Pakistan Research Repository Home

Title of Thesis

Idrees Ahmad Nasir
Institute/University/Department Details
University Of The Punjab/ National Cnetre Of Excellence In Molecular Biology
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
regeneration, cell suspension, gladiolus, fusarium oxysporum, cormels, cormlets, dendrograms, somaclonal variation, molecular variation, rapd analyses

Gladiolus is one of the most cultivated, economically important and common flowering plants worldwide including Pakistan. Fusarium corm rot caused by Fusarium oxysporum f. sp. Gladioli mass is one of the most serious diseases of Gladiolus; it can reduce corm and flower production. Management of Fusarium wilt is mainly through chemical soil fumigation and resistant cultivars.

Cormels pieces of four reported highly Fusarium susceptible Gladiolus cultivars (Friendship, peter pears, Victo Borge and Novalux) formed friable calli when cultured in-vitro on MS basal medium containing 2,4-D and BAP. The friable calli of each cultivar established cell suspensions in their respective callus maintenance gelrite free G2-medium. One week after the initiation of suspension cultures only few viable cells could be visualized under microscope. At initial stages of suspension cultures, two types of cells were noted. The first cell type consisted of elongated. And with on starch contents. The second type was round in shape and small in size as compared with the first type of cells.

Plantlets were regenerated from the all four cultivars of immature cormel slices when the cormel slices went through a cellus phase. Plant regeneration was obtained form the control callus, control cells suspension derived callus, Gamma irradiated callus and in-vitro selected FOG resistant cell-lines of Gladiolus CV-I. Cell suspension or cell suspension derived calli of CV-4 did not regenerate plants after 36 months. The calli derived from cell suspension with 15-30% PCV were found most suitable for regeneration of maximum number of plantlets. Gamma irradiated plantlets of CV-1 were weak and grew in clusters which could not be separated and died when individual plantlets were cultured on any of the media.

Freshly regenerated plantlets from all other type of calli, when transferred in the C-medium started elongation and multiplication of buds whereas, after sub-culturing in the medium containing 6% sucrose only cormlet multiplication was noted. The maximum rate of multiplication was noted during the first 4-6 weeks of shifting plantlets from regeneration medium to the cormlet induction medium. The differences among all selected cell-lines and control of CV-1 in the formation of cormlet under in-vitro condition were non-significatn as assessed by fresh weight of the cormlets. The cormlets of all types of plantlets formed under in-vitro condition showed 85-95% germination after breaking dormancy of 8 weeks at 4C.

Cell suspensions of all the susceptible Gladiolus cultivars were found highly sensitive to the Fusaric Acid. Gradual increase in F.A concentrations to the cell-suspension cultures above related to the toxin concentrations. On the basis of initial experiments, toxin concentration of 0.04mM was considered sub-lethal, so, used for the initiation and maintenance of selection experiments. The resistant callus-clumps obtained from cell suspension culture of callus cultures were maintained for callus initiatin on callus medium containing 0.04mM F.A., resistance to the toxin as compared with the control was noted. One albino plant was found from the VS2 generation in-vitro selected cell line. The albino plant was found highly susceptible to the FOG, therefore, died ager 3rd week of second application of the FOG conidia. At this stage all other susceptible plants were exhibiting very few to moderate symptoms of severity. The VS1 cormlets of all in-vitro selected cell lines were inoculated with a conidial suspension of FOG at a final concentration of 104 sproes/ml before planting and also sprayed with the same spore suspention when the height of plants was about 6cm in length for further characterization. The Cell lines. CAMB-G01, CAMB-G04, CAMB-G06 and CAMB-G09 showed the same response whether or not inoculated with conidia of the FOG. Plantlets of all the selected cell lines exhibited significant growth as compared with control after application of conidia of the FOG.

RAPD analyses of ten fusarium oxysporum f. sp. Gladioli (FOG) resistant and one Gamma irradiated in comparison with control was performed with total of 29 amplified reproducible fragments produced form 5 random decamer primers. The number of fragments per primer ranged form 4 in s 13 and R8 to 10 fragments in S19. The total size of the amplified products varied form 200bp to 1800bp. All the primers were found to be polymorphic and produced different percentages of polymorphism. Average number of fragments per primer was 6 form which 62% were found to be polymorphic fragemtns.

Similarity indices were generated through RAPD-PCR analyses using five primers. The dendrograms have been generated form the similarity indices data in the from of genetically related in-vitro selected cell-lines and control of Gladiolus CV-1. It has been documented that RAPD techniques can be successfully used to assess genetic variations, which usually arise during the in-virtro selection process at cell suspension or callus level.

Download Full Thesis
1421.23 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
153.82 KB
2 1 Introduction 1
75.16 KB
3 2 Literature Review 11
197.59 KB
  2.1 Corm Dormancy 11
  2.2 In-Vitro Production Of Cormlets 12
  2.3 Regeneration Of Plantlets 15
  2.4 Susceptibility Of Gladiolus To Diseases 17
  2.5 Callus Initiation 19
  2.6 Establishment Of Cell Suspension Cultures 22
  2.7 In-Vitro Selection For Disease Resistance 25
  2.8 Somaclonal Variation 28
  2.9 Molecular Variation 29
  2.10 Miscellaneous 32
4 3 Materials And Method s 39
107.39 KB
  3.1 Medium 39
  3.2 Plant Material And Initiation Of Callus 39
  3.3 Establishment Of Cell Suspension Cultures 41
  3.4 Plantlet Regeneration 42
  3.5 In-Vitro Selection At Cell-Suspension Level 42
  3.6 In-Vitro Selection At Callus Level 43
  3.7 Culture Conditions 45
  3.8 Phytotoxin 45
  3.9 Fungal Culture And Screening 46
  3.10 Characterization Of Plants Regenerated From Resistant Cell-Lines 46
  3.11 Preparation Of Fungal Culture And Disease Rating 47
  3.12 Application Of Gamma Irradiation 47
  3.13 In-Vitro Production Of Cormlets 48
  3.14 Acclimatization Of In-Vitro Produced Cormlets 48
  3.15 Packed Cell Volume 49
  3.16 DNA Extractions 49
  3.17 Photometric Quantification 50
  3.18 Quantification Of DNA By Agarose Gel 51
  3.19 Amplificatin Conditions And Analyzing 51
  3.20 Data Eveluation 52
5 4 Results 53
768.47 KB
  4.1 Callus Induction And Maintenance 53
  4.2 Establishment Of Cell Suspension Cultures 60
  4.3 In-Vitro Selection At Cell Suspension Level 63
  4.4 Selection Of Resistant Cell-Lines At Callus Level 71
  4.5 Palntlet Regeneration 77
  4.6 In-Vitro Cormlet Production 90
  4.7 Disease Rating Of Vs1 Plantlets At 0.4 Mm F.A. 91
  4.8 Disease Rating Of Vs1 Plantlets At 0.5 Mm F.A. 92
  4.9 Disease Rating Of Vs1 Plantlets Regenerated From VS1 Cormlets 92
  4.10 Selection Of Variant Cell Lines 95
  4.11 RAPD Analyses 101
  4.12 Cluster Analyses 103
6 5 Discussion 117
115.58 KB
  5.1 Callus Induction And Maintenance 117
  5.2 Cell Suspension Cultures 119
  5.3 Regeneration Of Plantlets 121
  5.4 Cormlet Production 122
  5.5 In-Vitro Selection 123
  5.6 RAPD Analyses 128
  5.7 Cluster Analyses 129
7 6 References 132
179.94 KB
8 7 Appendices 156
31.88 KB