运用离体诱变进行蔺草遗传改良的研究

摘要

Mat rush (Juncos effusus L.) is a cosmopolitan, emergent macrophyte inhabits fresh to brackishmarshes, wetlands and meadows．1t is an economical as well as ecologically important plant grownfor reclamations of land, mitigation and construction of wetlands, while its stem is used as rawmaterial for production of various products．Keeping in view the importance of mat rush plant aseries of experiments was conducted to induce genetic variations and to improve the quality of plantusing in vitro mutagenesis techniques．An efficient tissue culture protocol was developed in the firststep as a prerequsite of in vitro mutagenesis experiment．Plants were grown on MS (Murashige andSkoog, 1962) medium and effects of hormonal combinations and medium innovation on tissueculture system were investigated．Positive effects of additional benzylaminopurine (BA) in themedium were observed on callus proliferation, plant regeneration and its multiplication．0.5 mg/L concentration of BA in combination with 4mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) in themedium resulted in the highest callus induction (90.48％)．The effects of various growth regulatorsand their combinations with successive subculturing (medium replacement) were found to beessential to develop embryogenic calli and to regenerate shoots．Among different combinations ofgrowth regulators tested in the study, the combination of 0.1mg/L BA and 2mg/L 2,4-D in MSmedium was determined to be the best for callus proliferation．Medium innovation was also foundnecessary for successful plant regeneration．Our results suggested the importance of the ratio ofauxin (IAA) to cytokinin (BA and KT) in the manipulation of shoot regeneration in this plant．Themaximum plant survival frequency and multiplication rate (90.97％)were recorded in the presenceof 0.5mg/L BA in the 1/2 MS multiplication medium for the plant. An in vitro mutagenesis study was carried out using the tissue culture protocol developed in thefirst study to induce genetic variability in mat rush and to unveil the mutagenic effects ofethylmethane sulfonate (EMS at 0.1-1.0％for 12 and 24 h) and ultraviolet radiations (UV for 15, 30and 45 min) on physiological characteristics and ultra-structural responses of the plant．The basalculms of plants were treated with EMS and UV radiations and then transferred to 1/2 MS media togrow and multiply．Physiological and ultra-structural alterations of leaf were observed in the leavesof treated plants．A large number of mutants with changes in leaf colour, length, size and growth rates were found．With the increase in the level of mutagens, the activities of antioxidant enzymesincreased invariably, however, at their higher level these activities showed downward trend．Plantgrowth and biomass significantly reduced at the higher doses of both mutagens．Further study of theselected mutants showed greater reduction in chlorophyll contents as well as noticeable changes atthe ultra-cellular levels．Over all reduction in the size of cell and chloroplast, appearance of largenumber of oil bodies in chloroplasts and disruption of the thylakoid integrity were some of theobvious changes in resultant mutants．Present study suggested that both EMS and UV mutagenscaused mutation at functional and ultra-structural levels that can be effectively used to create geneticvariations in mat rush (Juncus effuses L.).

目录

英文文摘

声明

ACKNOWLEDGEMENTS

CHAPTER 1 Introduction

1.1 Mat rush (Juncus effusus L.)

1.2 Over view of the study

1.3 In vitro mutagenesis

1.4 Effect of mutagens on plants

1.5 Objectives

CHAPTER Literature Review

2.1 Mutagenesis for crop Improvement

2.2 Effects of Ems and UV radiations on plant growth

2.3 Chlorophyll deficient mutants

2.4 Chlorophyll biosynthesis inhibition

2.5 Effects of Ems and UV radiations on chlorphyll

2.6 Oxidative stress

2.6.1 Chloroplast damage

2.6.2 Antioxidative response of plants to EMS and UV radiations

2.7 Cchloroplast

2.7.1 Structure of chloroplast

2.7.2 Functions of chloroplast

2.7.3 Effects of mutation on chloroplast development

2.7.4 Chloroplast ultrastructural changes under mutation

2.7.5 Production of plastoglobuli (Lipid bodies)

CHAPTER 3 Use of BA benzylaminopurine) and medium innovation for callus induction and plant regeneration via somatic embryogenesis in mat rush (Juncus effusus L.)

3.1 Introduction

3.2 Materials and methods

3.2.1 Plant material

3.2.2. Seed sterilization and germination

3.2.3 Callus induction

3.2.4 Callus proliferation

3.2.5 Plant regeneration

3.2.6 Plant multiplication

3.2.7 Data analysis

3.3 Results

3.3.1 Callus induction and proliferation

3.3.2 Callus differentiation and plant regeneration

3.3.3 Plant multiplication

3.4 Discussion

3.4.1 Callus induction

3.4.2 Importance of subculturing

3.4.3 Plant regeneration

3.4.4 Effects of auxin / cytokinin ratio

3.5 Conclusion

CHAPTER 4 Physiological and ultra-structural changes in leaves of mat rush (Juncus effusus L.) mutants induced by ethylmethane sulfonate and ultraviolet radiation

4.1 Introduction

4.2Materials and methodsv

4.2.1 Plant material and mutagenic treatments

4.2.2 Determination of total soluble protein and antioxidant enzymes

4.2.3 Determination of chlorophyll (a+b) contents

4.2.4 Transmission electron microscopy

4.2.5 Data analysis

4.3 Results

4.3.1 Plant survival rate and growth parameters

4.3.2 Effects on total soluble protein and antioxidant enzymes

4.3.3 Effects on chlorophyll contents

4.3.4 Ultra-structure study

4.4 Discussion

4.4.1 Morphological and physiological parameters

4.4.2 Effects on total soluble protein and antioxidant enzymes

4.4.3 Effects on chlorophyll contents

4.4.4 Electron microscopy

4.5 Conclusion

REFERENCES