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