Trees

, Volume 23, Issue 5, pp 1019–1031 | Cite as

Large-scale field acclimatization of Olea maderensis micropropagated plants: morphological and physiological survey

  • Gina Brito
  • Armando Costa
  • Celeste Coelho
  • Conceição Santos
Original Paper

Abstract

Micropropagation allows large-scale plant multiplication and germplasm preservation, representing an added value in forest breeding strategies to combat desertification and/or protect endangered species. We developed a large-scale micropropagation protocol of Olea maderensis (a native endangered wild olive of Madeira Archipelago) using OMG medium (rich in Fe, Mg and Mn) supplemented with zeatin for elongation and with NAA for rooting. We now describe the performance of micropropagated plants during five-period field acclimatization: (1) in vitro, (2) growth-cabinet, (3) greenhouse, (4) open-greenhouse, and (5) field mountain in Porto Santo Island. One hundred OG4 plants were acclimatized, showing >95% surviving rates. During acclimatization, several physiological parameters were evaluated; water content remained higher in in vitro/greenhouse conditions, decreased in field leaves. Soluble protein contents decreased during the first acclimatization periods increasing thereafter. Membrane permeability slightly increased during the field acclimatization. Chlorophylls content increased in in vitro leaves, while during acclimatization, mostly chl b decreased, increasing chl a/chl b ratio. F0 decreased in first acclimatization periods, increasing thereafter, while the other parameters (Fv; Fm; Fv/Fm) decreased. Nutrient contents decreased in plants transferred to poor field soil conditions, reaching values similar to mother plant leaves. Overall, with the exception of PSII fluorescence, field acclimatized plants had similar values to mother plants, showing a good adjustment to stressful field conditions. This protocol is being used in large-scale micropropagation within a reforestation program, and is an example of R&D technologies with immediate application on protection of endangered ecosystems.

Keywords

Forestation Nutrients Olive micropropagation OMG medium Osmolality Photosynthesis 

Abbreviations

Elong

Elongation stage

Greenh

Greenhouse

In vitro Ac

In vitro acclimatization

M plant

Mother plant

NAA

α-Naphthalene acetic acid

OM

Olive medium

OMG

Modified olive medium

Op greenh

Open greenhouse

RH

Relative humidity

SE

Standard error

WH

Without hormones

Introduction

Olive is one of the most important fruit trees cultivated in the Mediterranean basin with a wide range of adaptability and comprises several economically important cultivars and wild olive genotypes. Olea maderensis (Lowe) Rivas Mart. and Del Arco (known as wild olive) (Brito et al. 2008) is an endemic and endangered tree native to the Madeira Archipelago and is a particularly important component of the arboreal climatic community (micro-forest) named Zambujal that occurs in dry infra-Mediterranean climate (Brito and Santos 2009).

In Porto Santo Island, a serious process of desertification and land degradation is occurring, with only a few isolated individuals of O. maderensis being found, and generally in inaccessible rifts, where they survive under adverse environmental conditions (Brito and Santos 2009). Propagation and preservation of this species is very important owing to its endangered status in the Archipelago. However, conventional propagation of this species is hampered by the poor germination rates and low seed production and by the poor ability of plant production by macrocuttings. Therefore, the optimization of micropropagation strategies (providing production of genetically identical plants of conventional propagation recalcitrant species) will allow to use this endangered species on reforestation programs in Porto Santo Island (Brito and Santos 2009).

In the last decades, the intense research activity developed in micropropagation of Olea genus was almost restricted to commercial cultivars (e.g., Briccoli Bati et al. 2006; Peixe et al. 2007; Rugini 1984; Zacchini and De Agazio 2004; Zuccherelli and Zuccherelli 2002). However, there is not enough knowledge for its use in a mass-scale nursery production (Peixe et al. 2007; Zuccherelli and Zuccherelli 2002). Furthermore, little is known about the micropropagation of other Oleaceae species/genotypes with environmental relevance (Brito et al. 2003, 2007b; Lopes et al. 2009; Lucchesini and Mensuali-Sodi 2004; Santos et al. 2003). We recently developed an efficient protocol for routinely micropropagate O. maderensis plants (Brito and Santos 2009).

The ultimate success of in vitro propagation in a reforestation program depends on a reliable acclimatization protocol, ensuring low cost and high survival rates (Hazarika 2006). In vitro protocols provide minimal stress and optimum conditions for shoot/plant multiplication (Hazarika 2006). As a consequence of these special conditions (e.g., high air humidity, low irradiance, low CO2 during photoperiod, high levels of sugars as carbon source and growth regulators), in vitro grown plantlets usually exhibit abnormal morphology, anatomy and/or physiology (Hazarika 2006; Pospíšilová et al. 1999; Premkumar et al. 2001). Under these conditions, in vitro plantlets can develop specific features (e.g., non functional roots and/or stomata) that are inconsistent with the development under greenhouse or field conditions. Also the mixo-heterotrophic mode of nutrition and poor mechanism to control water loss render micropropagated plants vulnerable to the transplantation shocks when directly placed in a greenhouse or field.

Understanding the physiological characteristics of micropropagated plants and the changes they undergo during the hardening process should facilitate the development of efficient transplantation protocols and will help to make decisions on, if necessary, adjusting environmental conditions (e.g., irrigation, soil fertilization). (Hazarika 2006). For example, water/osmotic stress is often the cause of micropropagated plants mortality and its monitorization is particularly important when acclimatization occurs in a degraded land as is the case (Brito et al. 2003). Also as the Island is exposed to high insolation levels (~2,242 h year−1), surveying photosynthesis-related parameters will give information on photooxidation risk and will allow to prevent it. Finally, nutrient analysis is a crucial approach when dealing with plant acclimatization to poor soils as it is the case of Porto Santo. Moreover, the physiological behavior of micropropagated olive plants in the field is still scarcely known, compared to plants from grafted or own-rooted cuttings (Briccoli Bati et al. 2006).

In this work we present a successful field acclimatization protocol of Olea maderensis, based on the controlled exposure to low relative humidity and high light intensity. In order to evaluate field performance of the micropropagated plants, physiological parameters were assayed on shoots/plants during in vitro and after transfer to ex vitro conditions. Therefore, we followed surviving and elongation rates and other physiological features (chlorophyll fluorescence, chlorophyll content, membrane integrity, water content, osmolality, soluble protein and mineral composition) in O. maderensis micropropagated plants from in vitro to field conditions.

Materials and methods

Micropropagation studies

Plant material, in vitro establishment and rooting

Cuttings from field five-grown of Olea maderensis adult trees (>30 years, genotype OG4 was collected in 2004 (Fig. 1a) and the others (OG9–OG12) were collected in 2007) were collected and disinfected according to Brito and Santos (2009). Disinfected cuttings (3-cm long) comprising one or two axillary bud(s) were placed on solid (0.7% agar, Agar agar, Duchefa Biochemie, The Netherlands) induction media OMG that is a modified OM medium (Rugini 1984), enriched with the double concentration of FeNaEDTA, MgSO4 and MnSO4 as described by Brito and Santos (2009). The sprouted shoots were then transferred to elongation/proliferation medium, consisting on the same basal medium (OMG) but supplemented with 9.12 μM zeatin (Brito and Santos 2009). For shoot proliferation and germplasm long-term maintenance, subcultures were performed monthly. Cultures took place at 22 ± 1°C, with a photoperiod of 16 h and an average light intensity of 45 μmol m−2 s−1.
Fig. 1

Stages of efficient micropropagation of Olea maderensis (genotype OG4). a An example of one of the few O. maderensis trees existing in Madeira Archipelago, used as mother plant; b cuttings with axillary buds sprouted, after 30 days on induction medium (OMGWH) (arrows axillary buds); c shoots on elongation stage (1-year-old subcultures) after 30 days on OMG medium supplemented with 9.12 μM of zeatin; d rooted shoots after 1 month in rooting medium (1/2 OMGWH) (arrows roots); e plantlets before acclimatization, showing a well-developed rooting system; f 1-month-old regenerated plantlet in in vitro acclimatization on a plastic vessel with peat:vermiculite (1:3) (plantlet with ~4.5 cm length); g 3-month-old acclimatized plants in greenhouse on plastic vessels with peat:vermiculite (1:3) (plants with ~10 cm length on average); h 9-month-old acclimatized plants in open greenhouse in Porto Santo Island (plants with ~35 cm length on average); i 2-year-old established plant in field (7 months after transfer to field, with ~90 cm length); j established plant, in field (1.5 years after transfer to field, with ~110 cm length); l mountain on Porto Santo Island (named ‘Pico do Castelo’) to where micropropagated plants were transferred (white arrow open space in forest where plants were cultivated); m field on ‘Pico do Castelo’ with micropropagated plants of Olea maderensis: black arrow forest zone with Pinus; white arrows micropropagated plants of Olea maderensis in field

For shoot rooting, the methodology described by Brito and Santos (2009) was followed. Briefly, apical segments with 2–3-cm long and with two or three nodes were incubated, for 5 days in dark, on half-strength OMG medium (1/2 OMG) with 3.22 μM NAA, and then transferred to 1/2 OMG medium without growth regulators (1/2 OMGWH) where they remained for at least 1 month. This procedure was carried out in three independent experiments with 20 apical segments in each experiment. After 1.5 months, several parameters were measured in shoots (n = 20): shoot growth, number of leaves, number of nodes, number of newly formed roots, length of roots and percentage of rooting shoots. Data concerning rooting only report to OG4 shoots (Table 1). Then, a total of 100 plants were used for acclimatization procedures.
Table 1

Growth parameters during rooting stage (shoot growth, number of new leaves and nodes) and rooting characteristics of O. maderensis OG4 shoots

Genotype

Shoot growth (cm)

No. of new leaves

No. of new nodes

Rooting shoot (%)

Number of roots (shoot−1)

Mean length (cm)

OG4

0.98 ± 0.09

1.81 ± 0.43

0.87 ± 0.16

84

5.94 ± 0.75

6.73 ± 0.36

Values are means ± SE (n = 20 shoots) obtained over 6 weeks of growth in OMGWH (from three independent experiments)

This micropropagation protocol was established first for OG4, and presently it is being used in micropropagation of all five genotypes (OG4 and OG9-OG12). However, as plants in the field are screened from 2006, data presented here concern only to the population of OG4 tree. Therefore, data concerning acclimatization procedures only report to OG4 plantlets.

Plant acclimatization

Acclimatization (one hundred OG4 plantlets) started when, at least, 2–4 roots with 5–10-cm length were developed and a five-period acclimatization strategy was followed:
  1. 1.

    In vitro acclimatization In vitro plantlets were transferred to sterile plastic vessels (120 ml volume) with a sterilised mixture of peat:vermiculite (1:3) wet with 1/2 OMG liquid medium (with no sucrose or growth regulators). These vessels were introduced inside glass vessels (650 ml volume) and maintained for 1 month in growth chamber conditions (see above).

     
  2. 2.

    Growth-cabinet acclimatization Plastic vessels with plantlets were removed outside the glass vessels and transferred to a growth-cabinet with the same temperature and photoperiod as before but with progressive decreasing of relative humidity (%RH) from 100 to 90% (longer spacing between fogging day after day, steadily decreasing %RH) and with an average of light intensity of 70 μmol m−2 s−1. Occasional treatments with 1 g l−1 of fungicide solution Benlate® were made. Plantlets were maintained on this growth-cabinet for 2 months.

     
  3. 3.

    Greenhouse acclimatization Plants were placed in a greenhouse, and subjected to low RH from 90 to 70% and to an average of light intensity of 70 μmol m−2 s−1, for 2–3 months.

     
  4. 4.

    Open greenhouse acclimatization Greenhouse acclimatized plants were transferred to an open greenhouse in Porto Santo Island, and placed on pots with peat:natural soil with vegetal organic matter (1:3), subjected to low RH from 70 to 50% and higher light intensity (200–400 μmol m−2 s−1, at noon). Plants remained in the open greenhouse for 10 months.

     
  5. 5.

    Field acclimatization Plants were transferred in December 2006 to the field (open spaces of Island mountains mostly reforested with Pinus species, Fig. 1l, m), with averages of RH from 60 to 40% and light intensity from 900 to 1,300 μmol m−2 s−1 (at noon). Plants were surveyed regularly for survival and morphological aspects.

     
In the two final acclimatization periods (4 and 5) plants were exposed to the uncontrolled climatic conditions of Porto Santo Island for an adjustment to the climatic/edaphic conditions. The soil of the selected site was characterized by Brito et al. (2007a), and it is an alkaline soil poor in water content, with low organic matter (0.6%), and rich in Fe, Mg, and Mn and Na, while other nutrients are in deficit.
To evaluate plant growth and performance during acclimatization, several parameters were measured in plants (n = 20) on the different acclimatization periods: plant length, number of leaves, number of nodes, and number of new axillary shoots. Average plant elongation rates were calculated for two stages (Table 2):
Table 2

Growth parameters measured during different periods of acclimatization of O. maderensis OG4 plants

Acclimatization periods

Age (months)

Plant length (cm)

No. of leaves

No. of nodes

No. of new axillary shoots

Elongation (cm)/month

In vitro

1

4.48 ± 0.14 a

8.19 ± 0.44 ad

5.24 ± 0.18 a

0.00 ± 0.00 a

2.36

Growth-cabinet

2

6.86 ± 0.36 ad

11.24 ± 0.81 af

6.67 ± 0.30 ad

0.00 ± 0.00 a

Greenhouse

5

16.29 ± 0.90 cd

23.35 ± 1.06 ef

12.53 ± 0.50 cd

2.06 ± 0.42 ad

Open greenhouse

9

35.47 ± 1.32 bc

39.35 ± 2.04 ce

21.71 ± 1.32 bc

2.94 ± 0.37 cd

5.99

Open greenhouse

15

79.59 ± 2.82 b

184.53 ± 5.71 bc

31.00 ± 0.83 b

17.18 ± 0.96 bc

Field

17

83.41 ± 2.91 b

210.94 ± 6.78 b

34.82 ± 0.99 b

23.00 ± 1.32 b

Values are means ± SE (n = 20 plants). For each parameter (column), means followed by the same letter are not significantly different (P < 0.05)

  1. 1.
    Elongation rates between in vitro and greenhouse acclimatization periods (controlled light intensity, RH and temperature):
    $$ {\text{Elongation}} = {\frac{{\left( {T_{f} \left( {\text{plant\;length\;greenhouse}} \right) - T_{i} \left( {{\text{plant\;length\;in\;vitro\;ac}}.} \right)} \right)}}{{{\text{no}}. {\text{\;of\;months}}}}} $$
     
  2. 2.
    Elongation rates between open greenhouse and field acclimatization periods (uncontrolled light intensity, RH and temperature):
    $$ {\text{Elongation}} = {\frac{{\left( {T_{f} \left( {\text{plant\;length\;field}} \right) - T_{i} \left( {\text{plant\;length\;open\;greenhouse}} \right)} \right)}}{{{\text{no}}. {\text{\;of\;months}}}}} $$
     

Physiological studies

Plant material

Leaves were collected and treated separately, for determination of the physiological parameters described below, from: (a) OG4 mother plant in winter; (b) OG4 shoots on elongation stage (1-year-old subcultures) after 30 days on OMG medium supplemented with 9.12 μM of zeatin; (c) OG4 plantlets on in vitro acclimatization (1-month-old plants); (d) OG4 plants acclimatized to greenhouse (5-month-old plants); (e) OG4 plants acclimatized to open greenhouse (9-month-old plants in Porto Santo Island); and (f) OG4 plants in field (17-month-old plants established in field—2 months after transplantation to field).

For water content, mineral composition and soluble protein content, results were averaged for groups of three leaves of three independent individuals (shoot or plant) on each stage of the micropropagation/acclimatization process. For chlorophyll content, results were averaged for groups of three leaves of six independent individuals (shoot or plant) on each stage of the micropropagation/acclimatization process. For fluorescence analysis (photosynthetic efficiency), results were averaged for groups of three leaves of nine independent individuals (shoot or plant) on each stage of the micropropagation/acclimatization process. For osmolality and membrane permeability, results were averaged for one leaf of three independent individuals (shoot or plant) on each stage of the micropropagation/acclimatization process.

Water content and osmolality

Water content was determined by the difference between fresh weight (FW) and dry weight (DW), after drying samples at 60°C until weight stabilization (during 1 week). For osmolality analysis, samples were submitted to freeze/unfreeze cycles to assure membrane rupture (Brito et al. 2003). Osmolality was determined by analyzing samples of leaves using an automatic osmometer Knauer (Berlin, Germany).

Chlorophyll and anthocyanin content

Leaf contents of chlorophyll a, b and anthocyanins were determined by following the procedures described by Sims and Gamon (2002), by homogenizing tissue in cold acetone/Tris buffer (80:20 volume; pH = 7.8). The absorbance of the extract solutions was measured with the Beckman DV68 spectrometer at 537, 647 and 663 nm. Chlorophylls and anthocyanins contents were calculated according to Sims and Gamon (2002).

Photosynthetic efficiency

Chlorophyll fluorescence was monitored using a Plant Efficiency Analyser (Hansatech Instruments Ltd). For the determination of fluorescence parameters, plants were stored in the dark for adaptation for 30 min in a growth chamber at 22 ± 2°C. Then, Photosystem II (PSII) fluorescence was monitored by illuminating leaves with a peak wavelength 650 nm and a saturating light intensity of 3,000 μmol m−2 s−1 (Santos and Caldeira 1999).

Chlorophyll basal fluorescence (F0), variable fluorescence (Fv), maximum fluorescence (Fm) and the ratio (Fv/Fm) were analyzed (Maxwell and Johnson 2000).

Soluble protein content

Tissue samples (0.5 g) were homogenized at 4°C in 1 ml of potassium phosphate buffer 0.05 M (pH = 7.8) containing 0.1 mM ethylenediamine tetraacetic acid, 5 mM cysteine, 1% (w/v) polyvinylpyrrolidone, and 0.2% Triton X-100 (Olmos et al. 1994). Homogenates were filtered and centrifuged at 8,000×g for 15 min, at 4°C. The supernatant was used to determine soluble protein content, following procedures described by Bradford (1976) and using a “total Protein Kit, Micro” (Sigma).

Membrane permeability determination by solute leakage method

The removed leaf was incubated in 5 ml of deionized water at 25°C on a rotary shaker (85 rpm). UV absorbing substances of the bathing solution were determined, after 24-h incubation (A280) and after autoclaving (A280), spectrophotometrically (Beckman DV68). The relative leakage ratio (RLR) was calculated as RLR = (A280/A280) and expressed as percent (Azevedo et al. 2005).

Mineral composition

Mineral content was determined in leaves, dried at 60°C until weight stabilization. Dried tissues were mineralized following procedures described by Azevedo et al. (2005) to determine the content of K, P, Ca, Mg, Mn, Fe, B, Cu, and Zn elements. Elemental contents were determined by induced coupled plasma spectroscopy (ICPS) using a Jobin Ivon JY70 Plus.

Statistical analyses

Variations among the measured growth parameters and among determined values in each physiological parameter of the plants on the different micropropagation stages were analyzed. One-way ANOVA was executed and a multiple comparison procedure (Tukey test) was used for a pairwise comparison. (SigmaStat for Windows Version 3.1, SPSS Inc., Richmond, CA, USA).

Results

Micropropagation and acclimatization

OMG medium successfully induced sprouting (>80%) in O. maderensis which was obtained in 1 month (Fig. 1b). After this period, the newly formed shoots were collected and transferred to elongation medium, and a stock material was obtained by subculturing shoots monthly. During elongation–proliferation stage shoots looked healthy, presented dark green leaves and good quality (Fig. 1c). Rooting was successfully achieved (>84%) (Table 1), and first roots appeared approximately after 4 weeks (Fig. 1d, e). During rooting stage, it was observed shoot growth, the appearance of new leaves and nodes (Table 1) and good quality of the newly formed roots (Fig. 1d, e). Plantlets had hard stems, dark green leaves, looked healthy and morphologically identical. This strategy allowed large-scale shoot multiplication and plant regeneration and simultaneous in vitro germplasm maintenance of this species for the last 5 years.

Plant acclimatization described here reports only to data of OG4 plants, which were transferred to field in 2006. Acclimatization was developed in five periods, being initiated in vitro (Fig. 1f), and after 1 month transferred to a growth-cabinet. O. maderensis showed excellent plant acclimatization results, with an average of 97% of plant survival in the growth-cabinet and greenhouse (Fig. 1g). Plants were transferred 5 months latter to an open greenhouse in Porto Santo Island, where they grew healthy for 10 months with survival rates of 100% (Fig. 1h). After this period plants were transferred to field in Porto Santo Island where they were established at a survival rate of 100% (Fig. 1i–m).

Some growth parameters were evaluated during the acclimatization stages (Table 2). In general, all plants showed healthy performance, looked morphologically identical and had active growth. The elongation rate in the first stage of acclimatization (from in vitro till greenhouse condition) was on average 2.36 cm month−1, lower than the elongation rate (5.99 cm month−1) from the open greenhouse to field conditions on Porto Santo Island. Also the mean values of the number of leaves and nodes per plant increased significantly (P < 0.05) during acclimatization. The number of new axillary shoots in each plant increased extraordinarily (P < 0.05) in the last stages of acclimatization (Table 2).

Physiological studies

Performance of plants during micropropagation stages was evaluated by analyzing several physiological parameters.

Figures 2, 3, 4, 5, 6, 7, and 8 present the results of the physiological studies performed during micropropagation/acclimatization process, and the sequence described in Figs. 2, 3, 4, 5, 6, and 7 refers to: M plant, mother plant; Elong, shoots on elongation stage (1-year-old subcultures) after 30 days on OMG medium supplemented with 9.12 μmol L−1 of zeatin; In vitro Ac, 1-month-old plants in in vitro acclimatization; Greenh, 5-month-old plants acclimatized to greenhouse; Op Greenh, 9-month-old plants acclimatized to open greenhouse in Porto Santo Island; Field, 17-month-old plants established in field (2 months after transplantation to field). In Fig. 8 the sequence 1–6 refers to: M plant, Elong, In vitro Ac, Greenh, Op Greenh, Field, in the same conditions described above.
Fig. 2

Water content of Olea maderensis OG4 leaves during micropropagation/acclimatization. Values are means ± SE (n = 3). Mean values with the same letter do not differ significantly at P < 0.05

Fig. 3

Osmolality in Olea maderensis OG4 leaves during micropropagation/acclimatization. Values are means ± SE (n = 3). Mean values with the same letter do not differ significantly at P < 0.05

Fig. 4

Concentrations of: a chlorophyll a (chl a), b chlorophyll b (chl b), c chlorophyll a/b ratio (chl a/b), d total chlorophyll (chl t), and e anthocyanin in Olea maderensis OG4 leaves during micropropagation/acclimatization. Values are means ± SE (n = 6). Mean values with the same letter do not differ significantly at P < 0.05

Fig. 5

Fluorescence parameters in Olea maderensis OG4 leaves during micropropagation/acclimatization. Values are means ± SE (n = 9). Mean values with the same letter do not differ significantly at P < 0.05. aF0, basal chlorophyll fluorescence; bFm, maximal chlorophyll fluorescence; cFv, variable component of chlorophyll (Fv = Fm − F0), and d ratio Fv/Fm

Fig. 6

Soluble protein concentrations in Olea maderensis OG4 leaves during micropropagation/acclimatization. Values are means ± SE (n = 3). Mean values with the same letter do not differ significantly at P < 0.05

Fig. 7

Membrane permeability (solute leakage) expressed as relative leakage ratio (RLR = A280/A280, expressed as percentage) in Olea maderensis OG4 leaves during micropropagation/acclimatization. Values are means ± SE (n = 3). Mean values with the same letter do not differ significantly at P < 0.05

Fig. 8

Contents of macronutrients (P, Mg, Ca, Na, and K) and micronutrients (Zn, Fe, B, Mn, and Cu) in Olea maderensis OG4 leaves during micropropagation/acclimatization. The sequence 1–6 refers to: M plant, Elong, In vitro Ac, Greenh, Op Greenh, and Field. Values are means ± SE (n = 3). Mean values with the same letter do not differ significantly at P < 0.05

Water content and osmolality

Leaves from plants in field (mother plant and acclimatized plants in field) had on average significantly (P < 0.05) lower water content values than in vitro elongation shoots and the other acclimatized plants (first periods of acclimatization) (Fig. 2). On the other hand, leaf osmolality was not statistically different among the several micropropagation stages (Fig. 3), showing that growth conditions did not affect this parameter.

Chlorophyll and anthocyanin contents

Chlorophyll content data are reported in Fig. 4a–d. Chlorophyll a (chl a) content of in vitro leaves (elongation stage 0.63 mg gfw−1) did not differ (P > 0.05) from those of the mother plant (0.57 mg gfw−1). However, during the first periods of acclimatization chl a level increased (P < 0.05; 0.68 mg gfw−1) with respect to mother plant leaves. When plants were transferred to field, chl a level decreased (P < 0.05; 0.59 mg gfw−1) to mean values close to the mother plant. Chlorophyll b (chl b) content increased (P < 0.05) in in vitro-grown leaves (elongation stage 0.70 mg gfw−1) and in first period of acclimatization (In vitro Ac, 0.64 mg gfw−1) when compared to leaves of mother plant. Chl b level decreased (P < 0.05) when plants were transferred to greenhouse (0.44 mg gfw−1), and this level slightly decreased till field condition. However, in plants acclimatized to open greenhouse and to field, chl b level did not differ (P > 0.05) from the one of the mother plant. In vitro-grown leaves had lower (P < 0.05) chl a/b ratio than mother plant leaves. Afterward an increase occurred (P < 0.05) during the acclimatization periods reaching values close to the mother plant. Chlorophyll a/b ratio from open greenhouse and field plants did not differ (P < 0.05) from those of mother plant. Total chlorophyll (chl t) increased (P < 0.05) in leaves on elongation stage, and this level was maintained in leaves on the two-first periods of acclimatization (In vitro Ac and Greenh), and then decreased (P < 0.05), when plants were transferred to open greenhouse and to field, compared to plants under in vitro acclimatization. Total chlorophyll content from open greenhouse and field plants did not differ (P < 0.05) from those of mother plant.

Concerning anthocyanin content (Fig. 4e) it significantly decreased (P < 0.05) in leaves during in vitro and greenhouse acclimatization periods with respect to mother plant leaves. However, anthocyanin contents from leaves in the elongation stage to field acclimatization did not vary (P > 0.05).

Photosynthetic efficiency

In the analysis of chlorophyll fluorescence parameters (Fig. 5a–d), basal fluorescence (F0) increased (P < 0.05) in the elongation stage (Elong) compared to mother plant, while the other parameters (Fm, Fv, Fv/Fm) decreased (P < 0.05). During acclimatization stage, F0 decreased (P < 0.05) on the first period (In vitro Ac), while in the subsequent periods until field condition it slightly increased although not significantly (P > 0.05). The other parameters (Fm, Fv, Fv/Fm) showed an increasing tendency (though not statistically significant, P > 0.05) during the first periods of acclimatization remaining stable until open greenhouse condition. When plants were transferred to field, F0 increased (P < 0.05), while Fm, Fv, Fv/Fm decreased (P < 0.05) compared with mother plant. However, Fv/Fm ratio was on average close to 0.8 in all stages of micropropagation (mean values between 0.77 and 0.84).

Soluble protein content

Soluble protein contents (Fig. 6) increased (P < 0.05) in leaves of the elongation stage, when compared to mother plant; but during the first acclimatization periods (In vitro Ac and Greenh) its levels decreased (P < 0.05). In open greenhouse and field acclimatized plants these values slightly increased compared to the two-first acclimatization periods (though not statistically different) but close to the values of the mother plant.

Membrane permeability

Relative leakage ratio (Fig. 7) did not undergo significant differences (P > 0.05) among first stages of micropropagation (Elong, In vitro Ac, and Greenh) and between these and mother plant. However, when plants were transferred to open greenhouse and to field in Porto Santo Island, membrane permeability increased when related to leaves of the first stages of micropropagation (Elong, In vitro Ac, and Greenh) and from mother plant.

Mineral composition

Macronutrient composition of Olea maderensis leaves changed through micropropagation/acclimatization stages (Fig. 8a–j). Magnesium (Mg) and sodium (Na) concentrations were maintained during the first stages of the micropropagation process till open greenhouse condition. However, when plants were transferred to field, significant (P < 0.05) differences were observed: Mg decreased compared to leaves on elongation stage and on first period of acclimatization; Na content also decreased compared to leaves of the first two acclimatization periods. Phosphorus (P) content was sensibly maintained between mother plant and the subsequent stages of micropropagation, till greenhouse condition. However, P increased in open greenhouse decreasing thereafter. Concerning to calcium (Ca) level it decreased in leaves on elongation stage compared to mother plant, but on the subsequent acclimatization periods its concentration was maintained. Contrarily, potassium (K) level significantly increased in leaves on elongation stage compared to mother plant, decreasing during the first period of acclimatization (P < 0.05) and being maintained till greenhouse condition. In open greenhouse, K level increased again (P < 0.05 compared to the in vitro acclimatization period. When plants were transferred to field, K content decreased reaching values similar to those of the mother plant.

Concerning micronutrients levels, iron (Fe) and copper (Cu) did not undergo significant differences trough micropropagation stages. However, Zinc (Zn), boron (B) and manganese (Mn) contents showed significant differences through micropropagation process. Zn content was higher in leaves from the elongation stage to the open greenhouse period being lower in leaves of both mother plant and field acclimatized plants. B and Mn levels were sensibly maintained (P > 0.05) through the micropropagation/acclimatization process decreasing only after plants transfer to field condition.

Discussion

Recently we described a protocol for large-scale micropropagation of Olea maderensis and demonstrated that this species needed basal modifications—Fe, Mg, Mn enriched OM medium, named OMG—to improve plant micropropagation rates (Brito and Santos 2009). This protocol is presently being used with other genotypes (OG9–OG12) with similar success rates of induction and multiplication, allowing large-scale plant production and long-term cultures maintenance (data not shown). As OG4 plantlets were transferred to field in 2006, only this genotype was used to assess the acclimatization process described in this work.

The good quality of the newly formed root system was necessary for a successful acclimatization (97% in greenhouse and 100% in mountain conditions). Also, this five-period acclimatization protocol yields high survival rates, based on the controlled exposure to low relative humidity and high light intensity. The first period (in vitro acclimatization) showed to be very efficient and facilitate transfer to ex vitro conditions. Hazarika (2006) and Pospíšilová et al. (1999) suggest that stimulating autotrophic characteristics under in vitro conditions may improve acclimatization ability and survival rates. During the whole mountain field acclimatization period (100 plants were transferred on December 2006) all plants survived, and looked morphologically identical, having an active growth without senescence signals, supporting the conditions established by Hazarika (2006) for a successful acclimatization to occur. In a wild olive acclimatization protocol previously used by our group, which consisted on one step greenhouse transfer, plants survival was much lower (~70%) and no plants survived to open greenhouse conditions (Santos et al. 2003).

Due to the hard conditions of the Island, and in order to follow the acclimatization and be able to detect physiological disorders that could be responsible for eventual plant senescence or death, parameters related with photosynthesis, hydric and nutrient status were followed.

In a detailed analysis, and concerning water content, it was observed that values of in vitro-grown leaves (elongation stage) are, as expected, higher than mother plant, once in vitro environments generally keep %RH values close to saturation (Brito et al. 2007b; Malda et al. 1999). In the first acclimatization periods, plants were not under water deficiency, suggesting the development of mechanism(s) to control water loss during transfer of plants to ex vitro. When plants are transferred to field, water loss naturally occurred most probably associated with the dry conditions of the field. Moreover, the possibility that, at this stage, leaves still present histo-anatomic differences from those naturally growing in field (e.g., cuticle thickness) should not be excluded. (Hazarika 2006; Malda et al. 1999; Pospíšilová et al. 1999). Osmolality did not undergo statistically differences during micropropagation/acclimatization meaning that despite the final water content in the field decreased, the final concentration of osmotically active solutes was maintained, suggesting an efficient osmoregulation ability of plants during the whole acclimatization process.

In vitro plantlets grow generally under low level of light, with plenty of sugar and nutrients to favor heterotrophic growth and in an atmosphere with high %RH (Hazarika 2003). Due to these factors, in vitro plants have low rates of photosynthesis and an incipient photosynthetic apparatus. After transfer to ex vitro conditions, most micropropagated plants develop a functional photosynthetic apparatus, although the increase in light intensity is not linearly translated in an increase in photosynthesis (Amâncio et al. 1999). Despite other studies (e.g., Amâncio et al. 1999; Hazarika 2003) indicate a reduction of the chlorophyll content during in vitro condition, our results shown an increase of chl a and chl b levels in vitro, which may be explained by the increase of Fe, Mg, and Mn levels (essential for photosynthesis) in the OMG medium (Brito and Santos 2009). Afterward acclimatization affected chlorophyll concentration, in particular in chl b, increasing chl a/b ratio. Similar results were obtained by Amâncio et al. (1999) with a decrease of chl b content and higher values of chl a/b ratio, when in vitro grapevine plants were exposed to high light intensity during acclimatization. These data suggest that when plants are transferred to ex vitro conditions at higher light irradiances, photoinhibition or even photooxidation of chlorophyll can occur (Amâncio et al. 1999). This explains the reduction of chl b and chl t during acclimatization of this wild olive. However, leaf blade of acclimatized plants did not revealed any stress signal such as chlorosis or dry spots. Moreover, the mean values of chlorophyll content of the field acclimatized plants are close to those of the mother plant, proving that plants are well adapted to field.

The reduction of anthocyanin contents during the two-first periods of acclimatization may be explained by the reduced luminosity, lower stress and lower differentiation/age of these leaves with respect to mother plant leaves. Anthocyanin production is a plant response to stressful situations including UVB, drought and nutrient stress (Chalker-Scott 1999) and have a protection role from photoinhibition (Steyn et al. 2002). The apparent increase of anthocyanins in field acclimatized plants may suggest a defense strategy of these plants to the high irradiance preventing photoinhibition.

In the elongation stage, leaf F0 increased, while Fm, Fv and Fv/Fm decreased compared to mother plant. However, F0 significantly decreased in first period of acclimatization, increasing slightly thereafter until field conditions, probably related with a switch to autotrophy condition and suggesting that PSII reaction center (chl a) was not deteriorated during last periods of acclimatization. Overall slight increases were observed in the other parameters (Fm, Fv and Fv/Fm), during acclimatization until greenhouse. Pospíšilová et al. (1999) described an increase of fluorescence parameters during de acclimatization of Nicotiana tabacum plants, confirming the results obtained to O. maderensis in first periods of acclimatization. However, when plants are transferred to field, F0 increased while the other parameters decreased. The reductions of Fm and Fv are probably due to the new plants’ environment, such as high light intensity, low relative humidity, strong wind and soil composition. The optimal quantum yield (Fv/Fm ratio) found for these plants was around 0.8, which is typical of non-stressed and healthy plants (Dodd et al. 1998; Fracheboud 2001; Seon et al. 2000) and it was found for other species (e.g., Oliveira et al. 2009; Santos et al. 2005; Seon et al. 2000). This ratio was maintained stable trough all the process, suggesting the stability of the photosynthetic machinery.

The presence of sugars in the medium may promote mixotrophy, leading to a downregulation of photosynthesis due to feedback inhibition of the Calvin cycle (Amâncio et al. 1999; Premkumar et al. 2001; Van Huylenbroeck et al. 2000). Among photosynthetic enzymes, Rubisco has deserved much attention, since it performs a dual role as a catalyst in carboxylation of CO2 and as a major storage protein being 40–80% of the total soluble leaf proteins (Premkumar et al. 2001). Both roles could be important in overcoming the critical acclimatization phase, when the mixo-heterotrophic behavior of the in vitro plants is shifted to an autotrophic functioning. Protein content decreased during the first acclimatization periods, increasing thereafter to values similar to mother plant. These data support that initial acclimatization is stressful, and part of the degraded protein is probably rubisco, as proposed by Carvalho et al. (2005) for micropropagated grapevines who recommended the evaluation of this enzyme during acclimatization.

Micropropagation and acclimatization process of O. maderensis membrane did not induce solute leakage, once membrane integrity is stable during this time. When plants are transferred to field, relative leakage ratio increased, revealing that these plants suffer some stress during the field acclimatization. Nonetheless, this stress apparently did not compromise plants performance and survival, allowing a good tolerance to the hard conditions of the field (e.g., dry soil, low relative humidity). It is well documented that cell membranes are the first targets of many plant stresses, and the maintenance of their integrity and stability under water stress conditions is a major component of drought tolerance in plants (e.g., Bacelar et al. 2006).

Concerning mineral composition, the values found for O. maderensis fit within those already described for in vitro shoots on OMG medium (Brito and Santos 2009). Through the micropropagation process, macro and micronutrients contents had a general increase during in vitro. When plants were transferred to the poor soils of the Island (Brito et al. 2007a), the level of nutrients decreased in relation to previous periods of acclimatization, and reached mean values close to those found for mother plant. None of the common visual symptoms of nutrient deficiencies (e.g., small chlorotic leaves, dead areas of leaf tips/leaves, bark lesions) (Clatterbuck 1999) were found in O. maderensis during micropropagation and acclimatization process, supporting no nutrient deficiencies and that plants were perfectly adapted to edaphic environment (e.g., magmatic nature soil highly rich in Fe, Mg, and Mn).

In conclusion, micropropagation is presently the only available process for large-scale propagation of the endangered species O. maderensis. The full integration of this methodology in an ongoing reforestation program of Porto Santo Island required a successful mountain field transfer protocol. We report here that the acclimatization to Porto Santo mountain conditions of 100 micropropagated plants (transferred on December 2006) was fully achieved (100%) and improved plant performance compared to the direct acclimatization protocol previously tested (Santos et al. 2003). The physiological parameters chosen here gave a global view of plants performance in the adverse conditions of the Island and confirmed that plants are not under severe stress, supporting that this multistep acclimatization protocol supports a large-scale plant implantation. This protocol is presently being used for this and other genotypes (OG9–OG12) to maintain the genetic patrimony of this endemic species and simultaneously integrate plants in field, contributing to fight desertification.

Notes

Acknowledgments

Authors thank Direcção Regional de Florestas and Secretaria Regional da Educação from Madeira Autonomous Region and Porto Santo Town Hall. Thanks are also due to Mr. L. Silva, Mr. Martinho, Mrs. I. Oliveira and Mr. J. Brito for assistance during acclimatization of plants in the Island and to FCT project (FCT, PNAT/1999/AGR/15011/99) that supported this work.

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Gina Brito
    • 1
  • Armando Costa
    • 1
  • Celeste Coelho
    • 2
  • Conceição Santos
    • 1
  1. 1.Laboratory of Biotechnology and Cytomics, CESAM, Department of BiologyUniversity of AveiroAveiroPortugal
  2. 2.CESAM, Department of Environment and PlanningUniversity of AveiroAveiroPortugal

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