Improved shoot multiplication and development in hybrid hazelnut nodal cultures by ethylenediamine di-2-hydroxy-phenylacetic acid (Fe-EDDHA)
Abstract
Garrison, W., Dale, A. and Saxena, P. K. 2013. Improved shoot multiplication and development in hybrid hazelnut nodal cultures by ethylenediamine di-2-hydroxy-phenylacetic acid (Fe-EDDHA). Can. J. Plant Sci. 93: 511–521. Micropropagation of hybrid hazelnut cultivars is difficult because of their recalcitrant nature. The current study assessed the effect of different iron sources on in vitro shoot multiplication and subsequent plantlet development from nodal explants of the cultivar Geneva. Two chelated forms of iron, ethylenediamine di-2-hydroxy-phenylacetic acid (Fe-EDDHA) and ethylenediamine tetraacetic acid (Fe-EDTA) were tested to determine the effect on shoot development. Shoots were longer and had a higher number of nodes when cultured on a modified NCGR-COR medium supplemented with 230 µM Fe-EDDHA, whereas shoots failed to grow on a medium with 460 or 690 µM Fe-EDTA. All plantlets grown in the presence of Fe-EDDHA had more chlorophyll, larger leaves, and higher dry weights compared with Fe-EDTA. Electron microscopy of in vitro grown tissues revealed that the form of Fe influenced the number of granal and stromal lamellae per chloroplast, the number of thylakoids per granum, and the overall chloroplast structure. Nodal explants originating from the proximal end of stems developed longer shoots with more nodes than those derived from the distal region. The use of double-phase culture medium produced plants with longer shoots and more nodes, although these exhibited hyperhydricity, showed greater morphological variation, and contained less chlorophyll. These results demonstrate the efficacy of the use of Fe-EDDHA in growth medium for improving micropropagation efficiency of hazelnut.
Résumé
Garrison, W., Dale, A. et Saxena, P. K. 2013. L'acide éthylènediamine di-2-hydroxy-phénylacétique (Fe-EDDHA) améliore la multiplication et le développement des pousses dans les cultures de nœuds de noisetier hybride. Can. J. Plant Sci. 93: 511–521. Il est difficile de multiplier végétativement in vitro les cultivars de noisetier hybride en raison de leur nature récalcitrante. La présente étude devait évaluer l'incidence de diverses sources de fer sur la multiplication des pousses in vitro et sur le développement subséquent de plantules à partir des explants de nœuds du cultivar Geneva. Les auteurs ont testé deux formes chélatées de fer, l'acide éthylènediamine di-2-hydroxy-phénylacétique (Fe-EDDHA) et l'acide éthylènediamine tétraacétique (Fe-EDTA), en vue d'en préciser les effets sur la croissance des pousses. Ces dernières sont plus longues et comptent un plus grand nombre de nœuds quand on les cultive sur un milieu NCGR-COR modifié, enrichi avec 230µM de Fe-EDDHA, alors que les pousses refusent de croître sur un milieu renfermant 460 ou 690µM de Fe-EDTA. Toutes les plantules cultivées avec du Fe-EDDHA présentaient plus de chlorophylle, des feuilles plus grandes et un poids plus élevé que celles cultivées avec du Fe-EDTA. L'examen des tissus cultivés in vitro au microscope électronique révèle que le type de Fe exerce une influence sur le nombre de lamelles granaires et stromales par chloroplaste, sur le nombre de thylakoïdes par granum et sur la structure générale des chloroplastes. Les explants des nœuds issus de l'extrémité proximale des tiges donnent des pousses plus longues présentant un plus grand nombre de nœuds que les explants tirés de la région distale. Le recours à un milieu de culture à deux phases donne des plants à pousses plus longues comportant plus de nœuds, bien que ceux-ci soient hyperhydriques, présentent plus de variations morphologiques et renferment moins de chlorophylle. Ces résultats prouvent l'utilité du Fe-EDDHA quand on l'ajoute aux milieux de croissance pour rendre la multiplication in vitro du noisetier plus efficace.
Hazelnuts, including Corylus avellana L. and C. americana M., are in the initial stages of industrial production in Ontario, Canada, because of their commercial potential for the fresh market and processing industries. At present, the major hazelnut producing regions in the world are Turkey, Italy, the United States, and Spain (Bacchetta et al. 2008). About 90% of hazelnut production is shelled and sold as kernels to processors, while the rest are sold fresh in the shells (Bacchetta et al. 2008). Hazelnuts have been found to contain taxanes, a group of compounds used to produce chemotherapy drugs (Bestoso et al. 2006) and oils from the nuts have been proposed as a source for biodiesel development (Xu et al. 2007; Demirbas 2008) further emphasizing the economic potential of this crop.
Hazelnuts are traditionally propagated from basal shoots or “suckers” from vigorous mother plants. This is labour-intensive, and the number of plants that can be produced is limited, as relatively few suckers are produced from a plant. Hazelnut cultivars can also be difficult to propagate by cuttings (Kantarci and Ayer 1994; Solar et al. 1994). Consequently, to establish plantations of hazelnuts in Canada by conventional stool beds large enough to satisfy local and international markets would be challenging and take many years. In vitro technologies such as micropropagation can be used to produce plants of existing and new cultivars in large numbers in order to distribute plants with greater expediency. Micropropagation allows plants to be multiplied exponentially in a short time period (George 2008), and since these are grown in aseptic culture conditions, plants are healthy and disease-free. However, large-scale propagation of hybrid hazelnuts requires a protocol where explants reproducibly respond to growing conditions that allow for consistent shoot growth and development.
Iron is an essential element required for growth and development of plants (McClendon 1976; Williams 1997). Iron was shown to be involved in many of the redox reactions in photosynthesis and respiration, and in the synthesis of chlorophyll (Raven 1988). However, plants cannot take up iron efficiently if it was not in solution (Hell and Stephan 2003). There are two common elemental states of iron, ferrous iron (Fe2+), which is relatively soluble but readily oxidized, and ferric iron (Fe3+), which is less soluble. For ferric iron to be taken up by plants, either the substrate must be ameliorated, or the plants must have mechanisms to improve its uptake (Hell and Stephan 2003). At pH 5.8, commonly used in plant tissue culture, unchelated ferric iron forms insoluble ferric oxides, which were shown to be unavailable to plant tissues (Hangarter and Stasinopoulos 1991). To enhance its solubility, Fe3+ has been used in chelated forms (Alvarez-Fernandez et al. 1996) as ethylenediamine di-2-hydroxy-phenylacetic acid (Fe-EDDHA) and ethylenediamine tetra acetic acid (Fe-EDTA) in micropropagation medium. Fe-EDTA is the form of iron contained in Murashige and Skoog medium (Murashige and Skoog 1962), Driver and Kuniyuki walnut medium (Driver and Kuniyuki, 1984), and Woody plant medium (Lloyd and McCown, 1980). Various studies on hazelnut have utilized medium with Fe-EDTA (Messeguer and Mele 1987; Diaz-Sala et al. 1990; Bassil et al. 1991; Berros et al. 1994; Diaz-Sala et al. 1994; Rey et al. 1994; Nas and Read 2001; Andres et al. 2002) even though it has been shown to be a less effective source of iron for plants grown in vitro (Van der Salm et al. 1994; Ciccotti et al. 2008). Fe-EDDHA has been substituted for Fe-EDTA in a modified Driver and Kuniyuki walnut medium to successfully multiply and root several C. avellana cultivars (Yu and Reed 1995). However, a comparative study of the effect of iron source on multiplication, morphology, and development of in vitro hazelnut shoots has not been conducted.
The objectives of this study were to determine whether hazelnut explants grew differently in two chelated iron sources by assessing the effect of these on shoot elongation, multiplication, leaf chlorophyll content, leaf area, and shoot dry mass accumulation. The same criteria were used to evaluate hazelnut explant growth in a two-phase or semi-solid medium. In addition, hazelnut explants were analyzed to determine if shoots grew differently depending on the position of the node from which explants were derived. The hybrid hazelnut cultivar Geneva, ecologically appropriate for eastern North America growing conditions, was used to study the parameters with an overall objective of developing a consistent, repeatable, and economically feasible micropropagation method for the growing hazelnut industry in Canada.
MATERIALS AND METHODS
Role of Iron Type on Shoot Development
Explant material collected from 1-yr-old greenhouse-grown hybrid hazelnut C. avellana L.×C. americana M. cv. Geneva (Grimo Nut Nursery, Niagara-on-the-Lake, ON) was used for this study. Fresh shoot buds were excised in May and June 2011. Nodal segments with single buds were surface disinfested in 70% ethanol for 1 min, followed by 3 min of rinsing in sterile deionized water. Shoot buds were further surface disinfested in 15% commercial bleach (5.5% sodium hypochlorite) solution with two drops of Tween-20 (Sigma-Aldrich, St. Louis, MO) for 10 min, followed by three rinses in sterile deionized water, each lasting 3 min. Only the youngest four to six buds from each shoot were used as explants. Shoots were excised into single node segments (10 mm) and placed into GA-7 culture vessels (Magenta Corporation, Chicago, IL) each containing 50 mL of medium and lids sealed with Micropore™ tape (Fisher Scientific Inc., Ottawa, ON). Six explants were placed in each GA-7 with six boxes used for each treatment, and the experiment was repeated three times. Explants were maintained in a growth room at 28°C with a 16-h photoperiod of 45 µmol m−2 s−1 provided by cool-white fluorescent lamps (Osram Sylvania Ltd., Mississauga, ON). Visual observations and data collection for each explant were made on day 35 of the culture period. Data for analysis included shoot height, number of nodes per shoots, number of shoots per explant, chlorophyll (Chl) content, and Chl a/Chl b ratio.
The basal medium consisted of modified NCGR-COR medium (Yu and Reed 1993, 1995) supplemented with 10 g L−1 myo-inositol, 200 mg L−1 glycine, 100 mg L−1 nicotinic acid (IBA), 100 mg L−1 thiamine (PhytoTechnology Laboratories, Shawnee Mission, KS), 17.6 µM benzylaminopurine (BA; Sigma-Aldrich, Oakville, ON), 0.014 µM indole-3-butyric acid (IBA), 0.29 µM gibberellic acid (GA3; PhytoTechnology Laboratories, Shawnee Mission, KS), and 30 g L−1 glucose. The pH of the medium was adjusted to 5.7 and 6 g L−1 agar (A360-500, Fisher Scientific Inc., Ottawa, ON) was added before autoclaving at 121 °C for 20 min. Iron was supplied either as ethylenediamene tetra acetic acid-ferric-sodium salt (Fe-EDTA) or ethylenediamine bis(2)-hydroxyphenylacetic acid (Fe-EDDHA) (PhytoTechnology Laboratories, Shawnee Mission, KS), at 0, 230, 460, or 690 µM.
To measure chlorophyll content, a minimum of 100 mg of fresh leaves was excised from shoots in each treatment. Twelve samples for each treatment were analyzed and the experiment was repeated three times. For each sample the fresh weight of leaves was recorded. Leaves were immediately placed in a 15 mL centrifuge tube (Fisher Scientific Co., Ottawa, ON) with 4 mL of 100% methanol added to each tube. Tubes were incubated in a 65°C water bath for 10 min and immediately stored at 4°C for 24 h. The tubes were then centrifuged for 5 min with the resulting supernatant filtered through a 0.45 µm filter system (Fisher Scientific Co., Ottawa, ON). For the chlorophyll content analysis, 1 mL of filtered extract was added to a cuvette and absorbance at both 652 nm and 665 nm were measured using a DU800 spectrophotometer (Beckman Coulter, Mississauga, ON). Chlorophyll content was calculated according to the method of Lichtenthaler (1987). The same methodology was used to prepare samples to determine the Chl a/Chl b ratio. Calculations of Chl a and Chl b were also performed according to the method of Lichtenthaler (1987).
For evaluation of leaf area, 12 shoots per treatment were measured and the experiment was repeated three times. Once explants were removed from the respective in vitro treatments, the leaves were immediately excised from each shoot in order to avoid desiccation, and measured using a LI-3000 Area Meter (LICOR, Inc., Lincoln, NE).
To evaluate the dry matter content of explants, 12 shoots per treatment were measured and the experiment was repeated three times. Fresh weight (FW) of explants were taken and then these shoots were immediately oven dried at 65°C for 48 h to obtain shoot dry weight (DW). Percentage dry matter content was calculated using the following formula: (DW/FW)×100.
The preparation of leaf tissue for electron microscopy was similar to the method used by Spiller and Terry (1980). Tissue from leaves was cut into 1.0 mm3 blocks and fixed for 2 h in a solution containing 4% glutaraldehyde and 50 mM Sorensen's phosphate buffer (pH 7.2). Samples were then rinsed twice in 50 mM Sorensen's phosphate buffer (pH 7.2), 10 min per rinse. Samples were placed post-fixation in 1% osmium tetroxide (OsO4) (Canemco-Marivac, Lakefield, PQ) in 50 mM Sorensen's phosphate buffer (pH 7.2) for 2 h and then rinsed in buffer for 10 min. The samples were then dehydrated in ethanol-distilled water solutions for 10 min each at 25, 50, 75, and 95% ethanol, which was followed by two transfers to 100% ethanol for 10 min each time. The samples were then embedded twice in 50:50 solutions of ethanol (Commercial Alcohols, Brampton, ON) and LR White resin (London Resin Co., Reading, England). Samples were then placed in pure LR White resin, stained with lead citrate for 2 min, and saturated with aqueous uranyl acetate for 7 min before encapsulation. Capsules were placed in a drying oven for 24 h before observation. The sections were examined in a LEO912AB electron microscope (Zeiss, Oberkochen, Germany) operating at 100 k in a zero-loss mode. The images were magnified in a range of 4000 to 10000×. The OSIS imaging system was used in conjunction with the iTEM software suite. Photographs were taken with an OSIS Cantiga 2K×2K digital camera.
Effect of Type of Media on Shoot Development
For the experiment assessing the effects of semi-solid and liquid medium on shoot development, four treatments were used. The treatments included medium with no growth regulators, semi-solid medium with growth regulators but no liquid, semi-solid medium containing growth regulators with 20 mL of liquid medium applied at day 14 after the initiation of the experiment, and semi-solid medium containing growth regulators with 10 mL of liquid medium applied at day 14 and 10 mL of liquid medium applied at day 28. Shoots from in vitro grown hybrid hazelnut C. avellana L.×C. americana M. cv. Geneva were excised into single node segments (10 mm) and placed into GA-7 vessels containing 50 mL of medium. Six explants were placed in each GA-7 with six boxes used for each treatment and the experiment was repeated three times. Data for analysis included shoot height, number of nodes per shoots, and number of shoots per explant. Twelve samples from each treatment were analyzed for chlorophyll content, Chl a/Chl b ratio, leaf area, and shoot dry weight and the experiment was repeated three times. Chemical composition of the basal medium was similar to that used for the iron experiment except the iron level for the media containing growth regulators was kept constant at 460 µM Fe-EDDHA. In double-phase medium, liquid was added on top of semi-solid medium. The liquid medium had the same composition as the semi-solid medium except for the absence of agar. Explants were maintained in a growth room at 28°C with a 16-h photoperiod of 45 µmol m−2 s−1 provided by cool-white fluorescent tubes. Visual observations and analysis were made on day 35 of the culture period.
Nodal Origin of Explant and Shoot Development
Shoots from in vitro grown hybrid hazelnut C. avellana L.×C. americana M. cv. Geneva were dissected into single node segments (10 mm) and immediately placed into GA-7 vessels containing 50 mL of medium. The six buds nearest the shoot tip were used and designated one (bud nearest the shoot tip or distal) through six (furthest from the shoot tip or proximal). Nodal explants were positioned horizontally and slightly depressed in the medium. Six explants were placed in each box with five boxes used for each treatment and the experiment was repeated twice. The basal medium consisted of modified NCGR-COR medium supplemented with 460 µM Fe-EDDHA. Explants were maintained in a growth room at 28°C with a 16-h photoperiod of 45 µmol m−2 s−1 provided by cool-white fluorescent tubes. Visual observations and analysis were made on day 35 of the culture period.
Experiments were conducted using a complete randomized block design. All statistical analyses were subjected to analysis of variance (ANOVA) using the general linear model (PROC GLM) procedure in SAS software ver. 9.3 (SAS Institute, Cary, NC) program package for Windows. In cases where the ANOVA indicated that the model was significant, multiple comparisons among means were performed using Turkey's multiple range test. The data were presented as means±standard error and different letters in the tables and figures indicate significant differences at P<0.05.
RESULTS
Role of Iron Type on Shoot Development
Shoot explants grew significantly better with Fe-EDDHA as the iron source when compared with Fe-EDTA (Table 1). Little or no growth occurred in medium containing either Fe-EDTA or no iron. Shoots were longer and had more nodes when Fe-EDDHA was used as the iron source (Fig. 1). In all Fe-EDDHA treatments, the number of shoots and the amount of chlorophyll were similar, with larger leaves and greater shoot dry weights occurring at 460 and 690 µM Fe-EDDHA. Plants grown on medium supplemented with 230 µM Fe-EDDHA produced almost two more nodes on average per explant than either of the other two Fe-EDDHA treatments. With Fe-EDTA as the iron source, explants exhibited appreciable growth only at 230 µM with the 460 and 690 µM treatments failing to yield shoots suitable for subculture. Levels lower than 230 µM (175 and 200 µM) were found to give poor and inconsistent results for both Fe-EDTA and Fe-EDDHA treatments (data not shown).
Fig. 1.
Table 1.
a–e Data represent mean values±SE and for means in the same column, different letters indicate significant differences among treatments using Tukey-Kramer test at P≤0.05. –, indicates no data available for that treatment.
Electron microscopy revealed distinct differences in chloroplast structure between the iron treatments. In the Fe-EDDHA treatments, hazelnut chloroplasts exhibited eight or nine thylakoids per granum with a large number of granal and stromal lamellae. With no iron or Fe-EDTA in the medium, there was a reduction in the number of grana per chloroplast and the number of thylakoids per granum (Fig. 2A, B). The internal structure of the leaf grown in the 230 µM Fe-EDTA treatment (Fig. 2B) lacked organization, with thylakoids absent in many intracellular spaces, and sporadic location of grana and lamellae. In contrast, the leaves excised from shoots grown in Fe-EDDHA (Fig. 2C–E) had chloroplasts with better organization of thylakoids and grana, a well-arranged network of lamellae, and fewer osmiophilic lobules.
Fig. 2.
Effect of Type of Medium on Shoot Development
Explants did not grow in semi-solid medium without growth regulators (Table 2). Those grown in double-phase medium had similar numbers of shoots, longer shoots with more nodes, and greater leaf area, but less chlorophyll and a lower dry weight than explants cultured in semi-solid medium. Hyperhydricity was observed in plants grown in double-phase medium, exhibiting enlarged and curled leaves and a greater propensity towards chlorosis. Many of these explants had thickened and curved stems, and highly variable internode lengths compared with plants from semi-solid medium (Fig. 3A–D). There were differences between the two double-phase systems as the application of 10 mL of liquid medium over two intervals produced shoots with higher dry weight than the one time 20-mL treatment. The 20-mL treatment resulted in twisted stems which made them more difficult to work with during subculture.
Fig. 3.
Table 2.
a–c Data represent mean values±SE and for means in the same column, different letters indicate significant differences among treatments using Tukey-Kramer test at P≤0.05. –, indicates no data available for that treatment. S-S No PGR (semi-solid no plant growth regulators); S-S PGR (semi-solid+plant growth regulators); S-S+PGR+20 mL (semi-solid+plant growth regulators+20 mL liquid plant growth regulators); and S-S PGR+2×10 mL (semi-solid+plant growth regulators+2 applications 10 mL each liquid plant growth regulators).
Nodal Origin of Explant and Shoot Development
Explants derived from nodes five and six had the longest shoots with the most nodes (Table 3). The shoots became progressively shorter with fewer nodes when explants were obtained from buds located nearer to the apex of the original shoot (Fig. 3E).
Table 3.
a–c Data represent mean values±SE and for means in the same column, different letters indicate significant differences among treatments using Tukey-Kramer test at P≤0.05. Node one is nearest the shoot tip through to node six, which is furthest from the shoot tip.
DISCUSSION
A major objective of hazelnut breeding programs is the rapid proliferation of clonal material for different hazelnut genotypes. Increased shoot and internode length allows for more precise handling of plant material during the subculture process with reduced damage to sensitive apical and axillary buds during the excision of stems into nodal explants. This study provides a model for the in vitro culture of hybrid hazelnut and demonstrates the impact of iron source on in vitro shoot multiplication. Fe-EDDHA at all levels was superior to Fe-EDTA in producing longer shoots and more nodes, and shoots were easier to subculture. Iron is an essential element for in vitro shoot development. When explants were not provided with iron they failed to develop shoots, and when provided with Fe-EDTA failed to develop shoots comparable to those subcultured in Fe-EDDHA.
Fe-EDTA has been the more widely used chelator in hazelnut micropropagation studies (Messeguer and Mele 1987; Diaz-Sala et al. 1990, 1994; Bassil et al. 1991; Yu and Reed 1993; Damiano et al. 2005; Bacchetta et al. 2008) despite the evidence of an improved culture response with Fe-EDDHA in a number of other species, including a few hazelnut cultivars (Van der Salm et al. 1994; Molassiotis et al. 2003; Ciccotti et al. 2008). The micropropagation system developed in this study demonstrates the benefits of incorporating Fe-EDDHA in the medium. Our results indicate that there may be reason to re-evaluate the routine use of EDTA over EDDHA in hazelnut, and possibly in many other recalcitrant species, since the rationale for the use of EDDHA and its effect on the physiology of tissue cultures remains a relatively unexplored area within micropropagation research.
The chelating agents EDTA and EDDHA were added to the medium to maintain the solubility of iron (Murashige and Skoog 1962). The stability of chelated iron at a pH range of between 5 and 6 was essential for iron availability and incorporation into hazelnut plant tissues. However, Fe-EDTA photo-oxidizes at a pH of 5.7 (Hangarter and Stasinopoulos 1991) and quickly forms insoluble ferric oxide, which is unavailable to plant tissues (Lindsay and Schwab 1982). A 45% loss of initial Fe concentration with chelated Fe-EDTA has been reported at a pH of 5.8 or less (Dalton et al. 1983). Thus, iron in the form of Fe-EDTA may have remained unavailable to the hazelnut explants, most likely as a result of its degradation by exposure to light, and resulted in a medium which was iron-deficient. Photo-oxidation of EDTA was also correlated with formaldehyde formation which was toxic to plant growth (Hangarter and Stasinopoulos 1991), and may also have been a factor in the poor growth of explants in the Fe-EDTA treatments. The superior performance of in vitro hazelnuts grown with Fe-EDDHA as the iron source can also be attributed to the reduced energy expenditure required for its uptake compared with Fe-EDTA. Alcañiz et al. (2005) reported that the effectiveness of Fe-EDDHA as an iron source was because of the lower energy requirement for its decomposition. Additionally, the enhanced shoot height with Fe-EDDHA was related to the increased length of time iron was available to explants. In a comparative study of different iron chelates, Fe-EDDHA retained more chelated Fe in solution than Fe-EDTA (Alvarez-Fernandez et al. 1997). With Fe-EDDHA, iron was available longer and plants required less energy to utilize it effectively for growth. Fe-EDDHA has been shown to be more photostable than Fe-EDTA (Molassiotis et al. 2003), which allows iron to be more available, and reverses the effects of chlorosis. Thus, it was not surprising that the replacement of Fe-EDTA with Fe-EDDHA provided superior results in the micropropagation of a number of plant species. The use of Fe-EDDHA resulted in greater in vitro shoot growth and increased chlorophyll content in rose rootstocks (Van der Salm et al. 1994). Rooting percentage, root number, and root length were significantly improved in Prunus rootstocks with the use of Fe-EDDHA compared with Fe-EDTA (Antonopoulou et al. 2007; Ul Hasan et al. 2010). Substituting Fe-EDDHA for Fe-EDTA in a modified Murashige and Skoog medium produced higher quality Malus microshoots (Ciccotti et al. 2008).
Hazelnut tissues in the Fe-EDDHA treatments exhibited higher chlorophyll content and more organized chloroplast structure. Micropropagated plants with higher chlorophyll content have a greater photosynthetic capacity which contributes to successful rooting and acclimatization (Kanechi et al. 1998). Hazelnut explants in both the Fe-stressed and Fe-EDTA treatments expressed poorly organized photosynthetic apparatus, and in the case where no iron was provided large starch granules displaced the grana, indicative of a reduced photosynthetic capacity (Lee et al. 1985). The displacement of photosynthetic apparatus by large starch granules also results in poorly organized grana. The large number of osmiophilic lobules that occurred when no iron was provided to the hazelnut explants were common to Fe-stressed shoots and potentially represent lipids and carotenoids that can accumulate when membranes were absent (Spiller and Terry 1980; Lee et al. 1985). The irregular chloroplast structure of explants grown in 230 µM Fe-EDTA was associated with lower chlorophyll content. Micropropagated plants increase their photosynthetic rate soon after their transfer to the ex vitro environment (Diaz-Perez et al. 1995; Kadlecek et al. 2001). Thus, sufficient chlorophyll levels in plantlets grown in vitro are important for their successful acclimatization. Explant leaves containing insufficient chlorophyll have a lower photosynthetic rate and this can reduce a plant's ability to survive transfer to ex vitro conditions. The higher Chl a/Chl b ratio in the 230 µM Fe-EDTA treatment may indicate Fe-stressed leaves. Spiller and Terry (1980) determined that Fe-stressed leaves of Beta vulgaris had lower chlorophyll content and a higher Chl a/Chl b ratio than leaves which were Fe sufficient. There was a greater loss of chlorophyll b than chlorophyll a and an associated reduction in light harvesting complexes with Fe stress. Fe deficiency interrupts energy transfer from the part of the antenna that was disconnected from the PSII reaction centres (Morales et al. 2001). Decreases in photosynthetic energy conversion efficiency with Fe stress have also been reported in Prunus persica and Pyrus communis (Nedunchezhian et al. 1997; Morales et al. 2000). Conversely, the use of Fe-EDDHA resulted in hazelnut explants with higher chlorophyll content, a more organized chloroplast structure, and a more balanced Chl a/Chl b ratio, all of which can result in superior ex vitro acclimatization compared with Fe-EDTA.
An essential requirement for successful acclimatization of explants was attainment of enough nutrient reserve to survive the stress of transition from in vitro to ex vitro conditions (Debergh et al. 1992; Van Huylenbroeck and Debergh, 1996). The greater leaf area and dry weight content of shoots grown in the Fe-EDDHA treatments increase their ability to acclimatize to the ex vitro environment. Leaf area at the beginning of the acclimatization phase can be a useful predictor of successful acclimatization. The leaf area of Solanum tuberosum transplants at the end of the acclimatization phase was positively influenced by the leaf area at the time of transfer to the ex vitro environment (Tadesse and Struik 2000). Vaccinium microshoots with higher dry weights at the time of transplanting continued to maintain their higher dry weights for several weeks post-transfer (Isutsa et al. 1994). Thus, it is advantageous for micropropagated plants to develop a larger leaf area and maximize their dry weight content to ensure their ex vitro survival.
There may be advantages with the use of liquid medium in a micropropagation system for hazelnuts (Diaz-Sala et al. 1990) and other crops including Pinus (Aitken-Christie and Jones 1987). Overlaying agar-solidified medium with liquid medium resulted in greater shoot multiplication in roses, and increased shoot elongation, fresh weight, and rooting percentage in Magnolia (Horan et al. 1995; Maene and Debergh 1985). The use of a double-phase culture system resulted in an increase in shoot multiplication rates and quality in Pyrus (Viseur 1987) and increased shoot height in Chimonanthus (Kozomara et al. 2008). However, our results indicate that the use of double-phase medium can be problematic for micropropagation of hybrid hazelnut and should be performed with caution because of the onset of hyperhydricity and irregular morphology. This was similar to the observations with Quercus suber in which greater shoot proliferation and elongation in double-phase medium was also accompanied by vitrification (Romano et al. 1992). Explants of Persea americana grown in semi-solid medium had a stomatal system with a more normal morphology, reduced stomatal density, and more prominent epicuticular waxes than those grown in double-phase medium, which produced hyperhydritic shoots with deformed stomata and low epicuticular waxes (de la Vina and Pliego-Alfaro 2001).
The nodal position within the source shoot can influence the rate of shoot multiplication and development in long-term micropropagation. Nodal explants of hazelnut derived further from the shoot apex developed longer shoots with more nodes than those from the distal portion. Similar results for the effect of nodal position on shoot length were also found in Quercus robur (Volkaert 1990), Rosa hybrida (Hsia and Korban 1996), and Ulmus americana (Shukla et al. 2012). Vitis rotundifolia explants derived from basal nodes produced better shoot proliferation than those from terminal nodes (Goldy 1991). In our study, there was no effect of bud position on proliferation in cv. Geneva as each node produced an average of one shoot per explant. However, the increased number of nodes per shoot resulting from buds five and six would ultimately result in an increased number of explants in each subsequent subculture and exponentially increase propagation rates. Successful in vitro axillary bud development requires the correct balance between plant growth regulators, specifically auxin and cytokinin, with the explant response being specific to each species and genotype. It may be that hazelnut basal nodes were more developed and less inhibited by auxin than the terminal nodes, which resulted in greater and earlier shoot development in vitro. A greater combined effect of auxin and cytokinin occurs in the basal nodes as endogenous auxin moves basipetally from the terminal to basal end of a shoot (George 2008) and can result in a larger morphogenic response in explants derived from these nodes. For commercial propagators, the understanding that some nodes are potentially more prolific than others will allow for more accurate prediction of subsequent shoot numbers and assist in the commercial development of hybrid hazelnut micropropagation systems.
The results from this study confirm that the type of iron chelate used in the basal medium was a critical factor in the successful micropropagation of hybrid hazelnut with the substitution of Fe-EDDHA for Fe-EDTA significantly improving in vitro shoot development. The use of Fe-EDDHA corrected many of the deficiencies associated with Fe-EDTA-containing medium. Double-phase medium has the potential to provide some advantages in vitro, but problems of hyperhydricity and altered plant morphology suggest that further research into the use of liquid medium was required. Bud position also has an influence on the shoot and nodal development of hybrid hazelnut and should be a consideration in the prediction of numbers of explants generated in a micropropagation system.
ACKNOWLEDGEMENTS
The study was funded by an OMAFRA/University of Guelph HQP Scholarship and the Ontario Centre of Excellence. The authors thank Grimo Nut Nursery and Butternut Farms for providing the plant material for the study. We thank Bob Harris for technical help with the electron microscopy.
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Information & Authors
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Published In
Canadian Journal of Plant Science
Volume 93 • Number 3 • May 2013
Pages: 511 - 521
History
Received: 30 August 2012
Accepted: 7 December 2012
Version of record online: 1 January 2013
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GarrisonWalter, DaleAdam, and SaxenaPraveen K.. 2013. Improved shoot multiplication and development in hybrid hazelnut nodal cultures by ethylenediamine di-2-hydroxy-phenylacetic acid (Fe-EDDHA). Canadian Journal of Plant Science. 93(3): 511-521. https://doi.org/10.4141/cjps2012-218