2. Organ culture
Organ culture can be defined as the organs or plant parts culturing in an artificial media or a culture
from isolated medium.
Any part of plant can serve as explants in organ culture-like shoot (shoot tip culture), root (for root tip
culture), leaf (for leaf culture), and flower (for anther, ovary, ovule cultures).
Most important types of organ culture for in vitro plant propogation are meristematic culture, shoot tip,
nodal culture of separate lateral bud , isolated root, and embryo culture.
The plant species can be rapidly propagated from small cuttings of the bulb by the technique of organ
cultured bulb can be harvested after some period of culture in MS medium where growth rate, alkaloid,
and microelements were reported to be many times (30-50) more in cultured bulbs.
3. Importance of organ culture
i) Organ culture provides an excellent experimental system to
define the nutrients and growth factors normally received by the
organ from other parts of the plant body and from its external
environment.
(ii) Organ culture is particularly valuable in studies of the
interdependence of organs for growth hormones and other growth
factors.
(iii) Cultured organs may be ideally suited for studying specific
problems in morphogenesis.
4.
5. Leaf culture
Leaf culture is the culture of excised young leaf primordia or immature young leaf
of the shoot apex in a chemically defined medium where they grow and follow the
developmental sequences under controlled conditions.
Principle:
Leaf primordia or very young leaves are excised, surface sterilized and inoculated
on an agar solidified medium. In culture leaf remains in healthy condition for a long
period. Leaves can be taken from aseptically grown plants for culture. Since leaves
have a limited growth potential, so in culture the amount of leaf growth depends
upon the stage of maturity at the time of excision. Leaf primordia or very young
leaf have more growth potential than nearly mature leaves.
6. Protocol:
(1) Detach vegetative bud or very young leaf from shoot apex at the vegetative
phase of the plant. Wash the explants thoroughly with running tap water.
(2) Immerse the leaf buds or young leaves in 5% Teepol for 10 minutes. Wash the
explants to remove Teepol.
3) Leaf buds or young leaves are surface sterilized by immersion in 70% v/v
Ethanol for 30 seconds. This treatment is followed by 10-15 minutes incubation in
sodium hypochlorite solution with 0.8% available chlorine. Rinse the explants 3-4
times in sterile distilled water.
(4) Excise the leaf primordia from the leaf bud with the help of surgical scalpel.
(5) Inoculate the leaf primordia or young leaf onto 20 ml of solidified medium in a
culture tube
7. 6) Incubate the culture at 25° C under 16 hrs. light.
8. Shoot tip culture/ 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.
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 ex- plants are thoroughly rinsed 4 times in sterile
distilled water.
Transfer each explants to a sterilized petri dish.
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.
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.
10. 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, develop root, transfer them to
hormone free medium.
The plantlets formed by this way are later transferred to pots containing
compost and kept under greenhouse conditions
11.
12. Root culture
Root culture can be defined as the culture of excised
radical tips of aseptically germinated seeds in a liquid
medium where they are induced to grow independently
under controlled conditions.
13. Protocol
Initiation of Isolated Root Culture:
(1) Seeds are surfaced sterilized by the conventional methods and
germinated on moist filter paper or White’s basal medium at 25°C
in the dark
2) When the seedling roots are 20 to 40 mm in length, 10 mm
apical tips (tip inoculum) are excised with a scalpel and each
transferred to 40 ml of liquid medium contained in 100 ml wide-
necked Erlenmeyer flasks.
(3) Flasks are incubated at 25°C in the dark.
14. Flower culture
Flower culture can be defined as the aseptic culture of
excised floral bud on a chemically defined nutrient
medium where they continue their development to
produce a full bloom in a culture vessel.
15. Protocol:
(1) Flower buds or mature flowers are collected from the healthy plants.
(2) Wash them thoroughly and dip them in 5% Teepol solution for 10 minutes and
wash.
(3) Transfer them to laminar air-flow cabinet. Surface sterilizes them by
immersing in 5% Sodium hypochlorite, wash with autoclaved distilled water.
(4) Using flamed forceps, transfer the flower bud or mature flower to culture
tubes containing 20 ml solid medium.
(5) Incubate the culture in 16 hrs. light at 25° C.
16. Ovule culture
Ovule culture is an elegant experimental system by which ovules are
aseptically isolated from the ovary and are grown aseptically on che-
mically defined nutrient medium under controlled conditions.
Importance of Ovule Culture:
Ovule culture is a boon for the plant breeders in obtaining seedlings from
crosses which are normally unsuccessful because of abortive embryos.
17. Embryo culture
Embryo culture is a technique for cultivating an embryo
under aseptic condition on a nutrient medium.
The method can be divided into two applications.
One is performed with mature embryos and helps
mainly in shortening the period of germination by
overcoming seed dormancy.
The other is performed with immature embryos and is
called early embryo rescue.
