Optimization of Hormonal Compositions of Media for In vitro Propagation of Apple (Malus ×domestica Borkh.) Cultivars

Examination of the effects of MS medium supplemented with various concentrations of indole-3-acetic acid (IAA) and kinetin revealed that the significantly highest shoot response was recorded for the ‘Princess’ cultivar with maximum shoot proliferation rate (65.25%), shoot number per explant (2.57), shoot length (7.28cm), and leaf number per shoot (6.15) after four weeks of culture. The root induction in micro shoots of three apple cultivars was observed after 20 days of culturing. The strength MS medium (full and half) containing 1.5 mg/L IAA significantly affected (at P<0.05, chi-square test) root induction in all three apple cultivars, especially rooting rate. However, there was no significant difference in root number and root length per micro shoot among the apple cultivars. Among the cultivars, significantly the highest rooting rate(48.30%), root number (6.25), and root length (4.15cm) were recorded for cultivar ‘Princess’ on full-strength MS medium.


INTRODUCTION
Traditionally, vegetative propagation methods (including cutting, budding, and layering) are time-consuming (about three years), with a low production rate. An additional consequence of these techniques is that the resulting germplasm cannot be guaranteed virus-and disease-free [1,2].
The tissue culture of the domesticated apple (Malus × domestica Borkh.) has a rich and extensive history spanning, approximately 60 years [3]. Micropropagation is a high-speed propagation method under aseptic conditions that has been effectively used for the production of thousands of healthy and disease-free apple cultivars in a limited space and short period [4]. The lack of a suitable regeneration system has been a major limitation to the production of transgenic plants. Therefore, the availability of a reliable regeneration system is a significant advance for the apple tree industry to allow it to utilize multiplication, genetic improvement, and cryopreservation processes. However, one of the challenges with in vitro regeneration of apples is its recalcitrant nature [3,4].
A consistent genetic background in a given cultivar can be maintained only by vegetative propagation, i.e., cloning since the apple genome is highly heterozygous. This would be important in the production of genetically uniform scions and rootstocks for commercial apple production [5]. Regeneration from the root under aseptic conditions is a biotechnological approach to producing large numbers of true-to-type, young plants in a short period of time without seasonal interruption. A root-based regeneration procedure is important because roots are: ideal organs to be preserved during cryopreservation, easy to culture and maintain, genetically stable over a long time, and produce true-to-type plants. A rapid in vitro vegetative propagation method offers an accelerated method of production of new tree fruit cultivars/rootstocks that may be extremely valuable for commercial and private nursery growers [6].
Apple has wider consumer acceptability in Ethiopia both as a fresh table fruit and value-added processed product. The scarcity of apple planting materials, the absence of large-scale apple orchards, and other factors hampered consumers' need for apple fruit despite the huge potential for apple production in Ethiopia. The productivity is very low compared to horticulturally advanced countries where orchards are established using apple trees grafted on dwarfing rootstocks predominantly. The main bottleneck for the adoption of highdensity plantations is the limited availability of clonally propagated dwarfing rootstocks. Therefore, the present study was planned to investigate the in vitro propagation of apple (Malus × domestica Borkh.) cultivars from nodal segments.

Sample Collection
The nodal segment explants were collected from three apple cultivars like 'Anna', 'Princess', and 'Dorsett Golden' from Girawa Research Site, Haramaya University, Ethiopia. The nodal segments close to the shoot tip were with sterile scissors at about 30cm including the meristematic shoot tip during the vegetative onset of the fall season (on 20 th October 2020). The actively growing shoot tips were used as the explant's sources for the successful organogenesis of the explants. The explant samples were brought to Plant Biotechnology Laboratory, Haramaya University, Ethiopia.

Explant Preparation and Surface Sterilization
The shoot tip nodal segment explants of mature apple cultivars were used as explant sources. Following the removal of leaves with sterile scissors, the shoot tip nodal segments 16 cm in length were washed with distilled water in 1000 ml beakers for 5 min. Then the explants were successively surface sterilized in 75% (v/v) ethanol (30 sec), and 0.1% (w/v) mercuric chloride with two drops of Tween-20per100mL of disinfectant solution(10min) in a laminar hood. To remove all traces of detergents and Tween-20 from the surface, the explants were rinsed in sterile double distilled water three times [7,8]. The exposed cut ends of the explants were trimmed off with sharp secateurs to eliminate all toxic effects of mercuric chloride. Four explants were used per treatment. All experiments were conducted in two replications.

Media Preparation
The Murashiege and Skoog (MS) medium was prepared as per the standard procedure [7]. The pH of the media was adjusted to 5.8 using 0.1NHCl and 0.1NNaOH, and finally, 0.8% (w/v)agar-agar (Merck, Germany) was added as a gelling agent. The prepared MS medium was autoclaved at 121°C and 2.2kpa pressure for 15-20min in 150mL Erlenmeyer flasks by dispensing 40mL of molten media into each flask plugged with aluminum foil. The sterilized medium was kept for two days before inoculation so as to check for possible contamination. The filter-sterilized vitamins and hormone supplements were poured into autoclaved MS basal medium.

Shoot Regeneration
The culture media for in vitro shoot induction consist of the basic salts and vitamins of basal MS medium at full strength supplemented with kinetin (KIN) at 1.0, 1.5, 2.0, and 3.0 mg/L; indole-3-acetic acid (IAA) at 0.5, 0.8, and 1.0 mg/L according to the protocol described by Dalal et al. [9] and James and Thurbon [10] with slight modifications in concentrations of PGRs. The media were also supplemented with 3.5%(w/v) sucrose and 8%(w/v) bactoagar. Ascorbic acid at 2g/L was used to minimize explants browning by adsorbing phenolics, in activating polyphenol oxidase and peroxidases. About 16 cm nodal segment explants were inoculated into culture jars containing 50 ml MS medium.
The cultured jars were sealed with non-absorbent cotton and placed in a growth chamber room at 26 ± 2°C, 16 h photoperiod, and high light intensity being 600 lux. The experiment was replicated twice. Two weeks later, the following parameters of shoot response were recorded-average number of shoots/explant, the average length of proliferated micro shoots/explant and the average number of leaves/micro shoots.

Rooting of Regenerated Shoots
The basic salts and vitamins of MS medium at full and half strength were used for rooting according to the procedure described by James and Thurbon [10] with slight modifications in concentrations of PGRs. At the end of the growing period, healthy regenerated shoots were excised, and transferred individually in glass jars (9 × 4.5 cm), each containing 30 ml basal medium supplemented with 0.2, 0.5, 1.0, 1.5 mg/L naphthalene-3 acetic acid (NAA), 3% (w/v) sucrose, and 1g/L bactoagar and cultured vertically. The pH of the rooting media was adjusted to 5.8 before the addition of agar. The culture jars were capped with aluminum foil and autoclaved at 121°C for 20 min, then left to cool and harden for five days before being used. The cultured jars were incubated at 25 ± 1C, with 16h photoperiod and high light intensity being 500 lux [2]. Each experiment was replicated two times. Data, including rooting rate, root number per plantlet, and root length per plantlet were recorded after five weeks of culturing.

Data Analysis
The data pertaining to shoot regeneration and rooting were subjected to one-way analysis of variance (ANOVA) and the differences among means were compared based on the least significance difference (LSD) test. Analyses were conducted using SAS software version 9. 2 [11].

Optimization of MS Medium for Shoot Proliferation of Apple Cultivars
The effects of MS medium supplemented with various concentrations of IAA and KIN on shoot proliferation rate, shoot number per explant, shoot length, and leaf number are shown in Table 1. The hormone-free basal MS medium was used as a control. In all experiments, no growth was observed on the basal MS medium. Significantly, the highest shoot response was recorded for cultivar 'Princess' with maximum shoot proliferation rate (65.25%), shoot number per explant (2.57), shoot length (7.28cm), leaf number per regenerated shoot (6.15) after four weeks of culture. The least shoot responses were recorded in cultivar 'Anna' with shoot proliferation rate (51.25%), shoot number per explant (2.15), shoot length (3.26 cm), and leaf number per shoot (4.40).
PGR combination of IAA (1.0mg/L) and KIN (3.0mg/L) was found to be the best for shoot proliferation. In the presence of IAA at 1.0mg/L, the shoot response was found to increase with an increase in KIN concentration. A similar study was conducted by Bhatt et al. [12] who reported that axillary shoot tips of M9 rootstock formed multiple shoots on MS medium containing 3mg/L 6-benzyladenine (BA), 2mg/L KIN, 3% (w/v) sucrose and 0.8% (w/v) agar-agar, with 85.7% of shoot apices forming multiple shoots. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4μM BA and.27μM thidiazuron(TZD) during the shoot multiplication phase, while 8.8 μM BA combined with 1.14µM TZD and 2.8µM gibberellic acid (GA 3 ) was found to perform the best during the shoot elongation phase for all genotypes [13].

Optimization of MS Medium for Root Initiation of Apple Cultivars
The root induction in micro-shoots of three apple cultivars was conducted by supplementing full or half-strength basal MS medium with IAA at different concentrations ( Table 2). Root induction was observed after 20 days of culturing. Significant differences in rooting rate, root number, and root length were observed among treatments. Significantly highest rooting rate (48.30%), root number (6.25), and root length (4.15cm) were recorded for the 'Princess' cultivar on full-strength MS medium.
The 'Dorset' cultivar displayed the lowest rooting rate (38.35), root number (4.10), and root length (2.45cm) on fullstrength MS media; and rooting rate (45.80), root number (4.34) and root length (3.90 cm) on half-strength MS media. As the concentration of IAA within the medium increases, the root response including root initiation rate, root number, and root length also increased with maximum values observed at 1.5 mg/L. No root induction was observed in the control medium (HF MS medium). It was also noticed that the root response increased as the concentration of the growth regulator increased from 0.2 to 1.5 mg/L. The half-strength MS medium was better for root induction than full strength MS medium.  For successful in vitro rooting, micorshoots were cultivated on basal MS medium supplemented with different IBA concentrations. Indeed, root development was apparent in the course of as brief as 5 days of exposure of microshoots to a rooting medium [14]. Afterroot initiation, the micro-shoots were re-implanted on half-strength MS medium for root development [14,15]. Renu et al. [14] reported the highest number of roots (7.66) as well as root length (22cm) in the medium containing 0.05μMNAA for the 'Golden Delicious' apple, while 'Red Fuji' showed no root induction in presence of NAA. On the other hand, successful in vitro propagation of apple rootstock 'G. 814' was achieved using 1 mg/L BAP at the multiplication stage and 1.5 mg/L IBA at the rooting phase [16].

CONCLUSION
The protocols developed in this study have the potential to be utilized for the generation of disease-free, quality planting materials and in crop improvement programs. PGR combination of IAA (1.0 mg/L) and kinetin (3.0 mg/L) was found to be the best for shoot proliferation of examined apple cultivars. The shoot responses were found to increase with an increase in kinetin concentration when it is combined with IAA at 1.00 mg/L. The root induction was observed after 20 days of culturing in all cultivars. Significant differences in rooting rate, root number, and root length were observed for all treatments. The comparison of the effect of full and half-strength MS medium on root induction at 1.5 mg/L IAA revealed significant differences (P < 0.05) in rooting rate among apple cultivars. Additional studies are required to identify the most efficient and cost-effective micropropagation protocol for studied apple cultivars.

AUTHORS' CONTRIBUTION
Zekeria Yusuf: initiation and design of the study, Lab experiment, data analysis; Bona Abdella: Lab experiment, data collection, and write-up of the document; Yohannes Petros: Analysis and interpretation of data. All authors contributed to the article and revised it critically for important intellectual content.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
The data will be available on request from the corresponding author [Z.Y].

FUNDING
This project was funded by a Haramaya University Research grant, under project code: HURG_2020_06_01_22.

CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or