I= IN VITRO SELECTION AND EVALUATION OF THE GENETIC PURITY OF REGENERATED BANANA PLANTS
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Title of Thesis
IN VITRO SELECTION AND EVALUATION OF THE GENETIC PURITY OF REGENERATED BANANA PLANTS

Author(s)
Bushra Ahmad Saeed
Institute/University/Department Details
Department of Biotechnology/ University of Karachi
Session
2006
Subject
Biotechnology
Number of Pages
172
Keywords (Extracted from title, table of contents and abstract of thesis)
genetic purity, banana plants, banana bunchy top virus disease, pentalonia nigronervosa, ploidy levels, shoot formation

Abstract
Banana is an important crop in Pakistan both economically and nutritionally and is grown mostly in the Sindh Province with an average annual production of 209,820 tonnes. However since 1988, Pakistan has been facing an epidemic, of Banana Bunchy Top Virus Disease (BBTVD)-a lethal and persistent disease transmitted by a black aphid, Pentalonia nigronervosa. This has resulted in drastic decrease, both in quality and quantity of banana fruits, and has greatly affecting the socio-economic status of the local farmers in the banana growing regions. Thus it is important to exploit modem Biotechnological approaches for replenishing the infected fields with clean and true to type banana plants on a large scale. It is important to ensure the trueness to type of the banana plants produced through micropropagation as it is reported that plants subjected to repeated subculturing are prone to develop variations. This study was conducted to develop a feasible means of determining the genetic stability of plants that were subjected to mass multiplication by micropropagation.

Plants collected from the vicinities of Sindh were subjected to virus indexing regimes and DAS-ELISA was used to check the status of the mother plants to be multiplied. To develop a cost effective protocol of micropropagation, sucrose was successfully replaced with table sugar, and agar with surgical cotton. From the series of experimentations, it was determined that the most suitable media for shoot proliferation in vitro was MS media supplemented with BAP (0.50 mg/L) in combination with KIN (1.00 mg/L), where the maximum number of shoots (9.15±0.59) were obtained using cotton as a support system over a 4 week period. Likewise, the length of shoots (89 ±11.42) was also revealed to be higher in this media. A suitable media (IBA 1.00 mg/L) for root initiation was optimized which produces (6.43 ±0.26) roots, and yielded a plant viability of 100% during acclimatization.

This study was than directed towards the development of optimum molecular markers for varietal characteristics. Four marker systems were analyzed, viz, RAPD, SSR, AFLP and Protein SDS-P AGE for estimating the genetic diversity among 15 cultivars under study. Similarity matrices were constructed and it was determined that by the utilization of microsatellites, a single primer combination, AAT-CAT produced an average number of 24 bands per variety, from which 20 polymorphic bands were selected for varietal analysis for the differentiation of all 15 varieties. The highest diversity index in the comparative assays of the markers employed were yielded by microsatellites (0.76) followed by AFLP (0.63), RAPDs (0.60), and the lowest diversity index was shown by protein electrophoresis (0.46). Genetic variations across various sub culture levels were studied and it was revealed that the micropropagated plants exhibited high degrees of polymorphism below the 81h sub culture onwards.

In conclusion, this study has demonstrated that SSR's, AFLP's and RAPD's could be used for the molecular characterization of Banana germplasm and its genetic relationships. The robust effectiveness of Microsatellites has been proven by the possibility of a very high band resolving power, maximum banding patterns and reproducibility of results. This strategy has shown to be effective and can be employed as a simple PCR based assay for easy routine screening for genetic stability of micropropagated banana plants, both in vitro and ex situ

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
468.2 KB
2 1 Introduction 1
1351.89 KB
  1.1 General Introduction 2
  1.2 Soil And Climate 2
  1.3 Ploidy Levels Of Banana 3
  1.4 Composition And Uses Of Banana 4
  1.5 Economic Significance Of Banana 6
  1.6 Banana Cultivation In Pakistan 8
  1.7 Banana Bunchy Top Virus 11
  1.8 Other Viruses Infecting Banana Crop 19
  1.9 Alternative Methods Of Banana Propagation 21
  1.10 Variation In Banana 25
  1.11 Methods For Evaluation Of Genetic Stability 27
  1.12 Diagnostic Techniques Of Viruses In Banana
  1.13 Objectives Of This Research Study 42
3 2 Materials And Methods 43
500.53 KB
  2.1 General Introduction 43
  2.2 Virus Indexing Of Banana Plants 45
  2.3 Preparation Of Explants For Micropropagation 49
  2.4 Regeneration Of Axillary Shoots And Roots 50
  2.5 Acclimatisation 50
  2.6 SDS-Page 50
  2.7 Molecular Marker Analysis 53
4 3 Molecular Markers 60
996.23 KB
  3.2 Protein Based Assay 61
  3.3 Evaluation Of DNA Based Molecular Assays 62
  3.4 Results, Separation Of 15 Banana Varieties Using RAPD€™s 73
  3.5 Discussion 97
5 4 Micropropagation 102
702.91 KB
  4.1 General Introduction 102
  4.2 Methodology 103
  4.3 Optimization Of Media For Maximum Multiple Shoot Formation In Banana 110
  4.4 Optimization Of Media For The Induction Of Root In Banana Plants Regenerated Via Tissue Culture 114
  4.5 Acclimatization 115
  4.6 Low Cost Alternatives For The Micropropagation Of Banana 117
6 5 Evaluation Of Genetic Purity 121
446.87 KB
  5.1 General Introduction 121
  5.2 Analyses Of Genetic Stability Of Micropropagated Banana Plants At Various Subculture/Cycle 122
  5.3 Results And Discussion 127
7 6 General Discussion 137
438.57 KB
  6.1 General Introduction 137
  6.2 BBTV Diagnostic Regime 138
  6.3 Micropropagation 138
  6.4 Selection Of Markers 140
  6.5 Genetic Variability 143
  6.5 Future Prospects 146
8 7 References Cited 149
550.33 KB
9 8 List Of Publications 172
31.22 KB