24 Feb 2022

Advancements in Coconut Tissue Culture (Part-2)

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Introduction

The coconut tree, also known as the tree of life, has several economical uses in our lives. Each part of the plant can be consumed as fresh or refined products.

Over the years tissue culture techniques have advanced many economical or horticulture plants, excluding coconut palm. The process for the development of techniques to increase the market of coconut has been slow.

There are several reasons for the slow advancements, including its heterogeneity, spread of diseases and pests (such as cadang-cadang), and reduced production with the aging of the plant.

In the previous part of this article, we’ve discussed all the useful coconut trees in our lives, challenges faced by the coconut field, and why tissue culture is a better approach for increasing coconut production.

This article provides you with a glimpse of the previously achieved results in coconut tissue culture and explains the methods used to increase coconut production in the past.

Examples of a few Advancements in Coconut Tissue Culture

The progress in tissue culture of coconut has been sluggish due to its recalcitrant behavior. And, to achieve this we need a viable protocol, that can be done by:

Optimization of the most effective, responsive, and suitable explant (both stage and type)
Preparation of suitable culture media
Supplementation of the media with suitable type and quantity of plant growth regulators.

Given below are some examples of successful coconut tissue culture experiments performed by culturists in the past:

By using somatic embryogenesis, it is possible to propagate cells from zygotic plumular tissue. Despite this, it is still difficult to establish a universal protocol for the production of plantlets at an affordable large scale.
Zygotic embryo culture has now enabled the rapid production of seedlings derived from tissue culture and the collection of rare germplasm. Recently, this technique has been improved to yield greater results across a larger range of cultivars.
An anther-derived embryogenesis technique is used to produce doubled haploid plants.
Using cryopreservation, coconut embryos can be preserved and then regenerated without causing morphological, cytological, or molecular changes.
Though attempts have been made to genetically modify coconut, the results have been limited.

Coconut Tissue Culture Techniques

Developing impact tools in the areas of breeding and genetic resources has been enabled by the application of tissue culture techniques in coconut propagation. The technique has been widely used in breeding programs, conservation, germplasm exchange, gene introgressions, and genotype cloning.

Given below is a brief on tissue culture techniques used for enhancing coconut production—explaining how they have been used to propagate the plant. You’ll learn what techniques and hormones have been used so far in coconut tissue culture processes.

Embryo culture

10-14 months old zygotic embryos (post-pollination) have been used to establish coconut culture, and among them, 12 months have shown greater success in ex vitro conditions.
Among the different types of media, Y3 (Eeuwens and Blake medium) media was used to grow coconut trees. The media contains half the amount of ammonium and nitrate nitrogen contents and a tenfold greater concentration of iodine, copper, and cobalt, in comparison to MS (Murashige Skoog Media).
More than 4% of sucrose was proved to be useful for embryo regeneration and the use of activated charcoal was found good to prevent tissue necrosis.
1.5–0.8 % agar was preferable to create solid media for the plant however, in most studies two-stage medium was preferred, in which liquid media was used for embryo germination followed by agar medium.
Supplementing the media with gibberellic acid (0.5 micromolar) is effective for embryo regeneration and root growth, certain auxin analogs such as NAA (naphthalene acetic acid) or IBA (indole-3-butyric acid) are effective.
The environmental condition suitable for the plant is a warm temperature (25–31 °C), first in the dark (for 5–8 weeks), and then in the light (45–90 micromol m-2 s-1) until first signs of germination.
A mixture of peat moss and soil (1:1, w/w) in black polyethylene bags has been effective to acclimate tissue-cultured plants.
Despite the success of embryo culture in many coconut cultivars and the fact that the technique can be used to collect and exchange germplasm, the number of mature plants in the soil can be very low. Thus, in this case, protocol optimization is required.

Clonal propagation via somatic embryogenesis (SE)

Somatic Embryos in coconut can be achieved by either using somatic tissues (e.g., immature inflorescences and ovaries) or easier to manipulate zygotic tissues (e.g., immature or mature embryos and embryo-derived plumules).
For callus culture, the Y3 and BM72 are effective, compared to MS and B5 media.
Sucrose (3–4 %) is essential to initiate coconut SE, while charcoal (0.1–0.3 %) has been extensively used to prevent oxidation of explanted tissues and calli.
In zygotic embryo-derived callus cultures, polyvinylpolypyrrolidone (PVPP) was shown to promote the SE rate.
Usually, a high concentration of auxin (especially 2,4-dichlorophenoxyacetic acid (2,4D) is used to form callus. However, it has a different working concentration depending on the cultivar and type of explant.
A cytokinin such as 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin (KIN), or 2-isopentyl adenine (2iP) at a concentration of 5–10 microMolar is often added to the callus proliferation and maturation medium.
The best conditions to form the callus are often dark for at least one month at 28°C after culture initiation.

Homozygote production via anther culture

In some plant species, double haploid plant production is an ideal technique to overcome lengthy breeding cycles.
Anther culture in the BM72 (Karunaratne and Periyapperuma) media with a high concentration of sucrose (9 %) is used to produce somatic embryo structures, with root and shoot apices.
Microspore callus production can be achieved by using a moderate concentration of 2,4-D (100 lM) with the addition of TDZ (9 lM) and NAA (100 lM).
AgNO3 (10 lM) and ABA (5 lM) are used together for the maturation of the somatic embryos.
It takes at least 10 weeks for the callus cultures to grow at 28 C in the dark

Germplasm conservation via cryopreservation

Cryopreservation of coconut tissues can be achieved through either using mature (11 months post-pollination) zygotic embryos and using a physical dehydration method; or using plumule tissues excised from mature zygotic embryos combined with a chemical dehydration method.
The cryopreservation involves four steps:

Pre-culturing of explanted tissues before drying.

Tissue dehydration

Tissue freezing

Tissue recovery involving thawing and plantlet production

The most commonly used agents for chemical hydration are sucrose, glucose, and glycerol, all at high concentrations ([10%, w/v).
A rapid freezing approach has been widely used for coconut tissues.
Y3 media has been most commonly used in the tissue recovery stage.
When applied alone or in combination, auxins (2,4-D, NAA, or kinetin) had little effect on embryo germination or plantlet recovery.
The addition of high doses of sucrose (4–6 %) is important for the germination of the recovered embryos.

There’s a huge scope to advance the coconut tissue culture processes for better yield and commercialization of coconut and its derived products. It’ll also open doors to the transfer of coconut germplasm around the globe.

How Plant Cell Technology Is Helping Culturists Worldwide In Their Tissue Culture Application?

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And, that’s not it! Plant Cell Technology also offers consultation services to culturists of all sizes that help to get instant solutions to your tissue culture problems.

So, visit plantcelltechnology.com today and find out more about our product and services and how they help you to excel in your tissue culture processes.

Happy Culturing!!

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