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Title of Thesis

Biochemical Characterization Of In Vitro Salt Tolerant Cell Lines And Regenerated Plants Of Sugarcane (Saccharum Spp. Hybrid)

Author(s)

NEELMA MUNIR

Institute/University/Department Details
Department Of Botany / University Of The Punjab, Lahore
Session
2009
Subject
Botany
Number of Pages
329
Keywords (Extracted from title, table of contents and abstract of thesis)
Biochemical, Characterization, Vitro Salt, Tolerant, Cell Lines, Regenerated Plants, Sugarcane, cultivars, crops, ascorbic acid, protein

Abstract
Soil salinity is one of the major abiotic stresses affecting growth and productivity of important crops worldwide. Sugarcane is a glycophyte crop of major economical value in tropical and subtropical countries of the world where salinity is an ever-increasing problem. Sugarcane plants grown under stressed conditions show not only arrested growth of various parts but also a decrease in sucrose content. During the recent years, tissue culture technique has gained importance in producing plants with improved salt tolerance through selection of salt-tolerant cells lines and their subsequent regeneration.
During the present work, protocols were established for callus induction, maintenance and regeneration of the two sugarcane cultivars (cv. SPF 234 and cv. HSF 240). It was observed that the best medium for callus induction and maintenance was MS medium supplemented with 13.5 然 2,4-D. Callus cultures at day 120 were shifted to the various regeneration media. It was observed that among the different media used, MS medium supplemented with 8.87 然 BAP and 0.5 然 TDZ was the best in terms of all the growth parameters studied for both the cultivars. However, the maximum regeneration frequency in cv. SPF 234 was greater (85 %) as compared to cv. HSF 240 (76 %). Best rooting (95 %) of the regenerated shoots in cv. SPF 234 was obtained on MS medium supplemented with 2 然 IBA at day 30. Almost similar results were recorded for in vitro-grown plants of cv. HSF 240 for percentage root formation on MS medium supplemented with 1 or 2 然 IBA with 94 and 93 % rooting respectively. Hardening of sugarcane plants was also successfully accomplished for both the sugarcane cultivars.
During the study, 60-days-old calluses were shifted to MS media containing various NaCl levels (0-160 mM). The effect of salt stress on sugarcane callus browning, necrosis and fresh weights was observed at day 90, 120 and 150. It was found that with an increase in salt concentration, callus necrosis also increased. Callus cultures at day 120 were transferred to the regeneration medium (MS + 8.87 然 BAP + 0.5 然 TDZ). The data for regeneration frequency, number of shoots per culture vessel, shoot length, number of roots per culture vessel and root length were recorded at day 30 upon transfer of callus cultures to the regeneration medium. The regeneration frequency was less in the plants regenerated from NaCl-treated callus cultures as compared to control (calluses grown on 0 mM NaCl concentration). It was found that callus cultures of cv. SPF 234 retained 82 % regeneration potential after treatment with 100 mM salt concentration but this frequency decreased sharply at 120 mM salt concentration and only 10 % callus cultures retained regeneration potential. It was also observed that 75 % of the callus cultures of cv. HSF 240 were capable of plant regeneration after 100 mM NaCl treatment. It was also observed during the present study that the number of regenerated shoots from callus cultures treated with various NaCl concentrations was generally greater as compared to the control calluses maintained at 0 mM NaCl.
The present investigation also highlights the changes in soluble protein contents and antioxidant enzyme (peroxidase, catalase and superoxide dismutase) activities in response to salt stress at day 90, 120 and 150. Quantitative analysis of the soluble protein contents during the present work indicated that soluble protein contents in the callus cultures of both the sugarcane cultivars (cv. SPF 234 and cv. HSF 240) significantly decreased in response to various NaCl treatments. Results of the present study also indicated that when salt was supplied to the growth media, generally there was a significant increase in peroxidase, catalase and superoxide dismutase activity of the callus cultures as compared to the control in both the cultivars.
During the present work, significant difference was observed in the soluble protein contents of the regenerated plants from callus cultures treated with different NaCl concentrations. An interesting observation during the present work was that the plants regenerated from salt-treated callus cultures had generally more antioxidant enzyme activities as compared to the control plants (regenerated from non-treated callus cultures).
Salt and water stress show high degree of similarity. An experiment was performed during the present work to study the possible effect of PEG in improving salt tolerance. Sugarcane calluses were treated with four different salt concentrations after giving 1 % PEG pretreatment. The salt concentrations consisted of a control (0 mM NaCl) and three other salt concentrations including one sublethal NaCl level, one above and one below the sublethal NaCl concentration for each cultivar. Data for fresh weights, callus necrosis, soluble protein contents and activities of peroxidase, catalase and superoxide dismutase were recorded at day 90 and 120. No significant change in fresh weights was recorded as a result of PEG pretreatment both at day 90 as well as 120. Less necrosis was observed in the callus cultures of both sugarcane cultivars after PEG pretreatment when subcultured on salt medium as compared to the non-pretreated cultures maintained at the same salt level. PEG pretreatment enhanced the biosynthesis of soluble protein contents in the callus cultures of both the sugarcane cultivars as compared to the non-pretreated controls maintained at the same salt level except in callus cultures of cv. HSF 240 at day 120. A comparison of the PEGpretreated and non-pretreated callus cultures of cv. SPF 234 at day 90 indicated that generally at each salt concentration, PEG-pretreated callus cultures had relatively greater peroxidase, catalase and superoxide dismutase activity as compared to non-pretreated callus cultures maintained at the same salt level. At day 120, only superoxide dismutase activity was increased as a result of PEG pretreatment. In callus cultures of cv. HSF 240, PEG pretreatment affected the peroxidase activity only at day 120 while a significant increase in catalase activity was recorded for this cultivar both at day 90 and 120. PEG-pretreated callus cultures of cv. HSF 240 at day 90 had greater superoxide dismutase activities as compared to their non-pretreated controls maintained at the same NaCl level but at day 120 the effect of PEG pretreatment on SOD activity was non-significant. PEG pretreatment had no effect on regeneration potential of both the cultivars.
Ascorbic acid pretreatment to callus cultures was not proven to improve salt tolerance of callus cultures rather it caused more browning and necrosis of the callus tissues. It was also observed that ascorbic acid pretreatment caused decrease in fresh weights of sugarcane callus cultures. A significant effect of ascorbic acid pretreatment was recorded on soluble protein contents, peroxidase and catalase activities in callus cultures of cv. SPF 234 at day 90 but ascorbic acid pretreatment had no significant effect on SOD activity of callus cultures at day 90. Interestingly, the exogenous supply of ascorbic acid to in vitro-grown sugarcane plants improved their salt tolerance as indicated by the studied morphological as well as biochemical parameters. It was also observed that non-pretreated in vitro-grown plants of both sugarcane cultivars could tolerate up to 100 mM NaCl level but the pretreated plants of cv. SPF 234 and cv. HSF 240 could survive up to 160 and 140 mM NaCl concentration respectively. Furthermore, much less yellowing of leaves in ascorbic acid-pretreated plants was observed as compared to non-pretreated plants at the same salt concentration. A significant increase in the fresh weights, number of shoots per culture vessel and shoot length was observed after ascorbic acid pretreatment in both the sugarcane cultivars. A general decrease in root length was recorded after ascorbic acid pretreatment in the plants of cv. SPF 234. Ascorbic acid pretreatment had a significant effect on soluble protein contents as well as activities of peroxidase, catalase and superoxide dismutase in cv. SPF 234. The same trends were observed in in vitro-grown plants of cv. HSF 240 for peroxidase and catalase activity but no significant effect of ascorbic acid pretreatment was recorded on soluble protein contents or superoxide dismutase activity.

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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 LITERATURE REVIEW

2.1 Tissue Culture Studies in Sugarcane

2.2 Biochemical Aspects of Salinity Tolerance in Plants
2.3 Effect of Salt Stress on Graminaceous Crops
2.4 Salt vs. Water Stress
2.5 Effect of Salt Stress in Sugarcane

9
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4 3 MATERIALS AND METHODS

3.1 Media Preparation

3.2 Sterilization
3.3 Plant Material
3.4 Culture Conditions
3.5 Biochemical Studies

3.6 Experimental Plan

3.7 Statistical Analysis

57
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5 4 CALLUS INDUCTION, MAINTENANCE AND REGENERATION OF SUGARCANE (cv. SPF 234 and cv. HSF 240)

4.1 Callus Induction and Proliferation of Sugarcane (cv. SPF 234 and cv. HSF 240)

4.2 Plant Regeneration From Callus Cultures of Sugarcane (cv. SPF 234 and cv. HSF 240)
4.3 Rooting of the Regenerated Shoots of Sugarcane (cv. SPF 234 and cv. HSF 240)

4.4 Hardening and Acclimatization of in vitro-grown Plants of Sugarcane (cv. SPF 234 and cv. HSF 240)

80
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6 5 EFFECT OF SALT STRESS ON CALLUS CULTURES OF SUGARCANE (cvs. SPF 234 and HSF 240)

5.1 Effect of Salt Stress on Morphological Characteristics of Sugarcane Callus Cultures

5.2 Effect of Salt Stress on Fresh Weights, Callus Browning and Necrosis in Sugarcane Callus Cultures
5.3 Effect of Salt Stress on Regeneration Potential of Sugarcane Callus Cultures

5.4 Rooting of the Regenerated Plantlets

98
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7 6 EFFECT OF SALT STRESS ON SOLUBLE PROTEIN CONTENTS AND ANTIOXIDANT ENZYME AVTIVITIES IN SUGARCANE CALLUS CULTURES AND REGENERATED PLANTS

6.1 Soluble Protein Contents (mg/g tissue) of Sugarcane (Saccharum spp. Hybrid, cv. SPF 234 and cv. HSF 240) Callus Cultures Maintained on 9 Different Salt Stress Treatments (0-160 mM NaCl) Supplemented to MS Medium at Day 90, 120 or 150 under Dark Conditions (27 2 蚓)

6.2 Peroxidase Activity (mg/g tissue) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2 蚓)
6.3 Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2 蚓)

6.4 Superoxide Dismutase Activity (units/mg protein) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2 蚓)

6.5 Soluble Protein Contents (mg/g tissue) of the Regenerated Plants of Sugarcane (cvs. SPF 234 and HSF 240)

6.6 Peroxidase, Catalase and Superoxide Dismutase Activities of the Regenerated Plants of Sugarcane (cvs. SPF 234 and HSF 240)

129
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8 7 ROLE OF POLYETHYLENE GLYCOL (PEG) IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE

7.1 Effect of Polyethylene Glycol (PEG) Pretreatment on Fresh Weights, Browning and Necrosis in Sugarcane Callus Cultures

7.2 Effect of Polyethylene Glycol (PEG) Pretreatment on Soluble Protein Contents of Sugarcane Callus Cultures
7.3 Effect of Polyethylene glycol (PEG) Pretreatment on Peroxidase Activity of Sugarcane Callus Cultures

7.4 Effect of Polyethylene glycol (PEG) Pretreatment on Catalase Activity of Sugarcane Callus Cultures

7.5 Effect of Polyethylene Glycol (PEG) Pretreatment on Superoxide Dismutase Activity of Sugarcane Callus Cultures

7.6 Effect of Polyethylene Glycol (PEG) Pretreatment on Regeneration Potential of Sugarcane (cv. SPF 234 and cv. HSF 240) Callus Cultures

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9 8 ROLE OF ASCORBIC ACID IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE

8.1 Effect of Ascorbic Acid Pretreatment on Fresh weight, Browning and Necrosis in Sugarcane Callus Cultures

8.2 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in Callus Cultures of Sugarcane (cv. SPF 234)
8.3 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in Callus Cultures of Sugarcane (cv. HSF 240)

8.4 Effect of Ascorbic Acid Pretreatment on Fresh Weight, Browning and Necrosis in in vitro-grown Plants of Sugarcane (cvs. SPF 234 and HSF 240)

8.5 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in in vitro-grown Sugarcane Plants (cvs. SPF 234 and HSF 240)

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10 9 LITERATURE CITED 230
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