Elsevier

Scientia Horticulturae

Volume 161, 24 September 2013, Pages 88-94
Scientia Horticulturae

l-methionine influences in vitro root regeneration, total chlorophyll, total carbohydrate and endogenous proline content in the sweet cherry rootstock MxM 14 (Prunus avium × Prunus mahaleb)

https://doi.org/10.1016/j.scienta.2013.06.012Get rights and content

Highlights

  • The 1 mg/L IBA plus 0.5 mg/L methionine treatment was proved to be ideal on rooting.

  • Methionine with 2 mg/L IBA had an inhibitory effect on shoot fresh and dry weight.

  • Optimum leaf chlorophyll content with 2 mg/L IBA plus 1 mg/L methionine.

  • IBA alone and with l-methionine doubled the carbohydrate concentration in leaves.

  • Increased proline concentration in leaves with low (0.5 mg/L) l-methionine.

Abstract

In the present study, the effects of indole-3-butyric acid (IBA) separately and simultaneously with l-methionine on the morphogenic and biochemical responses in the cherry rootstock MxM 14 (Prunus avium × Prunus mahaleb), from shoot tip explants, were investigated. Best rooting results were achieved with 1 mg/L IBA plus 0.5 mg/L l-methionine. The incorporation of the amino acid in the Murashige and Skoog (MS) medium, greatly enhanced the promotive effect of the auxin on rooting, exhibiting a synergistic action. The supplementetion of explants with l-methionine did not influence their shoot length, for each IBA treatment. On the other hand, l-methionine when combined with the highest IBA concentration (2 mg/L) had an inhibitory effect on shoot fresh and dry weight. l-methionine significantly affected chlorophyll, carbohydrate and proline concentration. The maximum leaf chlorophyll concentration was recorded with 2 mg/L IBA plus 1 mg/L l-methionine. IBA alone or combined with l-methionine doubled the leaf carbohydrate concentration. Moreover, proline concentration in leaves was significantly increased with low (0.5 mg/L) l-methionine, irrespective of IBA concentration. In roots, the combined effect of 0.5 mg/L IBA with 0.5 mg/L l-methionine and the 1 mg/L IBA plus 1 mg/L l-methionine treatment led to elevated levels of endogenous proline and total carbohydrates, respectively, in comparison to the control. Taking into account the different parameters examined, chlorophyll and carbohydrates appeared as the most accurate biochemical markers of the rooting process in the MxM 14 rootstock.

Introduction

MxM 14 (Prunus avium × Prunus mahaleb) is a clonal selection from an open pollinated population of P. mahaleb. Plants in the nursery show medium to high vigour and have a semi-spreading habit. Plants grafted on this rootstock are about 20% more vigorous than those grafted on CAB 6P (Prunus cerasus L.) and about 30% less vigorous than those grafted on cherry seedlings. All cherry cultivars grafted on MxM 14 present an earlier cropping, higher yield efficiency and better quality in comparison to those grafted on seedlings rootstocks (Dimassi-Theriou and Therios, 2006).

Amino acids can induce rhizogenesis. In shoot tips of Torenia fournieri grown in vitro, the amino acids glutamic acid, aspartic acid, alanine, glutamine, proline, serine and arginine induced rooting of explants in the presence of a-naphthaleneacetic acid (NAA) (Kamada and Harada, 1979). Furthermore, proline (10–200 mg/L) increased rooting percentange and number of roots per rooted explant of sweet cherry (P. avium L.) and sour cherry (P. cerasus L.), the root length however, was reduced (Baraldi et al., 1988). Pedrotti et al. (1994) found that after 30 days in a substrate containing IBA, l-asparagine had no effect on the root elongation of wild cherry, in comparison to the control (which was without amino acid), whereas root elongation was strongly inhibited by l-glutamic acid. The same authors also found that root length was likewise strongly inhibited with the autoclaving of l-glutamine, although no such inhibition on root elongation was observed when l-glutamine was applied with filtration (Pedrotti et al., 1994). The auxins IAA, IBA and NAA (1–2 μM), as well as the amino acids 5-oxyproline and tryptophan at 1.4 mM, reduced root elongation in wild cherry (Pedrotti et al., 1994).

Specific media components including amino acids have been found to play an important role on tissue culture systems of certain plant species (Benson, 2000). Amino acids have been used as an organic nitrogen source in in vitro cultures of several species as alfalfa, maize, sorghum, pineapple, rice and other monocots to enhance somatic embryogenesis and regeneration (Skokut et al., 1985, Claparols et al., 1993, Rao et al., 1995, Hamasaki et al., 2005, Grewal et al., 2006). It has been suggested that the positive effect of inorganic nitrogen, in comparison to that of inorganic sources is associated to enhanced mobility of the former at a lower energy cost than the later (Kim and Moon, 2007). Despite the fact that nitrate and ammonium salts have been universally used as N source in tissue culture media, numerous reports specify that reduced nitrogen forms, particularly amides and amino acids, e.g. glutamine, glutamic acid, proline, and alanine, can improve cell proliferation as well as regeneration in specific genotypes (Vasudevan et al., 2004).

Methionine, a sulphur containing amino acid, is a precursor of ethylene biosynthesis in plant tissues (Yang, 1985). Apart from its role as a protein constituent and its central role in the initiation of mRNA translation, methionine indirectly regulates a range of cellular processes as the precursor of S-adenosylmethionine (SAM) (Amir et al., 2002). SAM is the primary biological methyl-group donor and is also the precursor of plant metabolites such as ethylene, polyamines, vitamin B1 and the iron chelator mugineic acid (Sun, 1998). The substrate of SAM-dependent methyltransferases participates in both primary and secondary metabolism (Roje, 2006). Hence, as a donor for methyl groups, methionine through SAM regulates essential cellular processes such as cell division, synthesis of cell wall, synthesis of chlorophyll and membrane synthesis (Roje, 2006). In higher plants, SAM is also the precursor of the hormone ethylene, which regulates developmental stages (Matilla, 2000). Furthermore, SAM is the source of polyamines, spermidine and spermine, which play crucial roles in many aspects of plant growth, including cell proliferation and differentiation, apoptosis, homeostasis and gene expression (Kuznesov and Shevyakova, 2007, Pang et al., 2007).

Limited investigations have been carried out concerning the effects of various growth regulators and amino acids on cherry rooting in vitro. There is a scarcity of reported work regarding the effects of l-methionine in the culture medium on root proliferation of the cherry rootstock MxM 14, which is an important rootstock.

The aim of the present study was to test the possible effects of l-methionine on the rooting, total leaf chlorophyll (a + b) concentration, total carbohydrate and proline concentration both in leaves and roots of one commercial cherry rootstock, namely MxM 14.

Section snippets

Plant material and culture conditions

The effect of the amino acid l-methionine was studied in in vitro experiments employing the cherry rootstock MxM 14 (P. avium × P. mahaleb). Preliminary results indicated that certain levels of IBA and l-methionine combinations (0.5 mg/L IBA + 0 mg/L l-methionine, 1 mg/L IBA + 2 mg/L l-methionine and 2 mg/L IBA + 2 mg/L l-methionine) were not effective (unpublished data). Therefore, to avoid factorial treatments that required a greater number of explants, the following levels of IBA (mg/L) + l-methionine

Effect of l-methionine on rooting

The greatest values of the number of roots per rooted explant (11.15) (Fig. 1A), fresh (0.363 g) (Fig. 1C) and dry weight (0.036 g) (Fig. 1D) as well as rooting percentage (100%) (Fig. 1E) were recorded in the 1 mg/L IBA plus 0.5 mg/L l-methionine treatment (Fig. 2B and C) and these values were significantly different from the control (Fig. 2A). The greatest root length (54.76 mm) (Fig. 1B) was recorded in the combination of 0.5 mg/L IBA with 2 mg/L l-methionine (Fig. 2D). The rooting percentage was

Discussion

In this paper, it was found that the concentration of l-methionine and the auxin IBA interact, affecting in a different way rhizogenesis in vitro. In all the treatments, rooting always followed callus formation. To be more specific, l-methionine and IBA concentration, exerted different effects in the rooting characteristics and in various biochemical measurements.

A positive relationship was found between l-methionine concentration and rooting percentage for 0.5 and 1 mg/L IBA. Furthermore, in

Acknowledgements

We would like to express our sincere gratitude to Angelos Xylogiannis for kindly providing the MxM 14 (P. avium × P. mahaleb) plants; also our thanks to S. Kuti and V. Tsakiridou for technical assistance. The authors gratefully acknowledge the financial support of the Aristotle University of Thessaloniki.

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