Tissue Culture of Paphiopedilum species: An Effective Approach

Table of Contents

Paphiopedilum belongs to the family of Orchidaceae. The plants are known by the common name of “slipper orchids”, This is mainly due to the modified lip or pouch-shaped lip of the plant that gives it a slipper-like appearance.

Introduction

Paphiopedilum belongs to the family of Orchidaceae. The plants are known by the common name of “slipper orchids”, This is mainly due to the modified lip or pouch-shaped lip of the plant that gives it a slipper-like appearance.

The plant is among the highly valued or most commercially demanded orchids due to its spectrum of shape, size, and colors of flowers. Moreover, the plant also has attractive, marbled, and evergreen foliage.

ThePaphiopedilum species has around 96-100 species. They are most extended to the regions of India, China, Philippines, Southeast Asia, Malay Archipelago, New Guinea, and the Solomon Island.

Some species of the plants are also extinct to their overcollection and loss of favorable habitat. Thus, they are listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora.

In this article, we will learn about the propagation of the Paphiopedilum plant and how the tissue culture technique is an effective technique to grow these plants to fulfill commercial demands and protect the rare ones from getting extinct. You will also learn a procedure to tissue culture the P. rothschildianum using protocorm-like bodies.

Propagation of Paphiopedilum

Conventionally, Paphiopedilum is cultured through the division of the axillary bud from the mother plant. However, it's an inefficient, laborsome, and time-taking process. In one year, only one new growth can be observed in the plant after the flowering of a mature plant. Thus, the technique hampers the propagation of the plant on a commercial scale.

Also, germination of the lant through seed is also a time taking approach due to morphological and physiological characteristics. So, an alternative approach to the problem is the use of tissue culture in the propagation of the Paphiopedilum plant. The technique has been practiced on orchids for more than a century to form uniform clones of the plants and for many other purposes.

To define, tissue culture is an in vitro technique that utilizes the potential of plant cells, to divide and grow into any cell (termed as totipotent), to grow plants using a few tissues from any parts of the plants.

Tissue Culture of Paphiopedilum

Paphiopedilum is very popular among culturists for its availability in a variety of shapes, sizes, and colors of its flowers and attractive foliage. However, the growth of some species of plants is very slow. Thus, tissue culture is an effective technique to grow these plants on a commercial scale and meet people’s demands.

Here’s a procedure to culture Paphiopedilum rothschildianumusing protocorm-bodies like explant. It’s taken from Ng, C.-Y., & Saleh, N. M. (2010). In vitro propagation of Paphiopedilum orchid through the formation of protocorm-like bodies. Plant Cell, Tissue and Organ Culture (PCTOC), 105(2), 193–202. doi:10.1007/s11240-010-9851-0.

Plant Material

• Collect the 5-month-old capsule of the P. rothschildianum after self-pollination.
• Submerge the capsule in 95%(v/v) ethanol.
• Keep it on the flame for a few seconds under completely sterile conditions.
• Split the capsule longitudinally using a sterilized scalpel.
• Transfer the seeds onto a half-strength MS medium with 0.3% (w/v)Gelrite for seed germination.
• Maintain the germinated seedling on MS medium.
• Take the nodal explant of the germinated plant.
• Remove all the roots and leaves.
• Use the 5-mm portion of the basal part of the plant to induce callus.

Callus Induction

• Culture the nodal stem explants on 1/2MS supplemented with 170 mg/L NaH2PO4, 30 g/L sucrose, and solidified using 0.3% (w/v)Gelrite. Then, add plant hormones supplemented with 4 µM kinetin and 2 g/L peptone.
• Adjust the pH of the medium to 5.5–5.7 using 1 N NaOH or 1 N HCl.
• Autoclave the medium at 121 ℃ for 15 min at 1.05 kg cm-2.
• Place the nodal explant on a solid medium and culture them in the dark at 25±2 ℃.
• Subculture the induced callus onto a similar ½ MS medium every 8 weeks for further proliferation and development of the protocorm-like bodies (PLB).

Induction and Proliferation of Protocorm-like bodies (PLB) 

• Use the induced PLB to induce secondary PLBs.
• Culture the PLBs on half-strength MS medium supplemented with 4.0 µM kinetin.
• Keep the plants in dark for 8 weeks at 25 ± 2 ℃.
• After 8 weeks, subculture the PLB onto half-strength MS medium, plant growth regulator-free, and supplemented with 60 g/L banana homogenate (BH).
• Keep the cultures under continuous light (40 µmol m-2 s-1, provided by fluorescent lamps) at 25±2 ℃.

Plantlet Regeneration

• Culture the PLBs in clump on 1/2MS supplemented with 20% (v/v) coconut water.
• Separate the plantlets from the previous medium into separate single shoots of 1 cm and transfer them to another medium consisting of PGR-free ½ MS supplemented with 60 g/L potato homogenate for further development.
• After around 12 weeks you will observe leaves coming out of the plants and a developing root system.

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Happy Culturing!!