View of Effect of Different Plant Growth Regulators on Micropropagation of Some Pitaya Varieties

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2022, Vol. 12, No. 2, 183 – 190 http://dx.doi.org/10.11594/jtls.12.02.04

Research Article

Effect of Different Plant Growth Regulators on Micropropagation of Some Pitaya Varieties

Taner Bozkurt *, Sezen İnan, İjlal Dündar, Selda K. Özdemir

Tekfen Agricultural Research Production and Marketing Inc., Adana, Turkey

Article history:

Submission December 2021 Revised December 2021 Accepted March 2022

ABSTRACT

Pitaya belongs to the family Cactaceae and the genus Hylocereus. It is essential to develop tissue culture protocols according to the appropriate variety to spread pitaya commercial production and ensure healthy sapling production. This study aimed to determine the best plant growth regulators (PGRs) in the micropropagation process, and their effects on different pitaya cultivars were evaluated. Shoots of different pitaya cultivars were cultured in Murashige and Skoog (MS) basal medium supplemented with Indole-3-butyric (IBA), 6-benzyl amino purine (BAP), and gibberellic acid (GA3). The highest micropropagation coefficient was determined in Physical Graffiti variety cultured in MS medium supplemented with 2 mg/L BAP. The general evaluation based on the variety determined that the best micropropagation was in the Royal Red variety. In in vitro rooting studies, the best rooting variety was Royal Red (54.47%), followed by Siam Red (50.33%), Physical Graffiti (47.75%), and Seoul Kitchen (44.82%). It was determined that the Royal Red variety is grown in R2 (MS medium supplemented with 1 mg/l IBA) medium gave the best results in all criteria of shoot length (4.28 cm), root length (6.45 cm), and root formation on the face (74.48%). It was envisioned that these differences between the micropropagation, growth, and rooting of the cultivars used in the studies resulted from the cultivar characteristics.

Keywords: Pitaya, In vitro, Micropropagation, Royal Red, Seoul Kitchen, Siam Red, Physical Graffiti

*Corresponding author:

E-mail: [email protected]

Introduction

Pitaya (Hylocereus spp.) is a tropical and climbing cactus. It is a promising plant grown in different tropical and subtropical regions such as Southeast Asia and Central and South America [1]. The demand for its fruits is increasing day by day, and this fruit can be found in almost all exotic fruit markets in the world in recent years. The rapid recognition of the plant and its spread to the world can be explained by the appearance of its fruits, nutritional values and health benefits, and the commercial success of producing and export- ing countries such as Vietnam and Thailand [1].

Also known as dragon fruit, pitaya is also known as pitahaya or strawberry pear, or night- blooming cereus. The genus Hylocereus includes 16 species [2] and according to the color of the peel and pulp, H. undatus (red skin, white pulp),

H. polyrhizus (red skin and red pulp), H. costari- censis (red skin and red pulp), and H. megalanthus (yellow peel and white pulp) [3, 4]. Of these, H.

polyrhizus and H. costaricensis, in Central Amer- ica (Nicaragua), H. megalanthus, in South Amer- ica (Colombia and Ecuador), H. undatus, in South- east Asia (Vietnam, Thailand, Taiwan, and the Philippines), and parts of southern Mexico is cul- tivated [5, 6].

The pitaya's fleshy stems, epiphytic or hemi- epiphytic, are triangular (sometimes four or five), equipped with short spines, and lobed. They are long-day plants and bloom only at night. The flow- ers start to open at 19:00 and fully open at 22:00 [4].

Pitaya fruits are consumed in different ways as well as being consumed fresh. It is used in differ-

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ent food products such as desserts, yogurts, ice creams, marmalades, jellies, jams. Since pitaya fruits are rich in vitamins, minerals, and an excel- lent source of antioxidants, their consumption has become widespread in different ways. The fruits of pitaya varieties with red flesh color contain betacyanin, which is known as an essential antioxidant pigment [7, 8, 9]. In addition to betacyanin, the flesh of red pitaya contains high amounts of vegetable albumin, vitamins, and water-soluble fiber [10].

It has been reported to prevent diabetes and co- lon cancer due to its content's fiber, vitamins, and other active substances. Additionally, it is known to neutralize heavy metals, lower cholesterol and high blood pressure, and aid digestion. Pitaya varieties with red meat color are known to fight cancer, low blood pressure, and heart diseases, thanks to the lycopene they contain. The typical nutritional content for 100 grams of pitaya fruit is as follows; 83.0g water, 0.9g fiber, 0.012g carotene, 0.65mg iron, 0.61g fat, 0.68g ash, 36.1mg phos- phorus, 0.229g protein, 0.430mg niacin, 9.0mg acid ascorbic, 8.8mg calcium [11].

The production of the pitaya plant, whose cultivation is increasing, is mainly performed with seeds or stem cuttings. Although it varies according to the species, adverse effects such as low ger- mination rate, genetic expansions, and maturation within three years occur in seed production. Prop- agation with cuttings is the most widely used [12].

In propagation with cuttings, the plant can bloom after one year. Although production is made with seeds and cuttings, it cannot meet the market demand. In vitro tissue culture technique is used in pitaya production as an alternative method for dis- ease-free and faster production [13]. Plant tissue culture is a biotechnological tool for commercial- izing desirable elite plants.

Several biotechnological techniques can be performed on fruit crops to get better ones during the breeding process. One of the important plant biotechnology applications is planted tissue culture in fruit science. Plant tissue culture methods include several processes of clonal micropropagation [14]. Known technologies in plant tissue culture have been expanded to contain virus elimina- tion, somatic embryogenesis, organogenesis, production of haploid plants, and somatic hybridiza- tion [15]. It is also known that plant tissue culture studies can produce secondary plant metabolites [16, 17].

The development of effective tissue culture protocols is significant for rapid and massive proliferation and the conservation of genetic re- sources [4, 18]. Success in vitro micropropagation depends on culture medium components, plant explant type, genotype, and appropriate growing conditions. Therefore, new and updated protocols are great in plant tissue culture studies.

This study aims to evaluate the effects of different PGRs on micropropagation and rooting in in vitro propagation of different pitaya cultivars.

This article introduces new protocols to the literature and contributes to producing these commer- cially important pitaya varieties.

Material and Methods

All research studies were carried out in the biotechnology laboratories owned by Tekfen Agri- cultural Research Production and Marketing Inc., Adana, Turkey.

Plant material and Source of Explants

Plant materials were obtained from growers in Mersin, Turkey. Young shoots of Royal Red, Seoul Kitchen, Siam Red, and Physical Graffiti pitaya cultivars were used as explant sources for tissue culture.

Preparation of Explants and Surface Steriliza- tion

The explants were first washed with dishwash- ing liquid to remove their contamination. After re- moving the detergent residues, the explants were washed under running tap water for 20 minutes.

After these procedures, the explants were kept in 70% ethanol for 20 seconds and then in 1.5% (v/v) sodium hypochlorite for 20 minutes. The explants were finally washed thoroughly with distilled water.

Media and Culture Conditions

MS medium supplemented with 2.0mg/l BAP (Mc1) and 4.0 mg/l BAP (Mc2) was used for the micropropagation of plants. For rooting of plantlets, MS medium supplemented with 1 mg/l IBA (R2), 0.5mg/l GA3 (R3), 1 mg/l IBA + 0.5mg/l GA3 (R4) and hormone-free MS (R1) medium were used. Agar (7.5 g/l) was added to solidify the media, and the pH was adjusted to 5.8 using 1N HCl and 1N NaOH. Prepared media were auto- claved at 15 psi at 121°C for 20 minutes and dis- tributed in disposable sterile plastic containers.

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The explants, whose surface sterilization was completed, were cultured on MS basal medium supplemented with different plant growth regulators. All these cultures were incubated under 36 W cool white fluorescent lights in a light / dark pho- toperiod for 16/8 hours and at 25 ± 2°C. Time pe- riods of 3 subcultures were considered for micropropagation experiments. For rooting studies, a time period of 6 weeks was taken into account.

Acclimatization

Plants rooted in the in vitro conditions were removed under running water and cleaned of agar residues. After the cleaned plants were kept in 1g/l fungicide for 30 seconds, they were planted in plastic pots (5 cm x 6 cm width and depth) containing a 1:1 peat perlite mixture. After the plants were transferred to pots, their development was followed in greenhouses at an average temperature of 25 C.

Data Analysis

All trials were set up in a randomized plot de- sign with three replications. The obtained data were analyzed using the SAS-JMP statistical pro- gram and analysis of variance was performed. The differences between them were compared with the LSD multiple comparison test. Micropropagation coefficients were determined by considering the number of shoots per explant in micropropagation experiments. In this context, both cultivars and micropropagation media were evaluated. In rooting studies, root length (cm), % rooting, shoot length (excluding root) (cm), and callus formation per- centages were evaluated.

Results and Discussion

This study was conducted for micropropagation of different pitaya varieties in the media supplemented with different plant growth regulators.

When the cultivars were evaluated among themselves, the best cultivar was Royal Red with a micropropagation coefficient of 3.72. This was followed by Physical Graffiti (3.215), Siam Red (3.11), and Seoul Kitchen (2.88). When the mediums were evaluated among themselves, it was determined that the best medium was Mc1 with a micropropagation coefficient of 3.51, while Mc2 (2.95) medium was also found to provide successful micropropagation.

When the media were evaluated with pitaya varieties in the present study, the best result was determined in the Physical Graffiti variety reproduced in Mc1 medium with a micropropagation coefficient of 4.32. The lowest micropropagation coefficient was found in the Physical Graffiti variety reproduced in Mc2 medium with a coefficient of 2.11 (Table 1).

After micropropagation experiments, pitaya cultivars cultured in 4 different media were compared with their different properties (shoot length, root length, rooting percentage, and callus induction). In the general evaluation made according to the shoot lengths, the best medium was determined as R2 (2.86 cm), and no statistical difference was detected between the other mediums.

When the medium and cultivars were evaluated together, it was determined that the best result in terms of shoot length was the Royal Red variety developed in the R2 medium with 4.28 cm (Figure 1). It was determined that the lowest result was in

Table 1. The effect of media supplemented with different growth regulators on the micropropagation coefficient of pitahaya cultivars

Variety Medium Micropropagation coefficient

Code Content

Siam Red Mc1 MS + BAP (2 mg/L) 3.25 bc

Mc2 MS + BAP (4 mg/L) 2.97 bc

Royal Red Mc1 MS + BAP (2 mg/L) 3.69 ab

Mc2 MS + BAP (4 mg/L) 3.75 ab

Seoul Kitchen Mc1 MS + BAP (2 mg/L) 2.79 cd

Mc2 MS + BAP (4 mg/L) 2.97 bc

Physical Graffiti Mc1 MS + BAP (2 mg/L) 4.32 a

Mc2 MS + BAP (4 mg/L) 2.11 d

P<0.05*, P<0.01**, P<0.001***, LSD value was calculated according to the angle conversion value. Different letters (a–d) indicate significant differences according to the LSD test (p ≤ 0.05). LSD variety: 0.589***, LSD medium: 0.417***, LSD variety x medium: 0.834***

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Royal Red (1.05 cm) grown in R4 medium and (R4) Seoul Kitchen (1.05 cm) variety grown in the same medium, and it was determined that there was no statistical difference among them (Table 2).

The best results were obtained in Royal Red (3.56 cm) and Siam Red (3.25 cm) in evaluating varieties according to root lengths, and no statistical difference was observed between them. These were followed by Seoul Kitchen (1.48 cm) and Physical Graffiti (1.44 cm). Again, no statistical difference was found between these cultivars.

Consistent with these results among media, it was found that R2 (3.47 cm) and R1 (3.07 cm) media gave better results than other R3 (1.75 cm) and R4 (1.45 cm) media. In addition, it was determined that there were no statistically significant differences between them. In this context, when both medium and cultivars are evaluated together in terms of root length, the best result was determined in Royal Red (6.45 cm) cultivar grown in the R2 medium. In contrast, the lowest value was determined in Physical Graffiti (0.40 cm) cultivar grown in R3 medium.

Figure 1. Determination of rooting, callus formation, shoot length in pitahaya cultivars grown in different media.

(a) Royal Red rooted in R2 environment, (b) Physical Graffiti rooted in R4 environment.

Table 2. Effect of media supplemented with different growth regulators on shoot length, root length, % root formation and callus formation in pitaya cultivars

Medium Shoot Length (cm)

Root length (cm)

Rooting percentage (%)

Callus induction (%) percentage

Siam Red

R1 2.00 abc 3.51 bc 52 ef (46.16) 36 h (36.74) R2 2.73 abc 3.62 bc 88 ab (70.54) 48 fgh (43.84) R3 3.13 abc 4.53 ab 16 i (23.47) 20 i (26.45) R4 2.32 abc 1.34 cd 76 bc (61.17) 60 def (50.85)

Royal Red

R1 3.57 ab 4.61 ab 80 bc (63.55) 20 i (26.07) R2 4.28 a 6.45 a 92 a (74.48) 40 h (39.03) R3 2.65 abc 1.60 cd 24 hi (28.99) 70 bcde (56.92) R4 1.05 c 1.60 cd 60 de (50.87) 92 a (74.48)

Seoul Kitchen

R1 1.35 bc 1.81 cd 32 gh (34.33) 40 h (39.21) R2 1.50 bc 1.84 cd 84 abc (66.67) 44 gh (41.50) R3 1.55 bc 0.50 d 12 i (19.98) 72 bcd (58.07) R4 1.05 c 1.80 cd 72 cd (58.29) 56 efg (48.49)

Physical Graffiti

R1 1.63 bc 2.35 bcd 40 fg (39.14) 60 def (50.78) R2 2.95 abc 1.98 cd 88 ab (70.32) 76 bc (60.71) R3 1.30 bc 0.40 d 16 i (23.32) 62 cdef (52.00) R4 2.50 abc 1.06 d 72 cd (58.20) 80 b (63.60) P<0.05*, P<0.01**, P<0.001***, LSD value was calculated according to the angle conversion value. Different letters (a–i) indicate significant differences according to the LSD test (p ≤ 0.05). Shoot Length: LSD variety:

1.143***, LSD medium: 1.143***, LSD variety x medium: 2.28***, Root length: LSD variety: 1.198***, LSD medium: 1.198***, LSD variety x medium: 2.39***, Rooting Percentage: LSD variety: 4.901***, LSD medium:

4.901***, LSD variety x medium: 9,802***, Callus Induction Percentage: LSD variety: 4.414***, LSD medium:

4.414***, LSD variety x medium: 8.829***.

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In rooting experiments of Pitaya cultivars in different environments, % root formation values were determined and angle transformation was performed over these values and are shown in brackets in Table 2. When the findings are evaluated, the best rooting variety is Royal Red (54.47%), followed by Siam Red (50.33%), Phys- ical Graffiti (47.75%), and Seoul Kitchen (44.82%). When the environments were evaluated among themselves, it was concluded that the best environment was R2 (70%), followed by R4 (57.13%), R1 (45.79%), and R3 (23.94%), respectively. When both the medium and the cultivars were evaluated, the best result was found in Royal Red (74.48%) grown in the R2 medium. The lowest results were found as Royal Red (23.47%), Seoul Kitchen (19.98%), and Physical Graffiti (19.98%) developed in R3 environment, respectively.

In addition to these studies, calluses formed in plant varieties grown in nutrient media were also observed. The % callus formation values were determined and angle transformation was performed over these values and they are shown in brackets in Table 2. In this study, callus formation emerged as an undesirable situation. Considering the callus formation of Pitahaya cultivars, the least callus formation was detected in Siam Red (39.47%), followed by Seoul Kitchen (46.82%), Royal Red (49.12%), and Physical Graffiti (56.77%), respectively. Since callus formation is an undesirable feature, statistical results were interpreted in this direction. When the mediums were evaluated among themselves, the least callus formation was detected in the R1 medium (38.20%), followed by R2 medium (46.27%), R3 medium (48.36%), and R4 medium (59.36%), respectively.

When the medium and pitaya varieties are evaluated together, the least callus development was observed in Royal Red (26.07%) developed in R1 medium and Siam Red (26.45%) grown in R3 medium. The highest callus formations were observed in Royal Red (74.48%) variety grown in R4 medium, followed by Physical Graffiti (63.60%) grown in R4 medium and Physical Graffiti (60.71%) grown in R2 medium.

In a study with dragon plants, the effect of activated carbon on fungal contamination and callus culture, which is a problem in tissue culture, was investigated. While it was emphasized that activated carbon did not prevent fungal contamination, it was also reported that activated carbon reduced

tissue browning. On the other hand, it has been reported that it adversely affects the friability of calli [19]. On the other hand, no contamination problem was encountered in this study. In addition, although a particular protocol was not applied for callus culture, callus formations were observed.

While it was observed that the calli were soft and brown, it was predicted that it could adversely af- fect micropropagation in plants. It was determined that as the number of subcultures increased, calli could cause tissue death.

Investigation of suitable methods and nutrient media for micropropagation, which has an important place in the breeding of pitaya, will be effective in the cultivation of this plant. Since pro- ductions differ according to varieties and environmental conditions, the researchers will shed light on future studies. In a study conducted for this pur- pose, Gustavo Andres [20] studied the micropropagation of yellow pitaya (Selenicereus megalan- thus Haw) and found the highest proliferation coefficient to be 1.8 in T3 medium (MS + K (8 mg/L)). According to the study results in this article, it is a relatively low coefficient of proliferation. It is predicted that this situation may be caused by both different pitaya varieties and the difference between environments.

Zambrano-Forero [21], who conducted studies on the in vitro propagation of the yellow dragon plant (H. megalanthus), evaluated the different concentrations and combinations of two cytokines (BAP, Kinetin) and one auxin (IAA). The best results for shoot elongation (20.88 mm) and shoot proliferation (5.30 mm) were obtained in shoots cultured in MS medium supplemented with BAP 0.5 mg/L and BAP 1.0 mg/L, respectively. The best practice for root growth was Kinetin (KIN) 1.0 mg/L + IAA 0.3 mg/L. Although it has been reported that there is no significant difference between applications for root growth, it is seen that media containing BAP (0.5 mg/L) + IAA (0.3 mg/L) gives better results (7.89 mm). When the results are examined, it is seen that the medium containing only 1.5 mg/L KIN has a positive effect on the root length (7.05 mm). In the study in this publication, the values obtained were quite high and the root length was determined to be almost 10 times longer. In a similar study, Suman et al.

[22] investigated the effects of different plant growth regulators on tissue culture micropropagation of dragon fruit (H. undatus). Accordingly, they determined the least number of shoots (1.0 ±

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Figure 2. Images of pitaya varieties planted in plastic pots in the greenhouse, 15 months later 0.20) in the hormone-free MS basal medium used as the control medium. The highest root number (8.0 ± 0.5) and length (3.5 ± 0.06 cm) of explants were determined in MS medium supplemented with 3 mg/L BAP + 1 mg/L KIN + 0.2 mg/L NAA.

One of the methods frequently used in Pitahaya production is propagation with cuttings.

In a study using this method, Dano et al. [23] investigated the effect of commercial PGRs on the growth of cuttings of different pitaya genotypes ('Royal Red', 'Moroccan Red', 'Mexican White', and 'Hawaiian White'). No significant differences were detected in multifactorial trials with PGR.

On the other hand, in a one-factor experiment ex- amining the effect of genotype on root and shoot development, it was found that the red genotype (1.89 ± 0.34 new shoots for Moroccan Red and 1.44 ± 0.20 new shoots for Royal Red) was better in terms of shoot response than white genotypes.

However, the researchers suggested that the low number of shoots in the white genotypes may be due to ants' infestation. They reported that ants in- fested 14 of the 26 cuttings used and these were white genotypes. They also suggested that these may be secondary metabolites of white genotypes.

Similarly, in this study conducted in vitro, it was determined that the Royal Red variety was the best growing variety.

ElObeidy [24] reported that IBA positively af- fected pitaya cuttings. It found that it improved root count and root percentage. In the experiment performed on short cuttings (5 cm), it was determined that 10 mM IBA was more effective on rooting. When the related article is examined in detail, it is observed that the increase in IBA concentration (15 mM) has a negative effect on rooting. Experiments with higher concentrations will refute or strengthen this interpretation. In addition, although it was determined in the study that the si-

ze of the cuttings is also important in rooting pitaya, the IBA test was carried out in one size (5 cm).

Similarly, Ahmad et al. [25] determined that different concentrations (0-50-75-100 ppm) of IBA applied to pitaya cuttings had positive effects on rooting. In parallel with the increasing IBA concentration, an increase was observed in root characteristics (amount, length, diameter, fresh weight, and dry weight). The highest IBA concentration (100 ppm) has been determined that give better results. In this study, it was determined that IBA gave positive results on rooting in in vitro experiments. It was also determined that well-rooted plantlets had higher survival rates after transplant- ing into soil.

It has been determined that the plants cling to life and continue to grow during the acclimatization of the in vitro propagated pitaya cultivars to the external environment. It was determined that the Royal Red variety, which gave successful results in in vitro rooting, was also successful in acclimatization. On the other hand, it was observed that the growth progressed slowly in all cultivars (Figure 2).

Conclusion

This study evaluated in vitro propagation of 4 different pitaya cultivars with the potential for commercial production. While the best micropropagation was observed in the Royal Red variety, the lowest micropropagation coefficient was observed in Seoul Kitchen. When the media were evaluated, it was determined that the highest micropropagation was detected in the Mc1 (2 mg/L BAP + MS) medium, while the Mc2 (4 mg/L BAP + MS) medium also gave successful results in re- production. In rooting trials, the best shoot length, root length and percentage root formation were observed in Royal Red cultivar grown in R2 medium. In addition, it was determined that callus formation was low. It is clear that this study, which evaluates the efficiency of media in the in vitro production of different pitaya cultivars, will make significant contributions to the literature.

Acknowledgement

All experimental studies carried out in this research were realized with the support of Tekfen Agricultural Research Production and Marketing Inc.

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