INTRODUCTION:

Dragon fruit (Hylocereus costaricensis) is a perennial crop which grows in tropical and subtropical countries. Fruits which are edible have red-purple pigmented skin, while the color o pulp ranges from white to red and purple [1]. European Union, Asia, especially China is the largest import markets [2]. It is natural source of antioxidants and is generally and widely used in fruit salads [3]. In addition, it is also used as flavoring agent in beverages and food industries. Cross pollination is prerequisite for setting of fruits [4]. This becomes a hindering factor for large scale cultivation of dragon fruit even though superior planting materials are available.

Tissue culture or in vitro technique is an alternative method for large scale production of disease free improved high quality planting materials which is proved to be efficient and faster process in comparison with other conventional plant propagation methods [5, 6]. Though there were many studied on in vitro propagation of dragon fruit, only fewer reports are present on callogenesis. Also, efficient protocols for producing high quality planting material through tissue culture are very less [7,8]. In the present study we standardized the multiple shoot generation of red dragon fruit through callus for small and large scale tissue culture industries.

MATERIALS AND METHODS:

Collection of plant material and surface sterilization:

The dragon seeds were collected from fresh ripening fruit. The seeds were socked 3-4hrs in 4% sodium hypochlorite then washed with distilled water. Following this, seeds were washed with 70% ethanol for 2min and rinsed with distilled water for 3-4 times. Sterilized seeds were cultured on MS medium with 0.8% agar. Culture bottles containing seeds were incubated at 25±2⁰C under white fluorescent light with photoperiod of 16hrs light and 8hrs dark. Seedlings were obtained from culture bottles after four weeks. These stem cuttings were further used as explants for callus initiation.

Callus Initiation:

The good physically appeared plantlets were selected randomly. Plantlets were made into the small pieces under sterile environment and are used as explants. Explants are transferred to the MS medium supplemented with BAP and NAA in different concentrations. Culture bottles were maintained under white fluorescent light with photoperiod of 16 hrs light and 8hrs dark for two weeks. Bottles were regularly monitored for contamination and callus characteristics were measured.

Shoot Induction:

Whitish-yellow friable callus was carefully lifted using sterile forceps and is separated from the medium gently. Callus was cut into small pieces (5mm) and is cultured on MS medium supplemented with different concentrations of BAP and NAA (Table 1) to examine the capability of shoot initiation from callus. Culture bottles containing calli were incubated at 25±2⁰C under white fluorescent light with photoperiod of 16hrs light and 8hrs dark. Daily, culture bottles were monitored and optimum regeneration capability of callus was determined based on number of shoots regenerated.

Root Induction:

Shoots longer then 1.5cm was transferred into the rooting media, containing different concentrations of NAA in MS basal medium (Table 2). Sub culturing was carried out for every two weeks.

SEM analysis:

Six weeks old tissues were analysed using Scanning electron microscopy (SEM). The tissues were fixed with 3% of glutaraldehyde and 0.1M cacodylate buffers. The samples were then rinsed with cacodylate buffer minimum three time each with 10min interval, subsequently post fixed in cacodylate buffer along with 2% osmium tetroxide for 90min and dehydrated using acetone series 25%, 50%, 75%, 90% and 100%. The dried samples were fixed to two sides with carbon, observed the samples with SEM.

Statistical Analysis:

This experiment was carried in completely randomized design (CRD) with ten replicates in each experiment and the experiment was repeated thrice. Data was recorded at regular intervals. Data was analysed using ANOVA at 5% probability level. Values are expressed as mean of three replicates.

RESULTS AND DISCUSSION:

Success of in vitro propagation depends on type and concentration of plant growth regulators, explants selection and ex vitro plantlet survival [9]. When three months old seeds were utilized for raising of plantlets in vitro, seeds did not show any germination even though MS medium was provided with proper phyto harmones. This may be due to the loss of seed viability, indicating short seed viability period in red dragon fruit. Hence, freshly extracted red dragon fruit seeds were used. In the present work, stem cuttings were selected as ex plants and response of dragon fruit to BAP and NAA growth regulators was studied. The dragon fruit seeds are best for the production of disease free explants. The seeds were germinated and plantlets are as explants for regeneration. The explants (stem pieces) were directly grown on callus induction media (Figure 1a). At 3mg/l BAP and 0.5mg/l NAA good callus initiation was observed from explants (Figure 1b).

Fig 1 (a) Callus initiation from leaflet explants (b) Friable callus was formed (c) Shoots were initiated from the callus (d) Multiple shoots were formed (e) Root induction (f) Acclimatized young dragon fruit plants.

Good survival rate was recorded from stem explants. According to Thinesh and Seran [10], during indirect regeneration, high survival percentage and lowest contamination rate was observed from stem explants. Many studies reported using leaves as explants for callus initiation [11, 12]. Even under direct regeneration stem explants showed higher regeneration potential [13]. During incubation period, browning of explants was observed in few culture bottles. According to Ahmad, et al [14], phenolic compounds are released from cut surfaces of explants because of which tissue becomes dead and hence browning of tissue is observed. Among different concentrations used, 3.0mg/l of BAP and 0.5mg/l NAA revealed higher degree of callus formation than other combinations. Sub culturing is carried out regularly to avoid browning of calli. According to Meenashree et al., [15], there exists high phenolic content in explants because of which calli tend to become brown. After three weeks, whitish frabile calli started to produce multiple shoots. At 3.0mg/l BAP, callus formation was predominant while the same has been reduced when BAP concentration was increased to 3.5mg/l. Large multiple shoots were regenerated within 60 days (Figure 1d). Results are in accordance with Dahanayake and Ranawake [16]. Increased shoot proliferation from calli was also observed in soybean [17], wheat [18].

Table- 1 Effects of BAP and NAA on multiple shoots in Red Dragon fruit

S. No	BAP (mg/l)	NAA (mg/l)	Number of shoots from explants

1	1	0	1.2^abc
2	1.5	0	0.6^a
3	2	0	1^ab
4	2.5	0	3.2^e
5	3	0	4.2^hijk
6	3.5	0	4^efghij
7	4	0	3.4^efgh
8	4.5	0	3.2^ef
9	5	0	5.4^klm
10	1.0	0.5	1.4^abcd
11	1.5	0.5	3.2^efg
12	2	0.5	4.2^hijkl
13	2.5	0.5	8.4^p
14	3	0.5	13.2^r
15	3.5	0.5	8.4^pq
16	4	0.5	3.75^efghi
17	4.5	0.5	5.6^klmn
18	5	0.5	6.2^mno

Number of shoots- Values is mean three replicates. Means followed by different letters are significantly different at 5% probability

The regenerated shoots were grown up to 2.0cm height then transferred in to the rooting media the media contains NAA with different concentrations in MS basal medium. The brownish colored branched roots were observed after two weeks in the rooting media (Figure 1e). Among all different concentrations 3mg/l BAP and 0.5mg/l NAA resulted in efficient rooting (Table 2). Root induction was initiated regardless of shoot induction medium and the results are in line with Dahanayeke, et al [16] and Sauve, et al [17].

Table- 2 The effects of BAP and NAA on roots in Red Dragon fruit

S. No	BAP (mg/l)	NAA (mg/l)	Number of Roots Developed

1	3	0.1	1.4^b
2	3	0.2	0.8^a
3	3	0.3	2^efg
4	3	0.4	1.6^bc
5	3	0.5	4.8ⁱ
6	3	0.6	2.4^efgh
7	3	0.7	1.6^bcd
8	3	0.8	1.6^bcde
9	3	0.9	1.6^bcdef

Number of roots- Values is mean three replicates. Means followed by different letters are significantly different at 5% probability

Four weeks after rooting, plantlets were transferred in to the soil containing black colored polythene bags for primary hardening and observed daily (Figure 1f). Scanning electron micrograph of callus was produced from the dragon fruit with 3.0mg/l BAP and 0.5mg/l NAA (Figure 2). It shows the clusters of somatic embryos that were found as protrusions on the surface. These morphologies shows the different shapes viz., torpedo shapes, irregular surfaces and unorganized tissue types. This type of tissues demonstrated that have rapid growth and be highly friable.

Fig. 2 Embryogenic cultures of dragon fruit at 5 weeks of SEM images somatic embryos clusters and friable calli, the callus features including globular, unorganized and smooth structures.

Plant tissue culture is a selective method to produce disease free, virus free, herbicide resistant and high yielding plants material. Also, among different approaches, culturing plants in vitro, acts as a means of conserving commercial, rare and endangered plants rapidly [18]. Many researchers were developed suitable protocols for somatic embryogenesis for dragon fruit [19, 20]. However few reports were noted for the large scale production of dragon fruit material. In India red dragon fruit is rarely available fruit; in this study we developed best in vitro protocol for multiple shoot regeneration of red dragon fruit, which can be useful for larger scale multiplication in comparison with conventional propagation methods. Multiple shoot regeneration technology is economically profitable, faster and efficient method [17].

CONCLUSION:

Red dragon fruit explants produced16 shoots cuttings on MS basal medium supplanted with 3mg/l BAP and 0.5mg/l NAA within 60days. In this media explants were exhibits to a large extend well organized plantlets were produced. MS basal medium is supplemented with 3mg/l BAP and 0.5mg/l gave good rooting in dragon fruit plant. This protocol can be suitable for the large scale production of dragon fruit plantlets in in vitro condition.

REFERENCES:

1. Esquivel P., Stintzing F.C., Carle R. Comparison of morphological and chemical fruit traits from different pitaya genotypes (Hylocereus sp.) grown in Costa Rica. J. Appl. Bot. Food Qual. 2007a; 81: 7–14.

2. Le Bellec F. Pollinisation et fécondation d’ Hylocereus undatus et d’H. costaricensis à l’île de la Réunion. Fruits. 2004; 59: 411–422.

3. Rebecca O.P.S., Bovce A.N., Chandran S. Pigment identification and antioxidant properties of red dragon fruit (Hylocereus polyrhizus). Afr. J. Biotechnol. 2010; 9: 1450-1454.

4. Chang F.R., Yen C.R. Flowering and fruit growth in pitaya (Hylocereus undatus). J. Chinese Soc. Hort. Sci. 197; 43: 314- 321.

5. Choffe K.L., Victor J.M.R., Murch S.J., Saxena P.K. In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture. In Vitro Cell. Dev. Biol. 2000; 36: 30–36.

6. Dahanayake N., Ranawake A.L. Regeneration of dragon fruit (Hylocereus undatus) plantlets from leaf and stem explants. Trop. Agric. Res. Ext. 2011; 14: 85-89.

7. Lichtenzveig J., Abbo S., Nerd A., Tel-Zur N., Mizrahi Y. Cytology and mating systems in the climbing cacti Hylocereus and Selenicereus. American J. Bot. 2000; 87: 1058–1065.

8. Castillo R.M., Livera M.M., Alicia E., Brechú F., Márquez-Guzmán J. Compatibilidad sexual entre dos tipos de Hylocereus (Cactaceae). Rev. Bio. Trop. 2003; 51: 699–706.

9. Hartman H.T., Kester D.E., Davies Jr F.T., Geneve R.L. Plant Propagation: Principles and Practices. Prentice-Hall. London. 2002; 7: 886.

10. Thinesh A., Seran T.H. In vitro callogenesis from bud and stem explants of dragon fruit (Hylocereus undatus). Asian J Pharma Sci Tech. 2015; 5(4): 253-256.

11. Choffe K.L., Victor J.M., Murch S.J., Saxena P.K. In vitro regeneration of Echinacea purpurea L.: direct somatic embryogenesis and indirect shoot organogenesis in petiole culture. In Vitro Cell Develop Biol Plant. 2000; 36(1): 30-36.

12. Malathi R., Melchias G., Chandrasekar S., Cholarajan A. Role of Aluminium and Phosphorus on the Secretion and Behaviour of Acid Phosphatase in Callus Culture of Brassica juncea. Research J Engineering Tech. 2012; 3(4): 259-261.

13. Dahanayake N., Xiao-Lu Chen., Fu-Cheng Zhao., Yue-Sheng Yang., Hong Wu. An Efficient In Vitro Propagation System for Purple Cone-Flower (Echinacea Purpurea L). J of Tropical Agric Res and Extension. 2010; 13(2): 29-32.

14. Ahmad I., Hussan T., Ashraf I., Nafees M., Maryam, Rafay M, Iqbal M. Lethal effects of secondary metabolites. Am. Eurasian J. Agric. Environ. Sci. 2013; 13: 539-547.

15. Meenashree B., Kathiravan G., Srinivasan K., Rajangam B. Effect of Plant Hormones and Media Composition on Browning and Growth of Bacopa monnieri Callus Cultures. Research J Pharma Tech. 2017; 10(2): 497-500.

16. Dahanayake N., Ranawake A. L. Regeneration of Dragon fruit (Hylecereus undatus) plantlets from leaf and stem explants. Tropical Agric Res and Extension. 2011; 14(4): 85-89.

17. Sauve J.R., Mmbaga T.M., Zhou S. In vitro regeneration of the Tennessee coneflower (Echinacea tennesseensis). In Vitro Cell. Dev. Biol. Plant. 2004; 40: 325-327.

18. Rathnan Rahna K., Francis Jose., Irene Pius., Lamia Sathar., Nihil Jimmy., Shwetha Sasidharan., Ambili Mechoor. Comparative Study of Antimicrobial Activity of Leaves and Callus Extract of Ipomea turpethum against Antibiotic Resistance Pathogenic Microorganisms. Research J Pharma Tech. 2012; 5(6): 805-808

19. Yassen M.Y. Micropropagation of pitaya (Hylocereus undatus Britton & Rose) In Vitro Cell. Dev. Biol.Plant. 2002; 38: 427-429.

20. N Tel-Zur., S Abbo., D Bar-Zvi., Y Mizrahi ci. Clone Identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis. Scientia Hortic. 2004; 100: 279–289.

Received on 11.11.2018 Modified on 18.12.2018

Research J. Pharm. and Tech. 2019; 12(4): 1491-1494.

DOI: 10.5958/0974-360X.2019.00246.4