PROPAGATION OF BLACK CHOKEBERRY (ARONIA MELANOCARPA ELLIOT) THROUGH IN VITRO CULTURE

Wojciech Litwińczuk

ABSTRACT

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.

INTRODUCTION

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.

MATERIALS AND METHODS

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), PPM^TM (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 I^TM 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 ľmolˇm^-2·s^-1 PPFD and 26ą1°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 I^TM 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 ľmolˇm^-2·s^-1 PPFD and 21ą3°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.

RESULTS AND DISCUSSION

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.

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).

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.

CONCLUSIONS

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.

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