Shoot Tip Culture: Meaning, Principle, Protocol and Importance | Plant Tissue Culture

In the below mentioned article, we will discuss about the meaning, principle, protocol and importance of the shoot tip/meristem culture in different fields of plant science.

About Shoot Tip/Meristem Culture:

What is Shoot Tip Culture?

Shoot tip culture may be described as the culture of terminal (0.1-1.0 mm) portion of a shoot comprising the meristem (0.05-0.1 mm) together with primordial and developing leaves and adjacent stem tissue.

What is Meristem Culture?

Meristem culture is the in vitro culture of a generally shiny special dome-like structure mea­suring less than 0.1 mm in length and only one or two pairs of the youngest leaf primordia, most often excised from the shoot apex.

What is Mericloning?

Mericloning is a popular term. It is not applied in scientific usage. It refers to the in vitro vegetative propagation of orchids from excised shoot tips, axillary buds or floral organs.

What is Meristemming?

Meristem Ming is also a popular term. It is used to describe the in vitro clonal propagation of plants from various explant sources including shoot tips, leaf sections and calli.

Principle:

The excised shoot tips and meristem can be cultured aseptically on agar solidified sim­ple nutrient medium or on paper bridges dip­ping into liquid medium and under the appro­priate condition will grow out directly into a small leafy shoot or multiple shoots. Alterna­tively the meristem may form a small callus at its cut case on which a large number of shoot primordia will develop.

These shoot primordia grow out into multiple shoots. Once the shoots have been grown directly from the excised shoot tip or meristem, they can be propagated further by nodal cuttings. This process involves sepa­rating the shoot into small segments each con­taining one node. The axillary bud on each seg­ment will grow out in culture to form yet another shoot.

The excised stem tips of orchids in culture proliferate to form callus from which some organ­ised juvenile structures known as proto-corm de­velop. When the proto-corms are separated and cultured to fresh medium, they develop into nor­mal plants. The stem tips of Cascuta reflexa in culture can be induced to flower when they are maintained in the dark.

Exogenously supplied cytokinins in the nu­trient medium plays a major role for the devel­opment of a leafy shoot or multiple shoots from meristem or shoot tip. Generally, high cytokinin and low auxin are used in combination for the culture of shoot tip or meristem.

Addition of adenine sulfate in the nutrient medium also in­duces the shoot tip multiplication in some cases. BAP is the most effective cytokinin commonly used in shoot tip or meristem culture. Similarly, NAA is the most effective auxin used in shoot tip culture. Coconut milk and gibberellic acid are also equally effective for the growth of shoot apices in some cases.

Protocol:

(1) Remove the young twigs from a healthy plant. Cut the tip (1 cm) portion of the twig.

(2) Surface sterilize the shoot apices by incuba­tion in a sodium hypochlorite solution (1% available chlorine) for 10 minutes. The ex- plants are thoroughly rinsed 4 times in ster­ile distilled water.

(3) Transfer each explants to a sterilized petri dish.

(4) Remove the outer leaves from each shoot apices with a pair of jeweler’s forceps. This lessens the possibility of cutting into the softer underlying tissues.

(5) After the removal of all outer leaves, the apex is exposed. Cut off the ultimate apex with the help of scalpel and transfer only those less than 1 mm in length to the sur­face of the agar medium or to the surface of filter-paper Bridge. Flame the neck of the culture tube before and after the transfer of the excised tips. Binocular dissecting mi­croscope can be used for cutting the true meristem or shoot tip perfectly.

(6) Incubate the culture under 16hrs light at 25°C.

(7) As soon as the growing single leafy shoot or multiple shoots obtained from single shoot tip or meristem, develop root, transfer them to hormone free medium.

(8) The plantlets formed by this way are later transferred to pots containing compost and kept under greenhouse conditions (Fig 2.3).

Importance of Shoot Tip/Meristem Culture:

The uses of shoot tips and meristem in tis­sue culture are very varied and include mainly:

(i) Virus eradication,

(ii) Micro-propagation and

(iii) Storage of genetic resources.

Virus Eradication:

Many important plants contain systemic viruses which substantially reduce their poten­tial yield and quality. It is, therefore, important to produce virus free stocks which can be multi­plied. Generally, highly meristematic tissue of a virus infected plant remains free from virus due to their fast mitotic activity. Therefore, shoot tips and meristems of a virus infected plant are the ideal explants to produce a virus free stock.

This technique is also valuable for the mainte­nance of carefully defined stocks of specific vari­eties and cultivars in disease Free State. The size of the meristem explant is critical for virus erad­ication. Often so called meristem tip cultures have failed to eliminate virus infection because the explant contains shoot apices with vascular tissue instead of true meristem.

This technique, combined with heat treat­ment (thermotherapy) or chemical treatment (chemotherapy) has proved to be very effective in virus eradication. Heat treatment is done by placing an actively growing plant in a thermotherapy chamber. Over a period of two weeks the temperature is increased to 38°C inside the chamber and the plants are maintained at this temperature for two months.

After that period, the apical meristem is excised, surface sterilized and transferred aseptically to agar medium. Us­ing this technique 85% to 90% virus free plants have been obtained. Without heat treatment, shoot tips or meristem can be grown on chemotherapeutants added medium for virus eradication. Commonly used chemotherapeutants are 2, 4-D, mela-chife green, thiouracil etc.

Shoot tip or meristem culture has enormous horticultural value e.g. in the production of plants for the cut flower industry when stock plants of registered lines must be maintained in as near-perfect condition as possible. Any in­fection by virus that affect the growth or phys­ical characteristics of size and shape of flowers, is obviously very serious problem from commer­cial point of view.

Meristem culture technique to clean up the stocks could, therefore, avert a commercial disaster. Similarly, in the agricul­tural world, the production or yield of a crop can fall dramatically as a result of a viral infec­tion and render that particular variety no longer salable or commercial value. Meristem culture could be of value in restoring the original prop­erties of the variety by removing the infection.

Micro Propagation:

A sexual or vegetative propagation of whole plants using tissue culture techniques is referred to as micro-propagation. Shoot tip or meristem culture of many plant species can successfully be used for micro-propagation.

Storage of Genetic Resources:

Many plants produce seeds that are highly heterozygous in nature or that is recalcitrant. Such seeds are not accepted for storing genetic resources. So, the meristem from such plants can be stored in vitro. Besides the above-mentioned uses of shoot tip or meristem culture, it can also be utilized in various important fields of plant science such as:

Shoot Tip or Meristem Culture and Plant Breeding:

In many plant breeding experiments, the hybrid plants produce abortive seeds or nonvi­able seeds. As a result, it makes a barrier to crossibility in plants where non-viable seeds are unable to develop into mature hybrid plants. Shoot tip or meristem from such hybrid plant can be cultured to speed up breeding progra­mme.

Propagation of Haploid Plants:

Haploid plants derived from anther or poll­en culture always remain sterile unless and until they are made homozygous diploid. Meristem or shoot tip culture of haploid plants can be used for their propagation from which detailed genetic analysis can be done on the basis of morpholog­ical characters and biochemical assay.

Meristem or Shoot Tip Culture and Quarantine:

There are some strict regulations concern­ing the international movement of vegetative plant material. After thoroughly checking, the plant materials may be rejected by quarantine authority if the plant material carries some dis­eases.

Plantlets derived from shoot tip or meris­tem culture are easily accepted by the quarantine authority for international exchange without any checking. Therefore, using this technique, crop plants can be easily exchanged in crop improve­ment programmes that are based on materials from different parts of the world.