Influence of Root Zone Calcium on Shoot Tip Necrosis and Apical Dominance of Potato Shoot: Simulation of This Disorder by Ethylene Glycol Tetra Acetic Acid and Prevention by Strontium

The important roles of calcium on plant growth and development including cell division and cell elongation is well documented. The purpose of the present study was to determine the impact of root zone calcium on the growth and health of potato apical meristem and on the maintenance of apical dominance. For this purpose, single-node potato cuttings (Solanum tuberosum L. cv. Dark Red Norland) were grown in sterilized modified Murashige and Skoog (MS) media containing varying concentrations of calcium (1 to 3000 mM). After 13 to 30 d of growth, plantlets were harvested and data for height of the main shoot and for the number of axillary shoots produced were recorded. Plantlets were ashed and tissue calcium concentration was determined. Shoot height was retarded with decreasing concentration of calcium in the media. Calcium deficiency induced shoot tip injury and loss of apical dominance. Tip injury was followed by the development of axillary shoots. The number of axillary shoots increased from one to 21 as calcium concentration in the media decreased from 3000 to 1 mM. At calcium concentration of 1500 mM or higher, there was a single main shoot with no axillary shoots. Addition of ethylene glycol tetra acetic acid (EGTA), a calcium chelator, to the media with 2720 mM calcium (sufficient calcium) resulted in the development of shoot injury and in the formation of axillary shoots. Calcium deficiency injury symptoms were prevented by the addition of a calcium analog, strontium, to MS media deficient in calcium. Strontium has been reported to strongly bind to plant cell walls and the inclusion of strontium prevented injury in shoots of plants grown on calcium-deficient media. These results suggest that strontium is able to mimic the role of calcium in the maintenance of cell wall integrity and supports previous studies that showed that calcium deficiency results from cell wall collapse of the subapical cells. Calcium plays important roles in plant growth and development. Calcium ions are essential for cell wall strength and cell–cell adhesion (Marry et al., 2006; Marschner, 1995). Calcium bound to the outer surface of the plasma membrane maintains membrane stability and cell integrity (Hanson, 1984; Hirschi, 2004; Palta, 1996). Calcium is known to be transported in xylem making transpiration the main force for calcium transport (Busse and Palta, 2006; Clarkson, 1984; Kratzke and Palta, 1986). Thus, calcium deficiency symptoms are observed in tissues with a low transpiration rate including young expanding leaves, enclosed shoot tissues, fruits, underground tubers, and in portions of the plant principally fed by the phloem rather than the xylem (White, 2001; White and Broadley, 2003). A physiological disorder termed ‘‘shoottip necrosis’’ observed in in vitro culture of potato (Solanum tuberosum) has been hypothesized to be a calcium deficiency symptom (McCown and Sellmer, 1987; Sha et al., 1985). This condition is typified by browning and death of the shoot tip, loss of apical dominance, and axillary shoot development in an in vitro shoot culture. Transpiration is limited during in vitro culture by the high humidity that occurs in closed culture vessels. Therefore, uptake and transport of calcium ions, which is dependent on transpiration, is limited during in vitro culture (Williams, 1993). In addition to in vitro potato shoot culture, shoot-tip necrosis resulting from calcium deficiency has been observed in shoot cultures of Pistacia vera (Abousalim and Mantell, 1994). Although these authors were able to show a relationship between shoot-tip necrosis and the medium calcium concentration, they were not able to measure effects at the individual plant level owing to the interplant competition in the culture vessels. Calcium and strontium are closely related elements and have been shown to have similar behavior in plants (Mengel et al., 2001). Early studies by Queen et al. (1963) suggested that strontium can replace calcium during plant growth. Hutchin and Vaughan (1968) showed that plant uptake and distribution of these two elements is similar but not identical. Myttenaere (1964) found that strontium is deposited in the cell wall to a much greater extent than calcium. Results from these early studies suggest that strontium may be able to mitigate calcium deficiency symptoms, especially in cell wall development. No study to our knowledge has investigated the mitigation of calcium deficiency injury in shoot cultures using strontium. Recently, we have provided a detailed description of the injury by calcium deficiency at the cellular level in potato shoot cultures (Busse et al., 2008). We demonstrated that primary injury from calcium deficiency is localized in the expanding pith cells below the apical shoot meristem and injury is characterized by the collapse of the walls of subapical cells in potato. These results suggest that strontium may be able to prevent the cell wall collapse in potato shoot cultures grown in calcium-deficient media. In the present study, we provide further evidence for the role of calcium in shoot tip necrosis by 1) simulating the injury symptoms by including EGTA in the tissue culture media containing sufficient calcium; and 2) alleviating these symptoms by incorporating strontium in calcium-deficient media. By placing a single culture in each vessel, we were able to do studies at an individual plant level without interplant competition. Materials and Methods Plant material, medium, and growth conditions. Single-node cuttings from the second and third nodes of 1-month-old micropropagated potato (cv. Dark Red Norland) plantlets were cultured in vitro on MS media (Murashige and Skoog, 1962) containing a range of calcium concentrations in culture tubes (20 · 150 mm). Sucrose (3%) and 0.5 mM myoinositol were added and pH was adjusted to 5.60 ± 0.02. Agar Received for publication 29 Oct. 2010. Accepted for publication 23 June 2011. This research was supported in part by the College of Agriculture and Life Sciences, University of Wisconsin–Madison. We thank Carrin Carlson Stingl for her assistance with the strontium experiments and Laura VanderPloeg, senior media specialist, Biochemistry Department, University of Wisconsin–Madison, for her assistance with Figure 4. We also thank Dr. Sandra E. VegaSemorile, Assistant Scientist, Department of Horticulture, University of Wisconsin–Madison for her assistance with manuscript preparation. To whom reprint requests should be addressed; e-mail [email protected]. 1358 HORTSCIENCE VOL. 46(10) OCTOBER 2011 (0.7%) was added before autoclaving at (132 C) for 15 min. Cultures were placed under continuous light with about 60 mmol m s photosynthetic photon flux from cool white fluorescent lamps and 20 C (Steffen and Palta, 1986). Three separate experiments were conducted. The objective of the first experiment was to establish the root zone calcium concentrations required for the production and alleviation of calcium deficiency symptoms. The objective of the second experiment was to determine if the presence of EGTA will simulate the symptoms of calcium deficiency in media containing sufficient calcium. The objective of the third experiment was to determine if supplementing a calcium-deficient media with strontium would prevent the development of calcium deficiency symptoms. In the first experiment, single-node cuttings were cultured on MS media modified with calcium chloride (1.4, 6.8, 34, 68, 170, 850, 1360, or 3000 mM calcium) for 13 and 30 d. Each treatment had 10 replications. In the second experiment, agar media containing sufficient calcium (1360 and 3000 mM calcium) was used alone or supplemented with 5 mM EGTA to reduce calcium availability. Each treatment had five replications. In the third experiment, singlenode cuttings were cultured for 28 d in agar media containing deficient calcium (68 mM calcium) and supplemented with varying amounts of strontium chloride (0, 68, 204, 340, 476, 612, 748, 884, 1020 mM). Each treatment had 17 replications. In all the experiments, media calcium, strontium, and EGTA concentrations were fixed at the beginning of the experiment and the media was not replenished. Observation on the shoot growth and calcium and strontium contents. Plantlets were harvested after 2 to 4 weeks of growth in the media. Shoots from each tube were separated from their roots, weighed immediately, and the shoot length for the main shoot was recorded. The number of axillary shoots arising from the primary shoots per plantlet was counted. For this purpose, the axillary shoots visible with the naked eye (3 mm long or greater) were counted. In addition, shoots with dead tips were counted. For this purpose, shoot tips showing browning and necrotic lesions were counted as dead. Individual plants were analyzed for calcium and strontium content following the procedure of Ozgen et al. (2006). For this purpose, the samples were dried in an oven at 70 C, weighed, and ashed (450 C, 6 h). The ash was then dissolved in 2 N HCl. This solution was diluted with a lanthanum chloride (LaCl3.XH2O) solution and distilled, deionized water to obtain samples with a final concentration of 0.2 N HCl and 2000 mg L of lanthanum chloride. Calcium and strontium concentrations were determined by atomic absorption spectrophotometry (Varian Model Spectraa-20; Varian Associates, Inc., Sunnyvale, CA). Statistical analysis. As described previously, that data on the shoot tips alive were collected as two outcomes, dead or alive. These data were analyzed using the binomial option in frequency procedure to obtain 95% confidence intervals for the proportion alive (Version 9.2; SAS Institute, Inc., Cary, NC). Paired comparisons between strontium levels were made using Fisher’s exact test. The Bonferroni correction was applied to account for multiple comparisons. All other data were analyzed using a completely randomized design in the PROC GLM procedure of the Statistical Analysis System (Version 8.2; SAS Institute, Inc.). Means were separated using the least significant difference test at a = 0.05.