Download Generally it is believed that nuclear energy is used for destructive

Background Information
Generally it is believed that
nuclear energy is used for
destructive purposes only. But,
in fact it has more positive
(Peaceful)uses than its negative
uses.
Utilization of Radiation and
Radioisotopes
In Health Care


Nuclear medicine- for diagnosis and
treatment/ CT
Radiotherapy - Co-60 Machines, Gamma
Knifes, Linacs for external therapy and
sealed sources for Brachytherapy (radioactive implants
directly into the tissue)

X ray machines in radiology
Utilization of Radiation and
Radioisotopes
In Agriculture
 Using tracer techniques in management of
the soil, water and for researches of
optimization of cultivation;
 Industrial
Irradiation facilities for food
preservation and sterilization
 Gamma
Irradiators for plant mutation
breeding ( Induced mutation) -useful for crop
improvement
1. Crop improvement by mutation
techniques
Technical basis

Variation is the source of evolution

Spontaneous mutation rate is 1×10-8 ~ 1×10-5

Radiation can cause genetic changes in living
organisms and increase mutation rate up to 1×10-5 ~
1×10-2

Induced mutants are not GMOs, as there is no
introduction of foreign hereditary material into
induced mutants
Crop improvement by mutation
techniques
negative mutation
Mutant cultivars
-
Higher yielding
Disease-resistance
Well-adapted
Better nutrition
no mutation
Crop improvement by mutation
techniques
MUTANT VARIETIES
(2006)
Total Number : 2672
Plant Species :
170
Others 611
Legumes 203
Oil crops 198
Cereals 1206
Flowers 454
Sources: FAO/IAEA Mutant Varieties Database
Collaborative Research Programme on
Varietal Improvement of Potential Floriculture
SPP
for
International Floriculture Market
(1998-2003)
•
•
Funds Made Available by - CARP & Green Farms Ltd
Research Collaborators - Green Farms Ltd., HORDI & Fac.of Agri,UOP
Principal Researcher/Investigator(CARP/12/430/321)
W.D.C.J.Hewawasam
Collaborative Scientist 01 /Academic Supervisor - Prof. (Mrs) D.C. Bandara
Collaborative Scientist 02/ External Supervisor - Mr. W.M. Abeyrathna
New Phenotypes
of
Crossandra infundibuliformis var. Danica
through
In-vitro Culture and Induced Mutations
W.D.C.J. Hewawasam
Post Graduate Institute of Agriculture
University of Peradeniya
Introduction

New varieties

Induced Mutation Techniques +
In-vitro culture

Propagation and induction of
genetic variation

Improving selection technology

Accelerating breeding time
Introduction

Crossandra “Danica”

Natural chimera

Crossandra infundibuliformis

Higher export demand

Develop the plant for its
ornamental values
Objectives
1. To find the potential of using gamma radiation and
colchicine in combination with in-vitro culture for
creating new phenotypic variations in Crossandra
infundibuliformis var. Danica
2. To select novel and improved Crossandra
mutant lines with altered phenotypic characters
among the re-generated progenies and utilize
them to develop improved varieties/cultivars
Materials and Methods
Location of the Experiment
•Tissue culture division / HORDI-The basic
laboratory Experiments
• R & D section / Green Farms Ltd. - The
experiments under net house
Experimental Procedure
Mutagenic Treatment
Gamma radiation
Colchicine
Culture establishment-(M1V1)
1-month after-% cul. survival (ED 50)
Cultures in multiplication medium-(M1V2)
2 months after- Mean Shoot Length
- Av.No.of Shoots/explant
-Leaf abnormalities (%)
Cultures in Rooting Media-(M1V3)
-Time taken for root initiation
(1 months after)
-Av. No. of roots /explant
Rooted Plants (M1V3) in culture
Plant acclimatization and growing under 60% shaded net house conditions
(until blooming)
Different morphological characters were recorded on the basis
of visual observations prior to mutant selection –till 5 vegetative generations
Results and Discussion
•Effect of mutagenic agents in treated Crossandra
shoot tip cultures under in-vitro conditions
•Effect of mutagenic agents in treated Crossandra
shoot tip cultures under net house conditions
Results under in-vitro
conditions
Effect of Gamma irradiation on % survival
of cultures at 1 month after culturing
% survival of cultures
100
R2 = 0.99
50
0
0
3
6
9
Level of irradiation (Krad)
12
Effect of Colchicine on % survival of cultures
at 1 month after culturing
100
% survival of cultures
2
R = 0.98
50
0
0
0.03
0.06
Colchicine (%)
0.09
0.12
Estimated ED50 values for in-vitro
derived Crossandra shoots
By PROBIT ANALYSIS
•For gamma radiation 4.3 Krad
• For colchicine
0.04 %
Effect of Gamma radiation on mean shoot length
and mean no. of secondary shoots at 2 months
in culture
9
8
7
6
5
4
3
2
1
0
Mean shoot
length(cm)
Mean no. of
secondary
shoots /culture
0
3
6
Gamma (Krad)
9
Effect of Colchicine on mean shoot length and
mean no. of secondary shoots at 2 months in
culture
20
18
16
14
12
10
8
6
4
2
0
Mean shoot
length (cm)
Mean no. of
secondary soot
/culture
0
0.03
0.05
Colchicine (%)
0.09
Comparison of the difference in growth responses
of treated Crossandra shoots for 2 different mutagenic
Concluding Remarks
agents at 2 months in culture.
6 Krad
Gamm
a
CONTRO
L
0.05 %
Colchicin
e
X
60
Percentage shoots which
showed abnormal leaves
in mutagenic treated
cultures at 2 months in
culture
50
40
30
20
10
0
3
6
9
Gamma (Krad)
X
80
70
60
X=Abnormal
50
leaves %
40
30
=
20
10
0
0.03
0.05
0.09
Colchicine (%)
Treated cultures showed
one or more abnormal leaves
x100
Total no.of treated shoots
Effect of different levels of gamma rays and colchicine
on in-vitro rooting of treated Crossandra shoots in MS
medium
Treatment
Levels
Rooting %
Mean time
(weeks) to
initiate roots±SE
Mean no. of
roots/plant
±SE
Gamma
(Krad)
0
3
6
9
100
12
80
6
5.5±0.74 d
8.0±0.75 c
9.5±0.91 b
10.5±0.83 a
6.5±1.14 a
3.2±0.55 b
1.5±0.92 c
1.3±1.88 d
Colchicine
(%)
0
0.03
0.05
0.09
94
55
18
0
6.0±0.91 d
8.5±0.91 c
10.0±0.64 b
11.5±0.06 a
5.6±1.59 a
4.2±1.07 b
2.3±0.48 c
0.6±0.05 d
Effect of different levels of gamma rays and colchicine
on in-vitro rooting of treated Crossandra shoots in MS
+IBA (2 mg/l) medium
Treatment
Levels
Rooting %
Mean time
(weeks) to
initiate roots±SE
Mean no. of
roots/plant
±SE
Gamma (Krad)
0
3
6
9
94
55
20
8
7.20± 1.01 c
7.33± 0.61 c
8.86± 1.00 b
10.00±0.64 a
3.0±0.70 b
5.4±0.50 a
3.0±0.92 b
2.1±0.35 c
Colchicine (%)
0
0.03
0.05
0.09
90
53
18
0
6.8± 0.77
6.5± 0.51
9.0± 0.75
10.0±0.64
c
c
b
a
3.2±0.41 b
4.4±1.20 a
3.6±0.61 b
•
0.0
• = Basel callus development was observed instead of root initiation
Effect of gamma rays (6 Krad) and colchicine (0.09%)
on in-vitro rooted Crossandra at 9 weeks in culture
CONTROAL
GAMMA TREATED
0
6 Krad
COLCHICINE TREATED
0.09 %
Results under net house
conditions
Comparison of percentage survival of regenerated
Crossandra plants (M1 V3)at in-vitro rooting stage and
under net house conditions ( 4 months after
acclimatization)
Treatment
Levels
survival of
plants
under in-vitro
conditions (%)
survival of plants
under net house
conditions (%)
Gamma (Krad)
0
3
6
9
94
55
20
8
76
64
40
0
Colchicine (%)
0
0.03
0.05
0.09
90
53
18
0
95
68
42
0
Effect of different doses of gamma rays and colchicine
on plant height at 3 months after transfer to the normal
plant house conditions
Treatment
Dose/concentration Mean plant height
level
(cm±SE)
Gamma (Krad)
0
3
6
20.43 ±0.57 a
15.70 ±0.57 b
08.95 ±0.51 c
Colchicine (%)
0
0.03
0.05
18.20 ±0.63 a
13.60 ±0.66 b
11.57 ±0.63 c
Effect of different doses of gamma rays on
plant height of in-vitro derived Crossandra
plantlets growing under normal plant
house conditions-(3 months after acclimatization)
0 Krad
3 Krad
6 Krad
x
Gamma ray induced leaf
abnormalities observed
in plants growing under
normal plant house
conditions at 3 months
after transplanting
X-Represent the control leaf
Effect of gamma rays and colchicine on flowering
behavior of in-vitro derived Crossandra plants
10
9
8
7
Time (Months) 6
taken to full
5
blooming
4
3
2
1
0
Gamma (Krad)
Colchicine (%)
0 Krad/ 0 %
3 Krad/ 0.03 % 6 Krad/ 0.05 %
•
No visible changers in plant phenotypic
characters in in-vitro derived control
plant population.
But……..
Induced somatic mutations by gamma
irradiation and Colchicine
Mutagenic agent
Number of treated plantlets
which survived in in-vitro
multiplication and rooting stages
Mutation
rate
M1V1
M1V2
M1V3
3 krad Gamma radiation
60
240
960
1/960
0.03% Colchicine
60
195
585
5/585
0.05% Colchicine
60
180
370
2/370
Control (No treatment)
60
300
920
No
Comparison between normal and mutant
flowers
A
B
A)-Normal flower (Crossandra infundibuliformis var.Danica)
B)-Mutated flower (Crossandra infundibuliformis var.Savindi)
Characters of “Danica” and its
induced mutant “Savindi”
Charactor
Plant height (cm±SE)
Leaf length (cm±SE)
Leaf width (cm±SE)
Leaf shape (cm±SE)
Flower colour
Flower petal size (cm±SE)
Total flowers/plant (cm±SE)
Length of flowering spike (cm±SE)
Petiole length (cm±SE)
Time (months) taken to full bloom (cm±SE)
∗
Significant at p>0.05
“Danica”
“Savindi”
23.30 ± 0.18
13.00 ±1.97
5.40 ±0.56
Spatulate (wide)
Orange
7.35 ±0.73
6.00 ±0.97
6.00 ±0.98
6.25 ±1.25
6.35 ±0.41
18.00 ±0.83 ∗
7.67 ±2.44 ∗
4.48 ±0.24 ∗
0blanceolate (linear)
Pink
7.19 ±1.81
6.70 ±0.67
9.20 ±1.20 ∗
3.35 ±1.32 ∗
7.50 ±0.28 ∗
The number of vegetative shoots
multiplied in each vegetative
genaration of mutant Crossandra
“Savindi”
Vegetative
generations
V1
V2
V3
V4
V5
Number of
shoots in
generation
2
6
16
38
73
% plants survived
till flowering
100
66.66
75
76.31
89.04
Stability of phenotypic
characters (Yes/No)
Yes
Yes
Yes
Yes
Yes
Conclusions
•In-vitro induced mutagenesis using gamma
radiation and colchicine successfully introduced
new genetic variability in Crossandra
infundibuliformis var. Danica which could be in-vitro
propagated by apical shoot tips
• A new solid mutant line with altered phenotypic
characters was selected among gamma ray (3
Krad) treated,regenerated progenies and it is now
being assessed for its suitability for release as a
novel ornamental product
Con…..
Remark
• Much attention should be paid in the future
studies for the comparative analysis of original
Crossandra cultivar and there respective
induced mutants for better and clear
understanding of the origin and evolution of
somatic flower colour mutations at molecular
level.
Acknowledgements
★Supervisors
★Green Farms Ltd.
★HORDI
★ CARP