response to in vitro callus induction and shoot tip culture of pumpkin (cucurbita maxima duch.)
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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
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