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Title of Thesis

Regeneration of Chrysanthemum (Dendranthema Morifolium Plantlets through Tissue Culture)

Author(s)

Kashif Waseem

Institute/University/Department Details
Department Of Horticulture, Faculty Of Agriculture / Gomal University, D. I. Khan
Session
2008
Subject
Horticulture
Number of Pages
293
Keywords (Extracted from title, table of contents and abstract of thesis)
Regeneration, Chrysanthemum, Dendranthema, Morifolium, Plantlets, Tissue, Culture, auxins, phytohormonal, IAA, NAA, IBA, explants

Abstract
Regeneration of chrysanthemum (Dendranthema morifolium L.) plantlets was obtained by treating different explants viz: apical shoot tips,
nodal segments and leaf discs of chrysanthemum, with different concentrations and combinations of auxins (IAA, NAA, IBA & 2,4-D) and
cytokinin (BAP), for the formation of micro-shoots, which were sub-cultured for development of roots. Fully developed plantlets were successfully transferred to suitable growing media for acclimatization and their further growth and development.
Sterilization of the explants was obtained, by treating with 1.0% HgCl2 for three minutes plus 2-3 drops of tween-20 (a detergent) and then
rinsed thrice with double distilled water.
To check the effect of different concentrations and combinations of different auxins including IAA, NAA, IBA, 2,4-D and cytokinin including
BAP on the shoot proliferation of chrysanthemum explants, the parameters under study were, number of days to shoot initiation, shoot initiation percentage, number of shoots per explant, shoot length, leaves per shoot and nodes per shoot. For callus formation the parameters such as callus formation percentage, number of shoots developed from the callus, average shoot length, average leaves per shoot and average nodes per shoot were studied.
For the induction of roots of un-rooted chrysanthemum micro-shoots, the data was recorded for average days to root initiation, rooting
percentage, average roots per shoot and average root length, whereas for transplantation of these rooted plantlets to different growing media, the survival percentage was calculated.
When shoot tip explants were treated with different concentrations of auxins (IAA, NAA, IBA and 2,4-D) and cytokinin (BAP) for shoot proliferation, 1.0 mg/l BAP showed its superiority over all the other phytohormonal treatments, when used alone, as it took the minimum (4.0) days to shoot initiation, presented maximum shooting percentage (93.3%), maximum (4.1) shoots per explant, longer shoots (5.0 cm), higher number of leaves (11.0) and 5.5 nodes per shoot. It was followed by 0.1 mg/l IAA and 0.5 mg/l NAA, that took 4.0 days to shoot initiation, 86.6 and 80.0% shoot initiation, an average of 3.9 and 3.2 shoots per explant, 4.3 and 4.0 cm long shoots, an average of 10.0 and 9.5 leaves per shoot and an average of 4.8 and 4.5 nodes per shoot, respectively. While the maximum of 100% shoot initiation, maximum (11.8) shoots per explant, 6.0 cm long shoots, highest number of leaves per shoot (19.9) and maximum (6.5) nodes per shoot were recorded when MS media was supplemented with 1.0 mg/l BAP + 0.5 mg/l NAA, amongst all the hormonal treatments, used alone or in combinations.
Callus induction and its further organogenesis was only observed for 2,4-D. Maximum callus formation (73.3%) was observed when MS media was supplemented with 2.0 mg/l (2,4-D) and when sub-cultured in a shoot promoting media, it produced maximum shoots (6.6) per callus, maximum shoot length (3.9 cm), higher number of leaves per shoot (8.5) and maximum (4.5) nodes per shoot derived from the callus.
For nodal segments, 1.0 mg/l BAP excelled all the parameters compared to all phyto-hormonal treatments when used alone. Maximum (100%) shoot initiation, maximum (4.9) shoots per explant, longest shoot (5.8 cm), highest number of leaves per shoot (13.4) and maximum (6.3) nodes per shoot was observed for 1.0 mg/l BAP, followed by 0.3 mg/l IAA and 0.5 mg/l NAA, as they produced 80.0 % and 83.3% shoot initiation, 4.0 and 3.6 shoots per explant, 5.1 and 4.2 cm long shoots, 11.3 and 10.2 leaves per shoot and 5.6 and 4.7 nodes per shoot, respectively. The combination of 1.0 mg/l BAP + 0.5 mg/l NAA excelled all the hormonal treatments, used alone or in combination. This particular treatment had produced maximum (100%) shoot initiation, maximum (13.8) shoots per explant, longer shoot (7.4 cm), higher (21.2) number of leaves per shoot and maximum (7.9) nodes per shoot.
Callus formation was observed in MS media supplemented with different concentrations of 2,4-D, as the maximum (83.3%) callus was observed when MS media fortified with 2.0 mg/l 2,4-D and likewise the most positive results for in-direct organogenesis was reported for 2.0 mg/l 2,4-D, as it produced 10.5 shoots per callus, 4.2 cm long shoots, 9.0 leaves and 4.5 nodes per shoot.
As far as, the effect of different phyto-hormonal treatments, with regards to shoot proliferation from leaf disc explants of chrysanthemum is
concerned, a parallel situation was recorded as was previously observed in case of apical shoot tip and nodal segments. MS media supplemented with 1.0 mg/l BAP had showed its dominance by giving maximum (76.7%) shoot initiation, more (3.4) shoots per explant, longer shoots (3.8 cm), higher (9.5) number of leaves per shoot and maximum (4.3) nodes per shoot. It was followed by 0.1 mg/l IAA and 0.5 mg/l NAA, which gave 70% shoot initiation, 2.2 and 2.0 shoots per explant, 3.0 and 2.6 cm long shoots, 8.1 and 5.3 leaves per shoot and 3.5 and 3.1 nodes per shoot, respectively. A combination of 2.0 mg/l BAP + 0.5 mg/l NAA produced the over all better results for shoot proliferation compared to all other phytohormonal treatments, as it produced maximum (93.3%) shoot initiation, maximum (9.6) shoots per explant, longer shoots (5.0 cm), higher (16.5) number of leaves per shoot and maximum (5.5) nodes per shoot.
More callus formation was observed, for leaf disc compared to other explants, showing that leaf disc has much more potential for callus
induction and its further organogenesis than apical shoot tip and nodal segments. Callus formation was observed in almost all type of auxins used. The higher percentage of callus formation was found with application of 2.0 mg/l (2,4-D) that resulted in 100% callus formation, followed by IBA, NAA and IAA, respectively.
For in-direct organogenesis the callus already formed was cut into 1X1 cm2 pieces and sub-cultured on a shoot promoting media. The bestresponse was recorded on MS media supplemented with 2.0 mg/l 2,4-D, as it gave maximum (12.5) shoots per callus, maximum shoot length (4.7 cm), more (11.9) leaves per shoot and maximum (4.9) nodes per shoot.
The root initiation and all its other parameters under study were found positive when the un-rooted micro-shoots raised from all the chrysanthemum explants were sub-cultured on strength MS media supplemented with different concentrations of IBA, NAA and IAA. The best results regarding the rooting of micro-shoots was obtained on strength MS media fortified with 0.2 mg/l IBA, followed by 0.2 mg/l NAA and 0.2 mg/l IAA, respectively.
Minimum days (5.0) to root initiation, maximum (100%) root initiation, maximum (14.3) roots per micro-shoot and longest roots (9.0 cm) were noted for the micro-shoots raised from apical shoot tip explant of chrysanthemum, when sub-cultured in strength MS media fortified with 0.2 mg/l IBA. It was followed by strength MS media supplemented with 0.2 mg/l NAA and 0.2 mg/l IAA respectively.
For rooting of micro-shoots raised from nodal segments the maximum rooting (100%), maximum (16.0) roots per shoot and longest roots (11.0 cm) were found in strength MS media fortified with 0.2 mg/l IBA, followed by 0.2 mg/l NAA and 0.2 mg/l IAA, respectively.
A similar trend of results was recorded for the rooting of microshoots raised from leaf discs as was observed previously in apical shoot tip and nodal segments of chrysanthemum. Half strength MS media supplemented with 0.2 mg/l IBA excelled all the other phyto-hormonal treatments by taking minimum (5.4) days to root initiation, maximum (93.3%) rooting, maximum (11.3) roots per shoot and longer roots (8.1 cm), followed by 0.2 mg/l NAA and 0.2 mg/l IAA, respectively.
For the transplantation of these rooted plantlets to different growing media, a combination of sand : silt : leaf mold (1:1:1) showed its superiority over all the other growing media used, as it gave 83.3% survival percentage whereas the least response was observed in sand alone, that give 36.7% survival percentage for all the chrysanthemum plantlets.
It can be concluded that nodal segments showed much more positive response towards shoot proliferation, followed by apical shoot tip and leaf disc explants respectively, whereas, for callus induction and indirect organogenesis, leaf disc showed their superiority over the other two explants used.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 CONTENTS

 

 
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2

1

INTRODUCTION

 

1
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3 2 REVIEW OF LITERATURE

2.1 Work done in Pakistan
2.2 Work done in the World
2.3 Establishment of Aseptic conditions
2.4 Explant source
2.5 Growth regulators
2.6 Physical factors
2.7 Types of culture
2.8 Rooting
2.9 Transfer of plants to soil

6
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4 3 MATERIALS AND METHODS

3.1 Standardization of sterilization techniques
3.2 Regeneration of Chrysanthemum

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5 4 RESULTS & DISCUSSION

4.1 Standardization of sterilization techniques
4.2 Effect of different growth regulators on the shoot tip explant of chrysanthemum

4.3 Effect of different growth regulators on the nodal segments explant of chrysanthemum

4.4 Effect of different growth regulators on the leaf discs explant of chrysanthemum

4.5 Transplantation

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6

5

SUMMARY AND CONCLUSION

5.1 Summary
5.2 Conclusion

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7

6

LITERATURE CITED

 

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