Journal of Plant Physiology 168 (2011) 20282034

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Journal of Plant Physiology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . d e / j p l p h

Water relations in culture media influence maturation of avocado somatic embryosBeln Mrquez-Martn a , Rafael Sesmero a , Miguel A. Quesada b ,Fernando Pliego-Alfaro c,

, Carolina Snchez-Romero b

a IFAPA, Centro de Churriana, Cortijo de la Cruz s/n, 29140 Churriana, Mlaga, Spainb Departamento de Biologa Vegetal, Universidad de Mlaga, Campus de Teatinos s/n, 29071 Mlaga, Spainc Instituto de Hortofruticultura Subtropical y Mediterrnea La Mayora (IHSM-UMA-CSIC), Departamento de Biologa Vegetal, Facultad de Ciencias, Universidad de Mlaga, Campus de Teatinos s/n, E-29071 Mlaga, Spain

a r t i c l e i n f o

Article history:Received 23 March 2011Received in revised form 6 June 2011 Accepted 8 June 2011

Keywords:Gel strengthOsmotic potentialPersea americanaSomatic embryogenesisWater potential

a b s t r a c t

Application of transformation and other biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos capable of germination at an acceptable rate. In this

work, we evaluated the effect of different compounds affecting medium water relations on maturation of avocado somatic embryos. Culture media were characterized with respect to gel strength, water potential and osmotic potential. Improved production of mature somatic embryos was achieved with gelling agent concentrations higher than those considered standard. The osmotic agents such as sorbitol and PEG did not have positive effects on embryo maturation. The number of w-o mature somatic embryos per culture was positively correlated with medium gel strength. Gel strength was significantly affected by gelling agent type as well as by gelling agent and PEG concentration. Medium water potential was influenced by sorbitol concentration; incorporation of PEG to a culture medium did not affect medium water potential. The highest maturation results were achieved on a

medium gelled with 10 g l1 agar. Moreover, these somatic embryos had improved germination rates. These results corroborate the role of water restriction as a key factor controlling maturation of somatic embryos.

2011 Elsevier GmbH. All rights reserved.

Introduction

Avocado (Persea americana Mill.) is cultivated in most tropical and subtropical regions of the world for its fruits, which are appreci-ated for their high oil content and constitute a well balanced supply of proteins,

carbohydrates, minerals and vitamins (Ray,

2002). Avo-cado production is limited by several fungal diseases, such as the avocado root rot and the white root rot, Verticillium wilt, anthrac-nose and Cercospora spot.

Conventional breeding in avocado is hampered by its

long juve-nile period, and biotechnological approaches could therefore be useful for this species. An efficient regeneration protocol from sin-gle cells is a requisite for application of

biotechnological methods. In avocado, somatic embryogenesis (SE) is the method utilized for adventitious plant regeneration (Litz et al., 2005). Embryogenic

Abbreviations: B5m, maturation medium consisting of the MS formulation with the macronutrients of Gamborg et al. (1968); MSP medium, MS medium (Murashige and Skoog, 1962) supplemented with 0.1 mg l1 picloram; PEG, polyethylene glycol; s.e., somatic embryo; SE, somatic embryogenesis; w-o, white-opaque.

1 Corresponding author. Tel.: +34 952131947; fax: +34 952131944. E-mail address: ferpliego@uma.es (F. Pliego-Alfaro).

0176-1617/$ see front matter 2011 Elsevier

GmbH. All rights reserved. doi: 10.1016/j.jplph.2011.06.008

cultures derived from immature avocado zygotic embryos can be routinely obtained (Pliego-

Alfaro and Murashige, 1988; Witjaksono and Litz, 1999a). However, the low conversion rate of somatic embryos into plants is the main bottleneck in plant regeneration. As a consequence, although the introduction of specific genes and widening the genetic base following generation of somaclonal vari-ants are now feasible approaches for avocado breeding (Litz et al., 2005), the low conversion rate of the somatic embryos into plants is limiting the applicability of these techniques.

Embryo maturation is a key factor affecting plant conversion in somatic

embryogenesis systems (Ammirato, 1987). During the maturation stage, the somatic embryos undergo morphological and biochemical changes, such as storage product deposition, which are essential for subsequent plant development. Generally, accumu-lation of storage products causes the change from translucent to white-opaque color embryos (Cailloux et al., 1996). White-opaque appearance has been used as

maturity criterion in different species (Cailloux et al., 1996; Garin et al., 2000; Nrgaard, 1997), including avocado (Pern-Quesada et al., 2004; Witjaksono and Litz, 1999b).

Water relations between the embryo and its environment play regulatory role in embryo development, particularly during mat-uration (Adams and Rinne, 1980; Bradford, 1994). In conifer SE, it has been proposed that reduced water availability could stimulate

B. Mrquez-Martn et al. / Journal of Plant Physiology 168 (2011) 20282034 2029

a shift in the developmental program of the culture, from prolif-eration of cells and early somatic embryos to the

production of embryos at advanced stages ( Klimaszewska et al., 2000).

Water restriction has been a widely used strategy for improv-ing somatic embryo maturation both in gymnosperms and angiosperms. Water availability manipulation has been addressed through different means: addition of low molecular weight com-pounds (plasmolysing agents) such as inorganic salts, aminoacids and sugars; use of high molecular weight compounds (non-plasmolysing agents) such as polyethylene glycols (PEG) and dextrans (Stasolla et al., 2002) and drying process (Etienne et al., 1993). Although gelling agents are mainly used to create semisolid media, they can also influence the availability of water to tissues (Ziv, 1991).

Despite the wide application of various gelling agents, a few studies have been carried out to understand the effect on water availability of supplementing the culture medium with

compounds of different type ( Klimaszewska and Smith, 1997; Klimaszewska et al., 2000).

The aim of this study was to optimize maturation conditions of avocado somatic embryos by manipulating water relations through the addition of different compounds to a culture medium. In paral-lel, the culture medium physicochemical parameters such as water potential, osmotic potential and gel strength were determined with the objective of explaining the behavior of embryogenic tissues cultured under the different maturation conditions.

Materials and methods

Initiation and maintenance of embryogenic cultures

Avocado (Persea americana Mill.) embryogenic cultures were initiated from immature zygotic embryos, cultivar Duke-7, accord-ing to Pliego-Alfaro and Murashige (1988). One to two mm zygotic embryos were cultured on MS medium (Murashige and Skoog, 1962) supplemented with 0.1 mg l1

picloram (MSP) and 6 g l1 agar (A-1296, SigmaAldrich). Cultures were incubated in darkness at 25 1 C.

Maintenance of established embryogenic cultures was carried out on the same culture conditions used for initiation. Subcultures were carried out at monthly intervals.

Somatic embryo maturation

For maturation, embryogenic suspensions were initiated from cultures in maintenance conditions. Following the protocol of Witjaksono and Litz (1999a), 0.4 g of friable callus was inoculated in 40 ml liquid MSP medium in 100 ml Erlenmeyer flask. Suspen-sions were incubated on an orbital shaker (120 rpm) under low light intensity (5 mol m2 s1 ), provided by Grolux lamps (Syl-vania, Erlangen, Germany), and a 16 h photoperiod at 25 1 C. After 9 days they were sieved sequentially through 2 and 1 mm pore mesh. One hundred mg of the fraction that passed the 2 mm pore mesh and was retained in the 1 mm pore mesh were inocu-lated in 25 mm 150 mm test tubes containing 25 ml of maturation medium.

Maturation medium (B5m) consisted of the MS formulation with the macronutrients of Gamborg et al. (1968) (Pern-Quesada et al., 2004). Four embryogenic lines were

used for maturation experiments.

Maturation media were prepared by adding the appropriate components and adjusting the pH to 5.74, prior to adding the gelling agent. Media were heated in

an autoclave (121 C and 0.1 MPa) for 7 min to melt the gelling agent. Twenty-five ml of the molten medium were dispensed in 25 mm 150 mm test tubes which were

covered with polypropylene closures Kap-Uts. Media were steril-ized by autoclaving for 15 min at 121 C and 0.1 MPa.

Maturation experiments were carried out during three sub-cultures with successive transfers to fresh medium of the same composition at 57 week intervals. Incubation was in darkness at 25 1 C.

Twenty cultures (test tubes) were used per maturation treat-ment. Fresh weight increase and number of w-o somatic embryos produced per culture were recorded at the end of each subculture. As in previous studies (Cailloux et al., 1996; Pern-Quesada et al., 2004; Witjaksono and Litz, 1999b), white-opaque appearance of somatic embryos was used as a criterion of maturity.

Effect of gelling agent type and concentration

In this experiment, the effect of three gelling agents at differ-ent concentrations was studied on maturation of avocado somatic embryos. B5m medium supplemented with 30 g l1 sucrose was solidified with each of the following gelling agents: agar (A-1296, SigmaAldrich) at 6, 8, 10 and 12 g l1 , agargel (A-3301, SigmaAldrich) at 3.5, 5, 7.5 and 10 g l1 and gellan gum (Gelrite) (G-1910, SigmaAldrich) at 1.7, 3.4, 5.1 and 6.8 g l1 .

Effect of osmotic agent type and concentration

The effects of two osmotic agents of different nature (penetrat-ing and non-penetrating) were studied in the second experiment. Maturation medium containing 30 g l1 sucrose was supplemented with different concentrations of sorbitol (S-6021, SigmaAldrich) (2.5, 5 and 7.5% (w/v)) or polyethylene glycol (PEG), Mr 8000, (P-2139, SigmaAldrich) (2.5, 5 and 7.5% (w/v)). All media were gelled with 6 g l1 agar.

Germination of somatic embryos

White-opaque somatic embryos 4 mm developed in B5m gelled with 10 g l1 agar were cultured on M1 medium (Skene and Barlass, 1983) gelled with 1.7 g l1 gellan gum after partial excision of cotyledons, e.g., longitudinal cuts with removal of about one third of the cotyledons. Cultures were incubated under a 16 h light pho-toperiod provided by Grolux lamps (Sylvania, Erlangen, Germany) (40 mol m2 s1 ) at 25 1 C. Germination was carried out during

three subcultures of 5 weeks each.The germination experiment was repeated twice. Within

each experiment, 50 somatic embryos were used. Embryos were consid-ered germinated when shoot and/or root elongation was 2 mm.

Medium gel strength

Media were prepared as previously indicated and

dispensed in 25 mm 150 mm culture tubes until the liquid almost reached the top of the tube. After 24 h equilibration at 25 1 C, and according to

Klimaszewska and Smith (1997), the firmness of the medium gel was measured using the XT-plus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK) with a 4 mm-diameter stainless steel cylinder probe. The tests were always performed in the center of

the tube, in order to avoid edge effects. The trigger force was fixed at 3 g, the test distance depth at 2 mm, pre-test and test speed at 1 mm s1 and post-test speed at 5 mm s1 . Data of gel strength were obtained using the Texture Analyzer software (TEE32, v.3.0.1.0) as the maximum peak force. For each maturation medium, at least three replicates from different tubes were measured.

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Fig. 1. Effect of gelling agent type and concentration on maturation of avocado somatic embryos. Data represent the means of three subcultures. Different letters indicate significant differences at P = 0.05.

Water and osmotic potential of the medium

Media were prepared as indicated and 20 ml were dispensed in 90 mm Petri dishes. Petri dishes were sealed with parafilm and stored for 24 h in a culture chamber at 25 1 C. After equilibration, a cylinder was sampled from the central part of each Petri dish using an 8 mm-diameter cork bearer. The width of the slices was virtually constant, since in all cases exactly the same volume of medium was poured into the Petri dishes. Water potential was measured with a psychrometer (Beruto et al., 1999). Gel slices were cautiously put in a hermetic C-52 Sample Chamber connected to a HR33 T dew point microvoltmeter (Wescor Inc., Logan, USA). Samples were allowed to equilibrate for 2 min and the water potential was measured 30 s after the cooling stage.

For each maturation medium, at least three measurements were

carried out in different Petri dishes.

Osmotic potential was also determined using a psychrometer by adding 50 L of the medium without the gelling agent to 8 mm paper discs. As previously indicated, they were equilibrated for 2 min and then measured 30 s after the cooling stage. Three mea-surements of each sample were taken

Statistical analysis

In maturation experiments, the

data represent the average of three subcultures. Data were analyzed by ANOVA followed by com-parison of means using the least significant difference test (LSD). The degree of correlation between variables was estimated by using the Pearsons coefficient. The significance level used was 0.05 in all cases.

Results

Effect of gelling agent type and concentration

The gelling agent type had a significant influence on callus pro-liferation rate with values ranging from 0.67 g in agargel to 0.80 g in agar-gelled media (data not shown).

Number of w-o somatic embryos per culture was significantly affected by the gelling agent type (P = 0.0000) and concentration (P = 0.0000)

attaining the highest numbers on 8, 10 and 12 g l1 agar

and 7.5 g l1 agargel (Fig. 1). In general, better results were obtained with agar. Agargel, a blend of agar and gellan gum, gave rise to intermediate values, although more similar to those obtained with agar than with gellan gum. Gelling agent concentration was a crit-ical factor in the production of w-o somatic embryos. In general, concentrations higher than those considered standard enhanced maturation of somatic embryos in the three gelling agents tested. Nevertheless, somatic embryos obtained in a medium solidified with 12 g l1 agar were smaller than those obtained in slightly lower concentrations.

Effect of osmotic agent type and concentration

Osmotic agent type, concentration and the interaction type concentration, significantly affected culture growth during the maturation phase (data not shown). A greater fresh weight increase compared with the control was observed in a medium with 5% PEG (1.10 g vs. 0.96 g in the control). When sorbitol was added to culture media, significantly lower growth rates were obtained at all concentrations tested indicating a negative cor-relation between sorbitol concentration and the culture growth. Fresh weight was

only up to 0.32 g in the maturation medium supplemented with 7.5% sorbitol.

Osmotic agents in the maturation medium significantly influ-enced culture appearance (Fig. 2). PEG significantly increased the production of cotyledonary embryos. The higher the concentration of PEG in the maturation medium, the larger the somatic embryos developed. The somatic embryos were characterized by a beige-to-yellow color and hyperhidric appearance. White-opaque somatic embryos were rarely observed in PEG-supplemented media. Sor-bitol caused a deterioration of cultures. Calli became dark in color, losing the embryogenic characteristics even at the lowest con-centration (2.5% sorbitol). However, w-o somatic embryos were produced but embryo quality was significantly affected. Our results revealed a significant effect of the osmotic agent type (P = 0.0003) and concentration (P = 0.0000) on the number of w-o somatic embryos per culture (Fig. 3). There was also an interaction of osmotic agent type with osmotic agent concentration, which influ-enced this parameter (P = 0.0003).

In accordance with the previous results, B5m gelled with 10 g l1 agar was selected as the improved medium for maturation of avo-

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