium altered the nutrient status in explants tissues in our experiment. The concentration of boron, sodium, and chloride found in tissues indicated that under the levels of boron and NaCl applied in the medium uptake and accumulation was stimulated. Although B absorption is probably controlled by transpiration, under saline and excessive B conditions absorption may be passively diffused to the plants and the diffusion rates of B may be higher in salt sensitive plants (Danell et al., 2002). Recent papers have reported the induction of expression of the aquaporin isoform protein (PIP2-1) in salt-treated plants (Bastías et al., 2004) and considerable transport of B through plasma membrane aquaporin has been descripted (Dordas and Brown, 2001). In the present study, the accumulation of B in explant tissues was enhanced by increasing B in the medium, but uptake was not found to be stimulated under saline conditions. Similarly, other crops, like alfalfa, barley, and maize, responded independently to B and salinity conditions (Bastías et al., 2004). However, the application of B promoted the absorption of chloride and sodium from raspberry explants in the current study. An increase of membrane permeability caused by applied B under saline conditions has been reported in tomato. In contrast, membrane permeability was not found to differ in the presence of B applied under non-saline conditions (Alpaslan and Gunes, 2001).

Finally, from these results and taking into consideration the relative growth rate and relative performance of the explants, it can be deduced that excessive B conditions enhanced the NaCl inhibitory effect on raspberries grown in vitro. The results obtained in the present study should be taken into account when cultivating raspberries in soils with high levels of salt and boron.