This document discusses meristem culture and shoot tip culture techniques. It describes the three stages of meristem culture: establishment, multiplication, and root regeneration. Shoot tips less than 1 mm are excised and cultured on medium supplemented with hormones like cytokinins and auxins to promote growth. Meristem culture allows for virus elimination, micropropagation, genetic resource preservation, and facilitates international plant exchange. It is an effective method for producing disease-free plants.

1. Presented by:
Suman Rawte
Ph.D. Scholar
Department of Genetics and Plant Breeding
IGKV, RAIPUR

2. Morel and Martin (1952) developed the technique of meristem culture
for in vivo virus eradication of Dahlia.
Shoots of all angiosperms and gymnosperms grow by virtue of their
apical meristems.
The apical meristem is usually a dome of tissue located at the extreme
tip of a shoot and measures approx. 0.1 mm in diameter and 0.25 to 0.3
mm in length.
Meristem or shoot tip is isolated from stem by applying a V-shaped cut.
MS medium salts have been very satisfactory for such cultures though
White’s and Gautheret’s were the most widely used media during the early
days of meristem culture.
INTRODUCTION

5. STAGES OF MERISTEM CULTURE
Murashige reported that there are three stages of culture:
Stage 1 is the culture establishment stage when explant may develop into
single shoot or multiple shoots.
At this stage explant are supplements with cytokinin like BA, kinetin and
2iP.

6. In stage 2 the objective is to multiply the propagule and for this axillary
shoot proliferation is followed as it maintains higher genetic stability.
In axillary shoot proliferation, high levels of cytokinin are utilized to
overcome the apical dominance.

7. The stage 3 purpose is regeneration of adventitious roots from the shoots
obtain in stage 2
Numerous studies have indicated that NAA is followed by IBA,IAA, 2,4-D
and other auxins are used for induction of root generation.

8. Components of medium
 Inorganic nutrients (N2,P,Ca,Mg,S)
 Carbon source (sugar)
 Organic supplements including
Vitamins (Thiamine, nicotinic acid, panthonic acid, pyridoxine)
Amino acids (L-glutamine, L-asparagine, L-cysteine, L-glycine)
Complex organics (casein hydrolysate, coconut milk, yeast extract,
orange juice, tomato juice)
Plant growth hormones
Auxins (root)
Cytokinins (shoot)
Gibbrellins (internode elongation, meristem growth)
Abscissic acid (for culturing woody species)
Solidifying agent (agarose)
pH (optimum is 5.8) lower than 4.5 or higher than 7.5 greatly inhibit the
growth

9. PROTOCOL
Remove the young twigs from a healthy plant. Cut the tip (1 cm)
portion of the twig
Surface sterilize the shoot apices by incubation in a sodium hypochlorite
solution (1% available chlorine) for 10 minutes. The explants are
thoroughly rinsed 4 times in distilled water
Transfer each explants to a sterilized petri dish.

10. Remove the outer leaves from each shoot
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
Incubate the culture under 16hrs light at 25°C
As soon as the growing single leafy shoot or multiple shoots obtained from single
shoot tip or meristem, transfer them to hormone free medium to develop roots.
The plants are later transferred to pots containing compost and kept under
green house condition for hardening.

11. Application of Shoot-tip or Meristem
Culture
1. Virus Elimination
• Plants are often infected with more
than one type of virus, including
some even not known.
• A general term virus- free is used
by commercial horticulturist by this
method.

12. 2. Micro Propagation
• Asexual or vegetative propagation
(vegetative part) of whole plants using
tissue culture techniques referred to as
micro propagation.
3. 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.

13. 4. Use in Plant Breeding:
•In many plant breeding experiments the hybrid plants produce abortive
seeds or non viable seeds. As a result, it makes a barrier to crossibility in
plants where non-viable seeds are unable to develop into mature plants.
Shoot-tip or meristem from such hybrid plant can be cultured to speed up
breeding programme.
5. Quarantine
• Plantlets derived from shoot-tip or meristem
cultures 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.

15. List of the plants from which viruses have been eliminated by
meristem cultures

16. ADVANTAGES:
Lack of vascular tissue.
High auxin concentration.
Production of virus free plants
Facilitation of exchange between locations
Cryopreservation or in-vitro conservation of germplasm
DISADVANTAGES:
Isolation is difficult
Low survival rate & regeneration time for explants may be long(about 8
months for potato explant)
Removal of explant causes a setback in the growth of mother
plant.

17. CONCLUSION
It is very effective method of cloning of plant material and to
develop disease free clean plant stock. Shoot Tip Culture is a
part of plant tissue culture which is a sun-rise technology and
working as a catalyst of agricultural and industrial
development