Abstract
The purpose of this study was to improve the protocol of clonal micropropagation for effective mass production of the Zarya Alatau apple cultivar through the use of axillary buds. In Kazakhstan’s challenging climate, the Zarya Alatau apple thrives because of its unique traits, including fruit preservation until May, cold hardiness, and resistance to scab and powdery mildew. Micropropagation is essential for healthy mother tree establishment, and this research focused on key factors for successful in vitro propagation. The sterilization of explants was optimized: 1.6% solution of sodium hypochlorite effectively sterilized plant materials for 10 minutes. Nutrient media composition was evaluated for efficient shoot regeneration. The study examined axillary bud regeneration on Murashige and Skoog medium with different concentrations of hormones. A combination of 6-benzylaminopurine (0.5 mg/L) and gibberellic acid (0.5 mg/L) yielded optimal results, with shoots reaching 3.5 cm. Root induction was analyzed with varying indole-3-acetic acid (IAA) concentrations, and the best results were achieved with 1.5 mg/L IAA, resulting in an 85% rooting frequency. Adapting in vitro plants to ex vitro conditions is crucial given their sensitivity to environmental changes. Well-developed leaves and a robust root system are essential for successful acclimatization during transplantation into a soil substrate. This research provides valuable insights into the critical parameters for a successful transition of in vitro propagated plants to soil conditions, optimizing micropropagation practices.
Zaria Alatau is an apple of the Malus domestica cultivar developed by the Kazakh Research Institute of Fruit Growing and Viticulture. Zaria Alatau was obtained from seedlings of free pollinated Renet Orleans cultivar. It has been included in the State Register of the Republic of Kazakhstan since 1974. Zaria Alatau is classified as a late-winter apple cultivar and has favorable economic characteristics. It exhibits early entry into the fruiting stage, along with good resistance to scab, rot, and canker. Additionally, it has a high yield. The fruit of Zaria Alatau is characterized by a greenish-yellow color with a slight orange blush. Numerous subcutaneous dots are present across the fruit surface. The fruits of Zaria Alatau have a sweet-and-sour taste, are juicy, and possess a pleasant aroma. They are suitable for long-term storage and are commonly used in desserts due to their high pectin content (Sadovnik Ingo 2023).
Dobranszki and Teixeira da Silva (2010) noted that the tissue culture of apple has a rich historical background that has extended over a period of ∼60 years. The pioneering studies conducted by Elliott (1972) and Walkey (1972) marked the first instances of in vitro apple seedling production. Since then, numerous rootstock and graft genotypes have been successfully micropropagated. The micropropagation process is subject to multiple influencing factors, including genetic background of the tree, the type of tissue being used as an explant, the concentration of growth regulators, the environmental conditions during cultivation, the sterilization techniques, and the composition of the growth medium (Magyar-Tábori et al. 2011; Teixeira da Silva et al. 2019).
In recent years, several researchers have discovered suitable procedures for the micropropagation of apple trees (Duguma and Zakaria 2022; Shi et al. 2021). However, these protocols may not always be universally applicable to other apple genotypes due to varietal specificity. The reproducibility of these protocols in achieving optimal results remains uncertain.
In Kazakhstan, special attention was paid to the rational use of a unique variety of promising and local varieties not only to increase the production of fruits and berries but also to create new stable and high-yielding varieties of fruit crops on their foundation. In recent years, many farmers have begun to switch to growing local varieties of apples. The Zarya Alatau apple is a promising and beloved winter cultivar for many gardeners; it is highly decorative and has unique taste properties. Unfortunately, statistics show that it has become rare, and now is threatened with extinction. The purpose of this study was to find solutions that will help avoid the extinction of this species.
Materials and methods
Initiation of in vitro culture
In Spring 2022, shoots with axillary buds were collected from a 5-year-old Zarya Alatau apple trees from the orchard of the Kazakh Scientific Research Institute of Horticulture. The shoots (1.5 cm in length) were subjected to a thorough washing process. They were first washed in 1% a soapy solution for 30 min, followed by rinsing under running tap water for an additional 30 min. Next, they were immersed in a 3% hydrogen peroxide (local manufacturer, Kazakhstan) solution for 5 min, sterilized with 70% C2H5OH (local manufacturer, Kazakhstan) for 2 min and disinfected with varying concentrations of sodium hypochlorite (household bleach Fay, 000981; Kazakhstan) (1%, 1.6%, and 2.5%) for different durations (5, 10, and 15 min). Subsequently, the shoots were washed five times in sterile distilled water containing 10 mg/L of the antibiotic ceftriaxone (Chempharm, J01DD04), with each washing lasting for 5 min. Sterilized shoots with axillary buds were placed in test tubes (200 × 24 mm) containing Murashige and Skuga (MS) (M519, PhytoTech Laboratories, Lenexa, KS, USA) liquid basal medium with vitamins 4.4 g/L supplemented with 30 g/L sucrose (S391, PhytoTech Laboratories), 0.01 mg/L indole-3-butyric acid (IBA) (I538, PhytoTech Laboratories), 1 mg/L of 6-benzylaminopurine (BAP) (B3408-1G, Sigma, St. Louia, MO, USA) and 1 mg/L gibberellic acid (GA) (G500, PhytoTech Laboratories). The pH of the medium was adjusted to 5.6. It is known that Malus plants contain a high concentration of polyphenols (Richard and Tony 2011). When introducing apple shoots into in vitro culture, phenolic compounds are released into the nutrient solution, which can lead to plant death. To prevent necrosis, axillary buds were transferred to a fresh liquid medium once a day.
After 7 to 10 d, viable aseptic apple shoots ready for transplantation into tubes in a solid nutrient medium MS, supplemented with 30 g/L sucrose, 6 g/L agar (A111, PhytoTech Laboratories), 0.01 mg/L IBA, and different concentration of BAP (0.5, 1, and 2 mg/L) and GA (0.1 and 0.5 mg/L). The plants were grown at 23 to 25 °C under 3000 K LED laps with light intensity 41 to 42 μmol/m2/sec and a 16-h photoperiod. Subcultures were performed every 5 weeks. The multiplication coefficient was calculated based on the number of shoots obtained from each explant. Number of shoots, roots, and their length were recorded weekly and expressed as the mean value.
In vitro rooting and ex vitro adaptation
Plants measuring 3 to 5 cm in height were cultivated for rooting on half strength (2.2 g/L) MS with vitamins supplemented with 30 g/L sucrose, 6 g/L agar, various concentrations of indole-3-acetic acid (IAA) (1, 1.5, and 3 mg/L) and adjusted to pH 5.6. The effect of auxin on the formation of roots was evaluated after 30 d of cultivation.
Rooted plants with a height of 10 cm and 5 to 10 roots were transferred to pots (500 mL in volume) filled with a substrate (pH 5.5 to 7.0) containing a mixture of upland and lowland peat, river sand, and nitrogen (150 to 350 mg/L), P2O5 (30 mg/L), K2O (300 to 450 mg/L) (local garden shop, Kazakhstan). Before planting, the roots were washed with running water to remove any adherent substances. Plastic cups were used to cover the pots, maintaining a high humidity level for 2 weeks. After 7 d, the plastic cups were partially removed, and the plants were sprayed with water until new leaves emerged. After 2 weeks, the plastic cups were completely removed, and the plants were watered twice a week.
Data analysis
All experiments were repeated at different times, and three replicated cultures were used for each treatment. Observations on the number of branches, the number of roots and the length were recorded at weekly intervals. Analysis of variance (ANOVA) and the Duncan multiple difference test were performed in SPSS version 24 (IBM SPSS Statistics, Armonk, NY, USA).
Results
Sterilization of the material
The level of contamination in plant material was influenced by the concentration and duration of immersion in the household bleach. Higher concentrations and longer immersion times led to lower levels of contamination. The measurement of contamination was calculated as the percentage of contaminated, nonsterile explants relative to the total number of explants introduced into sterile culture. The formula used was as follows: Contamination Rate (%) = (Number of Contaminated Explants/Total Number of Explants) × 100. Treatment of explants with 1.6% of sodium hypochlorite for 10 min yielded satisfactory results, ensuring low contamination rate without causing harm to the explants (Table 1).
Rate of contamination of apple explants depending on the concentration of sodium hypochlorite and the soaking time.
The introduction of plants into aseptic conditions was a critical step in the process of micropropagation. Various factors to be considered when disinfecting plant material collected from the field for use as explants in vitro included the plant’s type, variety, and genotype, as well as the type of explant, the age and physiological state of the donor, and the specific disinfection procedure used (Silva et al. 2015; Teixeira da Silva et al. 2016).
Various chemical elements were used for the sterilization of plant raw materials, such as alcohol, petroleum ether, calcium hypochlorite, sodium hypochlorite, mercury chloride, and acetone. The specific concentrations and duration of immersion varied depending on the type of plant material. Mereti et al. (2002) achieved successful sterilization of strawberry plants by immersing plant tissues in a solution of sodium hypochlorite (1.5%) for 10 min. Carelli et al. (2002) demonstrated that satisfactory results can be obtained by immersing Rosa hybrida plant tissues in sodium hypochlorite (5%) for 20 min. Consequently, selecting appropriate methods and disinfectants for the initiation of plants in vitro is a crucial step in the micropropagation process. Numerous scientists successfully used various concentrations of HgCl2 for disinfection, as well as chlorine-containing bleach (Romadanova et al. 2017; Volgina et al. 1997).
Shoot multiplication
The release of phenolic compounds in the nutrient medium is a commonly observed phenomenon during the micropropagation of Malus plants. In this study, during the initial stages of cultivation, plants were transferred into test tubes containing a liquid MS medium daily for a period of 1 week to prevent tissue darkening caused by phenols. Filter paper bridges were used to hold the explants in the tubes (Fig. 1).
Introduction of Zarya Alatau apple cultivar’s explants into a liquid Murashige–Skoog medium with filter paper.
Citation: HortTechnology 34, 5; 10.21273/HORTTECH05464-24
The efficiency of the MS media composition was evaluated during the cultivation stage by determining the percentage of shoot germination. The results of this evaluation are summarized in Table 2.
The effect of plant growth regulators on the shoot development of axillary buds and differentiation of apple shoots on the Murashige–Skoog medium.
Axillary buds of the Zarya Alatau cultivar, cultured on plant growth regulator-free MS medium, showed a regeneration frequency of 12%, and the shoots grew to a height of 0.3 cm within 30 d. Shoot development of axillary buds was 70% on MS medium with the addition of 0.5 mg/L of BAP. However, increasing the concentration of BAP to 1 mg/L resulted in a shoot development decrease of axillary buds. Boudabous et al. (2010) showed that axillary bud sprouting from nodal explants of apple occurred with a frequency of 25.0% on MS medium supplemented with 0.5 mg/L BAP. In this study effective shoot development (87.0%) was observed on MS medium with a combination of 0.5 mg/L BAP and 0.5 mg/L GA, and shoots 3.5 cm long developed within 30 d. At a concentration of 2 mg/L BAP, the shoots were compact, and the leaves were smaller than other treatments. The best performing explants from previous phase were then used for next experiments. Al Maarri et al. (1986) obtained similar results in their study on clonal reproduction of quince, where they found that the rate of reproduction increases with an increase of BAP, while the rate of elongation decreases.
In vitro rooting of microshoots and adaptation to ex vitro
Multiple shoots were separated and transferred to a MS medium containing various concentrations of IAA (Table 3). The root development was observed starting from the 10th day. ANOVA revealed that the different concentrations of IAA had a highly significant impact (P < 0.05) on the induction, number, and length (Table 3).
The influence of IAA on the in vitro rooting of Zarya Alatau cultivar.
The root induction on the full-strength MS medium without auxin was observed only in 2% of plants. Thin roots emerged from the base of the shoots at a rate of 25% when the full-strength MS medium was supplemented with 1 mg/L IAA. Thick roots were induced with a high frequency of 85% on the half strength MS supplemented with 1.5 mg/L IAA. Further increase of auxin concentration to 3 mg/L decreased root induction by 24%.
Significant alterations in both the length and quantity of roots were observed when plants were cultivated in MS medium with different concentrations of IAA. At a concentration of 1.5 mg/L, the average number and length of roots recorded was 4.2 and 4.9, accordingly, and these values decreased with higher or lower amounts of IAA in medium.
In vitro plants display heightened sensitivity and vulnerability to environmental fluctuations, which may inflict harm if they are not gradually adapted to new conditions. Consequently, the process of adaptation in facilitating the successful transition of in vitro–rooted plants to ex vitro conditions.
For the successful transplantation of regenerating plants into a soil substrate, it is imperative that they possess well-developed leaves and a robust root system. In this study of rooting process of Zarya Alatau, it was observed that the development of the root system for this specific variety occurs within a period of ∼3 to 4 weeks.
This study elucidates the correlation between the survival rate of regenerating apple plants in a soil substrate and their initial sizes at the time of transfer to ex vitro conditions, as depicted in Fig. 2.
Biometric parameters of the Zarya Alatau cultivar after in vitro rooting: (A) in vitro rooting for 30 d; (B) in vitro rooting for 45 d; and (C) in vitro rooting for 60 d.
Citation: HortTechnology 34, 5; 10.21273/HORTTECH05464-24
The survival rate of apple trees was contingent on their biometric parameters (Table 4). Zarya Alatau in vitro rooting for 45 and 60 d, characterized by a size exceeding 5.0 cm and a well-developed root system, exhibited robust acclimatization when transplanted into a soil substrate. Conversely, regenerating plants characterized by comparatively low biometric parameters in vitro rooting for 30 d demonstrate a significantly lower survival rate of 22.5%.
Survival rate of Zarya Alatau regenerating plants according to biometric parameters during transplantation to ex vitro conditions.
Our findings demonstrate that the biometric parameters of plants have an impact on the viability of regenerating plants in the soil substrate, specifically in terms of plant height, root number, and root length. Well-developed plants with extensive root systems exhibited successful establishment. Our data indicate that apple plants with underdeveloped root systems, after 30 d on the rooting medium, are not suitable for transplantation from in vitro to ex vitro conditions. When comparing the biometric parameters of apple plants subjected to rooting for 45 and 60 d, there were significant differences in plant height and root system development levels. The Zarya Alatau cultivar after 60 d on rooting medium exhibited the highest plant height (8.1 ± 0.28 cm) and number (6.8 ± 0.12) and length (7.2 ± 0.31 cm) of roots per plant, resulting in a higher survival rate (78.8%) when transferred to ex vitro conditions (Table 4). Consequently, we recommend at least 60 d of root regeneration for a successful transition in vitro plants to soil conditions.
Discussion
Carelli and Echeverrigaray (2002), Mhatre et al. (2000), and Mereti et al. (2002) demonstrated that the addition of cytokinin to the nutrient medium increases the number of shoots. Several studies have shown a positive effect of a combination of cytokinin (BAP or kinetin) and auxin (IAA, NAA). Additionally, Yerbolova et al. (2013) found that for the Vitis vinifera cultivar Saperavi, the combination of 4.4 µM BAP and 0.05 µM NAA in the medium resulted in the highest number of plants (198 ± 2), compared with other hormone combinations, whereas the lowest number of plants was obtained with a combination of 8.8 µM BAP and 0.1 µM NAA (51 ± 1.7). Modgil et al. (1995) and Guan and De Klerk (2000), in their studies on in vitro apple propagation, also found that a combination of BAP and IBA during the induction stage was necessary and produced satisfactory results.
Numerous authors have recommended the dilution of the MS medium multiple times to optimize in vitro plant culture rooting (Fira et al. 2010; Sharma et al. 2000). Romadanova et al. (2018) discovered that the most favorable medium comprises half-strength MS, supplemented by 1.25 g/L of gelrite, 4 g/L of agar, and 0.25 mg/L of IBA.
Ahmad et al. (2003) reported that an increase in IBA concentration up to 3.0 mg/L resulted in enhanced root length in peach plants during in vitro regeneration. Nonetheless, at a higher concentration (4.0 mg/L), root development was inhibited.
This study revealed the most suitable protocol of clonal micropropagation for effective mass production of the Zarya Alatau apple variety with axillary buds on a basal MS medium supplemented with suitable concentrations of various growth regulators. This protocol can be used for micropropagation of the fruits of the Zarya Alatau apple tree to increase their productivity. An outline of the entire process of in vitro micropropagation of the Zarya Alatau apple is shown in Fig. 3. However, additional analysis is required to improve the protocol. It is also necessary to test the general agronomic characteristics of apple fruits, obtained from regenerated seedlings grown in tissue culture. The same test should be evaluated in other varieties of apple trees.
Schematic representation of micropropagation Zarya Alatau apple.
Citation: HortTechnology 34, 5; 10.21273/HORTTECH05464-24
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