Micropropagation of Chokeberry by In Vitro Axillary Shoot Proliferation

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 994)

Abstract

The black chokeberry—aronia (Aronia melanocarpa Elliot) is a shrub native to North America although nowadays well known in Eastern Europe. The fruits are regarded as the richest source of antioxidant phytonutrients among fruit crops and vegetables. Chokeberries can be easily propagated by seeds but this method is not recommended. Micropropagation is far more efficient than other conventional cloning methods like layering or softwood cuttings. Aronia clones are propagated in vitro through four- or three-stage method based on subculturing of shoot explants. The double diluted MS or full strength MS medium with elevated 50% Ca2+ and Mg2+ content are used in the initiation and proliferation chokeberry in vitro cultures, respectively. They are supplemented with 0.5–1.0 mg LBA, and 0.05 mg LIBA. The double-phase medium is recommended in the last passage before shoot rooting. The regenerated shoots could be rooted both in vitro on double diluted MS with 0.05 mg L−1 IBA or in vivo in peat and perlite substrate and subsequently grown in the greenhouse.

Key words

Aronia Axillary shoots Nodal explants Small fruits Tissue culture 

1 Introduction

The black chokeberry—aronia (Aronia melanocarpa Elliot) is a shrub native to North America although nowadays well-known, cultivated and utilized in Poland, Russia, and other countries of Eastern Europe. The fruits are rich in macro- and micronutrients (Ca, Fe, Mo, Mn, Cu, B, I, Co), vitamins (P, C, B2, B6, PP, E, proA), carbohydrates, cellulose, pectins, anthocyanins, proanthocyanidins, and catechins. The antioxidant effect of aronia fruits is much higher than cranberries, elderberry and blackberries, and even blackcurrants. Black chokeberry fruits are used in food industry and pharmacy. Chokeberries can be easily propagated by seeds. This method is not recommended because of late bearing of plants, vigorous and non-uniform growth, and plants being unsuitable for mechanical harvest. Black chokeberry is comparatively a young crop and only few cultivars or breeding clones are known and grown. As micropropagation is far more efficient than other conventional cloning methods, it should improve breeding and rapid propagation of new, valuable strains of Aronia. The studies on black chokeberry micropropagation were so far carried out by several authors (1, 2, 3, 4, 5, 6, 7). There are few reports concerning other applications of in vitro cultures: cryopreservation (8), and induction of colchiploids (9).

2 Materials

2.1 Preculture (Preparation of Source Material)

  1. 1.

    Young progeny of selected, well-evaluated, and bred plants, in plastic pots (ca 2 L volume) filled with universal potting medium consisting compost or healthy shoots of aforementioned plants (see Note 1).

     
  2. 2.
    “Forcing medium” (Table 1).
    Table 1

    Culture media based on the formulation of Murashige and Skoog (MS; (10)) used for micropropagation of black chokeberry

    Stages

    Shade-forcing parental shoots

    Initiation

    Proliferation

    Rooting in vitro

    Macronutrients

    25% MS

    50% MS

     

    50% MS

    N salts

    100% MS

    K salts

    100% MS

    P salts

    100% MS

    Ca salts

    150% MS

    Mg salts

    150% MS

    Micronutrients (without Fe salts)

    25% MS

    50% MS

    100% MS

    50% MS

    NaFeEDTA (mg L−1)

    9.1

    18.4

    55.1

    18.4

    Vitamins

    100% MS

    100% MS

    100% MS

    50% MS

    Myo-inositol (mg L−1)

    100

    100

    100

    Sucrose (g L)

    20

    20

    30

    20

    BA (mg L−1)

    0.5

    0.5

    1.0

    IBA (mg L−1)

    0.1

    0.05

    0.05

    0.05

    Agar (g L−1)

    5

    6

    6

    Other ingredients

    8-Hydroxyquinoline 140 mg L−1

    Glucose 5 g  L−1

    Arginine 200 mg L−1

    Fructose 5 g  L−1

    PVP 360 100 mg L−1

    Citric acid 210 mg L−1

    PPM™ 0.5 mL L−1

    pH

    5.6

    5.6

    5.6

    5.6

    MS macronutrients: NH4NO3 1,650 mg L−1, KNO3 1,900 mg L−1, CaCl2·2H2O 440 mg L−1, MgSO4·7H2O mg L−1, KH2PO4 170 mg L−1; MS micronutrients: NaFeEDTA 36.7 mg L−1, KI 0.83 mg L−1, H3BO3 6.3 mg L−1, MnSO4·4H2O 22.3 mg L−1, ZnSO4·7H2O 8.6 mg L−1, Na2MoO4·2H2O 0.25 mg L−1, CuSO4·5H2O 0.025 mg L−1, CoCl2·6H2O 0.025 mg L−1; MS vitamins: glycine 2.0 mg L−1, thiamine HCl 0.1 mg L−1, pyridoxine HCl 0.5 mg L−1, nicotinic acid 0.5 mg L−1

     
  3. 3.

    Fungicides (Bayleton 5 WP 0.1%, Topsin M 0.15%)

     

2.2 Surface Sterilization of Source Material

  1. 1.

    Tap water.

     
  2. 2.

    Ethanol 70% (v:v).

     
  3. 3.

    Commercial bleach solution (e.g., “ACE” bleach; 5% (v:v) NaOCl), diluted 1:5 (v:v) with tap water.

     
  4. 4.

    150 mL aliquots of autoclaved reverse-osmosis (or distilled) water in 300 mL screw capped jars.

     
  5. 5.

    Laboratory shaker or ultrasonic washer.

     
  6. 6.

    Shoots of parental plant as a source of explants.

     

2.3 Tissue Culture Facilities and Culture Media

  1. 1.

    Labware (autoclave, magnetic stirrer with heating, dry-heat sterilizer, microwave oven, pH meter, refrigerator, horizontal laminar flow bench).

     
  2. 2.

    Instruments (scalpels, forceps, glass bead sterilizers, or gas burners).

     
  3. 3.

    1 M HCl and 1 M NaOH.

     
  4. 4.

    Sterile Petri dishes (100  ×  15 mm) or sterile paper sheets (kept at 160°C for minimum 2 h) to prepare explants.

     
  5. 5.

    Sterile graduated (25 mL) pipettes with pumps, autoclavable and adjustable (1–5 mL) single-channel pipettes or other dispensers of liquid medium to make double-phase medium.

     
  6. 6.

    Media based on the formulation of MS (10) for (a) parental shoot forcing, (b) culture initiation, (c) shoot regeneration from stem nodal explants, (d) shoot elongation, and (e) root induction on regenerated shoots. Media formulations are given in Table 1.

     
  7. 7.

    15–75 mL capacity test tubes with caps (16  ×  75, 25  ×  150 mm) or 25–50 mL conical Erlenmayer’s flasks, aluminum packaging foil (0.04 mm thick) for culture initiation and first subcultures, 300–500 mL glass jars closed with autoclavable, transparent lids (or adequate culture vessels) for shoot proliferation and rooting, plastic, thermostable (0.1–3 L) beakers.

     
  8. 8.

    Culture room with air conditioning. The possibility of temperature (22–28°C), light intensity (30–50 μmol m−2 s−1 PPFD), and photoperiod (16/8; 4/2 h day/night) regulation is advantageous. The white growth shelves with white fluorescent lamps, preferentially with bottom-cooling system.

     

2.4 Acclimation of Regenerated Plants to Ex Vitro Conditions

  1. 1.

    Greenhouse or plastic tunnel with facilities or as a last resort well separated culture room.

     
  2. 2.

    Mist chambers (9% RH), multi-cell plug trays with transparent plastic covers/lids. Cell dimensions about 2  ×  3 cm (width, depth), transparent plastic foil.

     
  3. 3.

    Peat and perlite mixture (2:1 v:v).

     
  4. 4.

    SCOTTS Peters Plant Starter (10  +  52  +  10), calcium carbonate to regulate pH of peat mixture (5.6), fungicides (Previcur 607 SL, Rovral Flo 255 SC).

     

3 Methods

The genera Aronia and Malus belong to the subfamily Pomoideae. Thus, the modified micropropagation technique for apple rootstocks could be applied in chokeberry in vitro cultures. The protocol of Aronia propagation involves typical three or four stages. The first two stages are the same: the preparation of source material and establishment of axenic cultures (stage I), and multiplication of shoots (stage II) on solid medium with application of double-phase one in the last passage before shoot rooting. The double diluted MS or full strength MS medium with elevated 50% Ca2+ and Mg2+ content, supplemented with 0.5–1.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 indole-3-butyric acid (IBA) are used in the first and second stage, respectively. Regenerated shoots treated with IBA (3.0 g  L−1) could be rooted ex vitro in peat and perlite substrate (stage III  +  IV, 3-stage method) or rooted in vitro on double diluted MS with 0.05 mg L−1 IBA (stage III) and then acclimatized in vivo (stage IV, 4-stage method). Obtained microplants are transferred to the tunnel or greenhouse. Weaned, fast growing chokeberry plants could even be planted in the nursery field in the late spring the same year.

3.1 Preparation and Sterilization of Culture Media

  1. 1.

    All media used in the aronia micropropagation are the modifications of MS medium (10). They differ in concentration of macronutrients and auxins, cytokinins, and other substances (Table 1).

     
  2. 2.

    Prepare medium from stock solutions (or ready-made powdered one) and reverse-osmosis (or distilled) water. Add medium-specific substances and fix the final volume with water. Adjust the acidity (pH  =  5.6) with 1 M HCl and 1 M NaOH before addition of agar, as required.

     
  3. 3.

    When agar is dissolved after medium heating, dispense it into suitable containers, e.g., 2.5–10 mL aliquots into 15–75 mL test tubes for culture initiation, 50 mL aliquots into 300 mL jars for shoot multiplication and rooting, or 100 mL (without agar) into 250–300 mL screw capped jars to prepare double-phase medium.

     
  4. 4.

    Sterilize media by autoclaving at 121°C for 15, 20 or 25 min.

     
  5. 5.

    Store the media in the refrigerator up to 2 months if not used.

     

3.2 Plant Source Material and Surface Sterilization

  1. 1.

    Take rooted parental shoots (young plants) from nursery and plant them in plastic pots (ca 2 L volume) filled with universal potting medium containing compost. Place plants in the shade at room temperature. When plants are not available, collect shoots from parental plants at late winter / early spring. Clean shoots in tap water and spray with fungicides (Bayleton 5 WP, Topsin M). Immerse lower parts of shoots in “forcing medium” (Table 1). Place shoots in the shade at room temperature to obtain etiolated sprouts.

     
  2. 2.

    Tear off 5–10 cm sprouts; remove leaf blades (without petioles).

     
  3. 3.

    Wash shoots for 10 min under cold running tap water with detergent and surface sterilize them by immersion in 70% (v:v) ethanol for 1 min, followed by shaking them in a clean jar containing commercial bleach diluted with water (1:5 v:v) for 20 min. Use laboratory shaker or ultrasonic washer if available.

     
  4. 4.

    Wash shoots three times with sterile reverse-osmosis water for 5 min.

     

3.3 Culture and Maintenance of Explants

  1. 1.
    After sterilization, make nodal explants (about 10 mm long) using a sterile scalpel and place them individually on the initiation medium (Table 1) in small test tubes (Fig. 1a, b).
    Fig. 1.

    Propagation of chokeberry through in vitro cultures. (a, b) Initiation of in vitro culture, (c, d) maintained culture of aronia, (e) growth of in vitro cultures on double-phase “2” and solid “1” media, (f, g) rooted shoots in vitro, (h, i) regenerated plant of chokeberry. (e–g) Copyright by Wroclaw University of Environmental and Life Sciences (7).

     
  2. 2.

    Prepare 20 explants per genotype.

     
  3. 3.

    Excise new axillary sprouts which emerged in vitro. Make new explants (nodal sections with or without shoot tip, 6–10 mm long) and place again on initiation medium. Repeat it every 2–4 weeks to maintain active growth and select healthy cultures (see Note 2, Fig. 1c).

     

3.4 Multiplication of Shoots

  1. 1.

    Subculture explants (1 cm long) on proliferation (c) medium (Table 1) every 5–6 weeks from third-fourth passage. Use 300 mL capacity glass jars closed with transparent lids or adequate culture vessels. Place 12–16 nodal explants about 1 cm long in each vessel (see Notes 3 and 4).

     
  2. 2.

    In the last passage (before rooting) make double-phase medium (d, e media) to stimulate elongation of shoots. For this purpose pour 5–10 mL liquid (e) medium onto solid one (d) (Table 1), (see Note 5).

     
  3. 3.

    Grow the cultures at 26  ±  1°C and 16 h/8 h day/night photoperiod under cool-white light at about 20 (initiation)—30 μmol m−2 s−1 PPFD.

     

3.5 Acclimation of Regenerated Shoots to Ex Vitro Conditions

  1. 1.

    Place healthy shoots (1.5 cm long) onto rooting medium (see Notes 6 and 7; Table 1). After 2 weeks, root primordial and first short roots are visible. Clean roots carefully by removing agar with lukewarm tap water. Transfer rooted shoots into peat and perlite mixture (2:1 v:v; pH  =  5.8) watered with fertilizer “SCOTTS Peters Plant Starter” solution (0.8 g  L−1) and fungicide Previcur 607 SL (0.15%). Spray shoots with fungicide Rovral Flo 255 SC (0.1%) solution. Use multi-cell plug trays with transparent plastic covers/lids. Grow plants at high air relative humidity (95% RH) in 16 h/8 h day/night photoperiod under sodium light at 60–100 μmol m−2 s−1 PPFD at 20–24°C. Protect plants from direct intense solar radiation. After 2–3 weeks plastic lids should be gradually lifted up and finally removed. Transfer weaned, fast growing plants to the nursery tunnels or field.

     
  2. 2.

    The rooting in vitro stage could be omitted (see Note 8), by using 2 cm long shoots. Dip shoot base in water-ethanol (1:1 v:v) rooting solution of IBA (3.0 g  L−1). Place them directly in pots filled with peat and perlite mixture. Treat them with similar, aforementioned method.

     

4 Notes

  1. 1.

    Use cultivar supplied either by the breeder/owner or a reliable nurseryman. However, it is recommended to establish own collections of healthy parental plants.

     
  2. 2.

    Chokeberry clones readily adjust to in vitro conditions and change into rapidly growing cultures in second–third initiation passage (Fig. 1c, d). However, shoots harbor endogenous bacteria which contaminate medium even in fourth passage. Therefore addition of 0.5 mL L−1 PPM (plant preservative mixture) in the culture medium is recommended.

     
  3. 3.

    The propagation ratio is high. From one 2-node explant, more than 9 shoots (which could be divided into more than 20 new explants) or more than 6 shoots suitable for rooting can be expected (Fig. 1d). Thus more than 100 shoots from one jar may be obtained (Fig. 1e).

     
  4. 4.

    The chokeberry cultures at the proliferation stage can be easily stored in refrigerator for more than 1 year.

     
  5. 5.

    The double-phase medium strongly stimulates shoot elongation, but does not significantly influence shoot proliferation (Fig. 1e).

     
  6. 6.

    Although it is more complicated and expensive, the two-step procedure (rooting shoots in vitro and acclimation them in vivo) can be applied. The rooting passage lasts 2–3 weeks. More than 90% shoots root in vitro (Fig. 1f, g). A similar procedure may be applied during ex vitro rooting of the shoots. The survival rate reaches near 100% (Fig. 1h, i).

     
  7. 7.

    Shoots with tips root slightly better than nodal segments both in vivo and in vitro.

     
  8. 8.

    Rooting shoots ex vitro may be used. The IBA treatment of shoots does not improve efficiency of in vivo rooting (more than 70%) but promotes the further growth of obtained plants.

     

References

  1. 1.
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  2. 2.
    Zatyko JM, Molnar I (1990) Adventitious root formation of chokeberry (Aronia melanocarpa Elliot) influenced by the pH of medium. Fruit Sci Rep 17(1):21–27Google Scholar
  3. 3.
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  4. 4.
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  5. 5.
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  8. 8.
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  9. 9.
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  10. 10.
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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Faculty of Biology and Agriculture, Department of Plant Physiology and BiotechnologyUniversity of RzeszówRzeszówPoland

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