29 Dec 2020

Different Stages of Banana Tissue culture

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

The micropropagation of bananas started in the early 1970's by shoot tip culture. At that time, the sole purpose of in vitro propagation was the mass production of plants. In 1974, Berg and Bustamante cultured the species by using meristem culture in combination with heat treatment to obtain pathogen-free banana plants.

The in vitro propagation of banana and other plants involves three major steps:

1. Initiation of an aseptic culture
2. Multiplication of propagules
3. Rooting of plantlets and transfer to soil

Each step in the in vitro propagation of bananas depends on the specific media composition and addition of growth regulators. In this article, all the three steps of banana micropropagation are discussed. 

Three Stages of Banana Shoot Tip Culture

1. Initiation of Shoot Tip Culture

The initiation step includes the collection, disinfection, excision, and incubation of the explant. The first step is to cut the sucker of 40-100 cm in height from the source plant using a sharp knife. Then, cut a cube of tissue measuring 1-2 cm^3, containing the apical meristem, from the selected sucker.

Explant Disinfection 

Wash the cubes of tissues under running tap water, then rinse it with 95% alcohol for 15-30 seconds. Then, immerse the cubes in a solution of bleach (0.75% w/v sodium hypochlorite) for 15-20 minutes. Add liquid detergent (one drop per 50 ml of Tween 20, Teepol, or Tween 80) to the bleach solution by swirling it frequently.

Then, decant the bleach solution and rinse the cubical tissues with three changes of sterilized distilled water.

Explant Excision 

Put the disinfected block of tissue in a sterile petri dish, using sterile forceps. Then, remove superficial tissues of the block exposed to bleach. Excise the leaf primordia using a sharp scalpel and remove as much corm tissue you can. At last, you should have a 5 mm conical shoot tip.

After excision, immediately transfer the explant in a sterile solution of 50 mg/L cysteine to reduce or avoid the blackening of the explant and the medium.

Explant Incubation 

After the excision of the explant, dip it into cysteine solution and transfer it to the growth culture media which is a basic MS media supplemented with certain growth regulators. Two commonly used growth regulators in banana initiation media are auxin [0.175 mg/l indole-3-acetic acid (IAA)] and cytokinin [2.25 mg/l 6-benzyladenine (BA)]. The media is also supplemented with 30-40g/L sucrose. Further, the gelling agents are added to the media which either include agar (5-8 g/L) or Gelrite (2-4 g/L). It’s preferable to use Gelrite over agar in banana propagation because its higher transparency allows detection of contamination at early stages. Then, incubate the cultures at an optimal growth temperature of 28 ± 2°C in a light cycle of 12-16 h with a photosynthetic photon flux (PPF) of about 60 µE/m2s1.

2. Multiplication of Shoot Tip Cultures

The composition and components of the multiplication media are the same as the initiation media except for cytokinin, which is added in higher concentration. At the multiplication stage, a higher concentration of cytokinin BA (0.1-20 mg/L) in the media reduces the apical dormancy of the apical meristem. This stimulates the formation of axillary and adventitious buds from the explant.

After 6-12 weeks of culture initiation, the shoot is developed. Then, the shoots are subcultured repeatedly at an interval of 4-6 weeks in the multiplication media. The factors that affect the multiplication rate of the explant in culture include genotype, cytokinin concentration, shoot tip fragmentations, size of the explant, and culture duration.

3. Regeneration of Plants and Their Establishment

After proper shoot development, the plantlets are transferred to rooting media for the formation of roots. This step is essential because the plantlets are very small and if they are transferred to the soil without roots, they won’t survive. The shoot elongation is also achieved in this stage as the rooting media is advantageous for both shoot elongation and root formation.

At this stage, a higher concentration of cytokinin inhibits the root formation. The cytokinin BA is added in a concentration of 1 µM that is much lower than the multiplication media (0.1-20 mg/L). Auxin has a major role in root development and the most common auxins used in culture media include NAA, IAA, and IBA. The auxin NAA, in a concentration range of 0.2-1 mg/L, has been found effective in inducing rooting in banana cultivars.

After roots are developed, the plantlets either can undergo in vitro hardening before they are transferred to soil or they can be left on the rooting media for 2-3 weeks before directly transferring to the external environment. Special care is needed in this step as the tissue culture grown plants are delicate because of their growth in a highly sterilized environment. When transferred to the external environment, plants have to adjust to varying physiological conditions. So, carelessness in this step can result in significant losses of plants.

References

1. Vuylsteke R. D. (1998). Shoot tip culture for the propagation, conservation, and Distribution of Musa germplasm. Internation Institute of Tropical Agriculture, Ibadan, Nigeria.
2. Wong, W. C. (1986). In vitro propagation of banana (Musa spp.): initiation, proliferation, and development of shoot-tip cultures on defined media. Plant Cell, Tissue, and Organ Culture, 6(2). doi:10.1007/bf00180799
3. http://www.fao.org/3/ae216e/ae216e03.htm#
4. https://www.slideshare.net/sathes32/tissue-culture...