I= IN VITRO ORGANOGENESIS OF CHICKPEA (CICER ARIETINUM) AND ITS TRANSFORMATION BY AGROBACTERIUM
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Title of Thesis
IN VITRO ORGANOGENESIS OF CHICKPEA (CICER ARIETINUM) AND ITS TRANSFORMATION BY AGROBACTERIUM

Author(s)
A.K.M. Rafiul Islam
Institute/University/Department Details
University of the Punjab /Centre for Advanced Molecular Biology
Session
1991
Subject
Molecular Biology
Number of Pages
217
Keywords (Extracted from title, table of contents and abstract of thesis)
organogenesis, chickpea (cicer arietinum). agrobacterium, crop improvement, legumes, genetic engineering, callus, plant tissues

Abstract
The aim of the present investigation was to standardize techniques for in vitro culture of chickpea (Cicer arietinum) tissues and their ultimate use in plant genetic engineering. Conditions for in vitro culture of calli from hypocotyl, epicoty1, cotyledon and leaflet explants in Gamborg (B5) and Murashige and Skoog (MS) media supplemented with phytohormones have been established. In terms of fresh and dry weight 2,4-D with higher concentration of cytokinin yielded high amount of calli. Among the four explants studied hypocotyl gave maximum response for callusing. Morphogenetic expression of calli in different hormonal combinations has been studied and discussed. Although the calli derived fromdifferent explants underwent rhiogenesis but they failed to produce shoots.Both hypocoty1 and leaflet originated calli although showed some meristemoidal activities but only hypocoty1 derived calli underwent organogenesis to regenerate shoots. Pattern of shoot morphogenesis from hypocoty1 calli shows that regeneration of shoot buds required the presence of an organized meristematic region in the intact callus. The data obtained clearly demonstrate that various tissue explants exhibited specific and differential response attributable in endogenous levels of hormones and variations in the internal cellular organization. Changes in DNA, RNA and protein content were in correlation with the callus growth and are manifestation of metabolic activity associated with active and stationary growth phase of call.

Direct regeneration ability of three meristematic parts (hypocoty1, nodal explants and shoot apieces) of four genotypes of chickpea showed that the treatment combination not only produced pronounced variation in days to shoot initiation, frequency of shoot formation and shoot growth but also had a significant effect on number of shoot/explant and shoot length. The number of shoot/explant increased with the increase of concentration of BAP. Further increase in shoot number was observed with the additional of NAA or IAA. When two cytokinins (BAP and KIN) were applied together in the medium containing IAA maximum shoot number was achieved. The rate of increase or decrease in number of shoot/explant was negatively correlated with that of shoot length. Significant interactions were observed between genotypes and treatment combination.

The rooting experiment conducted with microcuttings of two-week-old seedlings of four genotypes showed that full strength MS medium with 1uM IBA improved rooting frequency, root quality, root number and root length. Therefore, all the regenerated shoots obtained directly form meristematic parts or indirectly via callus phase were transferred to same rooting medium for induction of roots to raise ful-fledged plants. The directly or indirectly regenerated shoots could be multiplied by repeated subculturing of stock cultures and in ths way they could be maintained for several months. The subculturing process might change the physiological state and gradually rejuvenate the cultures which in turn promoted better rooting.

All the four genotypes of chickpea were found to be susceptible to all virulent strains of A. tumefaciens and A. rhizogenes. The ability of the bacteria to form a compatible reaction varied greatly. The differences were also observed among the protocols used for transformation. Percent visual tumor formation and tumor sixe varied greatly among the genotypes. In co-cultivation system hypocoty1 explants were found more responsive than interenode and leaflet explants. In all conditions A281 produced large tumors as compared to A6 and C58. Autonomous growth of the transformed tissues in the absence of exogenous phytohormones, production of novel strain specific opines and presence of T-DNA genetic sequences demonstrate the stable transformation of chickpea tissues by Agrobacterium.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
144.42 KB
2 1 Introduction 1
421.34 KB
  1.1 Application Of Biotechnology To Crop Improvement 1
  1.2 The Chickpea Plant 4
  1.3 Tissue Culture Studies In Legumes 20
  1.4 Tissue Culture Studies In Chickpea 28
  1.5 Natural Plant Genetic Engineering Via Ti-Plasmid Of Agrobacterium 33
  1.6 Plant Transformation By Agrobacterium 38
  1.7 Aim Of The Present Investigation 40
3 2 Materials And Methods 42
221.97 KB
  2.1 Chickpea Tissue Culture 42
  2.2 Genetic Transformation Of Chickpea Tissues 55
  2.3 Biostatistical Analysis 65
4 3 Results 67
1176.93 KB
  3.1 Induction And Organogenesis Of Callus 67
  3.2 Induction Of Multiple Shoots On Hypocotyl Explant 96
  3.3 Induction Of Multiple Shoots On Axillary Bud 105
  3.4 Induction Of Multiple Shoots On Shoot Apices 113
  3.5 Induction Of Adventitious Roots On Microcuttings 120
  3.6 Multiplication Of Regenerated Shoots And Their Rooting To Raise Fulfledged Plantlets 126
  3.7 Genetic Transformation Of Plant Tissues With Agrobacterium 131
5 4 Discussion 166
271.78 KB
6 5 Summary 185
64.05 KB
7 6 References 190
326.04 KB