HRT4701 Chapter 8 - Rooting, Culture and Growth

HRT 4701 : PLANT MICROPROPAGATION
HRT4701
PLANT
MICROPROPAGATION
Semester 1, 2016-2017
1
Test 2 Notice
Date : 17 November 2016 (Thursday)
Time : 12.00 - 1.00 p.m.
Place : Lab D, Block D, Faculty of
Agriculture
Coverage : Chapter 5, 6 I and 6 II :
- General in vitro techniques
- Plantlet production
Including Lecture Handouts
- K 06, 07, 08.
Percent : 20%
2
HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique
Test 2
Chapter 5 –
General in vitro technique
3
General in vitro technique
Steps involved in plant tissue culture

1. Explant isolation
2. Sterilisation technique
3. Inoculation / culture
4. Subculture
5. Decontamination
4
General in vitro technique (cont.)
Sterilisation technique
a. Dry heat – hot air 160°C, no moisture –
e.g. oven
b. Steam – steam under pressure (121°C,
15 psi) e.g. autoclave
c. Filter – for heat labile compound in
solution (hormone, enzyme, vitamin) –
size 0.22µm
d. Chemical – heat labile – surface sterilise
e.g. Clorox, detergent, alcohol 5
HRT 4701 : PLANT MICROPROPAGATION Chapter 6 Plantlet Production
Test 2
Chapter 6 I and II –
Plantlet Production
6
Plantlet Production
7
HRT 4701 : PLANT MICROPROPAGATION Chapter 7 Micropropagation Stages
Starting materials for micropropagation
8
Previous Lecture
Chapter 7 –
Micropropagation Stages
9
Also refers to as :
in vitro growth phases
5 general stages in micropropagation procedures:
 Stage 0 : Donor plant selection
 Stage I : Establishment of sterile
culture
 Stage II : Shoot multiplication
 Stage III : Pre-transplant (Rooting)
 Stage IV : Transfer to natural
environment 10
HRT 4701 : PLANT MICROPROPAGATION Chapter 8 Rooting, Culture & Growth
Chapter 8
Rooting, Culture and Growth
11
Learning Outcome
Scope:
What is factors affecting success in tissue
culture
Learning outcome:
• To discuss different factors (biological,
substrate / nutrient (media), physical and
culture practice) and how it affect growth
in tissue culture
12
5 general stages in to successfully produce
plants via micropropagation:
 Stage 0 : Donor plant selection
 Stage I : Establishment of sterile
culture
 Stage II : Shoot multiplication
 Stage III : Pre-transplant (Rooting)
 Stage IV : Transfer to natural
environment 13
14
Rooting
Biological process in plants
Prepares plants for eventual growth
Precedes other vegetative growth
In vitro rooting number less
Usually induced by changes in
hormonal environment
Lower cytokinin and increase auxin
concentration
Spontaneous (natural) when conditions
favour – may occur in Stage II 15
In vitro rooting influenced by :

A. Genotype
B. Substrate (medium)
i. Auxin treatment
ii. Use of polyphenols
iii. Media effects
 MS media : full MS, ½ MS
 Micro elements
 Vitamin : Vitamin D
 Sucrose and N effects 16
In vitro rooting influenced by:(cont.)

C. Environment effects
i. Aeration and explant support
ii. Light effects
iii. Temperature effects
iv. Effects of activated charcoal
D. Explant effects
i. Wounding on explant
ii. Juvenility
17
Transfer
Procedure
 Clean plantlet of agar
 Dip in fungicide solution
- e.g. Benlate solution (0.1%)
 Plant in artificial medium
- e.g. peat moss, perlite, vermiculite etc.
- pH: 5.8 – 6.1
- irrigate
 Place in glasshouse / shade house
18
Acclimatisation
 Plantlet require skill handling
~ situation : in vitro :
- high humidity (~100%)
- low light
- sterile
~ plantlet requires adjustment in
glasshouse / field
 Main problem in acclimatisation :
Water loss
~ changes in plantlet morphology and
transpiration 19
Plantlet morphology and transpiration : (cont.)

Stomata stay open
Continuous transpiration –
Water loss
Epicuticular wax not normal
Transpiration protection depleted
Water loss due to guttation
Exudation of water from hydatodes
(organs connected to veins of leaves
and situated at leaf tips or vein ends on
leaf margins) 20
Cuticle
Waxy substance promotes development of
stomata
Plants in tissue culture do not have cuticle
21
Acclimatisation (cont.)
Rooted plantlets
deflasked and hardened
off by gradually
decreasing the humidity
Necessary – young tissue

culture plants have no
waxy cuticle to prevent
water loss
22
23
Plantlet morphology and transpiration : (cont.)

Photosynthetic process not normal
Process not stable
Plantlets use minimal CO2 in in vitro –
sucrose from medium
Other characteristics of plantlet :
Leaves, root – small
Palisade layer not developed
Few trichomes (hair, scales)
Less collenchyma
Vascular tissue reduced
Less chlorophyll 24
Preparation for acclimatisation :

Lower humidity
Stiffer agar
Decreasing salt level especially nitrates
Increase light intensity
Use anti-transpirant to reduce water stress
25
Transfer
Transplantation and after care
Usually done in glasshouse / greenhouse
Keep relative humidity high
Gradually increase light intensity and
lower relative humidity
Allows plant to harden and form cuticle
26
Transfer – Procedure :
Misting
Reduce shading 2-4 weeks after transfer
Fertilizer :
– 150 – 200 ppm fertilizer 20-20-20 (NPK)
or similar formulations
27
Culture and Growth

Factors affecting success in tissue culture
procedures :
o Biological
o Nutrient (Medium)
o Physical
o Culture practices
28
Biological factors
Explants
 Explant : part of plant isolated from
mother plant
 Obtained from selected mother plant
 Aseptic conditions – most important in
tissue culture
 Micro-organisms (bacteria, fungus)
affect culture growth and development
29
Biological factors (cont.)

Explant (cont.)
 Explant characteristics :
- Growth stage
- size, quality
- season when explant is excised
related to :
- nutritional status of explant
- presence of endogenous hormones in tissue
→ influence on regeneration
30
Biological factors (cont.)

Genotype
Most important factor
Differ between genus, species, variety
Herbaceous tissue – easier to culture than woody
species (soft stem)
Type of tissue
Explant from different tissues (even from same
plant) have different requirements
Direct organogenesis preferable
Juvenile better than matured
Explant size : 0.5 – 1 cm
Roots – difficult because of soil contact
31
Wild cinnamon
32
Culture media – Nutrient factors

Explant excised from mother plant
Nutrient supply cut off
Media supply nutrient
Media differ according to :
Culture stage – initiation stage,
multiplication stage, rooting stage
Basic media : Murashige & Skoog
Gamborg’s B5
Vacin & Went
Knudson C 33
Physical factors
Temperature, light intensity, pH of media,
aeration
– Temperature
optimum : 25 – 28 °C
Too high : effect on growth and development
high contamination
Too low : slow growth
– Light intensity
Light influences processes in organ formation
Requirements in terms of :
–Quality , quantity, duration 34
Physical factors (cont.)
– Light intensity (cont.)

Inflourescence light
Example : Duri-Lite, True-Lite, Vita-Lite
Light quantity
example :
Initial stage : 1000 – 3000 lux
Final stage : 3000 – 10000 lux
light duration : 12 -16 hr/day
35

– pH media
 Measure of acidity and alkalinity
 pH influences :
 Nutrient uptake
 Growth regulators
 Agar solidifying ability
 Growth at : pH 4.0 – 7.2
 pH optimum ~ differ
 Murashige & Skoog : 5.7 – 5.8
 Vacin & Went : 5.0 – 5.2
- before autoclaving 36

– Aeration
 Culture vessels sealed to prevent
contamination
 Allows exchange of air inside and outside of
culture vessel
 Important : no accumulation of gas
 Aluminium foil, cotton, plastic film etc. have
been used
 Others : bottle caps
37
Culture practices factors

Cleanliness
 Aseptic status – most important
 Laboratory design helpful
 Personnel movement determined
 Use of laboratory wear
 Decontamination of bad culture
 Record keeping
 labelling
38
Test 2 Notice
Date : 17 November 2016 (Thursday)
Time : 12.00 - 1.00 p.m.
Place : Lab D, Block D, Faculty of
Agriculture
Coverage : Chapter 5, 6 I and 6 II :
- General in vitro techniques
- Plantlet production
Including Lecture Handouts
- K 06, 07, 08.
Percent : 20%
39
Thank You
40

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HRT 4701 : PLANT MICROPROPAGATION

General in vitro technique

Steps involved in plant tissue culture

General in vitro technique (cont.)

Starting materials for micropropagation

Rooting, Culture and Growth

In vitro rooting influenced by :

In vitro rooting influenced by:(cont.)

Plantlet morphology and transpiration : (cont.)

Necessary – young tissue

Plantlet morphology and transpiration : (cont.)

Preparation for acclimatisation :

Culture and Growth

Biological factors (cont.)

Biological factors (cont.)

Culture media – Nutrient factors

Physical factors (cont.)

– Light intensity (cont.)

Physical factors (cont.)

Physical factors (cont.)

Culture practices factors

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