Collaborative Research Programme on Varietal Improvement of Potential Floriculture SPP to suit for ...
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Background Information
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.
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 Utilization of Radiation and Radioisotopes In Health Care
Utilization of Radiation and RadioisotopesUtilization of Radiation and Radioisotopes
In Agriculture Using tracer techniques in Soil & Water
Management , Crop Nutrition & Insect-pest control
Animal Production & Health - by RIA, ELISA, PCR, etc
Industrial Irradiation facilities for food preservation and sterilization
Gamma Irradiators for plant mutation breeding ( Induced mutation) -useful for crop improvement
Induced mutants are not GMOs, as there is no introduction of foreign hereditary material into induced mutants
- Higher yieldingHigher yielding- Disease-resistanceDisease-resistance- Well-adaptedWell-adapted- Better nutritionBetter nutrition
Mutant cultivarsMutant cultivars
Crop improvement by mutation techniques
no mutation
negative mutation
CHRYSANTHEMUM MUTANTS
(Source: Rumińska J.L.et al 2005-Holand )
Mutation breeding creates the world's most perfect orange
Source : http://io9.com/5791035/mutation-breeding-creates-the-worlds-most-perfect-orange
Gamma radiation Chamber –irradiation emitted from Cobalt-60 source .
Collaborative Research Programme on Varietal Improvement of Potential Floriculture
SPP to suit 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. BandaraCollaborative Scientist 02/ External Supervisor - Mr. W.M. Abeyrathna
Creating 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
Accelerating breeding time
Induced Mutation Techniques +
In-vitro culture
Propagation and induction of
genetic variation
Improving selection technology
Introduction
Crossandra “Danica”
Natural chimera
Crossandra infundibuliformis
Higher export demand
Develop the plant for its
ornamental values
Objectives
1.To develop a reliable protocol for in-vitro
propagation of Crossandra infundibuliformis var.
Danica through callus and shoot tip culture
2.To find the potential of using gamma radiation
in combination with in-vitro culture for creating
new phenotypic variations in Crossandra
infundibuliformis var. Danica
3.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
A: Developmental activities of tissue culture protocols in Crossandra “Danica”
1. Callus culture
% Explant survival% Callus initiationExtent of callus growth
Callus initiation
Callus proliferation
MS+Kin+2-4,D+2%Sucrose
Incubated in dark
%Callus proliferation
Extent of callus proliferation
Tender leavesImmature stems
MS+Kin+2-4,D +Sucrose
Incubated in dark
2%
3%
Con…
Plantlet regeneration via
callus phase
MS+NAA+BAP
Exposed to light
Callus proliferation
% Plantlet regeneration
2. Shoot tip culture
1/2MS+Kin+IAA
1/2MS+BAP+IAA
%Survival %RegenerationAv.No.of shoots/explant
Shoot tip establishment
Av.No. shoots/explant
Av.length of shoots
Multiplication of shoots Shoot elongationH/F-MS
Root induction
Acclimatization of plantlets
MS+IBAAv. No. roots/explant
%Rooted cultures
Room tem. 1week
Plastic pots-5,6 weeks
Normal plant house condition
MS+IBA+(0.2%ACH)
Apical & axillary shoot tips
B: In-vitro induced mutagenesis in Crossandra“Danica”using shoot tip culture
Experimental Procedure Mutagenic Treatment Culture establishment-(M1V1)
1-month after-% cul. survival (ED 50)
Cultures in multiplication medium-(M1V1) (M1V2) 2-3 months after- Av.Sec.Shoot Length/Plant - No.of Sec.Shoots/Plant,Phenotypic abnormalities Cultures in Rooting Medium-(M1V3)
(1 months after) Rooted Plants-(M1V3) in culture
Plant acclimatization and growing under 60% shaded net house conditions (until blooming) Changers in different morphological characters were recorded on the basis of visual observations prior to mutant selection –until 5 vegetative generations
Results and Discussion
Part 1- Developmental activities of tissue culture protocols in Crossandra “Danica”
Part 2-Effect of mutagenic agents in treated Crossdandra shoot tip cultures under in-vitro conditions
Part 3- Effect of mutagenic agents in treated Crossdandra shoot tip cultures under net house conditions
Part 1: Developmental activities of tissue culture protocols in Crossandra “Danica”
Effect of 2,4-D and kinetin (with 3% sucrose) on callus initiation in immature stems and tender leaves
2,4-D(mg/l)
Kin.(mg/l)
3% Sucrose in MS media
Immature stems Tender leaves
% explantsurvival
% callusinitiation
Callusgrowthranking
% explantsurvival
% CallusInitiation
CallusGrowthranking
1 1 90 66.6 + 80 0 -
2 1 100 0 - 70 57.14 +
3 1 80 0 - 40 0 -
5 1 80 0 - 50 40 +
1 2 80 87.5 + 60 100 +
2 2 70 42.85 + 40 75 +
3 2 50 0 - 20 0 -
5 2 40 0 - 020 0 -
1 3 70 85.7 ++ 20 0 -2 3 60 66.6 ++ 0 0 +
3 3 40 0 # 10 0 -
5 3 10 0 # 0 0 #
1 5 10 10 + 0 0 #
2 5 0 0 # 0 0 #
3 5 0 0 # 0 0 #
5 5 0 0 # 0 0 #
- No callus; + Poor callus; ++ Fair callus; +++good callus # explant degenerated
Each treatment consisted of 10 replicates
Effect of 2,4-D and kinetin (with 2% sucrose) on callus initiation in immature stems and tender leaves
- No callus; + Poor callus; ++ Fair callus; +++good callus
2,4-D(mg/l)
Kin.(mg/l)
2% Sucrose in MS media
Immature stems Tender leaves
% explantsurvival
% callusinitiation
Callusgrowthranking
% explantsurvival
% CallusInitiation
CallusGrowthranking
1 1 100 100 +++ 100 40 ++
2 1 100 60 ++ 90 0 -
3 1 90 0 - 80 0 -
5 1 50 100 + 100 0 -
1 2 100 70 ++ 100 55.5 +++
2 2 90 77.7 ++ 80 50 +
3 2 60 66.6 + 90 0 -
5 2 40 0 - 90 0 -
1 3 90 66.6 + 70 0 -
2 3 80 80 + 70 71.42 +
3 3 80 75 + 50 0 -
5 3 40 0 - 30 0 -
1 5 50 60 + 30 100 +
2 5 40 100 ++ 30 100 +
3 5 50 100 ++ 40 75 +
5 5 20 0 - 20 0 -
Each treatment consisted of 10 replicates
Initial stage of callus initiation in immature stem and tender leaf of Crossandra (2 wks)
+++ ++ + _ #
Good Fair Poor No Degenarated
Callus Growth Ranking - Immature Stems
Effect of different combinations of 2,4-D and kinetin (with 2% sucrose) on callus proliferation
- No callus; + Poor callus; ++ Fair callus; +++good callus
Growth regulators (mg/l)
Kinetin 2,4- D
% Callus
proliferation
Ranking of callus
proliferation
0.5 0.5 0 -2.0 0.5 0 -
3.0 0.5 0 -5.0 0.5 10 +
0.5 1.0 0 -2.0 1.0 30 +3.0 1.0 20 +5.0 1.0 40 +0.5 3.0 0 +2.0 3.0 10 +3.0 3.0 20 +5.0 3.0 60 ++0.5 4.0 0 -2.0 4.0 30 +3.0 4.0 30 +
5. 0 4. 0 90 +++
Each treatment consisted of 10 replicates
Ranking of Callus Proliferation
++++++_
GoodFairPoorNo
A- Roots initiated via callus phase as globular protrusions after 28 days in complete darkness
B- Elongated and branched roots in calli in the same medium after 45 days in sub culture
Effect of different combinations of NAA
and BAP on plantlet regeneration via callus phase
Growth regulators (mg/l)
NAA BAP
% callus survival
% shoot induction
1 1
20
2
1
0
3
1
0
4
1
0
1
2
60
2
2
2 20
3
2
0
4
3
0
1
3
0
2
3
0
3
3
0
4
0
Each treatment consisted of 10 replicates, ** Calli turned greenish in colour but plantlets could not be regenerated
1
2
3
4
4
4
4
4
00
0
0
000
0
**0
0
0
0
0
0
0
0
000
Effect of different combinations of cytokinin (Kinetin) and auxin (IAA)
on shoot tip establishment after 8 weeks in culture
CV(%) = 15.73
LSD(0.05) = 0.43
Growth regulators (mg/l)
Kinetin IAA %Survival
% bud breaking Mean Number of shoots/ex.p.
2 0 80 50 1.80±0.63
3 0 90 60 1.50 ±0.52
4 0 80 50 2.40 ±0.51*
5 0 60 66.67 1.70 ±0.48
2 1 70 80 1.50 ±0.52
3 1 60 25 2.00 ±0.00
4 1 60 66.67 1.60 ±0.51
5 1 60 80 1.20 ±0.66
2 3 70 66.67 1.40 ±0.51
3 3 60 33.33 1.70 ±0.48
4 3 60 50 1.30 ±0.48
Each treatment consisted of 10 replicates, * Significant at 0.05 level
Effect of different combinations of cytokinin (BAP) and auxin (IAA) on shoot tip establishment after 8 weeks in culture
* Significant at 0.05 level
Growth regulators (mg/l) BAP IAA
Survival % % bud breaking Mean Number of shoots/explant
2 0 80 75 6.00±0.51
3 0 80 100 6.40±0.51*
4 0 60 80 4.00±0.66
5 0 60 60 4.70±0.48
2 1 70 85.7 4.90±0.52
3 1 70 62.85 5.30±0.48
4 1 80 87.5 5.10±0.31
5 1 70 57.14 4.40±0.51
2 3 60 60 4.80±0.42
3 3 70 57.14 4.40±0.51
4 3 70 57.14 4.60±0.51
5 3 60 66.67 5.00±0 CV (%) = 8.82
LSD (0.05) = 0.13
Each treatment consisted of 10 replicates
Effect of the concentration of BAP on multiplication and growth of shoots of Crossandra var. Danica
* Significant at 0.05 level
BAP (mg/l) Mean no. of shoots per explant
Mean shoot length (cm)
0.0 4.70±0.48 0.50±0.06
0.5 6.00±0.81 0.70±0.06
1.0 6.80±0.42 0.70±0.06
1.5 9.80±0.42* 2.50±0.52*
2.0 7.00±0.47 1.40±0.51
2.5 2.30±0.51 0.40±0.04
CV (%)
LSD (0.05)
16.43
0.48
9.98
0.26
Each treatment consisted of 10 replicates
Shoot multiplication in 1/2MS+1.5mg/l BAP after 8 weeks in culture
An elongated shoot in a hormone free MS medium after 3 weeks in culture
Effect of different concentrations of IBA on in-vitro root formation in Crossandra
IBA mg/1 Rootability Remarks
Ave.No. of roots per explant
% rooted
0.0 5.6 90 Good root growth with no basal callus.
0.5 1.1 20 Poor root growth with basal callus.
1.0 - 0 No root growth with high callus formation at explant base.
2.0 - 0 No root growth with high callus formation at explant base.
5.0 - 0 Explant degenerated
Each treatment consisted of 10 replicates
A rooted shoot in a hormone free MS medium after 5 weeks in culture
Hardened roots with many lateral roots in modified rooting medium with 0.2% ACH after 3 weeks in culture
3 wks after acclimatization
12 wks after acclimatization
Healthy growth with 95%
survival rate
Normal plant house condition
part 01- Summery
MS + Kinetin (1mg/l) + 2.4,D (1mg/l) + Sucrose (2%)
MS + Kinetin (5mg/l) + 2.4,D (4mg/l) + Sucrose (2%)
2/1 MS + BAP (3mg/l)
2/1 MS + BAP (1.5mg/l)
MS Medium
MS + ACH (0.2%)
Part 2: Effect of mutagenic agents in treated Crossdandra shoot tip
cultures under in-vitro conditions
Effect of Gamma irradiation on % plant survival at 1 month after culturing
R2 = 0.99
0
50
100
0 3 6 9 12
Gamma (Krad)
% p
lan
t su
rviv
al
Estimated ED50 values for in-vitro derived Crossandra shoots
By PROBIT ANALYSIS
For gamma radiation 4.3 Krad
Effect of Gamma radiation on mean shoot length Effect of Gamma radiation on mean shoot length and mean no. of secondary shoots after 2 and mean no. of secondary shoots after 2 months in culturemonths in culture
0
1
2
3
4
5
6
7
8
9
0 3 6 9
Gamma (Krad)
Mean shootlength(cm)
Mean no. ofsecondaryshoots /culture
0
10
20
30
40
50
60
3 6 9
Gamma (Krad)
X
X=Abnormal leaves %
= Treated cultures showed one or more abnormal leaves
Total no.of treated shootsx100
•Modified equation from Yong Chong Koh and Davies,2000
Percentage shoots which showed abnormal leaves in mutagenic treated cultures after 2 months in culture
Some leaf abnormalities shown in Some leaf abnormalities shown in
gamma rays treated shoots after 2 months in gamma rays treated shoots after 2 months in multiplication mediummultiplication medium
ControlControl Leaf abnormalities in treated shootsLeaf abnormalities in treated shoots
Effect of different levels of gamma rays on in-vitro rooting of treated Crossandra shoots in MS +IBA (2 mg/l) medium
Treatment Treat. 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
7.33 ±0.61
8.86 ±1.00
10.00 ±0.64
3.0 ±0.70
5.4 ±0.50
3.0 ±0.92
2.1 ±0.35
Part 3:
Effect of mutagenic agents in treated Crossdandra shoot tip cultures 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 Treat. 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
Effect of different doses of gamma rays on plant height at 3 months after transfer to the normal plant house conditions
Treatment Dose/concentration level
Mean plant height (cm±SE)
Gamma (Krad) 0
3
6
20.43 ±0.57
15.70 ±0.57
08.95 ±0.51
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
Gamma ray induced leaf abnormalities observed in plants growing under normal plant house conditions,3 months after transplanting
x
X-Represent the control leaf
Effect of gamma rays and colchicine on flowering behavior of in-vitro derived Crossandra plants
01
2
34
5
67
8
910
Time (Months) taken to full
blooming
0 Krad/ 0 % 3 Krad/ 0.03 % 6 Krad/ 0.05 %
Gamma (Krad)
Colchicine (%)
Induced somatic mutations by gamma irradiation and Colchicine
Mutagenic agent
Number of treated plantlets which survived in in-vitro
multiplication and rooting stagesMutation
rateM1V1 M1V2 M1V3
3 krad Gamma radiation
0.03% Colchicine
0.05% Colchicine
Control (No treatment)
60
60
60
60
240
195
180
300
960
585
370
920
1/960
5/585
2/370
No
Characters of “Danica” and its induced mutant “Savindi”
Charactor “Danica” “Savindi”
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)
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 ±0.64 •
0blanceolate (linear)
Pink
7.19 ±1.81
6.70 ±0.67
9.20 ±1.20•
3.35 ±1.32•
7.50 ±0.28•
• =Significant at p>0.05 level by DMRT
The novel mutant with altered flower colour from 3 Krad gamma treatment
A B
A)-Original flower (Crossandra infundibuliformis var.Danica)
B)-Mutated flower (Crossandra infundibuliformis var.Savindi)
Vegetative generations
Number of shoots in generation
% plants survived till flowering
Stability of phenotypic characters (Yes/No)
V1
V2
V3
V4
V5
2
6
16
38
73
100
66.66
75
76.31
89.04
Yes
Yes
Yes
Yes
Yes
The number of vegetative shoots The number of vegetative shoots multiplied in each vegetative multiplied in each vegetative
genaration of mutant Crossandra genaration of mutant Crossandra “Savindi”“Savindi”
ConclusionsConclusions
Crossandra infundibuliformis var. Danica can be in-vitro propagated by using apical and axillary shoot tips
The tested callus culture media did not provide an optimistic protocol for plantlet regeneration of Crossandra via callus interphase
Con…..
In-vitro induced mutagenesis using gamma radiation 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
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
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 Culture establishment-(M1V1)
Gamma radiation
Colchicine 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
R2 = 0.99
0
50
100
0 3 6 9 12
Level of irradiation (Krad)
% s
urv
ival
of
cult
ure
s
Effect of Colchicine on % survival of cultures at 1 month after culturing
R2 = 0.98
0
50
100
0 0.03 0.06 0.09 0.12
Colchicine (%)
% s
urv
ival
of
cu
ltu
res
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 Effect of Gamma radiation on mean shoot length and mean no. of secondary shoots at 2 months and mean no. of secondary shoots at 2 months in culturein culture
0
1
2
3
4
5
6
7
8
9
0 3 6 9
Gamma (Krad)
Mean shootlength(cm)
Mean no. ofsecondaryshoots /culture
Effect of Colchicine on mean shoot length and Effect of Colchicine on mean shoot length and mean no. of secondary shoots at 2 months in mean no. of secondary shoots at 2 months in cultureculture
02468
101214161820
0 0.03 0.05 0.09
Colchicine (%)
Mean shootlength (cm)
Mean no. ofsecondary soot/culture
Concluding RemarksComparison of the difference in growth responses
of treated Crossandra shoots for 2 different mutagenic agents at 2
months in culture.
CONTROL6 Krad
Gamma
0.05 %
Colchicine
Some leaf abnormalities shown in Some leaf abnormalities shown in
gamma rays treated shoots at 2 months in gamma rays treated shoots at 2 months in multiplication mediummultiplication medium
ControlControl Leaf abnormalities in gamma rays Leaf abnormalities in gamma rays treated shootstreated shoots
0
10
20
30
40
50
60
3 6 9
Gamma (Krad)
0
10
20
30
40
50
60
70
80
0.03 0.05 0.09
Colchicine (%)
X
X
X=Abnormal leaves %
= Treated cultures showed one or more abnormal leaves Total no.of treated shoots
x100
Percentage shoots which showed abnormal leaves in mutagenic treated cultures at 2 months in culture
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)
Colchicine
(%)
0
3
6
9
0
0.03
0.05
0.09
100
12
80
6
94
55
18
0
5.5±0.74 d
8.0±0.75 c
9.5±0.91 b
10.5±0.83 a
6.0±0.91 d
8.5±0.91 c
10.0±0.64 b
11.5±0.06 a
6.5±1.14 a
3.2±0.55 b
1.5±0.92 c
1.3±1.88 d
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)
Colchicine (%)
0
3
6
9
0
0.03
0.05
0.09
94
55
20
8
90
53
18
0
7.20± 1.01 c
7.33± 0.61 c
8.86± 1.00 b
10.00±0.64 a
6.8± 0.77 c
6.5± 0.51 c
9.0± 0.75 b
10.0±0.64 a
3.0±0.70 b
5.4±0.50 a
3.0±0.92 b
2.1±0.35 c
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
0 6 Krad 0.09 %CONTROALCONTROAL GAMMA TREATEDGAMMA TREATED COLCHICINECOLCHICINE TREATEDTREATED
Effect of gamma rays (6 Krad) and colchicine (0.09%) on in-vitro rooted Crossandra at 9 weeks in culture
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)
Colchicine (%)
0
3
6
9
0
0.03
0.05
0.09
94
55
20
8
90
53
18
0
76
64
40
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 level
Mean plant height (cm±SE)
Gamma (Krad)
Colchicine (%)
0
3
6
0
0.03
0.05
20.43 ±0.57 a
15.70 ±0.57 b
08.95 ±0.51 c
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
Gamma ray induced leaf abnormalities observed in plants growing under normal plant house conditions at 3 months after transplanting
x
X-Represent the control leaf
Effect of gamma rays and colchicine on flowering behavior of in-vitro derived Crossandra plants
01
2
34
5
67
8
910
Time (Months) taken to full blooming
0 Krad/ 0 % 3 Krad/ 0.03 % 6 Krad/ 0.05 %
Gamma (Krad)
Colchicine (%)
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 stagesMutation
rateM1V1 M1V2 M1V3
3 krad Gamma radiation
0.03% Colchicine
0.05% Colchicine
Control (No treatment)
60
60
60
60
240
195
180
300
960
585
370
920
1/960
5/585
2/370
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 “Danica” “Savindi”
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)
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
Significant at p>0.05
Vegetative generations
Number of shoots in generation
% plants survived till flowering
Stability of phenotypic characters (Yes/No)
V1
V2
V3
V4
V5
2
6
16
38
73
100
66.66
75
76.31
89.04
Yes
Yes
Yes
Yes
Yes
The number of vegetative shoots The number of vegetative shoots multiplied in each vegetative multiplied in each vegetative
genaration of mutant Crossandra genaration of mutant Crossandra “Savindi”“Savindi”
ConclusionsConclusions 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