Dragon Fruit Tissue Culture: A Sustainable Approach for

Mass Propagation
Dr. Vangapandu Thriveni, Dr. Eggadi Ramesh Dr. Anant Tamang, and Prof. (Dr.) S.
Eswara Reddy
Summary:
Dragon fruit (Hylocereus spp.) is a tropical fruit known for its vibrant colors, unique
appearance, and numerous health benefits. With the growing demand for dragon fruit
globally, the need for efficient and sustainable propagation methods is essential to meet the
market requirements. Tissue culture, a technique that involves the aseptic culture of plant
cells, tissues, or organs, has emerged as a valuable tool for the mass propagation of dragon
fruit. This project aims to explore the tissue culture techniques for dragon fruit propagation,
optimize the protocols, and evaluate the potential of tissue culture for large-scale production.

Introduction : Dragon fruit (DF) is an exotic fruit that has recently entered the Indian market
and gained popularity in the agriculture sector and among the general public. It is known for
its mesmerizing color, shape, size, and the flesh of the fruit, which is loaded with small black
edible seeds. DF is cultivated in various regions of India and is attracting farmers and agri-
businesspersons due to its nutritious properties. It belongs to the climbing vine-like epiphytic
cacti plant species of the genus Hylocereus and Selenicereus, which are part of the family
Cactaceae and subfamily Cactoideae (Le et al., 2021). The genus Hylocereus and
Selenicereus are native to the forests of Mexico, northern South America, and Central
America (Mizrahi et al., 1997). One significant species of the genus is Selenicereus
megalanthus (Schum.) Britt. and Rose, also known as Yellow Pitaya or Pitaya amarilla
(Bellec & Vaillant, 2011; Perween et al., 2018). The most popular and commercialized
species are H. undatus (White flesh dragon fruit [WFDF]) and H. polaris (Red flesh dragon
fruit [RFDF]), which are grown in various parts of the world. Dragon fruit is a non-
climacteric fruit plants that climb on a single standing support system (1.5 to 2 m) made of
concrete, wood, or other materials (Liaotrakoon, 2013). It has many advantages including low
water and nutrient requirements, relatively less requirement of resources for establishing the
orchard and maintenance; multiple harvests of fruit in a year; potential to sustain a high yield
up to 20 years; high benefit to cost ratio; and high nutraceuticals and functional properties
(e.g. rich in antioxidants and fibres).
Overview of dragon fruit and its significance
Dragon fruit, scientifically known as Hylocereus spp., is a tropical fruit native to Central
America and is now cultivated in various regions around the world. It is also commonly
referred to as pitaya, named after the fruit of several cactus species from which it originates.
Dragon fruit is known for its striking appearance, with vibrant colors and a unique shape that
resembles a dragon's scales, hence its name.
Although the seed germination efficiencies for H. undatus range from 71 to 83
percent, seed propagation is not commercially viable due to the long juvenile period and
delayed fruit production. However, tissue culture offers a solution by enabling the production
of a large number of clonal plants using small plant material. Hylocereus undatus exhibits
branching in its younger sections, while its basal parts can only produce callus. The choice of

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appropriate explants and medium is crucial for achieving enhanced dragon fruit response in
vitro, flower buds from Mammillaria sp., and explants from meristematic tissue of
Selenicereus megalanthus. This study focused on developing an efficient and viable protocol
for in vitro establishment and vegetative propagation of two white-fleshed pitahaya varieties,
Hylocereus undatus, and H. megalanthus, using different-sized stem segments and shoot tips
as explants. Additionally, the study describes a successful regeneration protocol for
generating multiple shoots from in vitro-derived plants.
Review of Literature:
In dragon fruit tissue culture established a protocol for callus induction, shoot regeneration,
and acclimatization of plantlets, providing a foundation for large-scale propagation (Zhang et
al., 2019). A combination of cytokinins and auxins significantly enhanced the multiplication
rate and root induction, leading to efficient plantlet production (Chen et al., 2018). The
advantages of tissue culture in terms of rapid multiplication, disease-free plants, and year-
round production, emphasize its economic feasibility and potential benefits for dragon fruit
growers (Rahman et al., 2022).
Objectives:
 To develop an efficient tissue culture protocol for the mass propagation of dragon fruit
(Hylocereus spp.).
 To evaluate the growth and development of in vitro-propagated dragon fruit plants
under greenhouse conditions.
 To assess the genetic stability of tissue culture-raised dragon fruit plants using
molecular markers.
 To compare the agronomic performance of tissue culture-derived plants with
conventionally propagated plants.
 To analyze the economic feasibility of dragon fruit tissue culture as a sustainable
approach for mass propagation.
Rational and Scope:
1. Rapid multiplication: Tissue culture allows for the production of a large number of
clonal plants in a relatively short period of time.
2. Disease-free plants: Tissue culture provides a means to produce disease-free plants, as
the explants used for initiation are carefully selected and sterilized.
3. Year-round production: Tissue culture allows for the production of dragon fruit plants
throughout the year, irrespective of seasonal limitations.
The scope of dragon fruit tissue culture:
1. Callus induction: The initiation of callus from selected explants forms the basis for
subsequent regeneration and multiplication of plantlets.
2. Shoot regeneration: This step focuses on obtaining multiple shoots from the callus,
which can later be developed into complete plantlets.
3. Rooting and acclimatization: The in vitro regenerated shoots are rooted using suitable
hormonal treatments, and the rooted plantlets are then acclimatized to the ex vitro
conditions. This phase involves carefully transferring the plantlets to a greenhouse or
field environment, ensuring their successful adaptation and survival.

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 4. Agronomic evaluation: Tissue culture-derived dragon fruit plants can be evaluated for
their agronomic performance compared to conventional cuttings.
Research Methodology
Plant Material and Culture Initiation
The mother Dragon fruit plant (Hylocereus undatus & H. megalanthus), from which explants
were taken was obtained from the greenhouse (about 1 meter length from stem ) and shoot
tips.
Sterilization procedures to establish aseptic conditions
Surface sterilization of explants:
Areoles segments of 5 cm length were surface sterilize by submerging the explants into a
solution of 70% ethanol with continuous and gentle stirring for one min, transferring them to
100 ml conical flask containing 20% solution of commercial sodium hypochlorite with
continuous gentle stirring for seven min. The sterilant was decanted and the explants were
washed with three successive rinses of sterile distilled water under aseptic conditions. The
explants were then dried between two layers of sterile filter papers in a Petri dish. Using
sterile scalpel, the explants were cut into smaller pieces (2.5 cm length) containing buds.
Media Preparation and Culture Techniques
Culture medium:
The basal salts mixture of MS medium containing 30 g L−1 sucrose, solidified
with 7.0 g L−1 agar and pH adjusted to 5.7.
Treatments: For in vitro shoot (mini or micro-joints) induction, each of benzyl adenine
(BA), kinetin (Kin) and thidiazuron (TDZ) was added to MS culture media at 3
concentrations (0.5, 1.0 and 1.5 mg/l) to induce multiple shoot morphogenesis from explants.
Explants were subjected to cytokinin treatments for 12 weeks. The percentage of response
and the number of shoots per explant was counted after 2, 4, 6, 8, 10, and 12 weeks. After
that individual mini-joints were excised and transferred under aseptic conditions to the root-
inducing media. For in vitro root morphogenesis of the obtained mini-joints, MS culture
media was impregnated with indole butyric acid (IBA) at a concentration of 0.5 mg/l,
naphthalene acetic acid (NAA) at a concentration of 0.5 mg/l and a mixture of both IBA and
NAA (0.25mg/l each) with and without the addition of charcoal at 1, 2 and 3%. The rooting
treatment lasted for 8 weeks and after that regenerants were taken out of jars, washed and
transferred to pots for acclimatization after counting the number of rootlets obtained and
measuring the average root length.
Formulation of culture media with necessary nutrients and growth regulators
Exploration of different media types (e.g., Morishige and Skoog, Gamborg, etc.)
Study of growth regulators (e.g., cytokinins, auxins) for shoot initiation, multiplication, and
root development.
Cultural conditions: Cultures were generally incubated in a growth room under controlled
conditions, where the temperature was maintained at 25 ± 1 °C, day/night schedules 16/8 at and low
light intensity of 1500 lux using a white cool fluorescent lamp 120 cm long 40 watts.
Acclimatization: Rooted plantlets were taken out of the growth jars, and transferred to pots
containing (peat moss: sand: clay in a ratio of (1: 1:1 v/v). No, special measures were needed for
acclimatization which reached almost 100% success.
Micropropagation techniques (e.g., shoot tip culture, nodal segment culture)

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Recommendations for further research and implementation
By conducting this project, we aim to contribute to the advancement of tissue culture
techniques for dragon fruit propagation, thereby supporting the sustainable production of this
valuable fruit.
Expected Outcomes:
 A standardized and optimized protocol for the tissue culture propagation of dragon
fruit, enabling the production of a large number of high-quality plantlets.
 Healthy and vigorous dragon fruit plants derived from tissue culture, exhibit rapid
growth and enhanced tolerance to biotic and abiotic stresses.
 Genetic stability of tissue culture-raised plants confirmed through molecular marker
analysis, ensuring the preservation of desirable traits.
 Comparative evaluation of tissue culture-derived plants against conventionally
propagated plants, demonstrating their superiority in terms of growth rate, fruit yield,
and overall performance.
 Economic analysis revealing the viability and potential cost-effectiveness of dragon
fruit tissue culture as a sustainable approach for mass propagation, encouraging its
adoption by growers and nurseries.
Timeline: 2 Years
Cost estimation of tissue culture in dragon fruit can vary depending on various factors such as
location, scale of production, infrastructure, and the specific requirements of the tissue
culture facility. However, here's a general breakdown of the costs involved in setting up a
tissue culture lab for dragon fruit production:
Tissue Culture Supplies:
Culture vessels: This includes containers like test tubes, flasks, Petri dishes, and trays. The
cost will depend on the quantity and size required, but it could range from 50000/- or more.
Growth media and chemicals: This includes plant tissue culture media, plant growth
regulators, agar, nutrients, and other chemicals. The cost will depend on the quantity and
quality of the supplies, but it is in Rs.1,15242.00/-.
Expendable materials/items: Rs.16258/-
Acclimatization cost: Includes structural components like shade net houses, poly tunnels etc
required for acclimatization of the tissue culture-raised Dragon fruit plant population
(Rs.8500/-)
Plant Materials:
Source plants: You would need to obtain healthy mother plants of dragon fruit to initiate the
tissue culture process. The cost will depend on the source and the number of plants required,
but it could range from 32000/-.
Cost estimation of tissue culture Dragon (two year)
s.no. Input cost Requirement Rate Amount (Rs.)
a. Growing media & 115242.00/-
chemical cost
B. Culture vessels: 126000.00/-
(containers like test
tubes, flasks, Petri

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 dishes, and trays)
C Plant material cost 150 250/per plant 32000/-
D Expendable 16258/-
items/materials
E Skilled Personal 1 15000/month 180000/-
F Acclimatization cost 8500/-
Total cost of estimation 5,00,000/-
Expected Out comes No.
1 Mass multiplication of Dragon fruit plantlets (in no.) 100000 population
2 Research publications 3
3 Patents 1
The total cost of estimation for rapid multiplication of white-fleshed dragon fruit =
Rs.500000/-
All infrastructure facilities, equipment, utilities; electricity, water, gas supply, and regular
maintenance and calibration of equipment provided by Administrators of CUTM,
Paralakhemundi.
References:
Chen, J., Zhang, L., Yang, Y., & Li, M. (2018). Effects of plant growth regulators on in vitro
shoot proliferation and rooting of dragon fruit (Hylocereus spp.). In Vitro Cellular &
Developmental Biology - Plant, 54(3), 274-283. DOI: 10.1007/s11627-018-9902-y.
Rahman, M. M., Rauf, M. A., Sani, W., Aziz, M. A., & Mohamed, M. T. M. (2022).
Economic viability of tissue culture technology for dragon fruit (Hylocereus spp.)
production: A review. Scientia Horticulturae, 294, 110532. DOI:
10.1016/j.scienta.2021.110532.
Tan, W. Y., Lai, Q. X., Heng, P. C., Lee, C. T., & Chin, C. F. (2021). Agronomic Performance
of Tissue Culture-Derived Hylocereus sp. (Dragon Fruit) Compared to Conventional
Cuttings. Horticulturae, 7(2), 30. DOI: 10.3390/horticulturae7020030.
Yap, Y. S., Ho, C. L., Aziz, M. A., & Harikrishna, J. A. (2020). Genetic stability analysis of
tissue culture-raised Hylocereus sp. (dragon fruit) using RAPD markers. The Nucleus,
63(1), 65-73. DOI: 10.1007/s13237-020-00334-3.
Zhang, S., Huang, X., Guo, Z., Li, X., Liu, H., Huang, H., & Huang, Y. (2019). Establishment
of an efficient regeneration system for in vitro propagation of dragon fruit
(Hylocereus undatus). Plant Cell, Tissue and Organ Culture (PCTOC), 138(2), 333-
341. DOI: 10.1007/s11240-019-01708-3.