I= BIOCHEMICAL AND MOLECULAR MARKERS OF SOMACLONAL VARIANTS AND INDUCED MUTANTS OF POTATO (SOLANUM TUBEROSUM L.)
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Title of Thesis
BIOCHEMICAL AND MOLECULAR MARKERS OF SOMACLONAL VARIANTS AND INDUCED MUTANTS OF POTATO (SOLANUM TUBEROSUM L.)

Author(s)
Humera Afrasiab
Institute/University/Department Details
Department Of Botany/ University of the Punjab
Session
2006
Subject
Botany
Number of Pages
337
Keywords (Extracted from title, table of contents and abstract of thesis)
somaclonal variants, potato, physical mutagens, chemical mutagens, solanum tuberosum l, callogenesis, organgoenesis, regeneration, somatic embryogenesis, micropropagation, somaclonal variation

Abstract
Potato (Solanum tuberosum L.) is one of the important cash crops of Pakistan and is widely cultivated over an area of 109.7 thousand hectares, with an annual production of 1.9 million MT. Its high nutritional value and broad adaptability increases its consumption and demand day by day. It is propagated predominantly by asexual means (tubers) and propagation by true seed is primarily used for breeding purposes. In Pakistan, more than 95% of the seed requirement is met locally, and the seed so used is mostly uncertified and diseased, which results in significantly low production. Today non-availability of quality seeds of high yielding, well-adapted varieties remains the major constraint for low potato yields in Pakistan.

In recent years, tissue culture technology has provided a tool to complement conventional plant breeding. Plant tissue culture is recognized as a source to generate useful genetic variability (somaclonal variation) for crop improvement. Another source of generating variations in plants is by mutagenesis (physical and chemical). The combination of mutagenesis with in vitro techniques offers an efficient method for improving vegetatively propagated plants. These techniques allow induction of variation, selection and multiplication of the desired genotypes in a much shorter duration and smaller space than conventional methods.

In the present investigation tissue culture and mutagenesis techniques were applied to induce variations for improvement of yield and yield components in two well-adapted potato cultivars i.e. Desiree and Diamant. The variants and mutants produced were characterized on the basis of morphological, biochemical and molecular markers.

In the present study, response of different explants i.e. nodes, internodes and leaves were studied for callus induction. Among them, internodal explant gave better response for callus initiation and proliferation. The optimum temperature for callus growth was observed to be 24±1°C and better callus induction response was observed in dark conditions as compared to light. Effects of different auxins (2,4-0, NAA) and cytokinins (BAP, Kinetin) and their combination, supplemented in MS medium were also studied. In all the explants tried, maximum percentage of callus formation and proliferation was observed in MS medium supplemented with NAA (1.0mg/l) and BAP (0.5mg/1) as compared to 2,4-0 alone or in combination with BAP.

Two types of calli were formed in both the cultivars, smooth, compact and non-morphogenic and dry, nodular, compact and morphogenic. The later type of callus was green and was selected for subsequent studies. Sub-culturing at a proper interval of two weeks had a marked effect on callus proliferation and maximum number or plants regenerated from twelve weeks old calli. Callus incubated in MS medium supplemented with NAA (0.5mg/l) and BAP (2.0mg/l) showed best organogenesis response and plants were regenerated within 21 and 24 days in cvs. Desiree and Diamant, respectively.

In the present study, somatic embryo induction and formation from well-developed 12 weeks old calli of both the cultivars was also obtained. MS medium supplemented with 2,4-0 (2.0mg/l) and BAP (0.5mg/1) showed 70-75% embryo induction in cvs. Desiree and Diamant, respectively. MS medium containing 1.0mg BAP showed best regeneration from somatic embryos. However, rooting was observed in hormone free MS basal medium from shoots produced via organogenesis and somatic embryogenesis in both the cultivars. For acclimatization of regenerated plants, a combination of sand, soil and peat in equal ratio proved better than sand alone or sand and soil combination.

Conditions were also established for micropropagation of plants from apical and nodal explants of both the cultivars. Early shoot response was observed from both the explants in hormone free full strength MS basal medium as compared to media containing different auxins (BAP, NAA). Similarly, rooting was also observed in MS full strength medium in both the cultivars. To achieve maximum number of shoots from nodal explants, in vitro layering technique was also tried. Multiple shoots were obtained from stem pieces containing 2-3 nodes kept horizontally in full strength MS medium in jars.

The main objective of the present study was to get somaclonal variants and induced mutants of potato for desirable characters with special emphasis on yield and yield components in both the cultivars. For obtaining somaclonal variants, well-proliferated calli of both the cultivars (14-20 week old) were shifted to regeneration medium. The regenerated plants were analyzed on the basis of morphological characters for variation (A lbinos, viridis and others).

For mutation induction, gamma rays and ethyl methane sulphonate (EMS) was used. Ten week old, well proliferating calli of both the cultivars were exposed to 5, 10, 15, 20, 25, 30, 40 and 500y of gamma irradiation. An increase in callus weight in both the cultivars was observed at 5 to 20Gy after two weeks following irradiation. With further increase in the radiation dose, there was a decrease in callus weight accompanied with necrosis. Plants were regenerated from the irradiated calli and maximum number of regenerated shoots (8-10) was obtained at 20 Gy in both the cultivars. The regenerated plants showed variations in their morphological characters. Mostly albinos and viridis plants with other variations were obtained.

For obtaining chemical mutants, 10 weeks old calli of both the cultivars were treated with . chemical mutagen EMS at 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0%. The EMS had a drastic effect on callus proliferation and decrease in callus weight with increase in dose was observed as compared to control in both the cultivars. Plants were regenerated from the EMS treated calli and were analyzed for any visible variations. The regenerated plants showed morphological variations in form and texture of leaf, stem and internodal distances among others.

The plants regenerated from gamma and EMS treated calli were acclimatized and hardened in the glasshouse and were transferred to tunnels to obtain R1 generation. Tubers harvested from selected variant/mutant lines were further grown to obtain R2 generation to confirm the stability of variation under field conditions. The plants were screened on the basis of average shoot height, number of shoots, number of nodes/shoots, average tuber number, tuber size, tuber weight and number of eyes / tuber and only 26 variants/mutant lines were selected for further study. In case of cv. Desiree, 3 variant lines (SV I, SV2 and SV3), 6 gamma mutant lines (GM I, GM2, GM3, GM4 GM5 and GM6) and 4 EMS treated lines (EMS1, EMS2, EMS3 and EMS4) were selected. While in case of cv. Diamant, 4 variant lines (SV4, SV5, SV6 and SV7) 5 gamma mutant lines (GM7, GM8, GM9, GM 10 and GM 11) and 4 EMS treated lines (EMS5, EMS6, EMS7 and EMS8) were selected for further biochemical and molecular studies.

In cv. Desiree, among the selected variants lines, SV3 showed maximum number of tubers (11.8) followed by SV2 (10.8) and SVI (10.2) as compared to control (8.4). Similarly, average tuber weight and tuber size along with other morphological characters in the selected lincs also varied in comparison with control. In case of gamma mutant lines, the average tuber number was high in GMI (18.2) followed by GM2 (16.2) and GM5 (13) as compared to control (10.2). Similar increase in average tuber size and tuber weight and other morphological characters were observed in other mutants. In case of EMS treated lines, maximum average tuber numbers was observed in EMS2 (14.8) followed by EMS3 (14.4) and EMS I (12.6) in comparison with control (11.2) An increase in average tuber size, tuber weight and other morphological characters was also observed in all the mutants.

In cv. Diamant, among the selected variant lines, maximum tuber numbers along with tuber size and weight was observed in SV5 (13) followed by SV4 (11.2) SV7 (10.2) and SV6 (9.4) as compared to control (8.0). In case of gamma irradiated mutant lines, maximum yield of tubers was observed in GM8 (16.8) followed by GM10 (16.0) and GM11 (14.2) as compared to control (11.6). Similarly other mutant lines also showed variations from the control.. In EMS treated lines, maximum variation in terms of average tuber number was shown by EMS5 (14.4) followed by EMS8 (14.0) and EMS6 (12.8) as compared to control (11.0) along with other observed characters.

In the present study, all variants / mutants selected on the basis of morphological characters were also investigated by biochemical analysis. Various biochemical markers used in the present study were soluble protein content, peroxidases and esterases (qualitatively and quantitatively). In general an increase in soluble protein content, total peroxidase activity and total esterase activity in all the selected variants and mutants of both the cultivars was observed. Same results were obtained when qualitative study was conducted on PAGE (Polyacrylamide gel electrophoresis).

For molecular analysis, PCR based RAPD technique was used. A total of 24 random oligonucleotide primers were evaluated. for their ability to prime PCR amplification of genomic DNA of two cultivars of potato, Desiree and Diamant along with their somaclonal variants and induced mutants. About 65% of the bands revealed by primers were polymorphic among the variants and mutants of both the cultivars. Dendrogram of potato cultivars and their somaclonal variants and induced mutants generated by RAPD data using UPGMA method was constructed to estimate the genetic variability among the somaclonal variants and induced mutants in comparison with control.

In cv. Desiree, in case of selected variants, SV3 showed maximum genetic variation followed by SV2 while SV1 showed 80% genetic similarity with the control. In case of cv. Diamant, maximum genetic variation was observed in SV4 and SV5 followed by SV7 and SV6 as compared to control. In case of gamma treated mutant lines of cv. Desiree maximum genetic variation was shown by GM2 followed by GM1, GM5 and GM6 while, GM3 and GM4 showed minimum genetic dissimilarity from the control. In case of cv. Diamant two subgroups of genetic similarity was observed, GM7 and GM8 were present in one group and GM11 and GM9 were present in the second group showing genetic similarity among themselves and dissimilarity from the control followed by GM 10.

In case of EMS treated mutant lines of cv. Desiree, EMS3 and EMS4 showed maximum genetic similarity among themselves and maximum genetic dissimilarity from the control, while EMS I and EMS2 showed more genetic similarity with the control. In case of EMS treated mutant lines of cv. Diamant, EMS7 and EMS8 showed genetic similarity among themselves and maximum genetic variation from the control followed by EMS6, while EMS5 showed less genetic variation from the control.

The present study demonstrates that it is possible to produce useful variants both by gamma irradiation and EMS treatment in potato. The variants can be distinguished by using the RAPD-PCR technique. This technique provides a better evaluation of genetic diversions as compared to isozymes and protein analysis.

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
409.71 KB
2 1 Introduction 6
103.67 KB
3 2 Literature Review 12
721.17 KB
  2.1 Callogenesis 13
  2.2 Organgoenesis 16
  2.3 Regeneration 19
  2.4 Somatic Embryogenesis 22
  2.5 Micropropagation 25
  2.6 Somaclonal Variation 29
  2.7 Induced Mutations 38
  2.8 Chemical Mutagens 40
  2.9 Physical Mutagens 43
  2.10 Effects Of Gamma Rays 43
  2.11 Biochemical And Molecular Analysis 46
  2.12 Molecular Analysis 49
4 3 Materials And Methods 54
555.34 KB
  3.1 Taxonomic Status 54
  3.2 Crop History And Development 55
  3.3 Plant Characteristics 56
  3.4 Propagation Methods 60
  3.5 Agricultural Importance 61
  3.6 Procurement Of Seeds 65
  3.7 Callogenesis Organogenesis And Somatic Embryogenesis 66
  3.8 Micropropagation 70
  3.9 Induction And Selection Of Mutants 70
  3.10 Biochemical And Molecular Analysis 72
5 4 Results: In Vitro Studies Of Solanum Tuberosum L. Cvs . Desiree And Diamant 84
1472.97 KB
  4.1 Callus Induction From Different Explants Of S. Tuberosum Cvs . Desiree And Diamant 84
  4.2 Colour And Type Of Callus 92
  4.3 Effect Of Different Light Condition , Temperature And Time Of Sub Culturing On Callus Initiation And Proliferation 92
  4.4 Plant Regeneration (Organogenesis) From Callus 93
  4.5 Cultures Of Potato Cvs . Desiree And Diamant 97
  4.6 Regeneration Of Shoots From Embryogenic Calli 102
  4.7 Rooting Of Regenerated Plants 108
  4.8 Acclimatization Of Regenerated Plants 108
  4.9 Micropropagation Form Apical And Nodal Explants 114
  4.10 In Vitro Layering Technique For Micropropagation From Nodal Explants Of Potato 115
6 5 Results: Somaclonal Variation And Induced Mutational Studies In Solanum Tuberosum L. Cvs . Desiree And Diamant 120
1418.29 KB
  5.1 Selection Of Somaclonal Variants 120
  5.2 Selection Of Gamma Irradiated Mutants 124
  5.3 Selection Of Ems Treated Mutants 131
  5.4 Morphological Characters Of Selected Variants Lines 138
  5.5 Morphological Characters Of Mutants Selected After Gamma Irradiation 144
  5.6 Morphological Characters Of Mutant Selected After Ems Treatment 151
7 6 Results: Biochemical Analysis Of Somaclonal Variants And Inducted Mutants Of Solanum Tuberosum L. Cvs . Desiree And Diamant 157
726.66 KB
  6.1 Quantitative Estimation 157
  6.2 Qualitative Analysis 163
8 7 Results: Molecular Analysis Of Somaclonal Variants And Induced Mutants Of Solanum Tuberosum L. Cvs . Desiree And Diamant 179
621.62 KB
  7.1 Rapd Analysis Of Somaclonal Variants And Induced Mutants Of Potato Cultivars 179
9 8 Discussion 199
559.9 KB
  8.1 References 232
  8.2 Appendices 263