Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 106, Issue 2, pp 299–307

Organogenesis and encapsulation of in vitro-derived propagules of Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf]

Authors

    • Dipartimento DEMETRAUniversità degli Studi di Palermo
  • Maurizio Micheli
    • Dipartimento di Scienze Agrarie e AmbientaliUniversità degli Studi di Perugia
  • Benedetta Chiancone
    • Dipartimento DEMETRAUniversità degli Studi di Palermo
  • Laura Macaluso
    • Dipartimento DEMETRAUniversità degli Studi di Palermo
  • Alvaro Standardi
    • Dipartimento di Scienze Agrarie e AmbientaliUniversità degli Studi di Perugia
Original Paper

DOI: 10.1007/s11240-011-9921-y

Cite this article as:
Germanà, M.A., Micheli, M., Chiancone, B. et al. Plant Cell Tiss Organ Cult (2011) 106: 299. doi:10.1007/s11240-011-9921-y

Abstract

Due to widespread polyembryony, Citrus rootstocks are usually propagated by open-pollinated seed germination, although micropropagation offers many advantages. Encapsulation technology has recently attracted the interest of researchers in the field of plant propagation because it combines the advantages of zygotic or gamic seeds with those of micropropagation. In this study, we examined the encapsulation of Carrizo citrange uninodal microcuttings (3–4 mm long) and evaluated the influence of the calcium alginate coating, a short time storage at cold temperature, and different sowing substrates on the viability and regrowth of the explants. A secondary aim was to develop an efficient protocol to induce root formation in the microcuttings. The results showed that encapsulation did not negatively affect the viability, providing a satisfactory regrowth, and storage potential for 30 days at low temperature. No differences in viability and regrowth were detected between the two different sowing substrates tested (agar-solidified medium and paper filter). To optimize the production of microcuttings required to perform the encapsulation experiments, in a preliminary experiment we assessed different factors affecting the in vitro shoot regeneration from epicotyl segments (obtained from seeds of Carrizo citrange germinated in vitro), including the influence of in vitro organogenesis, explant orientation, cut surface contact with the medium, treatments with different growth regulators, and distance of the organogenic explants from the cotyledonary node. The highest organogenic response was obtained from segments horizontally cultured (particularly from the basal portion), from segments with the cut surface in contact with the medium and from explants cultured on medium supplemented with 6-benzyladenine. No significant difference in regeneration efficiency was found in response to the distance of the epicotyl portions from the cotyledonary node.

Keywords

Adventitious shootCytokininsExplant typeMicropropagationGermplasm storageSynthetic seed

Abbreviations

BA

6-benzyladenine

CSD

Cut surface down, in contact with the medium

CSU

Cut surface facing up

HP

Horizontal position

IBA

Indole-3-butyric acid

NAA

α-naphthaleneacetic acid

TDZ

Thidiazuron

VP

Vertical position

Introduction

Propagation of Citrus rootstock is performed through open-pollinated seed germination because most of the genotypes are highly polyembryonic and true-to-type plants can be obtained by nucellar embryos (Wutscher 1979). However, micropropagation is also an efficient propagation method, especially when shoot regeneration can be achieved by in vitro organogenesis, because it offers a number of advantages over conventional propagation methods, such as the possibility of obtaining a high number of true-to-type virus-free plants in relatively less time and in limited space (Barlass and Skene 1982). The in vitro system may be also useful in providing a source of in vitro adult-phase plant material for genetic transformation, pathology studies, and in vitro conservation (Marutani-Hert et al. 2010; Perez-Tornero et al. 2010). Thus, the development of an efficient, easy, and rapid procedure to propagate a large number of rootstocks is particularly important when there is a scarcity of mother plants for seed production and it is necessary to import seeds and plants (Starrantino and Caruso 1988).

Encapsulation technology has recently been attracting the interest of researchers in the field of plant propagation because it combines the advantages of zygotic or gamic seeds with those of micropropagation (such as high efficiency of propagation, reduced space requirement, ease of handling and transportability, storability, reduced size of the propagules and automation potentiality). As such, it represents a new and powerful tool in the plant nursery field as well as in approaches to germplasm conservation and plant material exchange (Standardi and Piccioni 1998; Micheli et al. 2003). Specifically, synthetic seed or artificial seed (or synseed), described as “artificially encapsulated somatic embryos, shoots or other tissues which can be used for sowing under in vitro or ex vitro conditions” (Aitken-Christie et al. 1995), is the main product of this technology. The application of encapsulation technology to in vitro-derived propagules of Citrus has resulted in the production of synthetic seeds from somatic embryos of Citrus reshni (Nieves et al. 1995), C. reticulata Blanco, C. clementina Hort. ex Tan., a lemon hybrid (Germanà et al. 1999, 2007), and Kinnow mandarin (Singh et al. 2007). Moreover encapsulation–dehydration technology has also been used for the in vitro conservation of several Citrus species, (Gonzalez-Arnao et al. 2003; Malik and Chaudhury 2006). Recently, encapsulation technology has been applied to different plant species using in vitro-derived unipolar explants (Pattnaik and Chand 2000; Sicurani et al. 2001; Micheli et al. 2007; Rai et al. 2009).

Troyer and Carrizo citranges [C. sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.] are the most widespread citrus rootstocks due to the productive and qualitative characteristics of the cultivar and, above all, to their tolerance to the Citrus Tristeza Virus (CTV) which, since the first serious epidemic observed in Argentina in 1930, caused the death of millions of sweet orange trees grafted onto sour orange (Bar-Joseph et al. 1989). The fight against CTV is currently one of the main priorities of the Italian Citrus industry because about 98% of plants are grafted onto sour orange (Citrus aurantium L.), which is very sensitive to this pathogen, so that the changeover to the use of CTV-resistant rootstocks, such as citranges, will be soon become an emergency measure. Consequently, there is a large demand for citrange rootstocks and for an efficient protocol for synthetic seed production. Improvements and increases in the production of citrange true-to-type and virus-free plants could be very advantageous for the citrus nursery industry.

The aim of the study reported here was to test, for the first time, the response of Carrizo citrange vitro-derived microcuttings to encapsulation. To optimize the production of the vitro-derived microcuttings required for the synthetic seed assembly, we also evaluated the influence of different factors (culture conditions and different explants types) on the in vitro organogenic response of Carrizo citrange epicotyl segments.

Materials and methods

Organogenesis

Plant material and culture conditions

Seeds of Carrizo citrange were extracted from freshly harvested fruits and air dried at room temperature for 1 day. Following removal of the tegument, the seeds were sterilized in 70% (v/v) ethanol for 5 min, followed by immersion in 1.2% sodium hypochlorite for 20 min. After three successive rinses with sterile distilled water, the seeds were placed on the solid germination medium, which consisted of MS (Murashige and Skoog 1962) salts and vitamin mixture supplemented with 30 g l−1 sucrose, 500 mg l−1 ascorbic acid, and 500 mg l−1 malt extract (solidified with 8.5 g l−1 plant agar; Micropoli, Cesano Boscone, Italy). Following adjustment of the pH to 5.8, the medium was autoclaved at 121°C for 20 min. The cultures were then incubated for 4 weeks at 27 ± 1°C in the dark, then transferred to a light condition (16/8-h light/dark photoperiod) with light supplied by cool-white fluorescent lamps (TMN 30 W/84; Philips, Suresnes, France) at a photosynthetic photon flux density of 35 μmol m−2 s−1. The experiments were conducted using epicotyls >5 cm excised from the vitro-derived seedlings.

Effect of explant orientation

To verify the effect of explant position and orientation on adventitious shoot formation, we cut each epicotyl (>5 cm) into five 1-cm-long portions and numbered each according to its position, with 1 indicating the proximal or basal (B) zone and 5 indicating the distal or apical (A) zone, with the latter referring to the cotyledonary node. Ten petri dishes containing 15 ml of organogenic medium [MS mineral salts and vitamin mixture, 30 g l−1 sucrose, 8 g l−1 plant agar (Micropoli), 0.1 mg l−1α-naphthaleneacetic acid (NAA), 1 mg l−1 6-benzyladenine (BA); the growth regulators used were the same as those reported by Bordòn et al. 2000] were prepared. In each petri dish, five segments were cultured in the horizontal position (HP) and five from another epicotyl were cultured in the vertical position (VP), with the numbers 1–5 always indicating the distance from the cotyledonary node (Fig. 1). The cultures of this and other experiments were subcultured monthly in the same medium and incubated in the growth chamber under the same culture conditions reported above. After 2 months, the number of adventitious buds (length up to 4 mm) and the number and the length of new shoots (length >4 mm) were recorded in all the trials.
https://static-content.springer.com/image/art%3A10.1007%2Fs11240-011-9921-y/MediaObjects/11240_2011_9921_Fig1_HTML.jpg
Fig. 1

Horizontally and vertically positioned epicotyl segments on organogenic medium. A apical zone, B basal zone, M middle zone

Effect of the cut surface contact with the medium

The effect of the cut surface of the explant being in contact with the organogenic medium on adventitious shoot formation was also evaluated. Five portions (1-cm long) of each epicotyl, as described in the previous experiment, were longitudinally split. In each petri dish, one-half of each portion was placed in contact with the medium, placing the cut surface down (CSD), while the other half of each portion was placed with the cut surface facing up (CSU). The composition of the medium, culture conditions, and number of petri dishes (10) were as reported above.

Effect of cytokinins and organogenic explant position

In this experiment, each epicotyl was cut into twenty 2-mm-long sections and cultured on medium [MS mineral salts and vitamin mixture, 30 g l−1 sucrose, 8 g l−1 plant agar (Micropoli), 0.1 mg l−1 NAA] in the same petri dish. As the aim of this experiment was to evaluate the influence of two cytokinins [BA and thidiazuron (TDZ)] on organogenesis, each of these cytokinins was supplemented to the medium at a concentration of 1 mg l−1 (same molarity). Ten petri dishes were prepared for each test medium. Each 2-mm section was numbered according to its distance from cotyledons to verify the possible effect of the position along the epicotyl.

Statistical analysis: single-factor experiments

To estimate the influence of each treatment (HP vs. VP; CSU vs. CSD; medium with BA vs. medium with TDZ), we performed the t test on the number of adventitious buds per segment and the number and length of shoots produced.

Encapsulation

Plant material, encapsulation procedure, and culture conditions

Proliferating shoots obtained from organogenesis were subcultured monthly in 500-ml glass jars containing 100 ml of medium (MS salts and vitamin mixture, supplemented with 30 g l−1 sucrose, 1 mg l−1 NAA, 10 mg l−1 BA, and 8 g l−1 agar). The pH was adjusted to 5.8 prior to autoclaving at 121°C for 20 min. Uninodal microcuttings, 3–4 mm long, without leaves and with two axillary buds were excised from the in vitro-proliferated shoots and subjected to an encapsulation procedure, which consisted of immersing the microcuttings in a sodium alginate (alginic acid sodium salt, medium viscosity; Sigma code A-2033; Sigma-Aldrich, St. Louis, MO) solution (2.5%, w/v) enriched with artificial endosperm (half-strength proliferation medium supplemented with 50 g l−1 sucrose). The alginate-coated propagules were then complexed for 35 min in a mixture of CaCl2 (1.1%, w/v) containing the artificial endosperm components. The hardened alginate capsules were washed three times, 15 min each time, in the sterile artificial endosperm solution (Micheli and Standardi 2005) and then sown in Magenta GA7 vessels (7 × 7 × 7 cm) containing 50 ml of the above-mentioned proliferation medium, without growth regulators. Five synthetic seeds were placed in each vessel, and six vessels were prepared for each treatment. The cultures were incubated in a growth chamber at 23 ± 1°C under a 16/8-h (light/dark) and a photon flux density of 40 μmol m−2 s−1. Data, recorded for all experiments at 45 days post-sowing, were collected on the following parameters: viability (explants with a green appearance, without necrosis or yellowing), regrowth (encapsulated microcuttings that produced shoots >4 mm), conversion (simultaneous extrusion of shoots and roots 4 mm long from encapsulated microcuttings), and callus frequency (monitored by the number of shoots with visible callus at the basal portion).

Effect of encapsulation, storage at low temperature, and sowing support

In order to evaluate the effects of encapsulation (factor E), a short period of storage at low temperature (factor C), and type of sowing support (factor S), we sowed 30 naked microcuttings and 30 encapsulated microcutting, either directly after encapsulation or after a storage time of 30 days at 4°C in the dark, in petri dishes containing 50 ml of artificial endosperm. All microcuttings (naked and encapsulated, stored at 4°C or not) were ultimately sown in Magenta GA7 vessels containing either 50 ml of agar-solidified medium or filter paper wetted with 10 ml of the same liquid sowing medium.

Effect of cold storage and rooting inductive treatment

In order to examine the influence of inductive rooting treatments on the viability, regrowth, and conversion of the synthetic seeds, microcuttings were excised directly at the end of the subculture or after an additional 7 days in the culture vessels at 4°C as a rooting inductive pre-treatment. In both cases, microcuttings were subjected to the rooting inductive treatment before or after the encapsulation, as described by Micheli et al. (2007). In brief, the encapsulation procedure consisted of immersing the microcuttings for 3 days, in the dark and at 23 ± 1°C, in 50-ml glass jars (10 microcuttings per jar) containing 15 ml of rooting solution (5 mg l−1 IBA and 15 g l−1 sucrose, pH 5.5). The synthetic seeds were then sown on the agar-solidified proliferation medium described above, maintaining the correct polarity of the microcuttings.

Statistical analysis: multifactor experiments

Factorial experiments were designed to quantify interactions and to identify the optimal combination of factors with the aim of improving the response of explants. With respect to the effects of encapsulation, storage, and sowing support, three factors were considered: (1) encapsulation, factor E (naked vs. encapsulated explants); (2) storage at 4°C, factor C (0 vs. 30 days); (3) sowing support, factor S (agar vs. paper). Four parameters were measured: viability and regrowth of encapsulated microcuttings, and number and length of new shoots; each parameter was analyzed by three-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Two factors were considered in the experiment on the effect of cold storage and rooting inductive treatments: (1) induction, factor I (pre-encapsulation vs. post-encapsulation) and (2) storage at 4°C, factor C (0 vs. 7 days). For this experiment, six parameters were measured (viability, regrowth, conversion, number and length of new shoots, and number and length of roots). Each parameter was analyzed by two-way ANOVA followed by Tukey’s test, with the exception of the data on root production. The very low rooting rate obtained in some cases prevented a complete ANOVA since the variance was not uniform.

Results

Organogenesis

Effect of explant orientation

After 30 days of culture, direct organogenesis was observed under all culture conditions. Adventitious buds were observed at both ends of explants (epicotyl segments) cultured in the HP (Fig. 2 top), while in those cultured in the VP, only the part of the explant dipped in the medium showed organogenesis (data not shown). An asynchronous development of buds and shoots was observed and, after 2 months of culture, 40% of apical and 64% of basal portions produced adventitious buds and shoots in explants cultured in the HP. The basal part appeared to be the most responsive to organogenesis, with the highest number of adventitious buds and shoots observed at the basal end of each epicotyl portion (HP 4.4, VP 1.8). Explants cultured in the HP yielded better results than those in the VP in terms of frequency of explants producing buds (92 vs. 84%), bud number per explant (7.3 vs. 3.4), and shoots per explant (2.8 vs. 1.5) (Table 1). Moreover, the shoots obtained from HP explants (Fig. 2 down) were significantly longer than those proliferated from VP explants (Table 1). For both HP and VP explants, no statistical differences were observed in morphogenic ability in terms of distance from the cotyledonary node (data not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs11240-011-9921-y/MediaObjects/11240_2011_9921_Fig2_HTML.jpg
Fig. 2

Adventitious buds (top) and shoots (bottom) produced by epicotyl segments cultured in the horizontal position

Table 1

Effects of the different factors tested on organogenesis from epicotyl segments of Carrizo citrange after 2 months of culture

Experiment

Treatmenta

Adventitious buds (n/segment)

Adventitious shoots

Number (n/segment)

Length (mm)

Effect of explant orientation

HP

7.3 a

2.8 a

9 a

VP

3.4 b

1.5 b

7 b

Effect of cut surface in contact with the medium

CSD

5.9 a

1.5 a

8 a

CSU

3.3 b

1.0 b

7 b

Effect of cytokinins

BA

1.4 a

0.2

5

TDZ

0.8 b

0.1

5

Within each experiment and column, values followed by different letters are statistically different at p ≤ 0.05 according to t test

aHP, Horizontal position; VP, vertical position; CSD, cut surface down, in contact with culture medium; CSU, cut surface facing up; BA, 6-benzyladenine; TDZ, thidiazuron

Effect of the cut surface contact with the medium

Adventitious buds appeared after 1 month of culture, with almost 30% of these subsequently developing into shoots. After 2 months of culture, explants with the CSD in contact with the medium produced a higher average number of adventitious buds than segments with the CSU (Table 1). CSD explants also produced a significantly higher number of buds (2.5) at the middle part of the epicotyl than at the basal and apical ends (1.8 and 1.7, respectively); in contrast, no differences among epicotyl portions were observed for CSU explants. As observed in the first experiment, no significant difference in shoot number and length was recorded with respect to the distance of the different explants from the cotyledonary node. About 2% of both CSD and CSU explants showed embryo formation, and in few cases indirect organogenesis was recorded.

Effect of cytokinins and organogenic explant position

Adventitious bud formation was slower in this experiment, compared with the previous ones. Moreover, only a low percentage of buds (nearly 13%) developed into shoots (data not shown). After 2 months of culture, however, epicotyl portions cultured on medium with BA produced a significantly higher number of buds (1.4) than those on medium with TDZ (0.8) (Table 1), regardless of their position relative to the cotyledons.

Encapsulation

Effect of encapsulation, storage at low temperature, and sowing support

At 45 days post-sowing, microcutting viability was very high, varying from 94% in naked microcuttings to 100% in encapsulated ones. The statistical analysis revealed an interaction between encapsulation (factor E) and storage (factor C) that could have determined a reduction in the viability of both naked and encapsulated microcuttings (Table 2). Regrowth (Fig. 3) varied from 28 to 56%, and no interactions were detected among the factors tested (encapsulation, storage, and sowing support). With respect to encapsulation (factor E), regrowth was statistically higher for the encapsulated microcuttings than for the naked ones. For the storage (factor C), microcuttings maintained for 30 days at 4°C showed a statistically higher regrowth value than the unstored ones (Table 2). There was no effect of sowing support (factor C, agar-solidified medium vs. wetted filter paper) on the parameters evaluated. The three factors compared (encapsulation, storage, and sowing substrates) had no significantly different effect on the number and length of shoots. From each microcutting, 1.1–1.2 shoots were obtained and, after 45 days, their length ranged from 4.5 to 4.9 mm. The two sowing supports compared (factor S) did not influence the parameters considered (Table 2).
Table 2

Effects of encapsulation, storage, and sowing support on the viability and regrowth of Carrizo citrange synthetic seeds after 45 days of culture

Factors/treatment

Viability (%)

Regrowth (%)

Shoots

Number (n)

Length (mm)

Encapsulation (E)

  Naked

94

33

1.2

4.5

  Encapsulated

100

51

1.1

4.9

Storage at 4°C (C)

  0 days

95

28

1.2

4.8

  30 days

99

56

1.1

4.7

Sowing support (S)

  Agar

97

43

1.2

4.8

  Paper

97

41

1.1

4.6

Statistical analysis of factorsa

  E

0.004

0.002

0.275

0.435

  C

0.033

<0.001

0.523

0.928

  S

0.879

0.533

0.092

0.758

  E × C

0.033

0.244

0.454

0.628

  C × S

0.541

0.849

0.523

0.899

  E × S

0.879

0.724

0.326

0.614

  E × C × S

0.541

0.244

0.517

0.682

aThree-way analysis of variance (ANOVA) (E, C, S), followed by Tukey’s test; p ≤ 0.05

https://static-content.springer.com/image/art%3A10.1007%2Fs11240-011-9921-y/MediaObjects/11240_2011_9921_Fig3_HTML.jpg
Fig. 3

Regrowth of encapsulated microcuttings

Effect of cold storage and rooting inductive treatment

In all of the conditions tested, the viability and regrowth of the microcuttings were 100%, and there were no statistical differences for the considered factors (cold storage and time of rooting inductive treatment; Table 3). There was an interaction on new shoot length, with the longest shoots obtained from microcuttings treated for rooting after the encapsulation and stored for 7 days at 4°C. The conversion was very low, and in two combinations it was equal to zero, thereby preventing any statistical analysis. The best performance of rooting (17%) was recorded in microcuttings stored for 7 days at 4°C and treated with the rooting solution after encapsulation (Table 4, Fig. 4).
Table 3

Effects of cold storage and rooting inductive treatments on viability, regrowth, number and length of shoots in Carrizo citrange synthetic seeds after 45 days of in vitro culture

Factors/treatment

Viability (%)

Regrowth (%)

Shoots

Number (n)

Length (mm)

Induction (I)

  Pre- encapsulation

100

100

1.49

6.6

  Post- encapsulation

100

100

1.53

7.8

Storage at 4°C (C)

  0 days

100

100

1.38

5.8

  7 days

100

100

1.65

8.8

Statistical analysis of factorsa

  I

0.844

0.220

  C

0.054

0.009

  I × C

0.589

0.024

aTwo-way ANOVA (I, C), followed by Tukey’s test p ≤ 0.05

Table 4

Effects of cold storage and time of the rooting inductive treatments on conversion and root production in synthetic seeds of Carrizo citrange after 45 days of in vitro culture

Treatments

Conversion (%)

Roots

Time of rooting induction

Cold (4°C) storage (days)

Number/explant (n)

Length (mm)

Pre-encapsulation

0

3

2

16

7

0

0

0

Post- encapsulation

0

0

0

0

7

17

2

20

https://static-content.springer.com/image/art%3A10.1007%2Fs11240-011-9921-y/MediaObjects/11240_2011_9921_Fig4_HTML.jpg
Fig. 4

Conversion from a microcutting submitted to rooting induction treatment after encapsulation and storage for 7 days at 4°C

Discussion

In vitro adventitious shoot regeneration of several Citrus species has been performed using different explants: shoot tips, stem and epicotyl segments (Kitto and Young 1981; Burger and Hackett 1986; Sim et al. 1989; Goh et al. 1995; Garcia-Luis et al. 1999; Bordòn et al. 2000; Moreira-Dias et al. 2000, 2001; Molina et al. 2007), roots and leaf sections (Sauton et al. 1982; Sim et al. 1989; Burger and Hackett 1986; Bhat et al. 1992; Gill et al. 1994; Goh et al. 1995), transverse thin cell layer (TCL) from stem internodes (Van Le et al. 1999), and reproductive organs (Germanà et al. 1994; Carimi et al. 1998). Published reports on organogenesis from epicotyls are contradictory in terms of the influence of the epicotyl region and of explant orientation (horizontal vs. vertical) on the morphogenic response of seedling explants (Costa et al. 2004; Garcia-Luis et al. 2006). In a number of studies on Citrus macrophylla and citranges Troyer and Carrizo, the best results were obtained from the basal part of epicotyl segments (Germanà et al. unpublished; Germanà et al. 2008; Moreira-Dias et al. 2000) and in the region nearest to the cotyledons (Burger and Hackett 1986; Sim et al. 1989; Garcia-Luis et al. 1999; Moreira-Dias et al. 2001), whereas in other studies, opposite results were obtained (Goh et al. 1995; Garcia-Luis et al. 1999, 2006; Bordòn et al. 2000; Costa et al. 2004). Specifically, the influence of explant orientation, polarity, and contact surface with the medium has been studied with the aim to increase the efficiency of in vitro regeneration in Troyer citrange (Garcia-Luis et al. 1999, 2006), Carrizo citrange (Cervera et al. 1998; Kayim et al. 2004), and Citrus macrophylla (Germanà et al. 2008). In our study, the production of adventitious buds from epicotyl segments was lower than those reported by Bordòn et al. (2000) in Troyer citrange and similar to those mentioned by Germanà et al. (2008) in Citrus macrophylla. Moreover, organogenesis was not affected by the segment position along the epicotyl.

As reported by several authors working on different Citrus spp. (Garcia-Luis et al. 1999; Bordòn et al. 2000; Moreira-Dias et al. 2000, 2001; Al-Bahrany 2002; Hassanein and Azooz 2003; Costa et al. 2004; Germanà et al. 2008) and in our study on Carrizo citrange, BA was the best cytokinin for inducing organogenesis. In this genotype, regeneration has been reported in epicotyl cuttings cultured in different positions (vertically or horizontally) and cut in different ways (longitudinally, transversally, and obliquely) (Moore et al. 1992; Cervera et al. 1998; Yu et al. 2002; Bespalhok Filho et al. 2003; Kayim et al. 2004; Peña et al. 2004; Duan et al. 2007). The results reported in our study are not in agreement with those ones reported by Duan et al. (2007) and Yu et al. (2002); specifically, they found that in terms of cut modes, epicotyl portions cut longitudinally had a better regeneration response than those cut transversally. In general, regeneration from the epicotyl portions was mainly direct due to the very low capability of citrange for callus production, as previously reported by Bordòn et al. (2000).

We report here the results of the first trials carried out on the encapsulation of Carrizo citrange microcuttings. These results showed that there is an interaction effect between encapsulation (factor E) and storage at 4°C (factor C) on the viability of Carrizo citrange microcuttings, thereby confirming previous results in different Citrus genotypes using encapsulated somatic embryos (Germanà et al. 1999). The positive effect of encapsulation on the regrowth of microcuttings that we observed is in agreement with results obtained in different woody species (Gardi et al. 1999; Micheli and Standardi 2005; Micheli et al. 2007). Moreover, the effectiveness of the cold temperature on the short-term storage of the synthetic seeds confirms previous results obtained in olive (Micheli et al. 2007), guava (Rai et al. 2008), aspen (Tsvetkov et al. 2006), and other woody species (Kinoshita and Saito 1990; Tsvetkov and Hausman 2005). Based on findings reported in other species (Sarkar and Naik 1998; Alvarez et al. 2002; Lucaccioni et al. 2005; Micheli et al. 2007), we conclude that the rooting inductive treatment tested in our study was able to achieve synthetic seed conversion, confirming the suitability of the adopted procedure, although further investigations are required to significantly improve the conversion rates of the Carrizo citrange synthetic seeds.

Conclusions

A biotechnological approach to propagate Carrizo citrange represents a valuable tool to quickly obtain a high number of virus-free plants. The results of our study on calcium-alginate encapsulation of vitro-derived microcuttings of Carrizo citrange confirmed the practical applicability of this technology for propagation and short-term storage of this important Citrus rootstock, although further studies are required to improve the conversion rates of the synthetic seeds. With the aim of optimizing propagation efficiency (in term of adventitious bud and shoot production obtained through in vitro organogenesis from epicotyl segments), we evaluated different aspects of this morphogenic process and found that the best combination to achieve a good response of direct organogenesis in Carrizo citrange was that of 1-cm long epicotyl segments cultured in the horizontal position. We also confirmed BA as a very effective cytokinin in Citrus organogenesis.

Acknowledgments

This work was supported by the project “CITRUS SYNSEED”, financed by the Assessorato Agricoltura e Foreste Regione Sicilia, XI Servizio Regionale allo Sviluppo.

Copyright information

© Springer Science+Business Media B.V. 2011