ABT 105

Experimental Techniques in
Agricultural Biotechnology
Exercise 2
Selected Techniques in
Plant Tissue Culture
Part II
Prof. Lilian F. Pateña
Associate Professor and
Head, Plant Cell and Tissue Culture Laboratory,
Institute of Plant Breeding, Crop Biotechnology Division
Crop Science Cluster, College of Agriculture, UPLB
 PLANT TISSUE
CULTURE
 technique wherein
different plant parts
(explants) are excised
from the parent plant
 and grown in a
defined medium
 under aseptic Mango
condition & controlled Patena 2015

environment
 PLANT TISSUE CULTURE
CULTURE MEDIA – solid, liquid or semisolid mixture
of nutrients containing energy
sources and inorganic salts to
supply growth needs.
Contains:
 water
 macronutrients and micronutrients
 vitamins
 growth regulators (auxin, cytokinins, GAs, ABA, etc)
 Sucrose
 Gelling agents (agar, gelrite, gulaman bar, etc)
 DEFINITION OF TERMS
PLANT TISSUE CULTURE
 Callus
 mass of unorganized parenchyma cells
derived from explants for use in biological
research and biotechnology

 Callus > 2 pathways

1) organogenesis
2) somatic embryopgenesis
 PLANT TISSUE CULTURE
RELATIONSHIP OF AUXIN AND CYTOKININ
Cytokinin
Leaf strip

Adventitious
Shoot

Root

Callus Auxin
 PLANT TISSUE CULTURE
Organogenesis: The process of
initiation and development of a
structure that shows natural
organ form and/or function.
Embryogenesis: The process of
initiation and development of
embryos or embryo-like
structures from somatic cells
(somatic embryogenesis).
 Virus-free Garlic
1
Garlic bulbs
Cloves separated
Washed

Pre-treatment (ref or RT)

5 Multiple shoots
2 index
Garlic bulbs under
thermotherapy
50oC (<1 hr)

Shoot tip (left);

meristem
(right) culture
6 Bulblets (Go) with shoot (left);
3 4 dormant (right)
1. Production of virus-free plants

Shoot Apical Meristem (SAM)

Excision of
SAM using a
microscope

Tunica-Corpus model of the shoot

apical meristem (growing tip). The
epidermal (L1) and subepidermal (L2)
layers form the outer layers called the
tunica[]. The inner L3 layer is called the
corpus. Cells in the L1 and L2 layers
divide in a sideways fashion, which
keeps these layers distinct, whereas
the L3 layer divides in a more random
fashion.
 ELISA Color Reaction:
None – negative, Yellow
- positive

Virus-free certification using ELISA

Patena 2013
 APPLICATIONS OF PLANT
TISSUE CULTURE
2. Micropropagation

Orchids
Patena 2013
APPLICATION OF PLANT TISSUE CULTURE:
2. Micropropagation of orchids

ORCHID EMBRYO CULTURE

Patena 2013
 APPLICATIONS OF PLANT TISSUE CULTURE
3. Tool in genetic transformation

Fig. 1. Nucellar tissue explants Fig. 2. Somatic embryos from

used in tissue culture of mango nucellar tissue explants of mango

Delayed ripening in mango

Patena 2013
 How is somatic embryogenesis initiated?

The morphological stages of

somatic embryo development
in alfalfa (Medicago sativa L.)

Somatic embryogenesis can be initiated in two ways (Sharp et al 1980 :

 by inducing embryogenic cells within the preformed callus, and
 directly from preembryonic determined cell, (without callus) which
are ready to differentiate into embryoids).
CULTURE ROOM FOR GROWTH AND DEVELOPMENT OF
GARLIC SHOOTS, ORCHID PLANTLETS AND MANGO
SOMATIC EMBRYOS

6.1.1. Slow growth

THINGS TO SUBMIT:
1. Scientific paper of 1 crop (PAS-style)
2. Materials & Methods, Results &
Discussion, References of 2 other crops

GRADES:
1. Attendance/Lab –performance,
2. 3 reports, &
3. 1 exam
 Enjoy plant tissue culture!

”The key to success is hard work and determination.”

Anonymous