Influence of ascorbic acid on growth and micropropagation of Aloe barbadensis Mill.

Behzad Kaviani ¹ ( Department of Horticultural Science, Faculty of Agricultural Science, Islamic Azad University, Rasht, Iran )
Aki Ramezani Sayad ² ( Department of Tissue Culture, North Region Branch of Iran (Rasht), Agricultural Biotechnology Research Institute of Iran (ABRII) )
Ahmad Majd ³ ( Department of Biology, Faculty of Sciences, Tarbiat-e-Moallem University, Tehran, Iran )
Mojtaba Khorrami Raad ⁴ ( School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne, Creswick, Australia )

تاریخ ارسال : 1400/04/24 تاریخ تایید : 1402/11/03

کلید واژه: Medicinal Plants, tissue culture, plant growth regulators, Vegetative growth, Aloe,

چکیده مقاله :

Ascorbic acid (AsA) has different roles in plant metabolism. Current research was done to evaluate the effect of various concentrations of AsA on growth and micropropagation of Aloe barbadensis Mill., an important medicinal and ornamental herb, for the first time. In this regards, results obtained from applying different concentrations of AsA on variables of aerial part length, length and number of root, number of propagule, brownness of medium, and fresh and dry weights of plants were analyzed after 8 weeks. Control plants showed slower growth in aerial parts than plants treated by AsA. Also, leaves were smaller in control plants. Fresh and dry weights in aerial parts were less in control plants than those of treated with AsA. There was a significant increase in produced numbers of propagules in different treatments compared with control treatment. Average number and length of produced roots in plants treated with AsA were more than those of produced in roots of control plants. In addition, brownness of medium and tissue cultures were reduced in plants treated with AsA, due to existence of different phenolic compounds in these plants. Totally, 80 mg L-1 AsA had the highest effect on induction of growth and development of A. barbadensis Mill. in vitro.

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Influence of ascorbic acid on growth and micropropagation of Aloe barbadensis Mill.

 

Abstract

Ascorbic acid (AsA) has different roles in plant metabolism. Current research was done to evaluate the effect of various concentrations of AsA on growth and micropropagation of Aloe barbadensis Mill., an important medicinal and ornamental herb, for the first time. In this regards, results obtained from applying different concentrations of AsA on variables of aerial part length, length and number of root, number of propagule, brownness of medium, and fresh and dry weights of plants were analyzed after 8 weeks. Control plants showed slower growth in aerial parts than plants treated by AsA. Also, leaves were smaller in control plants. Fresh and dry weights in aerial parts were less in control plants than those of treated with AsA. There was a significant increase in produced numbers of propagules in different treatments compared with control treatment. Average number and length of produced roots in plants treated with AsA were more than those of produced in roots of control plants. In addition, brownness of medium and tissue cultures were reduced in plants treated with AsA, due to existence of different phenolic compounds in these plants. Totally, 80 mg L-1 AsA had the highest effect on induction of growth and development of A. barbadensis Mill. in vitro.

Key words: aloe, medicinal plants, plant growth regulators, tissue culture, vegetative growth

 

Introduction

Aloe barbadensis Mill. (Liliaceae family) is an important medicinal-pharmaceutical plant. A. barbadensis has been widely grown as an ornamental plant, too (Duyff, 2017). This species is used in cosmetic and food industries (Uikey et al., 2021). Aloe spp. has some secondary metabolites. Gels prepared from aloe’s leaves are significant, due to variable applications in medicinal and cosmetic industries. Aloe is mainly propagated by suckers and of shoots which are too slow process for commercial plant production. One of the most suitable methods of propagation and breeding for the members of Liliaceae family is in vitro culture. Successful in vitro propagation of lilies rely on many factors such as type of culture medium and explant, as well type and concentration of plant growth regulators (PGRs) particularly auxins and cytokinins, (Taha et al., 2018; Youssef et al., 2019; Uikey et al., 2021). The successful application of tissue culture methods for micropropagation of some species of the genus Aloe has been presented (Hashemabadi and Kaviani, 2008; Uikey et al., 2021).

Ascorbic acid (AsA) or vitamin C has a necessary role in plant resistance against biotic and abiotic stresses (Lo’ay and EL-Khateeb, 2018; Martínez-Ortiz et al., 2019; Habibie et al., 2019; Chen et al., 2021), so that it is related to antioxidant features and providing a special redox status in symplastic and apoplastic compartmentations (Gonzalez-Reyes et al., 2003). In intracellular level, AsA engages with cell division and multiplication, and in extracellular level, it engages to defenses against pathogens, and adjusting cell elongation. In both cases, it considers that AsA is applied as a substrate for enzymes activity which adjust the processes like ascorbate peroxidase or some other peroxidases which affect on cell wall (Gonzalez-Reyes et al., 2003). It has proved that symplastic ascorbate is related to cell division and multiplication (Horemans et al., 2000). Cell division is a basic biological process promoted by molecular networks that are initiated in the terminal meristems. AsA is an effective molecular modulator involved in cell division (Kka et al., 2018). Generally, it has been accepted that ascorbate facilitate cell elongation through inhibiting enzymes engaged in strengthening cell wall.

The major method of non-sexual propagation for this plant is shoot production. This method is used for aloe culture in small level but in order to cultivate in wide level, tissue culture system seems necessary. There are no any reports related to the role of AsA on micropropagation of lilies family, particularly Aloe spp. Thus, this research, done for the first time, seeks to investigate the amount of growth and micropropagation of Aloe barbadensis Mill. in in vitro conditions influenced by different concentrations of exogenous AsA. 

 

Materials and Methods

 

Stem cuttings of Aloe barbadensis Mill. was obtained from the mother plants without any symptoms of disease growing in a greenhouse. Stem cuttings were washed under running tap water for 30 min. Stems containing buds were disinfected with 20% (w/v) sodium hypochlorite (NaOCl) for 15 min followed by three times rinses with sterile distilled water for 30 min. The surface disinfected stems were dissected into 5-7 mm segments of shoot tips and internodes as explants. Explants were cultured in Petri dishes containing basal MS (Murashige and Skoog, 1962) medium augmented with PGRs, kinetin (Kin) and 3-indoleacetic acid (IAA), both with concentration of 1 mg L-1 and different concentration of AsA (0, 4, 10, 60, 80, 100, 200, and 500 mg L-1). The content of 3% sucrose was utilized as carbon source and media were solidified with the concentration of 0.7% Agar-agar. The pH of the media was adjusted to pH 5.7 before autoclaving at 121°C and 102 kpa for 20 min. Five explants per jam jars were cultured and three replicates taken. Cultures were held in a growth chamber under 16 h photoperiods with light intensity of 2500 lux provided by cool-white fluorescent tubes at 26 ± 1°C and 70% relative humidity of the air.

Shoot length, root length, root number, propagule number, plant fresh and dry weights, and brownness of media were evaluated. Length of roots and stems were measured weekly. Regenerated seedlings were brought out of medium and evaluated.

The in vitro experimental design was conducted as a factorial with completely randomized block design (C.R.B.D) with five replications which was carried out through unequal repetition. Data were subjected to the analysis of variance (ANOVA) and means were compared by the Tukey’s test (p<0.05) using the SAS software package, version 9.1. Data processing of the results was done by an EXCEL.

 

Results

 

Current research evaluated the effect of different concentrations of AsA on growth and micropropagation of A. barbadensis Mill., an ornamental and medicinal plant. Studied characteristics were shoot length, root length; root number, number of produced propagules, fresh weight and dry weight. Our findings demonstrated that there are differences in the effect of various concentrations of AsA on these characters.

Effect of AsA on shoot length

The medium augmented with 500 mg L-1 AsA resulted in the highest shoot length (11.00 mm) (Table 1 and Fig. 1). The shortest shoot length (2.00 mm) was obtained in control plants. Data analysis showed that the effect of AsA was significant on the length of shoot (p≤0.01) (Table 4). Average of shoot length in plants treated with 10 mg L-1 of AsA was twice as much in comparison with control plants and it shows 1.95 times more in concentration of 4 mg L-1 AsA. This increase was approximately 2.8 times more in specimens treated with 60 mg L-1 AsA and 4.2 times more in specimens treated with 200 mg L-1 AsA compared to the control plants. The most increase in length was belong to the plants treated with 500 mg L-1 AsA which shows 5 times more growth in comparison with control plants (P<0.05). In higher concentrations than 500 mg L-1 AsA, the amount of growth decreases quickly (Fig 1). Decreasing of shoot length in plants treated with 80 mg L-1 AsA is due to high percentage of produced propagules in this concentration. In addition, in media supplemented with various concentrations of AsA, plants had natural appearance and green leaves during growth, while in media without AsA, leaves of some samples changed by color to yellow-brown.  

Effect of AsA on root length

The most increase in root length (18.50 mm) was observed in plants treated with 10 mg L-1 AsA (P<0.01) which was 3 times more than root length in control plants (Fig. 1). The least increase in root length (5.80 and 6.00 mm) was observed in control plants and plants treated with 600 mg L-1 AsA, respectively (Fig. 1). Media with more than 20 mg L-1 AsA produced plants with thicker roots and in media with more than 80 mg L-1 AsA increase in root thickness and broad and band-shaped roots were observed.

Effect of AsA on root number

The medium enriched with 500 mg L-1 AsA resulted in maximum root number (16.00) (Fig. 2). Minimum root number (5.70) was obtained in control plants. Data analysis showed that the effect of AsA was significant on the length of shoot (p≤0.01). Difference between the number of roots in plants treated with concentrations of 10 mg L-1 AsA and more was significant (P<0.05) than that of control plants (Fig. 3). The number of roots in plants treated with concentrations of more than 500 mg L-1 AsA was decreased and more than 600 mg L-1 AsA, rooting was inhibited. Also, if some roots appear out of culture medium conditions (ex vitro), their growth stops.   

Effect of AsA on the number of produced propagules

Our study on the effect of AsA on the number of produced propagules revealed that AsA had important effect on the number of produced propagules (Fig. 2). Statistical analysis of data showed that AsA had important influence on the number of produced propagules (P≤0.01). Maximum and minimum of propagules (7.70 and 1.70) were observed on medium containing 80 mg L-1 AsA and control, respectively (Fig. 2). The number of produced propagules in plants treated with concentrations of 10 mg L-1 AsA and more show significant difference (p<0.05) compared to the control plants. This amount increase gradually in higher concentrations and the most increase in the number of propagules was observed in concentration of 80 mg L-1 AsA (approximately 4.7 times more in comparison with control) (P<0.01). In concentrations more than 80 mg L-1, numbers of propagules decrease gradually (Fig. 2). By selecting internodes as explants in the same culture conditions, number of produced propagules could be increased to 19.8 propagules per explant in medium containing 80 mg L-1 AsA.

Effect of AsA on fresh and dry weight of plants

There is a significant difference (P<0.01) in amount of fresh and dry weight of plants treated with various concentrations of AsA, compared to the control plants. The freshest and dry weight (1.7 and 2.3 g, respectively) were observed in plants treated with 500 mg L-1 AsA (Fig. 3). The least fresh and dry weight (0.65 and 0.02 g) were observed in control plants and plants treated with 600 mg L-1 AsA, respectively (Fig. 3). In higher concentrations than 500 mg L-1 AsA, the amount of fresh and dry weight of plants decreases quickly. Also, in these concentrations, explants grew slowly and considerable decrease was seen in their fresh and dry weight. In concentrations more than 600 mg L-1 AsA, growth of samples was stopped.

Effect of AsA on brownness of plant tissues and media

Due to existence of various phenolic compounds in Aloe barbadensis Mill., which are secreted into medium, adding AsA as an antioxidant prevents medium and tissues to be brown and it also creates a more proper growth for cultured plants.

 

Discussion

 

Some reports have shown the effect of PGRs particularly auxins (NAA, IBA, 2,4-D, and IAA) and cytokinins (BA, Kin, and BAP) on in vitro propagation of Aloe spp. (Hashemabadi and Kaviani, 2008; Welehaweria, 2018; Shibiru et al., 2018; Niguse et al., 2020; Singh et al., 2020; Uikey et al., 2021). Explant used for this purpose mainly are stem disc, leaf segment, shoot tip, lateral shoot, and offshoots.

There are no any reports about the effect of AsA on in vitro micropropagation of lilies family, especially Aloe spp. Ascorbate regulates cell growth and development by controlling parameters such as biosynthesis of proteins rich in hydroxyproline, which is required for transition from G1 phase to G2 phase in the cell cycle, connections of cell wall glycoproteins and other polymers and redox reactions in cellular plasma membrane, which engaged in length increase mechanisms (Cordoba and Gonzalez-Reyes, 1994). Free radical of ascorbate induces high vacuolization of cells for elongation. This effect could be related to redox system activity, connected to plasma membrane (Cordoba and Gonzalez-Reyes, 1994). 

During conducted experiments, it was observed that emergence of root in explants happens 3 to 4 days’ sooner, roots are longer and have more branches in plants treated with different concentrations of AsA than those of control plants. Similar findings were reported on several other plants (Paciolla et al., 2001). These researchers showed that AsA stimulates cell multiplication in all root meristem and pericycle. Decrease in root length in high concentration of AsA in medium may be due to increase in dehydroascorbate concentration produced from ascorbate oxidation in medium. Potters et al. (2000) have reported that against stimulating role of ascorbate, oxidized ascorbate means dehydroascorbate prevents from cell multiplication. Results obtained by these experiments could be consistent with mentioned reports. It was clear previously that this form of oxidized ascorbate (dehydroascorbate) could be rather absorbed by plant cells (Horemans et al., 2000). Reduction of dehydroascorbate and changing to ascorbate in aerial parts of plants provides higher level of ascorbate for apical meristem of stem. Thus, as a result it increases cell division and multiplication and increase aerial parts growth, which is mentioned in this research. These findings are consistent with those reported by Horemans et al. (2000). In addition, amount of increase in fresh and dry weight, reported in this study is directly in relation with increase in ascorbate concentration in medium, so that decrease of ascorbate acid in medium reduces biomass production. Also, plant growth and development is controlled by plant growth regulators such as auxins, gibberellins and abscisic acid. With regard to increase of produced propagules and its most numbers in concentration of 80 mg L-1 AsA, it could be suggested that, in this concentration, cell division is increased and more callus is produced from one hand and on the other hand more cells are differentiated and new seedlings are produced. More studies about determining concentration of AsA in cellular differentiation from callus mass in medium without AsA and its comparison with produced calli under treatments, explains this process.

 

Conclusion

 

Ascorbic acid (AsA) or vitamin C is an organic molecule participated in cell division and proliferation. AsA plays positive roles in plants. This material is also an antioxidant that absorbs phenolic compounds especially from culture medium. Various phenolic compounds in A. barbadensis Mill. secrete into the medium. Adding AsA to medium prevents medium and tissues to be brown and it also induces a more proper growth for cultured plants. AsA at the concentration of 80 mg L-1 had the highest effect on shoot multiplication and root number of A. barbadensis Mill. in vitro.

 

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Fig. 1. The effect of different concentrations of AsA on length of aerial parts and roots of Aloe barbadensis Mill. plantlets cultivated on MS medium supplemented with 1 mg L-1 IAA and Kin. Maximum length of aerial parts and roots was obtained at 500 and 10 mg L-1 AsA, respectively

 

 

 

Fig. 2. The effect of different concentrations of AsA on the number of propagules and roots of Aloe barbadensis Mill. plantlets cultivated on MS medium supplemented with 1 mg L-1 IAA and Kin. Maximum number of propagules and roots was observed at 80 and 500 mg L-1 AsA, respectively

 

 

 

 

Fig. 3. The effect of different concentrations of AsA on fresh and dry weights of Aloe barbadensis Mill. plantlets cultivated on MS medium supplemented with 1 mg L-1 IAA and Kin. Highest fresh and dry weights were observed at 500 mg L-1 AsA

 

 

 

Fig. 4.  In vitro multiplication of Aloe barbadensisMill., on MS medium supplemented with 1 mg L-1 IAA and Kin. A. Produced propagules from internode explants treated by 80 mgl-1 AsA. B. Callus mass and regenerated plants. C. Shooting and rooting responses in vitro of explants. From right to left: control treatment, 0, 80, 100 and 200 mg L-1 AsA. D. Explants treated with 80 mgl-1 AsA. The best shoot multiplication and growth was observed at 80 mg L-1 AsA

 

 

 

Fig. 5. Clonal propagation of Aloe barbadensis Mill., on MS medium supplemented with 1 mg L-1 IAA and Kin. Treated by 4 (A), 10 (B) and 20 (C) mg L-1 AsA. Highest multiplication rate was obtained in 80 mg L-1 AsA