about tissue culture

1. PLANT
TISSUE
CULTURE

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

6. Totipotency

9. Cultured media
The artificial media used for the growth of explants contain sucrose, organic
salts, vitamins, growth hormones and some amino acids. Different nutrient
media like mS medium (Murashige and Skoog 1962), Er medium (Erikson,
1965) and B5 medium (Gamborg et.al. 1968) are used for the purpose of
tissue culture.
1. Agar Chemically, agar is a galactan, a complex
carbohydrate with galactose molecules which are
not metabolized by non-ocean microbes. Agar is
used to solidify the medium. Usually 6 gm/l of agar
is used. If a liquid medium is used for tissue culture,
no agar is required.

10. 2. Organic
Compounds
Organic Compound are used as the source of
carbon and energy. Sucrose is usually used in all
standard media.
3. Inorganic
compounds
Inorganic compounds used in culture media
include macronutrient and micronutrients. N, P,
K, Ca, Ng and S are the important
macronutrients that are used in the preparation
of culture media, whereas the micronutrients
include Bo, Mo, Cu, Zn, Mg, Fe etc. A
concentrated stock of macro- and
micronutrients is prepared and finally added to
the medium as and when required. To
overcome the problem of solubility, the stock
solution of iron is prepared in a chelated form,
namely the sodium salt of ferric ethylene
diamine tetra acetate (EDTA).

11. 4. Growth Hormones Hormones such as cytokinins, auxins and gibberellins are
used to regulate growth in tissue culture. Cytokinins
promotes cell division and regulate growth. The most
widely used cytokinins are adenine, kinetin, zeatin and
benzyl adenine. Auxins stimulate shoot elongation (IAA),
naphthalene acetic acid (NAA), and 2,4-
Dichlorphenoxyyacetic acid (2,4 D). Gibberelins are of less
importance. However, gibberellins are of less importance.
Howerver, gibberellic acid (GA#) is used in apical
meristem culture.
5. Vitamins Vitamins regulate the metabolic activities of cells. They
are required in minor quantities. Vitamin B(thiamine) is
used for all types of tissue culture. Nicotinic acid,
riboflavin, pyridoxin, ascorbic acid, biotin and
cyanocobalamin are also used in different cases.
6. Amino acids Amino acids such as L-aspartic acid, L-asperagin, L-
glutanic acid, L-glutamine, etc. are used in tissue culture
as source of nitrogen.

13. Use of Chemicals Chemicals such as Chromic acid, Mercuric chloride
(0.1%), sodium hyochlorite (0.5%), calcium
hypochlorite (0.5%) and alcohol (70%) are used for
the sterilization of glassware, work tables and
source materials of explants.
Use of oven A dry heat oven is used to sterilize glassware,
metallic instruments, etc. by hot air (200-300
degree Celsius for one hour)
Use of autoclaves Autoclaving is done to sterilize nutrient media,
distilled water, etc. with the help of steam (121
degree Celsius for 30 minutes)
Common Methods of Sterilization

14. Use of filtration Vitamins, hormones, etc.
are unstable at high
temperatures. So, they
are sterilized using seitz
filter, Millipore membrane
filter, etc.
Use of UV light UV light is used in the
incubation chamber to
make it germ-free.

19. In organ culture, any part of the plant (root, stem,
leaf, and flower) is used as an explant for
culturing purposes. This technique is extensively
used worldwide to retain or preserve the original
(as it is, in its natural environment) structure and
function of the particular part of the plant itself. It
also helps to study the growth, differentiation, and
development of the plant part.
Organ Culture

21. • In this type of culture, the embryo is isolated and cultured under in vitro conditions. Embryo
culture can be done either by using mature or immature embryos. The mature embryos are
obtained from ripe seeds and the immature embryos are obtained from the unripened or
hybrid seeds that failed to grow and couldn’t produce viable plants.
• The best thing about this culture technique is the absence of any surface or treatment stress
to the explant being cultured. This is because the whole ovule, seed, or fruit is surface
sterilized before obtaining the embryo. This protects the embryo from any damages that can
occur during the process of surface sterilization
Embryo Culture

22. Cell suspension culture is simply multiplying single cells
at a higher rate in a liquid medium. The liquid medium is
continuously agitated on an orbital shaker. This culture
method is used by scientists for studying cell growth and
development. It is also a popular tissue culture method for
many industries to extract certain components from plant
cells.
Cell Suspension Culture

23. The cool thing about plant tissue culture is that you can
grow complete plants from any part of the plant, even with
the seed. In this type of culture, you can use seeds
directly to grow plants. These plants will be uniform and
will grow much faster than in field conditions. This is also
a good method when seeds have a hard outer coating
and take longer to germinate in field conditions.
Seed Culture

24. Anther culture/ Pollen Culture
• The anther is actually the pollen bearing part of stamen. It is present in the
male reproductive part of the flower and contains pollen. These pollen
represent the desirable set of genes and also the desirable
characteristics. So when we use these pollen to produce plants using tissue
culture, it is called anther culture.
• One of the interesting facts about anther culture is that it produces haploid
plants. So what are haploid plants?
In an easy language, haploid plants are plants that are generated using only
one parent. These plants did not go through fertilization (crossing of an egg
and a pollen), which is a time saving advantage.
• Breeders use this method often to develop hybrids in a short duration of 2-
3 years.

25. • Plants have interesting tissues at the top of a growing system known
as meristem. These tissues have cells that do not have any specific
function yet, called meristematic cells.
• These cells have the ability to grow into any plant organ and perform
the necessary functions related to it. Interesting, isn't it? In plant tissue
culture, we exploit this ability of meristematic cells to develop plant
organs and later a whole functioning plant. Tissue culture using
meristem tissues is called meristem culture.
• Another interesting feature of meristem tissues is that they are free
from viruses and other microorganisms. This feature helps us to get
disease free plants using tissue culture. A lot of different plant species,
for example, banana, can provide healthy plantlets using meristem
culture.
Meristem Culture