Determination of Plant Regeneration Protocol of “Viking” Aronia Cultivar in the In Vitro Conditions

doi:10.21203/rs.3.rs-3004327/v1

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Determination of Plant Regeneration Protocol of “Viking” Aronia Cultivar in the In Vitro Conditions

https://doi.org/10.21203/rs.3.rs-3004327/v1

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In this study, in vitro micropropagation of 'Viking' aronia cultivar was investigated. For this purpose, different cytokinin and doses were tested in MS food medium for propagation medium. Except for cytokinins, low doses of IBA and GA were added and equal doses were used in all applications. For the rooting medium, different doses of IBA (0.125, 0.25, 0.5 and 1.0 mg l^-1) and activated charcoal (0.5 and 1.0 g l^-1) were tested in MS and ½MS mediums. A mixture of perlite and peat was preferred during acclimatization.

No plant death was encountered in the study, and 100% viability was detected in all applications. The highest number of shoots was detected in media containing TDZ; however, most of the shoots that can be used as micro cuttings were obtained from media containing BA and mT. The highest number of shoots occurred at 1.0 mg l^-1 doses of media containing BA and mT, and 16.83 and 21.76 shoots/explants were obtained, respectively.

100% rooting was achieved in all applications, including the control. The highest root number was detected in ½MS medium (10.17 roots/explant) containing 1.0 mg l^-1 IBA. Higher root number was obtained in ½MS medium compared to MS medium; However, it was determined that the root length decreased with the increase in the number of roots.

The acclimatization phase has resulted in high success, 98% of plants in ½MS medium and 92.4% of plants in MS medium survived.

aronia

in vitro

rooting

micropropagation

cytokinin

Aronia (Aronia melanocarpa) is a black, small-fruited, bush-formed, deciduous, perennial berry type of fruit originating from North America in the Maloideae sub-family of the Rosaceae family, the Aronia genus. Its green leaves turn red in autumn. A. melanocarpa plant is a species that can be sizeable to 90–180 cm, has a fruit diameter of 6–13 mm, and a fruit weight ranging from 0.5-2 g. Its fruits are in the form of clusters and there are 8 to 14 fruits in each cluster. Its mature plants yield 10–15 kg of fruit per plant (Kokotkiewicz et al. 2010; Engin, 2020). When the fruits are ripe, they can be harvested by hand or with a mechanical harvesting machine (Engin 2020). It is reported that it consists of three species: A. melanocarpa Elliot (black aronia), A. butifolia (red aronia) and A. prunifolia (purple aronia) (Hardin 1973). A. prunifolia is considered by many to be an inter species hybrid between A. arbutifolia and A. melanocarpa (Brand 2010). Recently, A. mitschurinii has been identified as a fourth Aronia species, a large-fruited species cultivated in orchards (Leonard et al. 2013; Brand 2016).

Aronia is a type of fruit that has a high tolerance to various environmental conditions and can be grown in a wide variety of climates (Rusea et al. 2019). Aronia is not drought tolerant. On the other hand, it can withstand temperatures below − 30°C (Engin 2020).

Aronia, besides being an ornamental plant, with preventive and curative properties diseases an important plant species with great potential as a new fruit species. Aronia, besides being an ornamental plant, with preventive and curative properties diseases, an important plant species with great potential as a new fruit species. as well as evaluating as an ornamental shrub, Aronia melanocarpa is the decorative fall coloration that makes it very popular (Hirvi and Honkanen 1985). They are also plants that require very little landscape maintenance (Brand 2016; Pırlak and Almokar 2018). It is a valuable landscape plant and small-grained fruit species that is becoming increasingly popular due to its high nutritional value and medicinal properties (Valcheva-Kuzmanova and Belcheva 2006; Kulling and Rawel 2008; Walther and Schnell 2009; Kokotkiewicz et al. 2010). There are aronia varieties with superior yield and fruit quality, and its cultivation is spreading all over the world (Rusea et al. 2019).

Aronia berries have higher anthocyanin content and antioxidant capacity compared to other fruits. The antioxidant effect of its berries is much greater than that of cranberries, elder-berries and blackberries, or even blackcurrants (Litwińczuk 2012). Its fruits are rich in vitamin, mineral and folic acid. Aronia has a healing effect on metabolic diseases due to its rich bioactive compounds. Its positive effects on diabetes, cardiovascular diseases, urinary tract infections, eye inflammation and diseases, and various viral diseases have been revealed as a result of studies conducted for many years. In addition, it has an antiproliferative effect on some cancer cells (Engin 2020).

Its black colored fruits are consumed fresh when fully ripe. In addition to its fresh consumption, it is widely used in the production of fruit juice, dried fruit, sauce, jam, sherbet, ice cream, cake and as a food supplement in the food industry (Engin 2020). Black aronia berries are used in the food industry and pharmaceutics (Litwińczuk 2012). Commercially, aronia is mainly used for juice. Apart from this, there are also uses such as food coloring, tea, syrup and fruit colorant. In Russia, apple and aronia juices are combined-mixed and fermented to produce red wine. In Europe the fruit juice, often by mixing with apple juice, gives a blush to the juice (Smith and Ringenberg 2003; Pırlak and Almokar 2018).

Aronia is a relatively new type of fruit in Turkey, but the growing demand for the fruit is encouraging many farmers to grow aronia. A fast and reliable clonal propagation protocol is needed to obtain sufficient seedlings from commercially important aronia cultivars to meet the demands of growers (Rusea et al. 2019).

Aronia can be easily propagated by seed. However, this method is not recommended due to genetic variation, late maturation of plants, vigorous and non-homogeneous growth, and plants not suitable for mechanical harvesting (Litwińczuk 2012). Except this traditionally, aronia plants can also be propagated by rooting green cuttings and wood cuttings (Rusea et al. 2019). The use of biotechnological methods such as micropropagation, other than traditional methods, provides high quality, disease-free and homogeneous growing plants. Thanks to modern and advanced developments in biotechnology, it has a great importance in the production of commercial plants, especially in the propagation of economically important plants. Biotechnology tools such as micropropagation and in vitro culture offer a valuable alternative in combating viruses and managing genetic resources. In vitro micropropagation often represents a fast, economical and safe technique for propagating valuable species and varieties of trees and shrubs (Şuţan et al. 2017). The easiest way to propagate is to use in vitro techniques, following micropropagation technology (Pırlak and Almokar 2018).

Plants obtained by micropropagation are mostly free of disease factors. Apart from that, micropropagation is the fastest clonal propagation method, the demands of the market can be met year-round with micro propagation, it is possible to produce continuously because it is independent of environmental conditions, and high number of plants can be obtained in a short time thanks to its high propagation coefficient (Nas 2021).

Black aronia is a relatively new crop and only a few cultivars or propagation clones are known and bred. Because micropropagation is much more efficient than other traditional cloning methods, rapid reproduction of new, valuable species of aronia must be developed. Few studies have so far been conducted on the in vitro propagation of aronia (Litwińczuk 2012; Rusea et al. 2019).

Being can done Aronia cultivation easily, being a very productive species, being can grown in different soil types, not to be damaged by late spring frosts due to late blooming, besides having many advantages such as resistance to diseases and pests, considering the importance of the its fruits in human nutrition, its positive effect on metabolic diseases and its features such as having a wide usage network and having a wide usage network, it is seen that aronia is one step ahead of many other fruit types (Engin 2020).

A fast and reliable clonal propagation protocol is needed to obtain sufficient seedlings from the commercially important the 'Viking' aronia variety to meet the growing demands of growers for aronia fruit. The main purpose of this study is to develop an in vitro clonal propagation protocol for the 'Viking' aronia variety.

2.1. Materiel

This study was carried out in the Plant Biotechnology Laboratory of the Department of Horticulture, Faculty of Agriculture, Selcuk University. In the study, unerupted axillary buds on cuttings taken from the “Viking” aronia cultivar seedlings were used as material.

2.2. Method

2.2.1. Surface Sterilization of Plant Material (explant)

Cuttings taken from the mother plant were rinsed in the laboratory environment under flowing tap water for half an hour. Then, with the help of pruning shears, the cuttings were divided into explants with one node in each explant and subjected to surface sterilization. Surface sterilization of the shoots was carried out in a sterile cabinet. For surface sterilization, the explants were first kept in 70% ethyl alcohol (EtOH) for about 3 minutes and then rinsed once with sterile distilled water. Afterwards, the explants were kept in a 15% commercial bleach solution containing 10 drops of Tween-20 per liter with occasional mixing for 20 minutes and then rinsed with distilled water three times.

2.2.2. In Vitro Propagation of Plant Material (explant) (Beginning Phase)

After surface sterilization and rinsing, the explants were cultured in the MS (Murashige ve Skoog, 1962) media that containing 1.0 mg l^− 1 Benzyladenine (BA) + 1.0 mg l^− 1 Gibberellic acid (GA₃) + 30 g l^− 1 sucrose and gelled with 7.4 g l^− 1 agar. After being in the in vitro medium, the cultures were daily controlled explant deaths, darkening, and microbial contamination occurring by controlling. After three weeks, explants free from microbial contamination and appearing healthy were subcultured. To ensure the continuation of the shoot culture, the newly obtained shoot clusters separating into individual shoots and the fresh shoots were transferred to the subculture medium (Fig. 2.1,2.2). After three subcultures, enough explants were obtained that could be used in the experiment.

2.2.3. Determination of the Effect of Different Cytokinin Sources and Doses on Propagation

To determine the culture medium that will provide the best micropropagation coefficient, shoot tip explants taken over 1.5-2.0 cm from plantlets cultured in the MS medium, cultured in the MS medium containing different cytokinin (Benziladenin, meta-Topolin, Kinetin, Tidiazuron) and its doses (0.5, 1.0 mg l^− 1), 0.01 mg l^− 1 IBA, 0.25 mg l^− 1 GA₃ ve 7.4 g l^− 1 agar. After the culture, the plants were checked from time to time and at the end of three weeks, the best culture medium that would provide the in vitro micro-propagation of the “Viking” aronia cultivar was tried to be determined.

In the study, the effects of the treatments made to examine the best growth (survival rate, shoot number per explant and shoot length) were examined with 3 replications and 15 plants in each replication.

Treatments made are as follows:

1- MS+ 0.5 mg l^-1 BA + 0.01 mg l^-1 IBA + 0.25 mg l^-1GA₃

2- MS+ 1.0 mg l^-1 BA + 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

3- MS+ 0.5 mg l^-1 mT+ 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

4- MS+ 1.0 mg l^-1 mT+ 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

5- MS+ 0.5 mg l^-1 Kin. + 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

6- MS+ 1.0 mg l^-1 Kin. + 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

7- MS+ 0.5 mg l^-1 TDZ + 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

8- MS+ 1.0 mg l^-1 TDZ + 0.01 mg l^-1 IBA+ 0.25 mg l^-1GA₃

2.2.5. Acclimatization of Rooted Plantlets

The plants propagated in vitro are more susceptible than plants grown outside. That's why they show drying when they are transfer to outside directly. To solve this problem, the rooted micro-shoots need to be gradually its acclimatization. This process was carried out according to Nas and Read (2004). The roots of the micro-shoots rooted as a result of the 30-day rooting experiment were washed under running tap water without damaging the roots to clean them from the nutrient medium. After root washing, the rooted plantlets were transferred to pet cups filled with a mixture of peat and perlite. In order to prevent the loss of the relative humidity of the environment, which will cause the plantlets to dry, the top of the pet glasses was closed with a transparent pet glass. Holes were drilled on the plastic cups after the second week for the plants placed in the climate room (24 ± 1°C, 16 /8 (light/dark)) to ripe. In this way, the relative humidity in the environment was reduced gradually, and after about two weeks, the pet glass was completely removed (Fig. 2.3).

The plants planted in plastic cups were kept in the laboratory for about two months and left in the greenhouse after reaching a length of 20–25 cm. After 2–3 weeks of acclimatization, the plants were planted in pots and left to grow outside environment (Fig. 2.4).

During the study, survival rate, number of shoot per explant, shoot length, rooting rate, root number and root length measurements were recorded. The findings obtained as a result of the study are given below.

The MS was used as a medium. The efficiency of the MS medium for the in vitro culture of Aronia has been emphasized in many published reports (Sivanesan et al., 2016; Borsai et al., 2017; Rusea et al., 2018; Imrak et al., 2021). The use of Woody Plant Medium (WPM), even if it becomes frequent in woody plants, including aronia (Kwak et al., 2015; Cheng, 2017), the MS medium is a selection with reliable results and is supported by many people. Writers such as Brand and Cullina (1990) reported that both the MS medium and WPM medium support the proliferation of strong shoot in A. Arbutifolia and A. Melanocarpa.

In this study, for the purposes of the surface sterilization, after the explants were first kept in 70% ethyl alcohol (ETOH) for about 3 minutes, which was rinsed once with sterile pure water. The explants then gave good results for decontamination as a result of rinsing with pure water three times after being mixed with a 15% commercial bleach solution, which contains 10 drops Tween-20 per liter for 20 minutes. Imrak ve ark. (2021) in surface sterilization, different doses of sodium hypochlorite (5%, 10%, 20%) in their study that tried argued that 10% sodium hypochlorite application for 10 minutes gives the best results for decontamination. Carried out the studies, the types and doses of cytokine used in the propagation of shoot differ between the plant species (Nas, 2021). The in vitro response of the axillary buds of the "Viking" variety has been significantly affected by both growth regulators and concentration. In the study, the highest number of shoots was detected in media containing TDZ; however, most of the shoots that can be used as micro-cutting was obtained from media containing BA and mT. With the increase in the number of shoot, the decrease in shoot lengths was observed. Brand and Cullina (1992) in different doses of BA (0.0, 0.4, 2.2, 4.4, 11.1 µM) in their study that used, most of the shoots that can be used as a micro-cutting have obtained from media containing 2.2 or 4.4 µM BA. Pırlak and Almokar (2018) obtained the highest shoot number from MS medium containing growth regulators at different concentrations (2.0 mg l^− 1 BA + 0.01 mg l^− 1 IAA + 0.1 mg l^− 1 GA₃). Polat and Eskimez (2022) best results in terms of number of plants per explant (4.56 numbers/explant), exile length (20.25 mm/explant) and knuckle number (4.72 points/explant) measured a medium containing MS + 1 mg l^− 1 BAP + 0,02 mg l^− 1 IBA + 1 mg l^− 1 GA₃ combination.

In the sources of all cytokines (BA, mT, Kin, TDZ), a dose of 1.0 mg l^− 1 has created a higher shoot than 0.5 mg l^− 1. On the contrary, the shoot lengths at a dose of 0.5 mg l^− 1 were higher than 1.0 mg l^− 1. As a result, the highest number of shoot was found in an medium containing 1.0 mg l^− 1 TDZ (31.88 units/explant) and the lowest number of shoots was found in medium containing 0.5 mg l^− 1 kinetin (1.78 units /explant). In the shoot length, the opposite result was obtained, the highest, medium containing 0.5 mg l^− 1 Kinetin (2.93 cm) and the lowest, was measured in a medium containing 1.0 mg l^− 1 TDZ (0.37 cm). The Benziladenin (BA) and the meta-Topolin (mT) showed similar reactions in terms of both the number of shoot and the length of the shoot (Table 3.1).

Table 3.1

During the propagation phase, the effect of different cytokinin and doses on the survival, number of shoot per explant and shoot length of the ‘Viking’ aronia cultivar
Type And Doses of Cytokinin (mg/l)	survival rate (%)	Number of shoot per explant	Shoot length (cm)
0.5 BA + 0.01 IBA + 0.25 GA₃	100.0 ^ns	10,70 e	2,44 b
1.0 BA + 0.01 IBA + 0.25 GA₃	100.0	16,83 d	1,78 c
0.5 mT + 0.01 IBA + 0.25 GA₃	100.0	9,67 e	2,44 b
1.0 mT + 0.01 IBA + 0.25 GA₃	100.0	21,76 c	1,84 c
0.5 Kin. + 0.01 IBA + 0.25 GA₃	100.0	1,78 f	2,93 a
1.0 Kin. + 0.01 IBA + 0.25 GA₃	100.0	2,65 f	2,01 c
0.5 TDZ + 0.01 IBA + 0.25 GA₃	100.0	28,90 b	0,40 d
1.0 TDZ + 0.01 IBA + 0.25 GA₃	100.0	31,88 a	0,37 d
LSD (%5)	0	1,20	0,31

Means in each column followed by different letters are different according to Duncan’s multiple range test (P < 0.05).

ns: not significant

In vitro rooting is the most important step in achieving success in micropropagation. In the woody plants such as fruit trees, the stage that most negatively affects the success of micropropagation is either the absence or insufficient of rooting. Rooting ability varies significantly with the salt strength of the medium and the IBA concentration (Nas, 2021). In our study, shoots in the ½MS medium had higher root numbers than those cultured in the full MS medium (except ½MS medium containing 1.0 g l^− 1 activated carbon) and increased root number with increasing IBA dose. Kwak et al. (2015) obtained the highest rooting rate from ½WPM (8.8 root/explant) containing 3.0 mg l^− 1 IBA. Sivanesan et al. (2016) obtained the highest rooted shoot rate (100%), the average root number per shoot (10.6) and the average root length (5.4 cm) from the ½MS medium fortified with 1.0 mg l^− 1 IBA after 30 days of culture. Irmak et al. (2021) obtained the best results for rooting from the ½MS plant medium combined with 2 mg l^− 1 IBA + 0.5 mg l^− 1 NAA. Polat and Eskimez (2022) reported that the best rooting medium was the medium containing the ½MS + 1 mg l^− 1 IBA. In this study, 100% rooting was achieved in all treatments, including the control. The use of IBA was found to be more effective than activated carbon in terms of root number. A higher number of roots were formed at all IBA doses than in the control. The number of roots formed in the control was found to be higher than the media containing activated carbon. A gradual increase in root number was detected with increasing IBA dose. In addition, as in the shoot, a decrease in root length was determined with an increase in the number of roots (Table 3.2).

The highest root number was determined in the ½MS medium (10.17 roots/explant) containing 1.0 mg l^− 1 IBA, and the lowest root number was determined in the MS medium (2.47 roots/explant) containing 0.5 g l^− 1 activated carbon. In the root length, higher values were noted in the MS medium in relation to the root number. Fewer roots were formed in the MS medium, and accordingly, the elongation of the formed roots was higher. The highest root length was observed in the MS medium containing 0.5 g l^− 1 activated carbon (7.27 cm), and the lowest root length was observed in the ½ MS medium containing 0.5 mg l^− 1 IBA (Table 3.2).

Table 3.2

Effect of Medium Density and Concentration of IBA and Activated Carbon on rooting
	Rooting Rate (%)		Root number		Root length (cm)
IBA and Activated Carbon Concentration	MS	½MS	MS	½MS	MS	½MS
0.0 mg l^− 1 IBA (Control)	100 ^ns	100 ^ns	3,97 d	4,07 d	4,18 b	3,23 b
0.125 mg l^− 1 IBA	100	100	4,30 cd	6,14 c	3,72 bc	2,87 bc
0.25 mg l^− 1 IBA	100	100	5,50 b	9,43 b	3,11 bc	2,20 cd
0.5 mg l^− 1 IBA	100	100	4,73 c	9,50 b	3,33 bc	1,73 d
1.0 mg l^− 1 IBA	100	100	7,73 a	10,17 a	2,21 c	2,15 cd
0.5 g l^− 1 activated carbon	100	100	2,47 f	3,13 e	7,27 a	5,81 a
1.0 g l^− 1 activated carbon	100	100	3,20 e	2,73 e	6,17 a	6,25 a
LSD (%5)	0	0	0,53	0,66	1,41	0,75

Means in each column followed by different letters are different according to Duncan’s multiple range test (P < 0.05).

ns: not significant

The final stage, acclimatization, resulted in high success, very insignificant number of plant deaths occurred. 98% of plants in the ½MS medium and 92.4% of plants in the MS medium survived. After the plants in the glass were planted in the pots, all the plants continued to grow healthily (Fig. 3.1).

We determined that every step of the in vitro micropropagation of aronia is easy. Litwińczuk (2012) reported that micropropagation was much more effective than traditional propagation methods such as seeds, dipping and cutting in the in vitro micropropagation work carried out by using axillary shoots of aronia.

Acknowledgments

This work was supported by Selcuk University BAP (Scientific Research Project) (22401040).

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Determination of Plant Regeneration Protocol of “Viking” Aronia Cultivar in the In Vitro Conditions

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Abstract

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1. INTRODUCTION

2. MATERIAL AND METHOD

2.1. Materiel

2.2. Method

2.2.1. Surface Sterilization of Plant Material (explant)

2.2.2. In Vitro Propagation of Plant Material (explant) (Beginning Phase)

2.2.3. Determination of the Effect of Different Cytokinin Sources and Doses on Propagation

2.2.5. Acclimatization of Rooted Plantlets

3. CONCLUSIONS AND DISCUSSION

Declarations

References

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