Plant tissue culture is a technique to grow plant cells, tissues or organs in a sterile nutrient medium. It allows for cloning plants with desirable traits, rapid plant production without seeds, genetically modifying plants, and reducing disease. Explants are placed on solid culture media containing inorganic salts, organic nutrients, vitamins and plant hormones. Specific protocols describe sterilizing and culturing rice, sugarcane and cotton plant materials.

2. What is plant tissue culture?
Plant tissue culture is a technique of growing
plant cells, tissues, organs, seeds or other plant
parts in a sterile environment on a nutrient
medium

3. WHY?
• The production of clones of plants that
produce particularly good flowers, fruits, or
have other desirable traits.
• To quickly produce mature plants.
• The production of multiples of plants in the
absence of seeds or necessary pollinators to
produce seeds.
• The regeneration of whole plants from plant
cells that have been genetically modified
• .The production of plants in sterile containers
reduces disease transmission

4. Plant tissue Culture Basics
• Explants are then usually placed on the surface
of a solid culture medium, but are sometimes
placed directly into a liquid medium, when cell
suspension cultures are desired.
• Culture media are generally composed of
inorganic salts plus a few organic nutrients,
vitamins and plant hormones.

5. Tissue culture for rice
• Healthy and mature seeds were selected by
physical appearance.
• Seeds were first washed with detergent and
then rinsed three times with simple tap water.
• For surface sterilization of seeds Clorox
(5.25% sodium hypochlorite) and ethanol was
applied. After the applications of Clorox and
ethanol seeds were rinsed thrice with
autoclaved distilled water.
• Finally the seeds were dried with autoclaved
filter paper, and then shifted to culture room

6. Basal Media Preparation
• M.S basal media were used for callus
initiation. These media were prepared
according to the ingredients .
• The exact amount of nutrients was dissolved in
the distilled water. Two types of growth
regulators 2,4-D alone or in combination with
BAP was used for callus induction respectively
was added in the media.

7. Continue…
• Sucrose at the rate of 3% and agar at the rate
of 0.7% was also added in the media. PH of
the media was adjusted at 5.78-5.80 with the
help of PH-meter.
• M.S. media were poured into the test tubes, it
was plugged properly and autoclaved at 20 lbs
pressure for 15 minutes in the autoclave
machine.

8. Continue…
• Inoculation of Sterilized Seeds The most
important step in tissue culture technique is
the inoculation of seeds. This operation was
performed in the laminar flow cabinet at the
culture room by UV light.
• Dried seeds were then inoculated into test
tubes under aseptic condition in laminar flow
unit

9. Continue…
• To minimize chance of infection the
instruments were dipped in disinfectant after
every operation. Growth Chamber under the
influence of 2000-lux light intensity for 16
hours photoperiod. Callus induction of rice
seeds were observed after 21- days.

11. Tissue culture for sugarcane
• Single buds from cane plant were sterilized in
a 20% commercial hypochlorite solution for 40
minutes, washed with water and planted in a
growth chamber at 37 °C for 15 days.
• When the seedlings were about 20-25 cm tall,
stem segments of about 4 cm, together with
meristem tip, were excised.

12. Continue…
• They were sterilized for 1 minute in 70%
alcohol and 20 minutes in 20% hypochlorite
solution. The segments were then washed 3
times with water under sterile conditions and
shoot tip was dissected as described before. A
spindle leaf near the meristem tip, 0.5-1.0 cm
in length, was used for callus culture.

14. Tissue culture for cotton
• Seeds of all varieties of Gossypium hirsutum
were treated with concentrated H2SO4 for 1-2
min.
• Seeds were thoroughly mixed with acid to
remove the lint completely, then washed with
tap water at least 4-5 times to remove the acid.
• For sterilization purpose approximately 500
seeds were dipped in distilled water with one
drop of Tween-20 per 100ml of water, shaken
well for 3 min.

15. Continue…
• The seeds were then treated with 0.1%
Mercuric chloride and 0.1% SDS for 3-5 min.
These seeds were washed with autoclaved
distilled water for 4-5 times.
• After sterilization procedure, the seeds were
placed on sterilized filter papers in Petri plates
and moistened with 2-3 ml of autoclaved
distilled water. The seeds were covered with
moist filter paper and then with lid of Petri
plate.

16. Continue…
• Plates were sealed with parafilm and kept at
30o C in dark for 72 hrs.
• Cultures were kept in a growth room at 16h
photoperiod. Seedlings were allowed to grow
for 10 days.