Have you heard about Hawaiian silversword (Argyroxiphium sandwicense)?
This long, narrow leaves plant is endemic to the islands of Maui and Hawaii. Unfortunately, the plant is listed as an endangered species because of the increase of hoofed animals (deer, giraffe, goat, etc.) on the islands. However, researchers are exploring tissue culture as a way to restore this plant in large numbers, giving it a chance to thrive once more.
Panama disease of banana, one of the most destructive plant diseases, is caused by fungus species Fusarium oxysporum forma specialis cubense. This fungus species grows in the vascular system of banana plants and blocks the passage of foods and nutrients. Because of this disease, fruits become small in shape and ripen prematurely.
Fortunately, Panama disease-resistant banana plants are being developed, and thanks to tissue culture, these plants can be produced in sufficient numbers and bring these newly cultured plants from the lab to the fields of farmers who are mainly depend on banana production.
These examples showing how vital the tissue culture process is becoming—not just for saving endangered plants, but for strengthening the economy and supporting a healthier ecosystem worldwide.
Totipotency and The Foundation of Tissue Culture
A concept that every living plant cell has the potential to develop into a complete plant was first introduced by T.H. Morgan (1901), an American scientist. He named the potential as Totipotency.
This concept came to reality by German scientist Gottlieb Haberlandt. In 1902, he successfully cultured dissected plant tissues on nutrient medium.
For this success, he is regarded as the Father of Tissue Culture.
What is Tissue Culture?
Tissue culture is a process in which dissected plant tissues are cultured on nutrient medium in aseptic condition for growth and development. Living cells, healthy tissues, and organs (e.g., anther, ovule, apical meristem, embryo) are used in this process to produce complete plantlets (young and small plants) by culturing.
Types of Tissue Culture
On the basis of the parts of plants that would be used to cultured, tissue culture process is divided into different types, such as:
- Organ culture
- Meristem culture
- Anther culture
- Micro propagation
- Protoplast culture
- Pollen culture
- Embryo culture
- Callus culture
Steps of Tissue Culture
You might think that for all different types of tissue culture, different steps may required. But the basic activities are similar and undifferentiated in all types.
1. Explant Selection
The part of plant selected for tissue culture is called explant. Explant must be selected from healthy and disease-free parental plants, otherwise newly produced plants will also carry the same disease. Surface sterilization is required of explant with sodium hypochlorite (NaOCl), bromine water, or 70% alcohol before transferring to the nutrient medium.
2. Culture Medium Preparation
For different types of tissue culture, different culture medium is needed. But the basic medium consists of some common elementary nutrients. Such medium is known as basal medium. This medium consists of:
a) an energy source (sugar)
b) amino acid
c) mineral nutrients
d) vitamins
e) hormones
Another necessary component is agar. Agar maintains the homogeneity (same state throughout the medium) and semisolid nature of the medium.
The pH should be maintained at 5.5–5.8.
3. Sterilization
When the medium is ready for the growth of selected explant, sterilization of the medium is the next step. The medium consists of vitamins and other components, which is convenient for developing parasites on it very fast and easily. Sterilization keeps the medium germ-free and unchanged in qualities.
Before sterilization, the prepared medium is poured into a closed container known as culture vessels (15 ml per 25×150 mm culture tube or 50 ml per 150 ml flask). After that, the culture vessels are plugged with non-absorbent cotton wool, wrapped with cheesecloth, and then the vessels and equipment are transferred to baskets and covered with aluminum foil. It is then autoclaved at 120°C under 2 atm for 15 minutes.
4. Inoculation
Inoculation is done under a laminar flow cabinet or tissue culture hood. A laminar flow cabinet is an enclosed bench work surface which is designed to prevent contamination.
The place used and hands of the inoculator are sterilized by 70% ethanol. If needed, the explant may also be sterilized with antiseptic chemicals. At the time of inoculation:
- With a spirit lamp, the mouth part of the culture vessel is heated and quickly unplugged.
- With sterilized forceps or wire loop, the explant is placed on the medium.
- Again, the mouth part of the culture vessel is warmed and plugged quickly.
Now, it’s time to see the formation of callus.
5. Callus Formation and Multiplication
When the explant starts dividing and forms an unorganized mass of cells, that is called callus. The inoculated culture vessels are kept in a growth chamber where temperature (17°–20°C), humidity (95–100%), and light intensity (1000–3000 lux; red and blue spectrum; 16 hours per day) are under control.
In this proper and suitable condition, the explant starts dividing and forms callus within a few days. After a certain period, continuously dividing callus needs to be transferred to a new medium. Because in that suitable culture medium, where some components are changed, numerous micro-plantlets grow from the callus. No roots may be seen in these micro-plantlets.
6. Development of Complete Plantlets
The micro-plantlets are separated from each other and placed on the rooting medium individually in each culture vessel. The medium is altered by changing the ratio of growth hormones, mainly two most important growth hormones, auxins and cytokinin.
Auxin stimulates the development of roots, and cytokinin stimulates shoot development.
At this step finally we get the complete plantlets from callus.
7. Transfer of Plantlets to Culture Pot
Culture pots are prepared with well-manured sterilized soil where the plantlets are placed from culture vessels. By alternately putting them inside or outside the growth chamber, the plantlets are acclimatized.
8. Transfer of Plantlets to the Field
When the pot plants are acclimatized, they become suitable for transfer to open fields. Thus, the plantlets are established in the cultivated field.
Advantages of Tissue Culture
- Only small amount of plant tissue is necessary for this process.
- New plantlets can be grown within a short amount of time.
- Tissue culture produces disease-free plants from a small, healthy tissue sample.
- Endangered plant species can be produced in large numbers from a single specimen.
- Tissue culture allows production and preservation of saplings year-round without waiting for specific seasons.
Disadvantages of Tissue Culture
- The equipment and materials required can be expensive, making it unaffordable for small-scale researchers.
- There is a risk of contamination at every step.
- Transporting plantlets is hard because they are very small and fragile.
- This process is expensive to carry out.
- Plants lose their ability to adapt over time because there are no new variations.
