Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 5
Issue 2
Available Online: http://www.ejpau.media.pl/volume5/issue2/horticulture/art-06.html


Wojciech Litwińczuk



The aim of the present researchers was to find out solutions which can improve efficiency or/and save costs of chokeberry micropropagation.

Key words: chokeberry, Aronia, micropropagation, in vitro, double-phase medium, arginine, IBA.


The black chokeberry is a shrub native to North America although nowadays well-known in Poland. It bears fruits which are rich in macro- and micronutrients (Ca, Fe, Mo, Mn, Cu, B, J, Co), vitamins (P, C, B2, B6, PP, E, proA), saccharides, cellulose, pectins and anthocyanins. Therefore fruits of black chokeberry are used in food industry and pharmacy [6]. Chokeberries can be easily propagated by seeds but this method is not recommended, because obtained plants come late into bearing. They also grow too vigorously, are not uniform and then are not suitable for mechanical harvest [2]. Black chokeberry is comparatively young crop and only few cultivars or breeding strains 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 multiplication in vitro were so far carried out by several authors [1, 7, 8, 9, 10, 11]. Because of that the aim of the present researchers was to find out solutions which can improve efficiency or/and save costs of micropropagation. The genera Aronia and Malus belong to the subfamily Pomoideae. Thus, some solutions which gave beneficial results in micropropagation of apple rootstocks, among others: application of double-phase medium to stimulate shoot elongation and/or proliferation [3] as well as addition of arginine to enhance shoot rooting in vitro [5] were tested in Aronia culture.


Experiments were carried out on in vitro cultures of black chokeberry (Aronia melanocarpa Elliot) cv ‘Nero’ and breeding clone ‘Galicjanka’ (‘Albigowianka’).

Etiolated shoots of mother plants were surface sterilized and node explants were placed on double diluted MS medium [4] supplemented with MS vitamins, sucrose (20 g dm-3), glucose (5 g dm-3), fructose (5 g dm-3), polyvinylpyrrolidone (PVP 360, 100 mg dm-3), PPMTM (0.5 ml dm-3), 6-benzylaminopurine (BA, 0.5 mg dm-3), indole-3-butyric acid (IBA, 0.05 mg dm-3) and solidified with Kobe ITM agar (5 g dm-3). Cultures were grown in 16 h/8 h day/night photoperiod under cool-white light (OSRAM L36W/20) at 22.8 µmol·m-2·s-1 PPFD and 26±1°C temperature at each micropropagation phase.

During proliferation stage MS medium with 50% addition of Ca2+, Mg2+ and Fe2+ salts as well as sucrose (30 g dm-3), BA (1.0 mg dm-3), IBA (0.05 mg dm-3) and Kobe ITM agar (6 g dm-3) was used. The double-phase (‘2F’) medium was obtained by pouring the liquid MS solution (10 ml/jar) on the medium solidified with agar (50 ml/jar) at the beginning of subculture. Composition of both solid (‘1F’) and liquid medium was the same. At the proliferation stage the 300 ml capacity glass jars (5 per treatment) closed with transparent plastic ‘Twist-off” caps and filled with solid (50 ml) or double-phase medium (50 ml + 10 ml) were used. Twelve nodal explants 1 cm long were planted in each jar. The subculture lasted 5 weeks. Thereafter the fresh weight of culture was determined and the numbers of short and long shoots (length about 5-15 mm and over 15&nbs p;mm, respectively) were counted and the electric conductance (EC) and the acidity (pH) of medium were measured for each jar separately.

The double diluted MS medium with addition of sucrose (20 g dm-3), arginine, proline or casein hydrolysate (100 and 200 mg dm-3), IBA (0.05 mg dm-3) and solidified with Kobe I agar (6 g dm-3) was used for rooting shoots in vitro. The 100 ml capacity Erlenmayer flasks (5 per treatment) covered with aluminium foil and containing 20 ml of medium were used. Twenty healthy shoots 1.5 cm long were planted in each flask. The rooting passage lasted 3 weeks. Thereafter the number of rooted shoots was counted and magnitude of root system was evaluated.

At least 54 of healthy shoots about 2 cm long were dipped in water-ethanol (1:1, v/v) rooting solution of IBA (0, 1.5 and 3.0 mg dm-3) and put down to peat and perlite (1:1, pH = 6.0) mixture watered with fertilizer ‘Peters Starter’ solution (0.8 g dm-3) and sprayed with solution of ‘Florovit’ (1%), ‘Previcur’ (0.15%) and ‘Rowral’ (0.15%). They were grown at high air humidity in 16h/8h day/night photoperiod under sodium light at 64.4 µmol·m-2·s-1 PPFD and 21±3°C temperature. The same conditions were maintained for adaptation of shoots rooted previously in vitro.

The data were subjected to ANOVA. The means were compared by LSD multiple test at the P < 0.05 significance level. The samples consisted of 60 cultures in proliferation stage, 100 and 54 shoots in rooting in vitro and in vivo stages, respectively and at least 64 plants during adaptation ex vitro.


I. Initiation stage. Both Aronia clones readily adjusted to in vitro conditions and changed into rapidly growing cultures in second – third initation passage. From many other fruit species studied by the author, among others: sour and sweet cherries, apples, mulberries, strawberries, blueberries, actinidias, only blackberries had similar high adaptation potential (data not presented).

II. Proliferation stage. The distinct relationship between growth of cultures and kind of medium was found (fig. 1). The fresh shoot weight of both Aronia cultures grown on ‘2F’ medium was significantly higher than ‘1F’ control (tab. 1). The double-phase medium strongly stimulated shoot elongation whereas did not significantly influence shoot proliferation. The total number of shoots of single culture remained similar both on ‘2F’ and ‘1F’ medium (tab. 1). The differences between two chokeberry clones in the growth intensity was not proved. Contrary to observation made by Litwińczuk [3] on ‘M 26’, ‘MM 106’ and ‘P 14’ apple rootstocks which behaved variously while were grown on ‘2F’ medium the reaction of both Aronia clones on tested media was generally the same. The only exception was the elongation of shoots which was relatively more intense on double-phase medium in case of ‘Nero’ cultures compared to ‘Galicjanka’ ones (tab. 1). Differences in the acidity of the medium were not found (tab. 2) which may indicate that nutrient preferences of both clones were similar on solid and double-phase media. However, the conductance of double-phase media at the end of passage was lower than in the control (tab. 2). It suggests that a liquid layer of double-phase medium, through diffusion improvement, favours the uptake of medium components by growing cultures. Similar observations were made by Litwińczuk [3] on cultures of apple rootstocks. However, it should be mentioned that double-phase medium was 20% richer in nutrients than solid one. Thus the better growth of cultures on double-phase medium might be also a result of better culture nourishment.

Figure 1. Growth of two chokeberry clones: ‘Galicjanka’ (V) and ‘Nero’ (Y) on solid (1) and double-phase (2) medium

Table 1. Chokeberry culture growth depending on clone and type of medium


Culture fresh weight

Number of short shoots
[5-15 mm]

Number of long shoots
[> 15 mm]

Total number of shoots
[>5 mm]

Mean length of long shoot

Total length of long shoots

A. Clone


355.9 a1)
405.0 a

3.1 b
2.2 a

6.5 a
6.5 a

9.6 a
8.8 a

33.3 a
35.2 a

231.7 a
240.1 a

B. Medium


330.0 a
429.2 b

3.0 b
2.3 a

5.8 a
7.2 b

8.8 a
9.5 a

30.7 a
37.5 b

183.9 a
285.0 b

A x B. Combination: Clone x Medium

Nero 1F
Nero 2F
Galicj. 1F
Galicj. 2F

291.0 a
420.7 b
369.0 ab
436.7 b

3.5 b
2.7 ab
2.5 a
2.0 a

5.3 a
7.7 b
6.3 ab
6.7 ab

8.8 a
10.4 a
8.8 a
8.8 a

28.5 a
38.0 c
32.9 b
37.1 c

159.8 a
303.6 c
208.0 ab
268.5 bc

A x B interaction SL2)







1) means in columns marked with diverse letter are significantly different at α = 0.05
2) SL – level of significance

Table 2. Acidity and electric conductance of media at the end of subculture depending on clone and type of medium



EC [mS]

A. Clone


5.2 a
5.0 a

4.0 a
3.6 a

B. Medium


5.0 a
5.1 a

4.0 b
3.6 a

A x B. Combination: Clone x Medium

Nero 1F
Nero 2F
Galicj. 1F
Galicj. 2F

5.2 a
5.1 a
4.9 a
5.1 a

4.4 b
3.6 a
3.7 a
3.5 a

A x B interaction SL



III-IV. Shoot rooting and adaptation stages. For rooting shoots in vitro six kinds of media supplemented with different doses of two aminoacids and casein hydrolysate were tested. In most cases the rooting efficiency was very high (tab. 3, fig. 2). The worst result was obtained while casein hydrolysate at 200 mg dm-3 was used (tab. 3). Rooting percentages of other media tested were similar. However, the best elongation of roots was observed on medium with arginine 200 mg dm-3. Similar result was achieved by Orlikowska [5] for ‘P 60’ and ‘P 2’ apple rootstocks. The clone-specific reaction on tested media was not proved. In general shoots of ‘Galicjanka’ rooted significantly better than ‘Nero’ ones (tab. 3). The higher regeneration potential of ‘Galicjanka’ clone was also confirmed while shoots of both clones were rooted in non-sterile conditions (tab. 4). The IBA auxine treatment of shoots did not improve rooting efficiency in vivo. However, auxine promoted the growth of obtained plants (tab. 4).

Figure 2. Rooted in vitro shoots of ‘Galicjanka’ clone

Table 3. Chokeberry shoot rooting in vitro depending on clone and kind of applied medium


Number of rooted shoots [%]

Number of roots

Length of the longest root

A. Clone


80.0 a

3.9 a

0.9 a


89.0 b

4.3 a

1.0 a

B. Substance

arginine 100 mg dm-3

86.8 b

4.0 a

1.2 cd

arginine 200 mg dm-3

93.8 b

4.1 a

1.5 d

proline 100 mg dm-3

91.3 b

3.8 a

0.8 ab

proline 200 mg dm-3

91.3 b

4.8 a

0.9 ab

casein hydrolysate 100 mg dm-3

83.8 b

4.4 a

1.1 bc

casein hydrolysate 200 mg dm-3

67.5 a

3.7 a

0.6 a

A x B. Combination: Clone x Substance

N arginine 100 mg dm-3

90.0 bcd

3.7 ab

1.0 bcde

N arginine 200 mg dm-3

90.0 bcd

4.2 ab

1.6 f

N proline 100 mg dm-3

86.7 bcd

3.3 a

0.8 abc

N proline 200 mg dm-3

80.0 bcd

4.5 ab

0.6 ab

N casein hydrolysate 100 mg dm-3

76.7 abc

4.1 ab

1.1 cde

N casein hydrolysate 200 mg dm-3

56.7 a

3.7 ab

0.5 a

G arginine 100 mg dm-3

84.0 bcd

4.3 ab

1.3 ef

G arginine 200 mg dm-3

93.3 cd

3.8 ab

1.2 def

G proline 100 mg dm-3

94.0 cd

4.1 ab

0.8 abc

G proline 200 mg dm-3

98.0 d

4.9 b

1.0 bcde

G casein hydrolysate 100 mg dm-3

88.0 bcd

4.6 ab

1.0 bcde

G casein hydrolysate 200 mg dm-3

72.5 ab

3.9 ab

0.7 abc

A x B interaction SL




Table 4. Chokeberry shoot rooting in vitro depending on clone and IBA concentration


Number of rooted shoots [%]

Height of plant
after 7 weeks [mm]

Height of plant
after 18 weeks [mm]

A. Clone


68.5 a

24.9 a

59.2 a


77.0 b

25.2 a

59.3 a

B. IBA concentration

0.0 g dm-3

70.4 a

22.0 a


1.5 g dm-3

76.9 a

27.0 b

61.8 ab

3.0 g dm-3

68.5 a

26.1 b

60.5 b

A x B. Combination of Clone and IBA conc.

N 0.0 g/dm-3

67.6 ab

22.1 a

57.9 ab

N 1.5 g/dm-3

78.7 b

26.8 b

59.7 ab

N 3.0 g/dm-3

59.3 a

25.9 b

60.0 ab

G 0.0 g/dm-3

73.1 ab

21.7 a

52.8 a

G 1.5 g/dm-3

75.0 b

27.3 b

64.1 b

G 3.0 g/dm-3

77.8 b

26.3 b

60.8 b

A x B interaction SL




Table 5. Comparison of the efficiency two schemes of Aronia shoot rooting and adaptation


Stage III
Number of shoots rooted
in vitro [%]

Stage IV
Number of plants adapted in vivo [%]

Stages III × IV
Number of obtained plants [%]

Stage III + IV
Number of shoots rooted
in vivo [%]


80.0 a
89.0 b

50.7 a
57.5 a

40.6 a
51.2 b

68.5 a
77.0 b

Adaptation of ex vitro shoots rooted previously in vivo was unsatisfactory since their percentage did not exceed 60 (tab. 5). Probably the unknown mistake was commited because Staniene et al. [9] reached near 100% plant survival rate. Thus the efficiency of two separate stages of micropropagation (III. Rooting shoots in vitro and IV. Plant adaptation in vivo) was at about 20% lower than for the single combined stage (III+IV. Rooting shoots in vivo) (tab. 5). As the last technique is easier and cheaper it should be recommended in chokeberry micropropagation.


  1. Both Aronia clones readily adjusted to in vitro conditions.

  2. The double-phase medium strongly stimulated shoot elongation whereas did not significantly influence shoot proliferation. The reaction of both Aronia cultures on tested media was generally the same.

  3. The best result for shoot rooting in vitro was achieved on medium supplemented with arginine (200 mg dm-3).

  4. Shoots of ‘Galicjanka’ rooted significantly better than ‘Nero’ ones both in vitro and in vivo.

  5. The IBA treatment of shoots did not improve rooting efficiency in vivo but promoted the growth of obtained plants.

  6. Rooting shoots ex vitro should be recommended in chokeberry micropropagation.


  1. Brand M. H., Cullina W. G., 1992. Micropropagation of red and black chokeberry (Aronia spp.). HortScience 27, 1, 81.

  2. Kleparski J., 2000. Aronia “czarny koń” naszego sadownictwa. [Aronia – driving force in our horticulture]. Hasło Ogrodnicze 11, 22 [in Polish].

  3. Litwińczuk W., 2000. Efficiency of a double-phase medium in micropropagation of semi-dwarf apple rootstocks M.26, MM.106 and P.14. J. Fruit Ornamental Plant Res. VIII, 3-4, 97-106.

  4. Murashige T., Skoog A., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiologiae Plantarum 15, 473-497.

  5. Orlikowska T., 1992. Influence of arginine on in vitro rooting of dwarf apple rootstocks. Plant Cell, Tissue and Organ Culture 31, 9-14.

  6. Orłow Ł., 2000. Aronia – atrakcyjny krzew. [Aronia – attractive bush]. Sad Nowoczesny 10, 36-37 [in Polish].

  7. Petrovic D. M., Jacimovic-Plavsic M. M. 1992. Aronia melanocarpa and propagation in vitro. In vitro culture. XXIIIrd International Horticultural Congress, Florence, Italy, 30 August 1990. Acta Horticulturae 300, 133-135.

  8. Ruzic D., 1993. In vitro rooting and subsequent growth of black chokeberry (Aronia melanocarpa) plants ex vitro. J. Fruit Ornamental Plant Res. I, 1, 1-8.

  9. Staniene G., Stanys V., Bobinas C., Duchowski P., Merkys A., 1999. In vitro propagation of non-traditional horticultural plants (Actinidia, Chaenomeles, Aronia). Zesz. Probl. Post. Nauk Roln. 468, 441-443.

  10. Velchev V., Mladenova O., 1992. Root formation and adaptation of micropropagated Aronia shoots. Rastenievdni Nauki 29, 5-6, 79-83.

  11. Zatyko J. M., Molnar I., 1990. Adventitious root formation of chokeberry (Aronia melanocarpa Elliot) influenced by the pH of medium. Fruit Science Reports 17, 1, 21-27.

Wojciech Litwińczuk
Department of Plant Production
University of Rzeszów
2 Ćwiklińskiej St., 35-601 Rzeszów, Poland
tel. (+48 17) 857 42 55 ext. 341 or 219
e-mail: wlitw@univ.rzeszow.pl

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