WELCOME TO MY

THESIS PRESENTATION

TitleResponse to in vitro callus induction and shoot tip

culture of pumpkin (Cucurbita maxima Duch.)

A thesis presented by

Bablu Chakma

Examination Roll No. 060736

Supervised By

Professor Md. Raihan Ali

Biotechnology and Genetic Engineering Discipline,

Khulna University, Khulna.

Background Pumpkin (Cucurbita maxima Duch.) is a member of the

Cucurbiteceae (gourd) family and the genus is Cucurbita which consist 13 species, including C. maxima (pumpkin).

The origin of pumpkins is not definitively known, although they are thought to have originated in North America.

It is widely cultivated vegetable all over the world.

It is a good sources of carbohydrates, fat, protein, mineral salts and vitamins.

Local name of pumpkin in our country is bilati kumra or misti kumra.

Objectives

To find out the effect of auxins in callus induction.

To determine the optimum doses of auxins in callus induction.

To study the effect of auxin along with cytokinin and coconut

water in callus induction and

To determine the effect of cytokinins in shoot development.

Materials and Methods

Plant Material

In this experiment, seeds of pumpkin were used as source of explants. The seeds were collected from the jhum farmers of Mahalchari, Khagrachari hill tracts. These seeds were germinated in vitro. Then the Hypocotyls, cotyledons and shoot tips of germinated seedlings were used as plant material.

Primary preparation

Seeds washed in running tap water

Seed coat removal Sterilized with 50%

Chlorox for 15 minute Washed in sterilized

distilled water

Cotyledon

Hypocotyl

Fig. 1

Culture media Murashigue and Skoog (MS) Media

Growth Regulators for callus induction 2,4-Dichlorophenoxyacetic Acid (2,4-D) α-Naphthaleneacetic acid (NAA) Picloram Coconut water

Growth Regulators for shoot formation Benzyladenine (BA) Kinetin (KN)

MethodsCollection of seeds from jhum farmers of Mahalchari

Media preparation and surface sterilization of seeds

Inoculation of seeds on to MS media for germination

Inoculation of explants onto MS media with different concentration and combination of growth regulators for callus induction

Inoculation of shoot tips onto MS media with different concentration of cytokinins for shoot formation

Data collection and analysis

Effect of different concentrations of 2,4-D, NAA and picloram in the formation of callus from

explants of cotyledon of Cucurbita maxima Duch.

Results

Explant Growth regulator mg/L

Days to callus initiation

% of explants formed callus

Callus intensity Colorof callus

Textureof callus

Cotyledon

MS (control) - - - - -

2,4-D

0.5

1.0

1.5

2.0

3.0

4.0

5-6

5-6

4-6

4-6

3-5

3-4

60

60

80

100

100

100

+

++

++

++

+++

+++

Cream

Cream

Cream

Cream

Cream

Cream

Friable

Friable

Friable

Friable

Friable

Friable

NAA

0.5

1.0

1.5

2.0

3.0

4.0

5-7

5-7

5-6

5-6

5-6

4-5

40

60

60

80

100

100

+

++

+

++

++

+++

Cream

Cream

Cream

Cream

Cream

Cream

Friable

Friable

Friable

Friable

Friable

Friable

Picloram

0.5

1.0

1.5

2.0

6-8

7-8

6-7

5-6

60

60

60

80

+

+

++

++

Cream

Cream

Cream

Cream

Friable

Friable

Friable

Friable

+ = Similar of the explants. ++ =Twice size of the explants. +++ = Larger size i.e. three or more times of the explants. - = No callus.

Effect of different concentrations of 2,4-D, NAA and picloram in the formation of callus from explants of hypocotyl of Cucurbita maxima Duch.

Explant Growth reguletor mg/L

Days to callus initiation

% of explants formed callus

Callus intensity Colorof callus

Textureof callus

Hypocotyl

MS (control) - - - - -

2,4-D

0.5

1.0

1.5

2.0

3.0

4.0

6-7

6-7

5-6

5-6

3-5

3-4

60

60

80

100

100

100

+

+

++

++

+++

+++

Cream

Cream

Cream

Cream

Cream

Cream

Friable

Friable

Friable

Friable

Friable

Friable

NAA

0.5

1.0

1.5

2.0

3.0

4.0

5-7

5-7

5-6

5-6

5-6

4-5

20

60

60

60

100

100

+

+

+

++

++

+++

Cream

Cream

Cream

Greenish

Cream

Cream

Friable

Friable

Friable

Friable

Friable

Friable

Picloram

0.5

1.0

1.5

2.0

6-8

7-8

6-7

5-6

40

40

60

80

+

+

+

++

Cream

Cream

Cream

Cream

Friable

Friable

Friable

Friable

+ = Similar of the explants. ++ =Twice size of the explants. +++ = Larger size i.e. three or more times of the explants. - = no callus.

Effect of 4.0 mgl-1 NAA in callus formation from cotyledon.

Effect of 4.0 mgl-1 2,4-D in Callus formation from hypocotyl and cotyledon.

Fig. 2

Fig. 3

Effect of 1.0 mgl-1 NAA in callus formation from hypocotyl

Effect of 2.0 mgl-1 2,4-D in callus formation from hypocotyl and cotyledon

Fig. 4

Fig. 5

Effect of different concentrations and combinations of auxins with cytokinins and coconut water in

callus induction from explants of cotyledon Cucurbita maxima Duch.

Explant Growth regulator mg/L

Days to callus initiation

% of explants formed callus

Callus intensity Colorof callus

Textureof callus

Cotyledon

MS (control) - - - - -

2,4-D+BA0.1+0.10.1+0.20.2+0.10.2+0.2

6-87-87-76-7

606080

100

+++++

+++

CreamCreamCream

Greenish

FriableFriableFriableFriable

2,4-D+5% CW0.5+5%1.0+5%2.0+5%

6-86-85-6

608080

++

++

CreamCreamCream

Friable FriableFriable

NAA+ 5% CW0.5 + 5%1.0+ 5%2.0 +5%

6-75-86-7

606060

++

++

CreamCream

Greenish

FriableFriableFriable

Picloram + 5% CW

0.5 + 5%1.0+ 5%2.0 +5%

7-86-75-6

606080

+++

CreamCreamCream

FriableFriableFriable

+ = Similar of the explants. ++ =Twice size of the explants. +++ = Larger size i.e. three or more times of the explants. - = no callus.

Effect of different concentrations and combinations of auxins with cytokinins and coconut water in

callus induction from explants of hypocotyl Cucurbita maxima Duch.

Explant Growth regulator mg/L

Days to callus initiation

% of explants formed callus

Callus intensity Colorof callus

Textureof callus

Hypocotyl

MS (control) - - - - -

2,4-D+BA0.1+0.10.1+0.20.2+0.10.2+0.2

6-87-87-76-7

606080

100

+++++

+++

CreamCreamCreamCream

FriableFriableFriableFriable

2,4-D+5% CW0.5+5%1.0+5%2.0+5%

6-86-85-6

606080

++

++

CreamCreamCream

Friable FriableFriable

NAA+ 5% CW0.5 + 5%1.0+ 5%2.0 +5%

6-75-86-7

606060

++

++

CreamCream

Greenish

FriableFriableFriable

Picloram + 5% CW

0.5 + 5%1.0+ 5%2.0 +5%

7-86-75-6

406060

+++

CreamCreamCream

FriableFriableFriable

+ = Similar of the explants. ++ =Twice size of the explants. +++ = Larger size i.e. three or more times of the explants. - = no callus.

Effect of 0.2 mgl-1 2,4-D + 0.2 mgl-1

BA in callus formation from cotyledon.

Effect of 2.0 mgl-1 2,4-D + 5% CW in callus formation from cotyledon.

Fig. 6

Fig. 7

Effect of cytokinin (BA) and (Kn) in different concentrations on shoot formation from Cucurbita maxima Duch.

Treatment mg/L Concentrations % Response Average mean of shoot (cm)

BA 0.5 - -

1.0 40 0.84

2.0 60 0.76

Kn 1.0 40 0.54

2.0 40 0.64

3.0 80 0.92

- = no response

Effect of 2.0 mgl-1 BA in shoot multiplication.

Effect of 3.0 mgl-1 Kn in shoot multiplication.

Fig. 8

Fig. 9

Conclusion Cotyledon derived calli are larger than other explants Hypocotyl derived explants take much time to produce callus 2, 4-D showed better results in callus induction than NAA and

Picloram 2, 4-D with BA and coconut water showed good response in

callus induction than NAA and Picloram with coconut water. At low concentrations NAA and Picloram showed lower

response of callus induction. Kinetin at high concentration showed high rate of shoot

formation then BA

Limitation Some facilities were not available in lab. during experimental

work

Recommendation

Pumpkin is an economically important vegetable crop, therefore further studies through tissue culture techniques will be supportive for breeders, farmers to obtain excellence in production.

THANKS TO ALL