High efficiency in vitro organogenesis from mature tissue explants of Citrus macrophylla and C. aurantium
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- Tallón, C.I., Porras, I. & Pérez-Tornero, O. In Vitro Cell.Dev.Biol.-Plant (2013) 49: 145. doi:10.1007/s11627-012-9476-6
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A simple and efficient protocol for obtaining organogenesis from mature nodal explants of Citrus macrophylla (alemow) and Citrus aurantium (sour orange) has been developed by optimizing the concentrations of the plant growth regulators, the incubation conditions, the basal medium and by the choice of explant. In order to optimize the plant growth regulator balance, explants were cultured in the regeneration medium supplemented with several N6-benzyladenine (BA) concentrations or with 2 mg l−1 BA in combination with kinetin (KIN) or 1-naphthaleneacetic acid (NAA). The presence of BA was found to be essential for the development of adventitious buds; the best results were obtained using BA at 3 and 2 mg l−1 for alemow and sour orange, respectively. The combination of BA with KIN or NAA in the culture medium decreased the regeneration frequency, with respect to the use of BA alone. The effect of three different basal media was rootstock-dependent. For C. macrophylla the best results were obtained with Woody Plant Medium or Driver and Kuniyuki Walnut Medium (DKW). However, for C. aurantium, although high percentages of regenerating explants were obtained independently of the basal medium used, the highest number of buds per regenerating explants was obtained with DKW medium. Attempts were made to identify the type of explants which had a higher regeneration ability using particular regions along the mature shoots of C. macrophylla. When nodal segments, where the buds were completely removed, and internode segments were compared, the highest percentage of responsive explants was obtained with nodal segments. The existence of a morphogenetic gradient along the shoot was observed and the organogenic efficiency was highest when explants from the apical zone were used. Incubation in darkness for 3 or 4 wk was essential for regeneration process in both rootstocks.
KeywordsAdult explantsAdventitious regenerationAlemowBasal mediumPlant growth regulatorsSour orange
Citrus is one of the most important genera of plants in terms of economic value and human nutrition, due to the high nutritional value of its fruits and the considerable commercial production of its fruit and fruit products (Barlass and Skene 1986; Chaturvedi et al.2001). In Citrus, the rootstock is essential in the growth, nutrition, and longevity of the tree and other characteristics of the scion’s performance, thereby playing an important role in the success of any orchard (Singh 2001).
During the last few years, large strides have been made in the citrus industry in terms of increases in production, acreage, and yield. Undoubtedly, sustainable development of the citrus industry is dependent mainly on a continuous supply of new and improved genotypes. The improvement of Citrus rootstock cultivars via conventional breeding strategies is normally hampered by several factors related to their reproductive biology, such as large tree size, polyembryony, high level of heterozygosity, and long juvenile period (Grosser and Gmitter 1990; Spiegel-Roy and Goldschmidt 1996). As alternatives, biotechnological techniques, such as plant tissue culture, mutagenesis, and genetic transformation approaches, are looked on as valuable strategies for improvement of Citrus rootstocks. Genetic transformation of Citrus rootstocks is a promising tool that enables the introduction of desirable traits without altering the genetic background (Molinari et al.2004; Tong et al.2009; Dutt et al.2010). However, transformation efficiencies are generally low and protocols are species or even cultivar-dependent. One of the main limitations of this technology is low plant regeneration frequencies, especially for many of the economically important citrus species (Peña et al.2004). An efficient system of regeneration via organogenesis is the first step in the successful introduction of genetic variation by genetic transformation of Citrus rootstocks.
In vitro regeneration in citrus has been achieved for several genotypes through different in vitro techniques, such as somatic embryogenesis from nucellar callus and protoplast cultures (reviewed by Gosal et al.1995), and the use of somatic hybrids (reviewed by Grosser et al.2000). More recently, the focus has been to derive efficient protocols for the recovery of plants through organogenesis of citrus rootstock genotypes (Bordón et al.2000; Germanà et al.2008; Dutt et al.2010; Silva et al.2010; Marques et al.2011). However, this process is usually limited to the use of juvenile plant tissue, such as epicotyl segments from seedlings germinated in vitro (Bordón et al.2000; Zou et al.2008; Dutt et al.2010) and internode segments from juvenile plants grown in greenhouses (Ghorbel et al.1998; Tong et al.2009; Silva et al.2010). Plants regenerated from these explants exhibit juvenile traits, which are undesirable for seedling production and genetic improvement since they require several years of maturation (juvenile period) before they can be evaluated for horticultural traits. Moreover, juvenile and adult tissues show marked differences in their responses to organogenesis induction in tissue culture, with a progressive loss of competence during the transition to the mature phase (von Aderkas and Bonga 2000). Tissues derived from adult plants are not normally used for in vitro culture, mainly because of the low responsiveness of woody plants to exogenous growth regulators, the failure of standard surface sterilization techniques, their low morphogenetic capacity (related to progressive repression or inactivation of gene activity during plant development), and the poor rooting of the shoots obtained (Almeida et al.2003; Cervera et al.2008; Mendes et al.2010). The development of a regeneration system for mature stem segments would bypass the juvenile phase and so accelerate the evaluation of tissue culture-derived events, and is essential in functional genomics studies (Almeida et al.2003; Cervera et al.2008; Rodríguez et al.2008). Plant regeneration from explants of mature citrus tissue has been reported only for a few sweet orange or mandarin cultivars (Cervera et al.1998; Almeida et al.2003; Cervera et al.2008; Rodríguez et al.2008; Oliveira et al.2010; Bassan et al.2011; Curtis and Mirkov 2012).
Among rootstocks, organogenesis has been achieved easily for juvenile explants of Carrizo citrange (Peña et al.2004; Germanà et al.2011); however, other rootstocks, such as Citrus limonia, Citrus volkameriana, Citrus macrophylla, and Citrus aurantium, proved to be recalcitrant (Bordón et al.2000; Azevedo et al.2006; Germanà et al.2008; Silva et al.2010; Marques et al.2011). To the best of our knowledge, plant regeneration via organogenesis from explants of mature citrus rootstocks has not been achieved before.
In the present study, we have evaluated the effect of the composition of culture media, incubation conditions, and explant type and origin, on in vitro organogenesis of two citrus rootstocks, alemow and sour orange, using mature tissues taken from in vitro-proliferated shoots, as an initial step in the development of transformation and mutagenesis protocols.
Materials and Methods
Adult explants were derived from in vitro shoot cultures of C. macrophylla and C. aurantium developed previously in our laboratory (Tallón et al.2012). Shoots were maintained in a multiplication medium composed of macronutrients, micronutrients and vitamins of Driver and Kuniyuki Walnut Medium (DKW; Driver and Kuniyuki 1984), 30 g l−1 sucrose, 25 mg l−1 phloroglucinol, 0.1 mg l−1 indolebutyric acid (IBA), 6 g l−1 agar (Pronadisa), 1 mg l−1N6-benzyladenine (BA) and 2 mg l−1 giberellic acid (GA) for C. macrophylla or 2 mg l−1 BA and 0.4 mg l−1 GA for C. aurantium. After addition of plant growth regulators and adjustment of the pH to 5.7 with 1 M NaOH, 100 ml of the medium were dispensed in 500-ml jars and sterilized in an autoclave at 121°C for 21 min. Cultures were grown at 25 ± 1°C with white light (55 μmol m−2 s−1) and a 16-h photoperiod.
General strategy for regeneration.
The explants were placed horizontally in contact with the regeneration medium (RM), which consisted of a basal medium with the same composition as the multiplication medium, with the exception of the plant growth regulators, unless stated otherwise. After addition of plant growth regulators and adjustment of the pH to 5.7, the medium was sterilized in an autoclave at 121°C for 21 min and then dispensed aseptically, 25 ml per sterile plastic Petri dish (9-cm diameter × 1.5-cm depth). The explants were incubated at 25 ± 1°C in darkness for 3 wk before exposure to light with a 16-h photoperiod, unless stated otherwise. The nodal explants were transferred to fresh medium every 4 wk.
Cytokinin level for optimal regeneration.
To test the optimum level of cytokinins in the regeneration medium, nodal segments of alemow and sour orange were cultured in RM supplemented with 1, 2, 3, 4 or 5 mg l−1 BA or 2 mg l−1 BA plus 0, 1 or 2 mg l−1 kinetin (KIN). Nodal explants of sour orange were selected from the apical region in the explant (see “Effect of explant type and regions along the shoot”).
Addition of NAA to the regeneration medium.
Nodal segments of alemow were cultured in the RM supplemented with 2 mg l−1 BA and different concentrations of 1-naphthaleneacetic acid (NAA; 0, 1 or 2 mg l−1).
Effect of the basal medium.
Nodal explants of alemow and sour orange were cultured on RM with macro and micronutrients and vitamins of the Murashige and Skoog Medium (MS; Murashige and Skoog 1962), Woody Plant Medium (WPM; Lloyd and McCown 1980) or DKW medium. All media were supplemented with 2 mg l−1 BA and 0.1 mg l−1 IBA. Nodal explants for sour orange were only selected from apical region (see “Effect of explant type and regions along the shoot”).
Effect of explant type and regions along the shoot.
In order to determine the most suitable type of explant for the regeneration of alemow, nodal and internode segments from the full explant, except the basal lignified segments, were used. For the experiment with nodal segments, explants were obtained from three different regions along the shoot: apical (the first three segments from the apex), middle (the second three segments from the apex), and basal (the last three segments in the explant; Fig. 1). All explants were cultured in RM supplemented with 2 mg l−1 BA and 0.1 mg l−1 IBA.
Effect of the dark period.
Apical nodal explants of alemow and sour orange were cultured in RM with 2 mg l−1 BA and 0.1 mg l−1 IBA, being incubated in darkness for 0, 1, 2, 3 or 4 wk before exposure to the light.
Experimental design and statistical analysis.
For each treatment, five replicates (Petri dishes) were prepared, each containing at least 10–12 nodal segments. After 8 wk of incubation, the number of nodal explants forming adventitious buds and the number of buds formed per responsive explant were recorded. The assessment of nodal explants was carried out with the aid of a stereomicroscope. Adventitious buds regenerating within the first 2 wk were eliminated, being considered as pre-existing meristems.
The effect of the treatments on the regeneration percentage was analyzed by means of maximum likelihood ANOVA. When a significant χ2 was obtained, specific maximum likelihood contrasts were designed to examine differences between treatments. ANOVA was used to analyze the effect of the treatments on the number of buds per responsive explant. The least significant difference test (LSD) was used to discriminate differences between various treatments. The values presented are means and their standard errors.
Results and Discussion
The success of a Citrus regeneration protocol relies on a variety of factors, including the choice of medium, hormone addendum, incubation conditions (Durán-Vila et al.1989; Bordón et al.2000; Moreira-Dias et al.2000, 2001; Molina et al.2007; Khan et al.2009) and the explant surface in contact with the culture medium (García-Luis et al.2006), without disregarding the enormous influence of the genotype (Barlass and Skene 1982; Moore 1986; Bordón et al.2000; Rodríguez et al.2008).
Cytokinin level for optimal regeneration.
It has been reported that cytokinin levels are critical for callus formation from the cutting zone of explants and the subsequent initiation of buds from those calli (Moreira-Dias et al.2000). Application of BA was critical for callus and bud formation from epicotyl cuttings of Poncirus trifoliata (L.) Raf. (García-Luis et al.1999). In C. macrophylla and C. aurantium adult explants, 1 mg l−1 BA produced regeneration percentages that were lower than with 2 or 3 mg l−1 BA. Similarly, Cervera et al. (2008) found that for internode segments from adult explants of clementine, 3 mg l−1 BA produced higher numbers of shoots per explant than did 1 mg l−1.
Concentrations of BA exceeding 2 or 3 mg l−1, respectively, for mature nodal segments of sour orange and alemow, decreased the regeneration rate (Fig. 2); this effect was described also by Ghorbel et al. (1998) for juvenile explants of alemow and sour orange. The toxic effect of high concentrations of BA has been reported also for other citrus genotypes such as “Troyer citrange”, “Cleopatra” and “Clementine” mandarin (Moreira-Dias et al.2000; Molina et al.2007; Cervera et al.2008).
The addition of BA to the regeneration medium has been considered essential for shoot formation, but in most cases its effectiveness has not been compared with that of other cytokinins. It has been observed that while BA strongly induces the formation of adventitious shoots, KIN allows normal shoot growth (Hu and Wang 1983). In epicotyl and internode segments of sour orange juvenile explants, Silva et al. (2008, 2010) found that the addition of BA to the culture medium in combination with KIN induced a better organogenic response than KIN alone. Further, in epicotyl cuttings of Troyer citrange, shoot formation was higher in the presence of BA compared with KIN (Molina et al.2007).
Frequency of responsive explants and number of buds per regenerating explant of alemow and sour orange nodal segments cultured in a medium with 2 mg l−1 BA and three KIN concentrations (0, 1 and 2 mg l−1)
KIN (mg l−1)
Responsive explants (%)
Number of buds per explant
48.72 ± 4.64 a
2.19 ± 0.16 a
29.55 ± 6.96 b
1.38 ± 0.14 b
30.23 ± 7.09 b
1.37 ± 0.36 b
52.03 ± 3.04 a
2.46 ± 0.14 a
40.82 ± 7.09 a
1.75 ± 0.20 b
39.22 ± 6.91 a
1.35 ± 0.18 c
Addition of NAA to the regeneration medium.
Supplementation of the medium with a combination of cytokinin and auxin has produced contrasting results regarding Citrus organogenesis. While some authors reported that NAA in combination with BA was fundamental for callus and bud formation (Bordón et al.2000; Ghorbel et al.2000; Almeida et al.2003), others observed marginal or even detrimental effects with the addition of auxin (Moore 1986; García-Luis et al.1999; Moreira-Dias et al.2000; Marques et al.2011). Such apparently contradictory results could be due to the use of different Citrus genotypes (Bordón et al.2000; Almeida et al.2003).
Frequency of responsive explants and number of buds per regenerating explant of alemow cultured in a medium with 2 mg l−1 BA and three NAA concentrations (0, 1 and 2 mg l−1)
NAA (mg l−1)
Responsive explants (%)
Number of buds per explant
48.72 ± 4.64 a
2.19 ± 0.16 a
33.33 ± 6.67 b
1.17 ± 0.13 b
20.41 ± 5.82 c
1.20 ± 0.20 b
It has been reported previously that unlike most juvenile explants, explants from mature citrus plants require balanced combinations of BA and NAA in the culture media for maximum shoot regeneration (Almeida et al.2003; Rodríguez et al.2008). In contrast to these reports, in our study, the best results in mature explants of alemow were observed when BA was used alone.
Effect of basal medium.
While many studies have concentrated on the influence of plant growth regulators in plant tissue culture, the effect of nutrients has received much less attention. However, the success of in vitro tissue culture is strongly dependent on the chemical composition of the culture medium (Ruzic et al.2004). Macronutrient salts, namely N, P, K, Ca, Mg and S, are indispensable for the growth of higher plants in vitro (George et al.2008).
The composition of the media used for in vitro culture of Citrus is usually based on the nutrients and vitamins of MS and MT media (Ghorbel et al.1998; Silva et al.2008; Marques et al.2011). Recent studies from our group proposed the use of Driver-Kuniyuki medium (DKW) for the in vitro culture of lemon and citrus rootstocks (Pérez-Tornero et al.2010; Tallón et al.2012), and WPM has been used successfully for tissue culture of recalcitrant woody species (Lloyd and McCown 1980). In our study, the best results for mature explants of alemow were obtained when DKW or WPM medium was used as the regeneration medium. The DKW and WPM media contain more sulfate and less chloride (11.95 and 7.18 mM SO42−, respectively; 2.02 and 1.31 mM Cl−, respectively) than MS (1.46 mM SO42−; 5.99 mM Cl−). The high Cl− concentration of the MS medium might have produced toxic effects in alemow adult explants since explants growing in MS produced fewer responsive explants and less buds per responsive explant (Fig. 3); these results match those of Teasdale (1987) for pine suspension cultures. However, there was greater stimulation of morphogenesis for explants cultured in MS medium, compared with WPM medium, for mature tissue of sweet orange and rangpur lime (Oliveira et al.2010); but, WPM induced larger shoots than did MS basal medium. Similar observations were reported for “Pera” (Kobayashi et al.2003) and “Clementine” mandarin (Cervera et al.2008), using explant mature tissue, but these results are not consistent with those obtained for mature explants of C. macrophylla in the present study.
In sour orange, although similar regeneration percentages were observed for all media (Fig. 3), the number of buds per regenerating explant increased when the DKW medium was used. The Ca2+ concentration in the DKW basal medium (9 mM) is three times higher than in WPM or MS (3 mM). Calcium ions are involved in in vitro morphogenesis and are required for many responses induced by plant growth substances, particularly auxins and cytokinins (George et al.2008). In Torenia stem segments, adventitious bud formation induced by cytokinin seems to be mediated, at least in part, by an increase in intracellular Ca2+ (Tanimoto and Harada 1986), and exogenous Ca2+ enhanced the formation of meristemoids, the first phases of outgrowth into organs, in tobacco explants (Capitani and Altamura 2004). Higher Ca2+ levels might be necessary to increase the bud production by adult nodal explants of sour orange.
Effect of explant type and regions along the shoot.
Most protocols for in vitro organogenesis of citrus rootstocks have been developed for epicotyls or internode segments from juvenile explants (Molina et al.2007; Tong et al.2009; Germanà et al.2011; Marques et al.2011). However, in some cultivars the number of responsive explants is low, even when young and “highly-responsive” explants are used.
In the natural environment, the wounding of plants by pruning, grazing or mechanical abrasion often stimulates callus formation and/or regeneration. Different authors have suggested that wounding promotes the transfer of endogenous hormones to the location of the injury, locally producing a more-suitable level of growth promoters for regeneration (Norizaku et al.1985; Park and Son 1988). In our study, when alemow nodal segments were used, the wounded regions in the explant were more abundant than in internode explants and this might have increased the accumulation of growth promoters in the explant, hence improving the number of responsive explants.
Protocols for in vitro organogenesis from mature citrus material are based on the use of internode segments (Rodríguez et al.2008; Oliveira et al.2010; Curtis and Mirkov 2012) and very few studies have been carried out using alternative sources of plant material. Paolo et al. (2004) observed that the regeneration percentages for sweet orange leaf petioles or midribs were double that achieved with internode segments.
To the best of our knowledge, little attention has been paid to variations arising from differences in the morphogenic potential of explants collected from the diverse regions of citrus explants. It is very probable that variations in the levels of endogenous growth substances are often responsible for the success or failure of organogenesis (George et al.2008). In fact, there are conflicting results in the literature regarding the effect of the region of seedlings on the morphogenic response. Whereas in some cases, the best results were obtained from the basal part of the segment (García-Luis et al.2006) and in regions nearest to the cotyledons (García-Luis et al.1999; Moreira-Dias et al.2000; Germanà et al.2008; Marques et al.2011), other authors observed a higher organogenic potential at the apical edge of the wound (Bordón et al.2000; Costa et al.2004).
When different regions (apical, middle or basal) along the shoot were used to select nodal segments for C. macrophylla adult explants, significant differences (P < 0.001) were observed in the number of responsive explants. The existence of a morphogenetic gradient along the shoot was observed. Nodal segments from the apical region exhibited the highest regeneration ability (52.14% regenerating explants) and explants from the basal region the lowest (23%; Fig. 4). Significant differences in the number of buds per regenerating explant were observed too (P < 0.05), and the best results (1.9 buds per regenerating explant) were obtained with explants from the apical region, since those from the middle or basal region produced about one bud per regenerating explant (Fig. 4). The organogenic response increased with the distance of the explant from the basal node of the shoot. A similar response was reported by Costa et al. (2004), who found that the regions of the epicotyl farthest from the cotyledons of two different Citrus species (C. limonia Osb. and C. paradise Macf.) were more organogenic. One may speculate that the apical portion of a branch possesses different nutrient or endogenous plant hormone concentrations when compared with the basal portion, which may affect adventitious bud induction (Mok 1994). Our results differ from those of Germanà et al. (2008) for epicotyl explants of alemow and Marques et al. (2011) for internodes of sour orange juvenile explants. In both of these cases, the highest regeneration response and number of buds were obtained when segments from basal regions were used. A gradient in the organogenic response was reported also for seedling explants along the Troyer’s citrange epicotyl axis, with a maximal response at the base, near the cotyledons (Goh et al.1995; García-Luis et al.1999; Moreira-Dias et al.2001).
Effect of the dark period.
In most protocols of Citrus rootstock organogenesis, incubation is performed in the light (Ghorbel et al.1998; Germanà et al.2008; Silva et al.2008; Tong et al.2009; Roussos et al.2011). In the few cases in which different lighting conditions were compared, shoot formation was higher either in the light (Moreira-Dias et al.2000; Silva et al.2008) or in the dark (Molina et al.2007; Dutt et al.2010; Bassan et al.2011). Genotypic differences may account for some of these contrasting results (Bordón et al.2000).
Durán-Vila et al. (1992) were the first to report that culturing explants in the dark greatly enhanced the regeneration of adventitious shoots from internode segments of “Pineapple” sweet orange seedlings. Since long exposures to darkness in some cases produced etiolated shoots that showed abscission, bleaching, and finally death, more recently other authors have shortened the explant incubation in darkness to 2–4 wk, depending on the citrus species (Cervera et al.2008; Dutt et al.2010; Oliveira et al.2010; Marques et al.2011). In our study, explant incubation in darkness for 4 wk decreased the regeneration percentage and the number of buds per regenerating explant in C. macrophylla; however, the best results in C. aurantium were obtained with 4 wk in darkness (Fig. 5), and these results are in agreement with those of Tavano et al. (2009) or Silva et al. (2010) for juvenile explants of sour orange.
Different studies of organogenesis using juvenile explants of alemow showed that the best results occurred when the culture was carried out with a 16-h photoperiod (Ghorbel et al.1998; Germanà et al.2008). These results disagree with ours since when adult explants of alemow were cultured directly with a 16-h photoperiod, the lowest number of responsive explants or buds per regenerating explant was obtained (Fig. 5). For sour orange, contrasting results can be found in the literature. While Ghorbel et al. (1998), Silva et al. (2008) and Roussos et al. (2011) obtained better results when explants were cultured in the light, compared with the dark, other authors observed that 3 or 4 wk in darkness was necessary for the optimal organogenesis of juvenile explants of C. aurantium (Silva et al.2010; Marques et al.2011; Schinor et al.2011); the latter results match those obtained in our study with mature tissue explants. Exposure to a period of darkness may modify the proportions of endogenous hormones like cytokinins and auxins, which then interact more effectively with exogenously applied growth regulators in the culture medium, leading to promotion of shoot regeneration (Miguel et al.1996; Gentile et al.2002).
In this study, an efficient in vitro organogenesis system for alemow and sour orange using mature nodal explants has been established. Explants from the two citrus rootstocks showed similar responses on the regeneration medium. A small amount of white friable compact callus developed at the wound surface and calli differentiation led to the formation of adventitious buds (Fig. 6). Most of the buds developed from calli by indirect organogenesis, but sometimes a few buds were produced directly from the tissue.
The protocol was optimized by varying the concentration of plant growth regulators, the incubation conditions, the basal medium and the type of explant used. Addition of BA and induction in the dark were indispensable for explant regeneration and the type of explant or basal medium had a great influence. Several combinations of BA and other plant growth regulators, like NAA or KIN, decreased the explant regeneration capacity.
In most reports on Citrus organogenesis, juvenile explants derived from seedling tissues have been used as the source of explants. We have established a highly efficient and simple protocol for the regeneration from nodal segments derived from mature tissue from alemow and sour orange: for both rootstocks, high regeneration percentages—above 50%—were obtained. As far as we know, this is the first report on organogenesis from citrus rootstock adult explants.
This organogenesis protocol could be used for the successful introduction of genetic variation by genetic transformation or mutagenesis of alemow and sour orange. In vitro mutagenesis and selection experiments, using alemow and sour orange mature explants, have already been performed successfully in our laboratory using this protocol.
The authors thank Mr. Fernando Córdoba for technical assistance in the laboratory. This work was supported by the Ministerio de Ciencia e Innovación, through the project AGL2007-65437-C04-04/AGR.