Plant growth regulators on the micropropagtion of Actinidia cultivars

Reguladores de crescimento vegetal na micropropagação de cultivares de Actinidia

ABSTRACT

Actinidia Lindl., commonly known as kiwifruit, is a valuable berry crop. The area of commercial kiwifruit plantations is increasing; the global production of kiwifruit is about 0.62% of the total production of major fruit crops. The use of biotechnological methods, which can significantly accelerate the propagation of quality planting materials, is considered to be relevant for the propagation of this crop. In this study, we optimized the culture medium composition at the micropropagation stage for the effective cultivation of promising cultivars of A. arguta, A. kolomikta, and A. polygama. We investigated the features of Actinidia morphogenesis depending on the genotype, the concentration of 6-Benzylaminopurine (0.5, 0.8, and 1.0 mg L^-1), and plant growth regulators (6-Benzylaminopurine, meta-topolin, and 2-isopentenyladenine at a concentration of 0.5 mg L^-1) in the media Quoirin and Lepoivre. Actinidia arguta (multiplication rate of 8.0) and A. polygama (6.8) developed faster at the micropropagation stage compared to A. kolomikta (4.9). The studied Actinidia representatives were cultured most effectively on Quoirin and Lepoivre media supplemented with 0.5 mg L^-1 meta-topolin, compared to the media containing 0.5 mg L^-1 6-Benzylaminopurine and 0.5 mg L^-1 2-isopentenyladenine. The use of meta-topolin in the medium contributed to the increase in various morphometric traits, such as the height of microshoots (up to 28% depending on the species), their number (up to 52%), and their multiplication rate (up to 42%). We also recorded a high morphogenic capacity of the investigated species.

Index terms:
A. arguta; A. kolomikta; A. polygama; cytokinin; regeneration.

RESUMO

A Actinidia Lindl., vulgarmente conhecida como kiwi, é uma frutífera valiosa. A área de plantio comercial de kiwis está a aumentando; a produção global de kiwis é de cerca de 0,62% da produção total das principais culturas frutícolas. A utilização de métodos biotecnológicos, que podem acelerar significativamente a produção de mudas de qualidade, é considerada relevante para a propagação desta cultura. Neste estudo, optimizámos a composição do meio de cultura na fase de micropropagação para o cultivo eficaz de cultivares promissoras de A. arguta, A. kolomikta e A. polygama. Investigámos as características da morfogénese de Actinidia em função do genótipo, da concentração de 6-Benzilaminopurina (0,5, 0,8 e 1,0 mg L^-1) e dos reguladores de crescimento de plantas (6-Benzilaminopurina, meta-topolina e 2-isopenteniladenina a uma concentração de 0,5 mg L^-1) nos meios Quoirin e Lepoivre. Actinidia arguta (taxa de multiplicação de 8,0) e A. polygama (6,8) desenvolveram-se mais rapidamente na fase de micropropagação em comparação com A. kolomikta (4,9). Os representantes de Actinidia estudados foram cultivados mais eficazes nos meios Quoirin e Lepoivre suplementados com 0,5 mg L^-1 de meta-topolina, em comparação com os meios contendo 0,5 mg L^-1 de 6-benzilaminopurina e 0,5 mg L^-1 de 2-isopenteniladenina. A utilização de meta-topolina no meio contribuiu para o aumento de vários traços morfométricos, tais como a altura dos micro rebentos (até 28%, dependendo da espécie), o seu número (até 52%) e a sua taxa de multiplicação (até 42%). Foi registrado, também, uma elevada capacidade morfogénica das espécies investigadas.

Termos para indexação:
A. arguta; A. kolomikta; A. polygama; citocinina; regeneração.

INTRODUCTION

The genus Actinidia Lindl. includes about 70 species cultivated around the world (Ferguson; Huang, 2007; POWO, 2023). Most Actinidia species have edible fruits widely used in various branches of the food industry, especially in canning (Ma et al., 2019; Kozak et al., 2020) and in the wine industry (Zakharenko, 2010; Park et al., 2013). Actinidia flowers and fruits are promising raw materials for the cosmetic industry (Chen et al., 2010; Kim et al., 2023). Different parts of the plants (leaves, vines, roots, and especially fruits) contain many types of bioactive compounds (triterpenoids, polyphenols, vitamin C, carbohydrates, amino acids, and minerals), which have laxative, anti-diabetic, antioxidant, anti-inflammatory, and other health-promoting effects (Abe et al., 2010; Chang et al., 2010; Ciacci et al., 2014; Wojdylo et al., 2017; Li et al., 2018; Panishcheva; Motyleva; Kozak, 2021). Therefore, these plants are used in traditional medicine and also as pharmaceutical raw materials in folk medicine (He et al., 2019; Ma et al., 2021).

The area of commercial kiwifruit plantations is increasing; the world production of kiwifruit is about 0.62% of the total production for major fruit crops (Rey et al. 2020). The most common commercially cultivated Actinidia spp. (including Actinidia chinensis Planch., Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) are grown in subtropical regions. However, there are only three frost-resistant species that can be cultivated in places with cold winters, which include A. arguta (Siebold et Zucc.) Planch. et Miq., A. kolomikta (Rupr. Et Maxim.) Maxim., and A. polygama (Sieb. et Zucc.) Maxim. (Chat, 1995). According to Avery (1991), A. arguta and A. polygama can withstand temperatures up to -31 °C without injury, while A. kolomikta has greater resistance to winter frosts (up to -40 °C). These species are cultivated in Russia.

The most representative collection of Actinidia in Russia of about 200 samples is available in the Federal Horticultural Center for Breeding, Agrotechnology, and Nursery (Kozak et al., 2020). Some valuable and unique genotypes are represented by a small quantity.

The vegetative propagation of unpopular cultivars is difficult because of the lack of the required number of donor plants. Therefore, innovative biotechnological methods need to be applied for mass propagation of high-quality planting material. The first micropropagation protocol of Actinidia (A. chinensis) was developed by Harada (1975), and it was further improved by Standardi (1980). Most studies on Actinidia were based on the in vitro culture of A. deliciosa (Mitrofanova; Mitrofanova, 2000; Nasib; Ali; Khan, 2008; Thakur et al., 2022;).

Some studies, however, have also investigated the micropropagation of other species, such as A. arguta (Hameg; Gallego; Barreal, 2017; Hameg et al., 2020), A. kolomikta (Kovac, 1993), A. polygama (Takahashi et al., 2004), and A. melanandra (Debenham; Seelye; Mullan, 2016). These researchers investigated the effect of macronutrients, plant growth regulators, and genetic characteristics on the in vitro shoot regeneration of these species (Hameg; Gallego; Barreal, 2017; Malaeva; Molkanova, 2021; Molkanova; Krakhmaleva; Kozak, 2022).

In this study, we optimized the content of the growth medium at the multiplication stage for the effective culture of the promising cultivars of A. arguta, A. kolomikta, and A. polygama. One objective of this study was to select the most optimal cytokinin and its concentration for better growth and development of plantlets of the studied Actinidia cultivars and species.

MATERIAL AND METHODS

All experiments were conducted in the Laboratory of Plant Biotechnology at Tsitsin Main Botanical Garden of the Russian Academy of Sciences (MBG RAS), Moscow, Russia. The generally accepted methods of plant biotechnology (Butenko, 1964) developed in the Laboratory (Molkanova et al., 2018) were used in this study.

Plant material

Cultivars of three Actinidia species with valuable features and in demand for mass production were selected for this study. All plant materials for in vitro initiation were obtained from the donor plants of the collection of the Federal Horticultural Center for Breeding, Agrotechnology, and Nursery located in the village of Mikhnevo, Moscow Region, Russia. Seven cultivars were selected from the in vitro gene bank of the Laboratory of Plant Biotechnology at MBG RAS. These cultivars were developed earlier while starting the institutional research project (male А. arguta cultivar, ‘Rebristaya’, ‘Komandir’. ‘Zemlyanichnaya’, ‘Izobilnaya’, male А. polygama cultivar, and ‘Beta’). Eight cultivars were introduced into the in vitro culture as part of the implementation of the grant (‘Solnechnyj’, ‘Zolotaya Kosa’, ‘Taezhnyy Dar’, male А. kolomikta cultivar, ‘Moma’, ‘Pamyati Kolbasinoy’, ‘Osennyaya’, and ‘Perchik’). In total, 15 promising cultivars of three Actinidia species were used in this study (Table 1).

Table 1:
The main characteristics of the studied Actinidia cultivars.

Species	Cultivar	Sex	Fruit color	Fruit mass (g)	Fruit taste
A. arguta	Male form	male	-	-	-
	‘Solnechnyj’	male	-	-	-
	‘Zolotaya Kosa’	female	yellowish green, green	8.0	sweet, with apple flavor
	‘Rebristaya’	female	green	7.6	sweet and sour, with pineapple flavor
	‘Taezhnyy Dar’	female	green	7.6	sweet and sour, with strong pineapple flavor
A. kolomikta	Male form	male	-	-	-
	‘Komandir’	male	-	-	-
	‘Zemlyanichnaya’	female	yellowish green	2.8	sweet, with strawberry flavor
	‘Izobilnaya’	female	yellowish green, with light longitudinal stripes	3.6	sweet and sour, with strong pineapple flavor
	‘Moma’	female	yellowish green	2.7	sweet, with pineapple flavor
	‘Pamyati Kolbasinoy’	female	olive green, with light longitudinal stripes	4.3	light sweet and sour, with pineapple flavor
A. polygama	Male form	male	-	-	-
	‘Beta’	female	orange, to dark orange	3.7	like bell pepper, with pepper aroma
	‘Osennyaya’	female	dark orange	4.5	like bell pepper, with pepper aroma
	‘Perchik’	female	orange	2.7	like bell pepper, with pepper aroma

Surface sterilization

The explants (apical and lateral buds) were first washed under running tap water for 15 min, then submerged in 2% fungicide solution for 10 min and 70% ethanol for 1-2 min. The explants were rinsed for 5-7 min in 5-7% calcium hypochlorite solution, and 1-2 drops of Tween 20 were added. After adding each reagent, the explants were washed 4-5 times in sterile distilled water (Molkanova; Krakhmaleva; Kozak, 2022).

Culture initiation

The initiation medium QL (Quoirin and Lepoivre, 1977), solidified with 6.6 g L^-1 agar (Roeper, Germany), containing 30 g L^-1 sucrose and 0.3 mg L^-1 6-Benzylaminopurine (6-BAP) (Sigma, USA), was used for shoot induction (Molkanova; Krakhmaleva; Kozak, 2022). The medium was adjusted to pH 5.8 with potassium hydroxide and sterilized in an autoclave (WAC-60; Daihan Scientific, South Korea) using pressurized saturated steam (P = 101 kPa) at 121 °C for 20 min.

Sterilized explants were grown in test tubes on the initiation medium. Explants were planted on the medium in a laminar flow cabinet, following the rules for working with sterile material. The explants were cultured at 23 ±2 °С and a 16-h photoperiod under cool-white light fluorescent lamps (light intensity 3,000-3,500 lux).

The duration of the initiation stage was 14 days. Shoots obtained after initiation were propagated and transferred to the multiplication medium. Later explants were subcultured every 30-35 days, before which, the multiplication rate (expressed as the product of the number of microshoots and the number of nodes per explant) was calculated.

Shoot multiplication

Explants (~1.0-1.5 cm long) with two nodes raised in vitro were transferred into 250 mL glass jars containing 50 mL of the QL medium supplemented with 6.6 g L^-1 agar (Roeper, Germany), 30 g L^-1 sucrose, and various cytokinins. Two experiments were conducted. The first experiment was designed to determine the optimal level of 6-BAP; 0.5, 0.8, and 1.0 mg L^-1 6-BAP were tested. In the second experiment, the effect of different cytokinins (6-BAP, meta-topolin (mT) (Duchefa, Netherlands), and 2-isopentenyladenine (2-iP) (Sigma, USA)) on plant morphogenesis was investigated at the concentration that was found to be optimal in the first experiment (0.5 mg L^-1). The explants for the second experiment were isolated from plants obtained as a result of the first experiment. The medium supplemented with 0.5 mg L^-1 6-BAP was used as a control. The pH of all media was adjusted to 5.8 before the experiment, and then, the media were sterilized in an autoclave (WAC-60) using pressurized saturated steam (P = 101 kPa) at 121 °C for 20 min.

During this stage, the explants were cultured at 23 ±2 °С and a 16-h photoperiod under cool-white light fluorescent lamps (light intensity of 3,000-3,500 lux).

All experiments were repeated in triplicate; for each treatment, 10 explants were used for each variant. After 30-35 days of culture, the data on the number of microshoots per explant, the length of the microshoots, the frequency of spontaneous rhizogenesis, and the multiplication rate (expressed as the product of the number of microshoots and the number of nodes per explant) were calculated.

Statistical analysis

All data were expressed as the mean ±SD. The data were analyzed using PAST 2.17c (PAleontological STatistics), SPSS Statistics 23, and Microsoft Office Excel 2010. The differences between the mean values of multiple groups were determined by Duncan’s multiple-range test. All differences were considered to be statistically significant at p < 0.05.

RESULTS AND DISCUSSION

Culture initiation

During the in vitro initiation of the explants, the main and axillary shoots developed after 14 days. After 45 days, some genotypes showed induction of the development of adventitious buds and shoots. The multiplication rate of the representatives of the genus Actinidia changed significantly during in vitro culture. However, all studied species showed a general pattern of change, in which during the first and second subcultures the multiplication rate increased and reached the highest values by the third to fifth subcultures, and then, it decreased from the fourth to sixth subcultures. Actinidia arguta showed the highest multiplication rate in the third subculture, A. kolomikta showed the highest multiplication rate in the fifth subculture, and A. polygama showed the highest multiplication rate in the fourth subculture.

A similar pattern was found in other cultures. The micropropagation of lavender and essential oils rose during nine subcultures showed a similar increase in the multiplication index by the third to fourth passage; further subcultures were characterized by a decrease in this parameter (Yegorova et al., 2019; Yegorova et al., 2021).

Shoot multiplication

While optimizing the clonal micropropagation technique, the biological traits and details of the in vitro regeneration of each taxon must be considered. They depend on several factors, which include the genotype, epigenetic traits of explant cells, the physiological state of intact plants, the composition of the medium, and culture conditions (Hameg; Gallego; Barreal, 2017; Mitrofanova, 2018; Molkanova et al., 2018).

Some studies showed that the determination of the morphogenetic potential of most taxa mostly depends on the genetic traits of the plants (Mitrofanova, 2018; Molkanova et al., 2018; Marino; Bertazza, 1990). The regeneration capacity of the explants of different Actinidia species varied significantly; A. arguta and A. polygama developed faster than A. kolomikta at the micropropagation stage (Figure 1), which correlated with their growth dynamics during introduction. These findings matched the results of our previous study (Molkanova et al., 2014). However, in this study, we found that the traits of the genus affected the multiplication rate of the representatives of the genus Actinidia more than the traits of the cultivars (62.7% vs. 28.4%).

Figure 1:
Effect of the genotype on the multiplication rate of the representatives of the genus Actinidia grown in the QL medium with 0.5 mg L^-1 6-BAP. The mean values followed by the same letters in the column indicate that the differences were not significant, as determined by Duncan’s test (p ≤ 0.05).

The genetic traits of cultivars are not only indicators of the ability of explants to micropropagate, but they also control their ability to micropropagate. Thus, the variation in the limits of the regeneration capacity of explants and the number of formed micro-plantlets were determined genotypically (Figures 2 and 3).

Figure 2:
The multiplication rate of the representatives of the genus Actinidia in the QL medium with 0.5 mg L^-1 6-BAP is presented. The mean values followed by the same letters in the column indicate that the differences were not significant, as determined by Duncan’s test (p ≤ 0.05).

Figure 3:
The representatives of the genus Actinidia with high morphogenetic potential are shown. A - A. arguta ‘Zolotaya Kosa’, B - A.kolomikta ‘Komandir’, and C - A. polygama male cultivar. Scale bars correspond to 1 cm.

The multiplication rate of A. kolomikta cultivars varied from 4.6 to 5.5 and was lower than the multiplication rate of the cultivars of A. arguta and A. polygama. The cultivars of A. arguta and A. polygama had similar multiplication rates (6.3-8.4 and from 6.0-7.7, respectively). Actinidia arguta cv. ‘Zolotaya Kosa’ had the highest multiplication rate (10.1).

Plant growth regulators are essential components of the medium for clonal micropropagation. Cytokinins are important plant growth substances that can regulate morphogenesis in plant tissue and organ cultures. They affect various plant growth processes. When added to the medium, these compounds promote cell division, induce the differentiation of shoots, and remove apical dominance. The type of cytokinin used and its concentration significantly affect shoot formation and the quality of plantlets. The synthetic cytokinin 6-BAP is most commonly used in micropropagation. Other naturally occurring cytokinins, including mT and 2-ip, also have high physiological activity and are effective for the micropropagation of some plants; however, they are mostly used for research purposes (Van Staden; Zazimalova; George, 2008). A commonly used and effective cytokinin at the micropropagation stage for some Actinidia representatives is 6-BAP, used at concentrations of 0.5-2.0 mg L^-1 (Marino; Bertazza, 1990; Akbaş et al., 2007). It can be added to the medium along with auxins, gibberellic acid, or other plant growth regulators (Hameg; Gallego; Barreal, 2017). The differences in the response of different species and cultivars of Actinidia to growth regulators can help determine the relevance of the studies to optimize the micropropagation procedure and better understand the effect of various plant growth regulators, especially cytokinins, on shoot induction and development.

The concentration of 6-BAP in the medium showed a lower effect on the changes in the multiplication rate of the representatives of Actinidia than the cultivar traits of the studied species (2.6 and 9.2% vs. 69.2-85.4%). Thus, no significant influence of the hormone composition of the medium and interaction of the studied factors were found on the multiplication rate of A. arguta.

Our results showed that increasing the concentration of 6-BAP increased the number of microshoots for most of the studied species, whereas the multiplication rates changed for only A. kolomikta and A. polygama (Table 2).

Table 2:
The effect of different concentrations of 6-BAP on the morphometric traits of the representatives of the genus Actinidia.

Species	6-BAP (mg L-1)	Length of shoot (cm)	Number of shoots per explant (unit)	Multiplication rate
A. arguta	0.5 (control)	1.7 a*	1.9 b	8.0 a
	0.8	1.6 a	2.0 ab	8.0 a
	1.0	1.6 a	2.2 a	8.2 a
A. kolomikta	0.5 (control)	1.6 a	1.4 b	4.9 b
	0.8	1.6 a	1.5 a	5.1 b
	1.0	1.6 a	1.6 a	5.4 a
A. polygama	0.5 (control)	2.5 a	1.7 b	6.8 b
	0.8	2.6 a	1.9 a	7.1 a
	1.0	2.5 a	1.8 a	7.2 a

• *Means of each species, followed by the same letters in the column, do not differ by Duncan’s test (p ≤ 0.05).

When the concentration of 6-BAP was increased from 0.5 to 1.0 mg L^-1 in the medium, the number of microshoots and multiplication rate increased slightly (by 1.1 times). Therefore, we suggest using 0.5 mg L^-1 6-BAP while growing the Actinidia representatives to reduce cytokinin accumulation in the tissues of explants and increase their commercial efficiency.

Some researchers have reported that 6-BAP often negatively affects plant development due to the formation of stable compounds (6-benzylaminopurine-9-glucosides). The accumulation of these compounds inhibits the development of tissues (Yablonskaya; Knishkajte; Romanova, 2014). Actinidia spp. may develop microshoots with hyperhydricity when cultured on a medium containing this plant growth regulator (Marino; Bertazza, 1990; Saeiahagh et al., 2019).

An analog of 6-BAP, known as mT, can affect the in vitro shoot multiplication of A. deliciosa (Prado; Herrera, 2005), Actinidia chinensis var. chinensis (Saeiahagh et al., 2019), and other plant species, including Maytenus emarginata (Willd.) Ding Hou (Shekhawat et al., 2020), Pogostemon cablin (Blanco) Benth. (Lalthafamkimi et al., 2020), Allamanda cathartica L. (Khanam et al., 2020), Ribes grossularia L. (Kucharska et al., 2020), Scaevola taccada (Gaertn.) Roxb. (Shekhawat et al., 2021), and Opuntia stricta Haw. (de Souza et al., 2019).

Malaeva (2020) showed that 2-ip (3.0-5.0 mg L^-1) positively affects the multiplication rate of some representatives of Actinidia.

While culturing Actinidia representatives on media containing different cytokinins (6-BAP, mT, and 2-ip), a significant change in the multiplication rate was found. Actinidia kolomikta (77.2%) and A. arguta (64.1%) had the greatest effect of growth regulators on the morphogenetic capacity of the studied plants. The effects of various plant growth regulators on the morphometric traits of promising cultivars of the genus Actinidia are presented in Table 3.

Table 3:
A list of the morphometric traits of promising Actinidia cultivars used for evaluating the effects of different plant growth regulators at 0.5 mg L^-1.

Species	Cultivar	Plant growth regulator	Length of shoots (cm)	Number of shoots per explant (unite)
A. arguta	Male form	6-BAP (control)	1.7±0.4 b*	1.8±0.6 b
		mT	2.2±0.5 a	2.1±0.7 a
		2-ip	2.4±0.4 a	1.1±0.3 c
	‘Solnechnyj’	6-BAP (control)	1.4±0.4 c	1.7±0.5 b
		mT	2.3±0.6 a	2.3±0.8 a
		2-ip	1.7±0.3 b	1.2±0.4 с
	‘Zolotaya Kosa’	6-BAP (control)	1.5±0.4 b	2.1±0.5 b
		mT	2.4±0.6 a	2.7±0.6 a
		2-ip	2.4±0.6 a	1.1±0.3 c
	‘Rebristaya’	6-BAP (control)	1.7±0.4 b	1.5±0.5 b
		mT	1.8±0.5 b	2.2±0.6 a
		2-ip	2.5±0.6 a	1.1±0.3 c
	‘Taezhnyy Dar’	6-BAP (control)	2.0±0.5 c	1.8±0.4 b
		mT	3.0±0.7 a	2.2±0.4 a
		2-ip	2.7±0.7 b	1.2±0.4 c
A. kolomikta	Male form	6-BAP (control)	1.6±0.7 a	1.6±0.5 b
		mT	1.4±0.4 a	2.3±0.5 a
		2-ip	1.4±0.5 a	1.2±0.4 b
	‘Komandir’	6-BAP (control)	1.1±0.4 b	1.9±0.6 b
		mT	1.4±0.3 a	2.3±0.6 a
		2-ip	1.5±0.4 a	1.1±0.3 c
	‘Zemlyanichnaya’	6-BAP (control)	1.5±0.5 b	1.6±0.6 b
		mT	1.8±0.2 a	2.0±0.0 a
		2-ip	1.8±0.5 a	1.2±0.4 c
	‘Izobilnaya’	6-BAP (control)	2.0±0.5 b	1.0±0.2 b
		mT	2.6±0.7 a	2.0±0.5 a
		2-ip	1.6±0.5 c	1.0±0.0 b
	‘Moma’	6-BAP (control)	1.8±0.5 c	1.1±0.3 b
		mT	3.0±0.6 a	2.3±0.8 a
		2-ip	2.4±1.0 b	1.0±0.0 b
‘Pamyati Kolbasinoy’	6-BAP (control)	2.1±0.8 a	1.4±0.5 b
	mT	1.7±0.4 b	1.9±0.6 a
	2-ip	2.4±0.6 a	1.1±0.3 b
A. polygama	Male form	6-BAP (control)	1.9±0.5 a	1.9±0.6 ab
		mT	2.0±0.5 a	2.1±0.5 a
		2-ip	2.0±0.6 a	1.8±0.6 b
	‘Beta’	6-BAP (control)	2.6±0.7 a	1.4±0.5 b
		mT	3.0±0.6 a	2.0±0.7 a
		2-ip	3.3±1.0 a	1.3±0.1 b
	‘Osennyaya’	6-BAP (control)	3.1±0.8 b	1.5±0.5 a
		mT	3.5±1.1 a	1.5±0.5 a
		2-ip	3.8±0.9 a	1.2±0.4 b
	‘Perchik’	6-BAP (control)	1.9±0.6 a	2.1±0.4 a
		mT	1.8±0.5 a	1.9±0.4 b
		2-ip	1.9±0.7 a	1.6±0.6 c

• *Values of each cultivar are mean ±SD, followed by the same letters in the column do not differ by Duncan’s test (p ≤ 0.05).

The growth of A. arguta and A. kolomikta on media supplemented with mT and 2-ip promoted the increase in the height of the microshoot (A. arguta: 2.3 cm and 2.3 cm vs. 1.7 cm (6-BAP); A. kolomikta: 2.5 cm and 1.9 cm vs. 1.7 cm). The cultivars of A. polygama showed no significant differences in the height of the microshoots grown on these media (2.6 cm (mT) and 2.7 cm (2-ip) vs. 2.4 cm (6-BAP)). Similar effects of mT on the height of micro-plantlets were reported by other researchers who studied various horticultural species (Moyo et al., 2018; Kucharska et al., 2020; Nowakowska et al., 2020). Other studies found that 2-ip might have different effects on the length of the microshoot, but for some tree crops, 2-ip can positively affect shoot growth compared to 6-BAP (Debnath; Kenneth, 2001; Faisal et al., 2006; Singh, 2020; Wulandari et al., 2021; Haida et al., 2022).

The studied plant growth regulators could be arranged according to their effect on the ability to promote shoot formation in the following order: mT > 6-BAP > 2-ip. Some cultivars had no significant differences in the number of microshoots formed during cultivation on media containing 6-BAP and mT. Also, 0.5 mg L^-1 2-ip did not significantly affect the formation of new microshoots for the cultivars of A. arguta and A. kolomikta. When 2-ip was used, the number of microshoots ranged from 1.0 ±0.0 to 1.2 ±0.6 units per explant.

The cultivation of samples on the medium containing mT resulted in a significant increase in the multiplication rate of A. arguta (by 1.3 and 1.7 times) and A. kolomikta (by 1.5 and 1.7 times) compared to cultivation on media containing 6-BAP and 2-ip. Actinidia polygama also showed an increase in the multiplication rate when grown on the medium with mT; however, no significant differences were recorded when the plants were grown on the medium with 6-BAP (Figures 4 and 5).

Figure 4:
The change in the multiplication rate of the representatives of the genus Actinidia grown on media with different plant growth regulators with a concentration of 5 mg L^-1. Different capital letters indicate significant differences between the tested growth regulators for each species, as determined by Duncan’s test (р ≤ 0.05); different lowercase letters indicate significant differences between species grown on the same medium, as determined by Duncan’s test (р ≤ 0.05).

Figure 5:
Microshoot development in the representatives of the genus Actinidia on media supplemented with different cytokinins at a concentration of 0.5 mg L^-1; A. arguta cv. ‘Taezhnyy Dar’ was grown on the medium with 6-BAP (A), mT (B), and 2-ip (C); A. kolomikta cv. ‘Moma’ was grown on the medium with 6-BAP (D), mT (E), and 2-ip (F); male cultivar of A. polygama was grown on the medium with 6-BAP (G), mT (H), and 2-ip (I). Scale bars correspond to 1 cm.

Our results showed that the variability limits of the regeneration capacity of explants and the number of formed plantlets depend not only on the genotype but also on the composition of the medium.

Rhizogenesis is an important stage of the micropropagation development technique. It may occur spontaneously and can also be induced. Spontaneous rhizogenesis is interpreted by some researchers as the “hormonal autonomy” of plant cells under in vitro conditions (Gamburg; Leonova; Rekoslavskaya, 1974). When plants under in vitro conditions are unable to synthesize hormones necessary for root formation, auxin needs to be added to the medium to induce root formation. High concentrations of cytokinins (generally used at the micropropagation stage) may inhibit or delay root formation and prevent root growth even in the presence of auxins. Therefore, one or more subcultures in a cytokinin-free medium may be required for the efficient rooting of micro-plantlets (Van Staden; Zazimalova; George, 2008). Thus, spontaneous root formation is a cheaper and less labor-intensive clonal micropropagation technique, as there is no need for a separate rooting stage and no additional costs of applying chemical reagents. These inexpensive options can contribute to the efficiency of micropropagation and the popularization of the in vitro propagation protocol for mass production (FAO, 2004).

Spontaneous root formation at different frequencies was noticed during the culture of Actinidia representatives on different media (Figure 6). Thus, the studied Actinidia species represent crops that can be easily rooted without the use of auxins.

Figure 6:
The frequency of spontaneous root formation of three Actinidia species when using different plant growth regulators at a concentration of 0.5 mg L^-1. The mean values of each species followed by the same letters in the column indicate that the differences are not significant, as determined by Duncan’s test (p ≤ 0.05).

The studied species can be arranged according to the frequency of spontaneous root formation in the following order: A. arguta (52.8%) < A. kolomikta (68.0%) < A. polygama (95.6%). This allowed us to conduct micropropagation and rooting in one subculture period, which increased the efficiency of in vitro cultivation.

In this study, we developed effective techniques of clonal micropropagation for promising Actinidia cultivars. These techniques might facilitate the propagation and in vitro long-time preservation of this crop.

CONCLUSIONS

In this study, we found that the regeneration capacity of the studied Actinidia cultivars depended on the genotype, growth regulators, and their concentrations in the medium. The efficiency of explant culturing on the QL medium supplemented with 0.5 mg L^-1 mT contributed to the increase in the height and number of microshoots, and also the multiplication rate. We also found that mT induced adventitious shoot formation at the explant base and the induction of bud development on microshoots.

ACKNOWLEDGMENTS

This study was funded by a research grant № 22-16-00074 of the Russian Science Foundation. Acknowledgment to scientific researcher Dr. N.V. Kozak from the Federal Horticultural Center for Breeding, Agrotechnology, and Nursery collection for providing the mother plant material for the study.

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Publication Dates

• Publication in this collection
  01 Dec 2023
• Date of issue
  2023

History

• Received
  23 June 2023
• Accepted
  06 Oct 2023