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TITLE
DEDICATION
DECLARATION
CERTIFICATE
ACKNOWLEDGEMENT
ABBREVIATIONS
LIST OF TABLES
LIST OF FIGURES.
CONTENTS
1. INTRODUCTION
1.1 Cycads
1.1.1 Zamia furfuracea L
1.1.2 Cycas revoluta Thunb
1.1.3 Cycas circinalis L
1.2 Cycad Tissue Culture
1.3 Objectives of the present study
2. REVIEW OF LITERATURE
2.1 In vitro Studies in non cycad Gymnosperms
2.2 In vitro studies in cycads
2.3 Histology
2.4 Histochemistry and Histoenzymology
2.5 Biochemistry
2.6 Isozyme
3. MATERIALS AND METHODS
3.1 Source & Choice of Plant materials
3.1.1 Zamia fur furacea L
3.1.2 Cycas revoluta Thunb Cycas circinalis L
3.1.3 Cycas circinalis L.
3.2 Establishment Of Cultures
3.2.1 Glassware & instruments
3.2.2 Basal Media
3.2.3 Preparation of Culture Media
3.2.4 Sterilization
3.2.5 Explant Preparation and Surface Sterilization
3.2.6 Inoculation and Incubation
3.2.7 Sub Culture
3.3 Callogenesis
3.4 Somatic embryogenesis
3.5 Histology, Histochemistry and Histoenzymology
3.6 Biochemical Quantification
3.7 Electrophoretic Techniques
3.8 Statistical analysis
TABLES
Table 1 Chemical composition of various nutrient media used.
Table 2 Plant Growth regulators and other Supplements used in cultures.
Table 3 Staining methods followed for general histology, histochemistry and histoenzymology
4. RESULTS
4.1 Zamia furfuracea L
4.1.1 Callogenesis
4.1.2 Embryogenesis in Zamia furfuracea
4.1.3 Indirect Organogenesis
4.1.4 Direct Organogenesis
4.1.5 Non - Organogenic Combinations
4.1.6 Histology
4.1.6.1 General Histology of Plant Organs
4.1.6.2 Ontogeny of callus
4.1.6.3 Histology of Non - organogenic Callus
4.1.6.4 Histology of somatic embryogenesis
4.1.6.5 Histology of regenerated structures
4.1.7 Histochemical Localization of metabolites and enzymes
4.1.7.1 Callus
4.1.7.2 Embryogenic callus
4.1.7.3 Indirect organogenic callus
4.1.8 Isozyme analysis in explants, callus and in differentiated organs
TABLES
Table 4 In vitro Response (%) of different basal media with out PGR to different explants of Zamia furfuracea on different Basal media without PGR after 45 days
Table 5 Effect of Auxins on callus induction in Zamia furfuracea explants on B5 + MS basal medium % of response after 4 weeks
Table 6 Effect of 2, 4-D on callogenesis from young rachis explants of Zamia furfuracea on B5 + MS medium
Table 7 Effect of NAA on callogenesis from young rachis explants of Zamia furfuracea on B5 + MS medium
Table 8 Effect of IAA on callogenesis from young rachis explants of Zamia furfuracea on B5 / MS medium.
Table 9 Effect of 2, 4-D + Kin on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 10 Effect of NAA+ Kin on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 11 Effect of IAA+ Kin on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 12 Effect of 2, 4-D+ BAP on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 13 Effect of NAA+BAP on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 14 Effect of 1AA+BAP on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 15 Effect of 2, 4-D+2iP on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 16 Effect of NAA+2iP on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 17 Effect of IAA+2iP on callogenesis from young rachis explants of Zamia furfuracea on B5+MS medium.
Table 18 Treatments for organogenic response in Zamia furfuracea on B5+MS medium supplemented with different combinations of PGR.
Table 19 Organogenic response in Zamia furfuracea callus on B5 + MS medium supplemented with 10% CW, 200 mg / l G1. 100 mg / l CH & 3% Sucrose after 120 days
Table 20 a, b. Effect of additives on Somatic Embryogenesis in Zamia furfuracea callus on the optimum Medium composition - B5 + MS medium supplemented with Kin 1.0 + 2iP 1.0 + NAA 0.5
Table 21a, b Effect of additives (% response & average no. of embryoids) on Indirect organogenesis in Zamia furfuracea on the optimum Medium composition - B5 + MS medium supplemented with Kin 1.0 + 2iP 1.0 + 2, 4D 0.5
Table 22 a, b Histochemical localization of metabolites & enzymes in the callus from young rachis of Zamia furfuracea.
FIGURES
Fig.1 Zamia furfuracea L.
Fig. 2 Callogenesis in Zamia furfuracea
Fig. 3 Non Organogenic calli in Zamia furfuracea.
Fig. 4 Darkening of Non Organogenic calli in Zamia furfuracea
Fig. 5 Induction and maturation of somatic embryos in Zamia furfuracea
Fig. 6 Maturation of somatic embryos in Zamia furfuracea
Fig. 7 Induction and maturation of shoot buds from callus in Zamia furfuracea
Fig. 8 Histological examination of the explants in Zamia furfuracea
Fig. 9 Histological examination of calli of Zamia furfuracea
Fig.10 Xylogenesis in Non organogenic calli of Zamia furfuracea
Fig.11Resin and Druses in the non-organogenic calli of Zamia furfuracea
Fig.12 Ontogeny of somatic embryos in Zamia furfuracea
Fig.13 Histology of in vitro shoot and root of Zamia furfuracea
Fig.14 Electrophoretogram showing isozymes of explants & in vitro cultures of Z.furfuracea
Fig. 15 Zymogram showing isozymes of explants & in vitro cultures of Z. furfuracea
Fig. 16 Callus growth index in young rachis explants of Z. furfuracea
Fig. 17 Callus growth rate in young rachis explants of Z. furfuracea
Fig. 18 Starch profile in callogenesis from young rachis explants of Z. furfuracea,
Fig. 19 Soluble sugar profile in callogenesis from young rachis explants of Z.furfuracea
Fig. 20 Protein profile in callogenesis from young rachis explants of Z, furfuracea
Fig. 21 Resin profile in callogenesis from young rachis explant of Z. furfuracea
Fig. 22 Starch profile in Embryogenic cultures of Z. furfuracea (E1-E2)
Fig. 23 Starch profile in Embryogenic cultures of Z.furfuracea (E3-E4)
Fig. 24 Soluble sugar profile in Embryogenic cultures of Z. furfuracea (E1-E2)
Fig. 25 Soluble sugar profile in Embryogenic cultures of Z. furfuracea (E3-E4)
Fig. 26 Protein profile in Embryogenic cultures of Z. furfuracea (E1-E2)
Fig. 27 Protein profile in Embryogenic cultures of Z. furfuracea (E3-E4)
Fig. 28 Resin profile in Embryogenic cultures of Z. furfuracea (E 1-E2)
Fig. 29 Resin profile in Embryogenic cultures of Z. furfuracea (E3-E4)
Fig. 30 Starch Profile in indirect organogenic cultures of Z.furfuracea.
Fig. 31 Soluble sugar profile in indirect organogenic cultures of Z.furfuracea.
Fig. 32 Protein profile in indirect organogenesis cultures of Z.furfuracea
Fig. 33 Resin profile in indirect organogenesis in Z.furfuracea
Fig. 34 Starch Profile in non organogenic cultures of Z. furfuracea
Fig. 35 Soluble sugar profile in non organogenic cultures of Z.furfuracea.
Fig. 36 Protein profile in non organogenic cultures of Z.furfurcea
Fig. 37 Resin profile in non organogenic cultures of Z.furfuracea
4. 2 Cycas revoluta Thunb
4.2.1 Callogenesis
4.2.2 Organogenesis in Cycas revoluta
4. 3 Cycas circinalis L
4.3.1 Callogenesis
4.3.2 Organogenesis In Cycas circinalis
4.3.3 Germination of zygotic embryos in Cycas circinalis
4.3.4 Embryogenesis from megagametophyte of Cycas circinalis.
4.3.5 Adventitious shoot induction from mega gametophyte of Cycas circinalis
4.3.5a Seedling cultures of Cycas circinalis
4.3.6 Effect of additives in organogenic response
4.3.7 Histology
4.3.8 Histochemical localization
4.3.9 Biochemical Analysis of non-organogenic cultures
4.3.10 Biochemical profile during Embryo genesis from megagametophyte
4.3.11 Biochemical analysis during Organo genesis from megagametophyte
4.3.12 lsozyme
TABLES
Table 23 Response (%) of different explants of Cycas revoluta on different Basal media without PGR after 45 days.
Table 24 Effect of Auxins on callus induction in Cycas revoluta explants on B5+MS basal medium % of response after 4 weeks.
Table 25 Effect of 2, 4-D on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 26 Effect of NAA on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 27 Effect of IAA on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 28 Effect of 2, 4-D + Kin on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 29 Effect of NAA + Kin on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 30 Effect of IAA + Kin on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 31 Effect of 2, 4-D and BAP on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 32 Effect of NAA + BAP on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 33 Effect of IAA+ BAP on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 34 Effect of 2, 4-D + 2iP on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 35 Effect of NAA + 2iP on callogenesis from young rachis explants of Cycas revoluta on B5 + MS medium
Table 36 Effect of IAA+2iP on callogenesis from young rachis explants on B5 / MS medium in Cycas revoluta
Table 37 In vitro Response (%) of different explants of Cycas circinalis on different Basal media without PGR after 45 days.
Table 38 Effect of Auxins on callus induction (% of response) in Cycas circinalis explants on B5+MS medium after 4 weeks.
Table 39 Effect of 2, 4-D on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 40 Effect of NAA on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 41 Effect of IAA on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 42 Effect of 2, 4-D + Kin on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium.
Table 43 Effect of NAA + Kin on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 44 Effect of IAA +Kin on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 45 Effect of 2, 4-D +BAP on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 46 Effect of NAA + BAP on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 47 Effect of IAA + BAP on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 48 Effect of 2, 4-D + 2iP on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 49 Effect of NAA + 2iP on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 50 Effect of IAA and 2iP on callogenesis from young rachis and megagametophyte explants of Cycas circinalis on B5 + MS medium
Table 51Treatments for organogenic response in megagametophyte explant of Cycas circinalis on B5 + MS medium supplemented with different combinations of PGR.
Table 52 PGR Treatments which showed organogenic response in mega gametophyte explants of Cycas circinalis on B5 + MS medium supp lemented with 10% CW, 200 mg / l GI. 100 mg / l CH, 20% Megagametophyte extract and 3% Sucrose after 120 days
Table 53 a, b Effect of additives on somatic embryogenesis in Cycas circinalis callus on optimum medium composition - B5 + MS medium supplemented with I mgL-1 BAP+0.5 mgL -1 NAA + 20% megagametophyte extract.
Table 54 a, b Effect of additives on shoot induction from megagametophyte of Cycas circinalis on optimum medium composition - B5 + MS medium supplemented with I mgL-1 Kin +0.5 mgL-1 BAP + 0.5 mgL-1 2, 4-D + 20% megagametophyte extract.
Table 55 a Histochemical localization of metabolites and enzymes in the callus from young rachis of Cycas revoluta & Cycas circinalis
Table 55 b Histochemical localization of metabolites and enzymes in embryogenic and organogenic cultures from megagametophyte explant of Cycas circinalis
FIGURES
Fig. 38 Cycas revoluta Thunb.
Fig. 39 Cycas circinalis L
Fig. 40 Callogenesis in Cycas revoluta & C. circinalis
Fig. 41 Induction of shoot bud in C. revoluta, In vitro germination of embryo of C. circinalis & Embryogenic calli of C. circinalis
Fig. 42 Adventitious shoot induction from megagametophyte of C. circinalis
Fig. 43 Maturation of adventitious shoot buds from megagametophyte of C. circinalis, In vitro germination of seeds of C. circinalis
Fig. 44 Histological examination of explants of Cycas species
Fig. 45 Histological observation of somatic embryogenesis in C. circinalis
Fig. 46 Electrophoretogram showing isozymes of explants & in vitro cultures of Cycas species
Fig. 47 Zymogram showing isozymes of explants & in vitro cultures of Cycas species
Fig. 48 Callus growth index in young rachis explant of C. revoluta
Fig. 49 Callus growth rate in pattern in young rachis explants of C. revoluta.
Fig. 50 Starch Profile in callogenic cultures in young rachis explants of C. revoluta.
Fig. 51 Soluble sugar callogenic cultures from young rachis explants of C. revoluta.
Fig. 52 Protein profile in callogenic cultures from young rachis explants of C. revoluta.
Fig. 53 Resin profile in callus cultures of young rachis explants of C. revoluta.
Fig. 54 Callus growth index in young rachis explant of C. circinalis
Fig. 55 Callus growth rate in pattern in young rachis explants of C. circinalis
Fig. 56 Starch profile in callogenic cultures from young rachis explants of C. circinalis.
Fig. 57 Starch profile in callogenic cultures from megagametophyte explants of C. circinalis
Fig. 58 Soluble sugar callogenic cultures from young rachis explants of C.circinalis
Fig. 59 Soluble sugar profile in callogenic cultures from megagametophyte explants of C. circinalis
Fig.60 Protein profile in callogenic cultures from young rachis explants of C. circinalis.
Fig. 61 Protein profile in callogenic cultures from megagametophyte explants of C.circinalis.
Fig. 62 Resin profile in callogenic cultures from young rachis explants of C.circinalis.
Fig. 63 Resin profile in callogenic cultures from megagametophyte explants of C. circinalis.
Fig. 64 Starch profile in non organogenic cultures of C. circinalis
Fig. 65 Soluble sugar profile in non organogenic cultures of C.circinalis.
Fig. 66 Protein profile in non organogenic cultures of C.circinalis.
Fig. 67 Resin profile in non-organogenic. cultures of C.circinalis.
Fig. 68 Starch profile in organic cultures of C.circinalis.
Fig. 69 Soluble sugar profile in organic cultures of C.circinalis.
Fig. 70 Protein profile in organic cultures of C.circinalis.
Fig. 71 Resin profile in organic cultures of C. circinalis
5. DISCUSSION
5.1 Callogenesis
5.2 Caulogenesis
5.2.1 Somatic Embryogenesis
5.2.2 Indirect Organogenesis
5.2.3 Direct organogenesis
5.2.4 Organogenesis from megagametophyte of Cycas circinalis.
5.2.5 Effect of additives
5.2.6 Effect of light on embryogenesis / organgensis
5.2.7 Maturation of proembryos
5.2.8 Histology
5.2.9 Histochemical Localization
5.2.10 Metabolic Profile during cell differentiation
5.2.11 Isozyme analysis
6. SUMMARY
REFERENCES