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20123
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Subject: Plant Breeding × End date: 2012 × Start date: 2012 × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    STUDIES ON SOMATIC EMBRYOGENESIS AND GENETIC TRANSFORMATION IN MAIZE
    (Punjab Agricultural University, Ludhiana, 2012) Dhillon, Navjot Kaur
    The present investigation entitled, “Studies on somatic embryogenesis and genetic transformation in maize” was undertaken using maize inbreds viz., LM 5, LM 6, LM 13, LM 15 and LM 16. Callus induction was studied on ten media compositions based on MS (1962) salts using three explants, viz., immature embryos, mature embryos and split seeds. Among five maize inbreds thus investigated, LM 13 was most responsive to tissue culture. Highest (88.7%) callusing was observed from immature embryos cultured on MS + 2,4-D (3.0 mgL-1 ) + Picloram (10.0 mgL-1). Whereas, the maximum (88.6%) callus induction using mature embryos was observed on MS + Picloram (10.0 mgL-1) + BAP (0.5 mgL-1). Subcultured calli exhibited frequent somatic embryogenesis and the highest (72.6%) somatic embryogenesis was observed in inbred LM 13 on MS + Picloram (10.0 mgL-1) + BAP (0.3 mgL-1). The addition of proline, casein hydrolysate, silver nitrate and sucrose enhanced somatic embryogenesis in all the calli. Whereas, the cefotaxime did not have much effect on somatic embryogenesis. Embrogenic calli, upon their transfer to regeneration medium, exhibited shoot bud/shoot and root regeneration. The maximum (71.6%) shoot regeneration in LM 13 from immature embryo-derived calli was achieved on MS + Proline (3.0 mgL-1) + BAP (3.0 mgL-1) + Kin (0.5 mgL-1) + Gelrite (2.0%). Whereas, the mature embryo-derived calli of same inbred cultured on same regeneration medium exhibited poor (26.6%) shoot regeneration. Genetic transformation was attempted with particle gun (Bio Rad) using two target tissues viz. embroygenic calli and immature embryos. The bombardment was done using plasmids carrying glyI, glyII genes (for abiotic stress tolerance) and Cry1A(c) gene (for insect resistance). The bombarded tissues were selected using hygromycin (35 ppm) for two cycles of selection of two weeks each. In case of glyII gene bombardment, a total 146 plants were regenerated from which 42 plants (43.29%) were GUS positive. Further, the PCR analysis revealed the presence of glyII gene in 6 plants (4.10%). Whereas, in case of Cry1A(c) gene, a total 171 immature embryos were bombarded from which 2 plants (1.16%) were found to be PCR positive. The tissue cultured regenerated plants from all the bombarded tissues were grown in the transgenic glass house for collection of seeds. None of the plants carrying glyII gene set any seed. Whereas, four seeds have been collected from a plant carrying Cry1A(c) gene.
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    ThesisItemOpen Access
    Genetic Appraisal of Derived Brassica juncea Amphiploids
    (Punjab Agricultural University, Ludhiana, 2012) Gupta, Mehak
    ABSTRACT Derived Brassica juncea (AABB) lineages were analyzed to study the genetic changes that follow allopolyploid formations. Most of genotypes showed normal fertility with normal 18II chromosome configuration during meiosis1. Disomic chromosome substitution was indicated for DJ 15, DJ 18, DJ 19-2, DJ 21, DJ 38, DJ 58, DJ 61, DJ 92, DJ 97, DJ 103 and DJ 113. These substitution lines were expected to involve A/ C substitutions. Some individual plants showed novel phenotypes like determinate growth habit, terminal siliquae; however, there appeared to be no direct correlation between the examined molecular alterations and deviant phenotypes. There was a significant increase in sequence loss across generations within both subgenomes. There was a bias towards losing more sequences from the B. rapa (A) subgenome. We did not find any evidence of total sequence loss on the phenotypic means. Diversity analysis could also nicely resolve the test progenies into three very distinct groups, determinate juncea, crop Brassica parents and dipoid donors and indeterminate type of derived B. juncea. A total of 134 markers could be resolved on 18 linkage groups with an LOD score value greater than 3.0. The total genetic length of the map was 1010 cM with an average distance of 7.53 cM between adjacent loci. Resynthesis of new polyploid plants by derived resynthesis rout led to immediate and striking reactions of genomes to evolution. Gene loss, gene gain, gene movement and chromosome structural changes such as translocation and inversion were indicated and these collectively contributed to the process of diploidisation. A large number of hybrids were developed by hand crossing the set of derived B. juncea genotypes with natural B. juncea testers namely PBR210, RLC1, CBJ002 and AMH2. Heterosis over best commercial check PBR210, during 2010-2011 ranged from -76.5 to 103 percent with an average of -8.66 percent for the hybrids involving PBR 210 as male parent. The corresponding ranges for RLC1, CBJ002 and AMH2 were: -68.4 to 82.7 percent,-11.09 (60 hybrids), -83.6 to 79.6 percent, -3.96 (70 hybrids) and -78.3 to 59.0 percent,-14.53 (64 hybrids). During 2011-12, corresponding range and means for hybrids for involving same male parents were: PBR210 (-42.8 to 102.7; 16.87 for 35 hybrids); RLC1 (-52.7 to 104.8; 14.97 for 34 hybrids); CBJ002 (-55.9 to 117; 23.73 for 35 hybrids) and AMH2 (-51.8 to 69.6; 8.23 for 34 hybrids). Many of the putatic heterotic combinations will find their use in developing commercial hybrids. The concept of derived juncea developed by Brassica group at PAU stood validated by these studies.
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    ThesisItemOpen Access
    EVALUATION OF CULTIVATED AND WILD ORYZA SPECIES FOR IRON UPTAKE UNDER AEROBIC CONDITIONS AND VARIATION IN IRON TRANSPORTER GENES
    (Punjab Agricultural University, Ludhiana, 2012) Rahul Kumar
    The cultivation of transplanted rice in northwest plains of India has lead to depletion of ground water to almost alarming level. Replacement of rice with other water requiring crop or changed production technology that will require less water has become essential. Cultivation of direct seeded rice under aerobic conditions is an alternative production technology that is expected to use less water, but it will require an altogether a different ideotype, one of the stresses the aerobic rice face is iron deficiency induced chlorosis. The present study focused on evaluation of cultivated and wild Oryza species for iron uptake under aerobic conditions and variation in iron transporter gene. Three hundred twenty Oryza accessions including six Oryza glaberrima, 105 O. rufipogon, 193 O. nivara, one barthii and 15 cultivated Oryza sativa genotypes were evaluated for iron deficiency induced chlorosis (IDC), days to 50% flowering; leaf area index and SPAD value. Some genotypes were tolerant to IDC throughout the growth period while other showed varying degree of chlorosis at the different stages. Based on the IDC score, 59 accessions including 49 tolerant and 10 susceptible were short listed for molecular studies and further evaluation. Basmati 370, Basmati 386, Lemont, PAU 201in cultivated varieties and O. nivara (CR 100003) and O. rufipogon (CR 100343) in wild species performed best under aerobic conditions with minimum or no chlorosis and desirable SPAD reading and LAI. These could be useful in aerobic rice breeding programme. In a hydroponics experiment O. rufipogon acc. CR 100001, O. nivara acc. CR 100127 and Basmati 370 showed better tolerance to IDC along with good SPAD value and high iron content in leaves. Microsatellite markers developed from 23 iron homoeostasis gene sequence were used for characterizing the 59 selected genotypes. Based on the variation in the 23 Fe homoeostasis genic SSRs markers, the 59 genotypes were clustered into three groups, significance of which is discussed.