EFFICIENT IN VITRO PROPAGATION OF VACCINIUM VITIS-IDAEA L. PLANTS ON THE DOUBLE PHASE MEDIUM
DOI:10.30825/5.EJPAU.160.2018.21.4

Marzena Mazurek, Aleksandra Siekierzyńska
Department of Plant Biotechnology and Physiology, Faculty of Biology and Agriculture, University of Rzeszow, Poland

ABSTRACT

The aim of the presented study was to investigate the impact of double phase medium in comparison with single phase medium on the growth and development of axillary and adventitious shoots in the lingonberry in vitro culture. Two-node explants were prepared from shoots collected from mother  in vitro cultures and were placed on single phase and double phase Zimmerman and Boome medium. For double phase medium, 5 ml of liquid medium was added. The composition of the liquid medium was the same as solid one minus agar. The liquid medium was added after positioning the explants horizontally.
   Shoots of axillary origin were used as explants. At the end of each of 3 passages, the number of axillary and adventitious shoots was determined. We demonstrated in presented research that the double phase medium significantly stimulated multiplication and elongation of axillary and adventitious shoots in comparison with single phase medium. Mean number of shoots was about twice higher for double phase medium. Application of double phase medium is a simple method which does not require special equipment. With a minimal increase of costs, it significantly increases the productivity of cultures. Therefore, it may be used successfully in micropropagation of lingonberry plants.

Key words: adventitious shoots, axillary shoots, lingonberry culture, micropropagation.

INTRODUCTION

The lingonberry (Vaccinium vitis-idaea L.) is woody, evergreen dwarf shrub distributed worldwide in northern temperate, boreal, and subarctic areas. This is a very popular fruit crop in many northern countries. The fruits of lingonberry are widely used for juice, sauce, preserves, candy, jelly, syrup, ice cream, pickles, wine, and liqueurs production [5, 20]. Other uses include extraction of arbutin from leaves to be used as a medicine for stomach disorders [20]. The fruits are rich in antioxidants and anthocyanins, therefore they are useful in medicine and industry [1, 19, 20]. Ogawa et al. [12, 13] proved that extract of lingonberry containing polyphenols in high amounts has a protective effect against UV and blue light-induced retinal photoreceptor cell damage mainly through inhibition of intracellular reactive oxygen production and activation of pro-apoptotic proteins.

The lingonberry plants are reproduced by seeds and rhizomes [20]. Cultivars of this crop are usually cloned by stem cuttings to maintain their genotype. However, such kind of propagation is very slow and inefficient [11]. Conventional propagation has some limitations as many genotypes do not positively respond to root inducing growth regulators [6, 7]. As an alternative, in vitro culture techniques allow rapid mass propagation of elite genotypes independent from seasonal influences [6, 11]. Many studies were focused on the optimization of micropropagation of Ericaceous plants [1, 2, 11, 14]. In general, the studies focused on improvement of shoot multiplication efficiency or elaboration of the method of axillary shoot development. The researchers were concerned on the effect of various cytokinins [1, 11, 14], auxins [9, 10], explants orientation [2], and cultivars [14]. Generally, solid media are used for micropropagation of plants. Some studies indicated that the double phase medium (2F) clearly improved the shoot proliferation and elongation in cultures of many plants species, like apple [8], conifer [15] and pineapple [17]. The 2F medium improved also multiplication rates, shoot quality, increased yield of axillary shoots (AX) and reduced vitrifications symptoms in comparison to a single phase medium (1F) in pears [16, 21].

These studies were focused mainly on regeneration potential of the plant species and did not investigate the occurrence of adventitious (AD) and axillary shoots (AX) of obtained regenerants [7, 14]. The occurrence of AD shoots was taken into consideration mainly in the case of cultures regenerated from leaf explants [11], but less in the case of cultures regenerated from shoots explants [3]. AD shoots may have an increased frequency of somaclonal variation [2]. To our best knowledge, the research on lingonberry micropropagation focused on the influence of double phase medium and its impact on the type of shoots formation were not been previously determined. Therefore the aim of the presented study was to investigate the impact of the 2F medium in comparison with the 1F medium on the development of lingonberry in vitro cultures and evaluation this technique in micropropagation procedure.

MATERIALS AND METHODS

Experiments were carried out on in vitro cultures of Vaccinium vitis-idaea L. 'Runo Bielawskie' (polish breeding clone). Two-node explants were prepared from shoots collected from mother in vitro cultures. The same type of explants originated from the middle part of shoots were used. Although the mother cultures consisted of axillary and adventitious shoots, only the shoots of axillary origin were used as explants. Therefore, fragments of shoots growing from axillary meristems were used. The Zimmerman and Boome medium [22] was supplemented with: glycine (2 mg dm^-3), pyridoxine (1 mg dm^-3), niacin (1 mg dm^-3), thiamine (2 mg dm^-3) and ascorbic acid (200 mg dm^-3), sucrose (25 mg dm^-3), fructose (5 mg dm^-3), L-cysteine (5 mg dm^-3),  6-γ,γ-dimethylallylamino purine (2iP) (10 mg dm^-3), indole-3-acetic acid (IAA) (4 mg dm^-3) and solidified with BIOCORP LAB-AGAR TM agar (8 mg dm^-3). The medium pH was adjusted to 5.0 before autoclaving. The 330 ml capacity glass jars were filled with 30 ml of medium for single phase and double phase medium as well. Then the glass jars were closed with transparent plastic ‘Twist-off” caps. They were autoclaved at 121ºC for 22 min. For double phase medium, 5 ml of previously autoclaved liquid medium was added. The composition of the liquid medium was the same as solid one minus agar. The liquid medium was added after horizontal positioning of the explants.

Every type of medium (1F and 2F) consisted of 5 repetitions (jars) with minimum 8 cultures individually. The cultures were grown for 8 weeks under two cool white fluorescent lamps (OSRAM) at approximately 47 µmol m^-2 s^-1 with a 16/8 hour day/night photoperiod. The temperature was set at 26°C. The cultures were grown through 3 successive passages. At the end of each passage, cultures were taken out from the jar and the number of axillary (AX) and adventitious (AD) shoots was determined. The AD shoots were shoots developed from callus.

Then the ratio of AX shoots was calculated according to following formula: 100% x number of AX shoots/ total number of shoots (both AX and AD ones). Within axillary shoots were distinguished: number of short axillary shoots (short AX; 3–14 mm length), number of long axillary shoots (long AX; > 15 mm length); total number of axillary shoots (short AX+ long AX) and the maximum length (cm) of axillary shoot (max AX). The AD shoots were analyzed similarly. The callus size in diameter (cm) was also measured. Statistical analysis

Collected data were subjected to an ANOVA, LSD mean separation test at α=0.05 using Statistica 9.0 computer software. Data presented as percentage were analysed after arcsin transformation (ratio of AX shoots in culture) or subjected to the test on the difference between two proportions (the number of viable explants, number of cultures developed exclusively AX shoots). The ratio of AX shoots was calculated according to following formula: 100 % × number of AX shoots/number of shoots (both AX and AD ones). Correlation for particular traits from single and double phase medium in subsequent passage was assessed using Spearman’s rank correlation.

RESULTS

The impact of studied media on the development of lingonberry in vitro cultures was clearly visible (Fig. 1). The growth on 2F medium was more intense than on 1F. On the other hand, the growth of callus in the case of the 2F medium was only slightly increased (Tab. 1). The differences in the shoot morphology or size and color of leaves and the vitrification of cultures were not observed (Fig. 1). The microbial contamination was also not observed under both conditions. The 2F medium much more than 1F medium stimulated growth and multiplication of AX and AD shoots, both short and long ones. Mean number of every kind of shoots was about twice higher on 2F medium in comparison with 1F medium (Tab. 1). Both proliferation and elongation of axillary (max AX) and adventitious (max AD) shoots on 2F medium was higher, but they were more distinct in the case of adventitious shoots. The analyses of every successive passage separately indicated that 2F medium also better-stimulated growth and multiplication of AX and AD shoots in comparison with single phase medium in particular passages (Tab. 2). Such phenomena (better growth of shoots) were observed in every passage although they were not statistically proven in the case of every analysed trait. The statistically significant differences proved better growth on 2F medium than on 1F were: long AX shoots in the first and last passage, short AX shoots in the third passage, long AD shoots in every passage and short AD shoots in the second passage. The maximum length of shoots was significantly higher on 2F medium in every passage in the case of AD shoots but for AX ones in the first and third passage (Tab. 2). The performed analysis indicated that 2F medium had relatively less impact on growth and development of callus in the second and third passage but not in the first (Tab. 2). Additionally, the number of viable explants on 2F medium was statistically higher than in case of 1F ones only in the first passage (Tab. 2). Obtained data indicated that 1F medium more stimulated growth and development of cultures consisted only of axillary shoots (Tab. 1). The Spearman’s rank correlation analysis indicated positive increasing correlation for each of obtained and analyzed traits in the case of subsequent passages for the 1F medium (Tab. 3). The statistically significant relationship indicated the positive relationship between number of long axillary shoots (long AX), the maximum length of axillary shoots (max AX) and callus size in every subsequent passage (Tab. 3). For the cultures growing on 2F medium, Spearman’s rank correlation analysis demonstrated also positive correlation for each of analysed traits in subsequent passages, except short adventitious shoots.

DISCUSSION

The aim of the presented study was to investigate the impact of the 2F medium in comparison with the 1F medium on the growth and development of axillary and adventitious shoots of lingonberry in vitro culture and evaluation such technique in micropropagation of this species. We showed that the growth of lingonberry cultures on 2F medium was much stronger than on 1F ones. The similar observation was obtained by Viseur [21] and Rodriguez et al., [16] in pear, by Litwińczuk [8] in apple rootstock, by Pullman and Skryabina [15] in conifer and by Scherwinski-Pereira et al., [17] in pineapple. Generally few studies were devoted to the impact of the kind of medium on the shoots type and the adventitious shoot development from shoots explants [3]. The phenomenon of occurrence of adventitious shoots is common in cultures of many species. Such shoots are suspected to be the main source of somaclonal variation [4]. In the presented study we demonstrated distinct differences in the proliferation and elongation of AD and AX shoots on the tested media. Both types of shoots grew more intensively on 2F medium than on 1F ones. However, the elongation and proliferation of AD shoots were relatively stronger than AX ones on 2F medium (Tab. 1). Therefore the risk of confusion of AX and AD shoots as well as the probability of somaclonal changes increases while the 2F medium is used. Therefore to efficient micropropagation of lingonberry plants using 2F medium requires to reduce growth and multiplication of AD shoots. The generally better growth of lingonberry cultures on 2F medium presented in this research might be a result of better culture nourishment. The observation made by Smith and Spomer [18] indicated that a liquid layer of 2F medium, through diffusion improvement, favors the uptake of medium components by growing cultures. This confirms the fact that in the case of 2F medium all explants started to grow while on 1F some of them (5%) died (Tab. 1). The greater number of long shoots obtained on the 2F medium can make this method useful in ex vitro rooting. Shoots that have not elongated sufficiently may root not efficiently, and consequently become more susceptible to death or have a decreased growth rate in the acclimation (authors observation, data not shown). Moreover, manipulation with too short explants, placing them in substratum is troublesome and may be harmful while auxins is used for rooting stimulation. Because of dissimilarity in growth and elongation of shoots in subsequent passages (Tab. 2) axillary shoots after third passage were observed as the longest and therefore will be convenient for ex vitro rooting. Application of double phase medium may be a simple method for the improvement in vitro shoot multiplication of Vaccinium vitis-idaea L. The optimization of lingonberry micropropagation in comparison to another Vaccinium species, was described only in a few studies.

The micropropagation of lingonberry plants is not efficient enough. Usage of double phase medium can increase micropropagation efficiency. The advantage of double phase medium is ease of preparation. Double phase medium requires preparing only one type of medium with the same ingredients and plant growth regulators as dedicated for 1F. This method does not require specialized equipment, what is an additional advantage. As a 2F medium stimulated stronger growth both AX and AD shoots than the normal solid medium it could be useful in mutagenesis, transformation and other breeding methods where the variability of the starting material is recommended.

CONCLUSIONS

1. The shoots growth of lingonberry cultures on 2F medium was usually stronger than on 1F ones, but it was more distinct in the case of adventitious shoots. 
2. This method significantly increases the productivity of cultures. Therefore it may be used successfully in micropropagation protocol of lingonberry plants.

Accepted for print: 26.09.2018

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