KULTUR JARINGAN

TANAMAN
TIM DOSEN MK BIOTEKNOLOGI
FAKULTAS PERTANIAN - UB
2013

TWO MAJOR AREAS OF PLANT

BIOTECHNOLOGY

Plant tissue culture

(plants cloning)

Recombinant DNA
technology (gene cloning)

Plant Tissue Culture

A Requirement for Transgenic Development

Callus
grows
A plant part
Is cultured

Shoots
develop

Shoots are rooted;

plant grows to maturity

http://us.123rf.com/450wm/bannerwega/bannerwega1212/bannerwega121200056/16989069-experiment-of-planttissue-culture.jpg

http://extension.missouri.edu/explore/images/mg0003art35.jpg

What Is plant tissue culture?

the culture of plant seeds, organs, tissues, cells, or protoplasts on
nutrient media under sterile conditions.

Tissue culture produces clones, in which all product cells have the
same genotype (unless affected by mutation during culture)

Teknik bagaimana mengisolasi bagian-bagian tanaman

(organ, sel, polen, protoplasma dll) ditumbuhkan secara
tersendiri, dipacu untuk memperbanyak diri, akhirnya
diregenerasikan kembali menjadi tanaman lengkap dalam
suatu lingkungan yang aseptik dan terkendali.

HISTORY

1960S and
1970s

1950S
1930S

1902
Single plant
cells could
be culture

Plant growth
regulator
(PGR)

PGR, somatic
embryo from
cells; virus free
plant

MS medium;
pollen culture;
protoplast fusion

1970s and
1980s

Develop
techniques to
introduce
foreign DNA
into plant cell

What conditions do plant cells need to

multiply in vitro?
Freedom from
competition
Nutrients and removal
of waste products

A controlled
environment

TISSUE CULTURE APPLICATIONS

Micropropagation

Germplasm preservation
Somaclonal variation
Embryo culture

Haploid & dihaploid production

In vitro hybridization protoplast fusion

Plant genetic engineering

TYPES OF TISSUE CULTURE

THREE FUNDAMENTAL ABILITIES

OF PLANTS
TOTIPOTENCY
The potential or inherent capacity of a plant cell to develop into an entire plant if suitably
stimulated.
It implies that all the information necessary for growth and reproduction of the organism is
contained in the cell
-Kapasitas potensial atau melekat sel tanaman untuk berkembang menjadi seluruh pabrik jika
sesuai dirangsang.
- Ini menyiratkan bahwa semua informasi yang diperlukan untuk pertumbuhan dan reproduksi
organisme yang terkandung dalam sel

DEDIFFERENTIATION
Capacity of mature cells to return to meristematic condition and development of a new
growing point, follow by re-differentiation which is the ability to reorganize into new organ
Kapasitas sel dewasa untuk kembali ke kondisi meristematik dan pengembangan titik tumbuh
baru, diikuti dengan re-diferensiasi yang adalah kemampuan untuk menata kembali ke organ
baru

COMPETENCY
the endogenous potential of a given cells or tissue to develop in a particular way

 Cultured tissue must contain competent cells (cc) or

cells capable of regaining competence
(dedifferentiation).
e.g. an excised piece of differentiated tissue or
organ (Explant) dedifferentiation callus
(heterogenous) redifferentiation (whole plant) =
cellular totipotency.

 Tanaman adalah organisme multiseluler yang kompleks

dengan organ-organ yang mempunyai fungsi berbeda.

Kemajuan ilmu pengetahuan memungkinkan,

organisme multiseluler yang kompleks tersebut
dikembalikan pada ground state pada level sel-sel
dengan potensi genetik yang sama.
Sel-sel yang tidak terorganisir tersebut dapat
dimanipulasi.

Apabila diperlukan, sel-sel tersebut diatur kembali

menjadi tanaman yang lengkap melalui manipulasi
lingkungan tumbuhnya.

BASIS FOR PLANT TISSUE CULTURE

1957

Skoog and Miller

demonstrated
that two
hormones affect
explants
differentiation.

Two Hormones
Affect Plant
Differentiation:
Auxin: Stimulates
Root Development
Cytokinin:
Stimulates Shoot
Development

Generally, the ratio

of these two
hormones can
determine plant
development:
Auxin Cytokinin =
ROOT DEVELOPMENT
Cytokinin Auxin =
SHOOT
DEVELOPMENT
Auxin = Cytokinin =
CALLUS
DEVELOPMENT

Callus formation
1. Meristems
2. Leaf sections

De-differentiation

3. Bulb sections
4. Embryos

Explants

Re-differentiation

Callus

5. Anthers

6. Nucellus

Protoplasts

Development
Suspension cells

Organs (leaves, roots, shoots, flowers,...)

What is required?
Appropriate
tissue

(some tissues culture better

than others)

A suitable
growth
medium

Medium is containing energy

sources and inorganic salts to
supply cell growth needs.

Aseptic
(sterile)
conditions,

as microorganisms grow much

more quickly than plant tissue and
can over run a culture

Growth
regulators
Frequent
subculturing

This can be liquid or semisolid

or solid

in plants, both auxins & cytokinins are needed.

Subculturing to ensure
adequate nutrition and,

to avoid the build up of waste

metabolites

EXPLANTS
A SMALL PIECE OF THE DESIRABLE PLANT
IS SELECTED.
GENERALLY MERISTEMATIC TISSUE OR
INTERNODAL SEGMENTS OF THE PLANT
IS SELECTED FOR MICROPROPAGATION.

THE SELECTED PLANT TISSUE IS CALLED

AS EXPLANT.

EX-PLANT SOURCE
Usually, the younger, less differentiated
explant, the better for tissue culture
Different species show differences in
amenability to tissue culture
In many cases, different genotypes within a
species will have variable responses to tissue
culture;
The only limitation is that each plant is
propagated differently and not every plant
will respond the same way.
Each genus, species and variety may require
a different tissue which will obtain the best
results.

CULTURE MEDIUM
THE TISSUE SO COLLECTED IS
PLACED IN A CULTURE
MEDIUM / NUTRIENT MEDIUM
FOR THE MULTIPLICATION OF
CELLS.

NUTRIENT MEDIA FOR PLANT TISSUE

CULTURES
A NUTRIENT MEDIUM is defined by its mineral salt
composition (macroelements and microelements),
carbon source, vitamins, plant growth regulators
and other organic supplements.
pH determines many important aspects of the
structure and activity of biological
macromolecules. Optimum pH of 5.0-6.0 tends to
fall during autoclaving and growth

Functions of medium

Provide water

Provide mineral
nutritional needs

Provide vitamins

Provide growth
regulators

Access to
atmosphere for
gas exchange

Removal of plant
metabolite
waste

MACROELEMENTS
Nitrogen
(N)
Sulfur (S)

Potassium
(K)

Magnesium
(Mg)

Phosphorou
s (P)

Calcium
(Ca)

MICROELEMENTS
Iron (Fe)
silicon (Si)

Manganese (Mn)

aluminum (Al),

Zinc (Zn)

Nickel (Ni),
Boron (B)
Iodine (I)

Cobalt (Co)

Copper (Cu)
Molybdenum (Mo)

FUNCTION OF NUTRIENTS IN PLANT GROWTH

ORGANIC COMPOUNDS
SUGAR (CARBON
SOURCE)
Sucrose, glucose,
fructose, maltose
20 to 40 g/l

VITAMINS
thiamine (vitamin
B1)
nicotinic acid
(niacin)
pyridoxine (B6)
myo-inositol

Other Compounds
Activated charcoal
It is used where phenol-like compounds are a problem,
absorbing toxic pigments and stabilizing pH.
Also, to prevent oxidation of phenols PVP
polyvinylpyrrolidone), citric acid, ascorbic acid, thiourea
and L-cysteine are used.

Plant growth regulators!

Auxin
Cytokinine

SUPPORT SYSTEMS
Agar (from seaweed)
Agarose

Gelrite (Phytagel) (from bacteria)

Mixtures (Phytagar)
Mechanical (bridges, rafts)

Media Formulations
Many available

Differ in salt concentrations

Differ in presence or absence of salts

3. PLANT GROWTH REGULATORS

(Body building Plants)
AUXINS:

CYTOKININS:

induces cell division, cell elongation, swelling of

tissues, formation of callus, formation of
adventitious roots.
inhibits adventitious and axillary shoot formation
2,4-D, NAA, IAA, IBA, pCPA

shoot induction, cell division

BAP, Kinetin, zeatin, 2iP

GIBBERELLINS:

plant regeneration, elongation of internodes

GA3

ABSCISIC ACID:

induction of embryogenesis
ABA

Plant growth regulators used in plant tissue culture media

Normal concentration range is 10-7 ~ 10-5M

Morphogenetic response of thin cell layers

explants of tobacco

TERIMA KASIH

TUGAS PER KELOMPOK

1. Pilih salah satu TOPIK (lihat di tipe-tipe Kuljar)

2. Dibuat Makalah
3. Format :
4. Pustaka minimal 5 buah
5. Dibuat PPT-nya dan digabungkan semua
kelompok dalam 1 (satu) CD
6. Dikumpulkam pada minggu ke-3
7. Di presentasikan mulai minggu ke-4

Format makalah
1. Pendahuluan
Latar Belakang
Tujuan (sesuai topik masing-masing)

2. Metodologi

Jenis Eksplan
Sterilisasi (teknik dan bahan sterilant)
Media
ZPT
SubKultur

3. Kesimpulan
4. Daftar Pustaka

Format makalah
Diketik di kertas A4
Spasi 1,15
Huruf Tahoma 10