Name of the student

Suhasini Huddone

Admission Number

H-2005-16-M

Name of the Major Advisor

Dr S. V. Bhardwaj

Year of Completion of Degree

2007

Title of the thesis

Comparative analysis of coat protein gene of Lily symptomless virus infecting Lilium species with other carlaviruses

Abstract

Lilium crop falls prey to a number of insects, pests and pathogens, especially viruses leading to drastic reduction in economic returns. The Lily symptomless carlaviru is prevalent in Solan district of H.P. as confirmed by serological detection using DAS-ELISA. It is a positive sense RNA virus and is one of the major limiting factors for cultivation of Lilium throughout the world. Comparative analysis of most variable region of its genome i.e. coat protein (ORF 5) was done, which could be helpful in designing knowledge based strategies such as coat protein mediated resistance in Lilium plants against this virus, studies on coat protein (CP) gene sequences of members of the genus Carlavirus depicted that the test virus, Lily symptomless virus isolate LSV-Oh; AJ748277 shared 17-98% and 1-98% homology at nucleotide and amino acid levels, respectively, with 78 other carlaviruses of the world present in NCBI database till date. Two already known nucleotide motifs, C/TTTAGGT (potential ribosome recognition sequence) and AATAAA (Polyadenylatiopn signal motif) and three of the amino acid motifs, R/KFAG/AFDxFx₂Vx₃AA, Hx_4-8Dx_15-20TGG (similar to motif found in cellular serine protease) and T-G-G(-X-X-G), were searched for in the multiple sequence alignments of CP nucleotide and amino acid sequences. The C/TTTAGGT and AATAAA motifs were not conserved in any of the LSV isolates under study in the CP nucleotide sequence alignments, whereas, all the amino acid motifs were highly conserved in most of the alignments of translated CP sequences. Interestingly, a conserved GDD sequence similar to on present in RNA dependent RNA polymerase (RdRp) motif  of viral replicase gene was also found in multiple alignment of carlaviral CP gene amino acid sequences from Canada (all Potato virus M isolates). Phylogenetic analysis was performed with carlaviral CP gene sequences by UPGMA, Neighbor-joining, parsimony and Maximum likelihood methods. These analysis clearly showed that the test LSV CP gene sequence shared its most recent common ancestries with native LSV isolates from Palampur and two of the LSV isolates, Lanzhou (DQ531052) and Yunnan (AY326460), from China. All the carlaviral sequences under study were shown to evolve simultaneously except for the Chrysanthemum virus B, which exhibit lot of variation differences with the other carlaviruses.

Name of the student

Anita Kumari

Admission Number

H-2006-02-M

Name of the Major Advisor

Dr. D.K. Srivastava

Year of Completion of Degree

2009

Title of the thesis

Plant regeneration and Agrobacterium - mediated gene transfer studies in brinjal (Solanum melongena L.)         

Abstract

The research work was conducted to standardize a protocol for plant regeneration and genetic transformation in brinjal. Plant regeneration studies were carried out using two types of explants viz hypocotyl and cotyledon. The cotyledon explants showed high frequency of shoot regeneration (77.77%) on MS medium supplemented with 2.5 mg/l Kn and 0.4 mg/l IAA as compared to hypocotyl (50%) on MS medium supplemented with 2.5 mg/l BAP and 0.5 mg/l IAA. MS medium supplemented with 0.10 mg/l IAA was found to be best for root regeneration (81.81%). The brinjal plantlets were able to regenerate within 6-7 weeks. Regenerated plantlets were acclimatized. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β-glucuronidase (gus) gene in binary vector (pBI 121) system along with kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for co-cultivation experiment to transfer gus and npt-II genes in brinjal cells. After cocultivation only the transformed cells were able to grow on selective shoot regeneration medium (50 mg/l Kanamycin and 500 mg/l Cefotaxime) whereas control explants died on the selective medium. Transformation experiment could be scored as early as four weeks after selection. Seven calli obtained on the selective medium were GUS positive. Putative transgenic shoots were obtained, which were able to grow on the selective medium containing 50 mg/l Kanamycin.

Name of the student

Balbir Singh

Admission Number

H-2006-04-M

Name of the Major Advisor

Dr. S.K. Sharma

Year of Completion of Degree

2008

Title of the thesis

 “In vitro plant regeneration in asiatic hybrid lily”      

Abstract

In the present investigation entitled “In vitro plant regeneration in asiatic hybrid lily”, the sterilized explants (tepal, ovary and filament) were inoculated on MS medium supplemented with different concentrations of growth regulators. The number of days for bulblet initiation decreased significantly when MS medium was supplemented with growth regulators. The highest per cent of explant forming bulblets and maximum number of bulblets were observed with tepal explant on MS medium supplemented with 0.5 mg l-1 NAA + 1.0 mg l-1 BA each. Tepal explant also gave the maximum average fresh weight per bulblet with MS medium supplemented with 1.0 mg l-1 0.5 mg l-1 BA followed by ovary and filament. It was also observed that cultivar Tuscana was able to produce maximum fresh weight per bulblet with tepal explant. The maximum per cent bulblet (79.27%) forming roots and maximum roots per bulblet (4.70) was observed with 1.5 mg l-1 NAA + 1.5 mg l-1 BA and 2.0 mg l-1 NAA + 2.0 mg l-1 IBA respectively. The maximum length of roots (45.25 mm) was observed with 0.5 mg l-1 NAA + 0.5 mg l-1 IBA. Cocopeat was found to be the best rooting mixture giving (75.30%) survival of plantlets. 

Name of the student

Ekta Bhardwaj

Admission Number

H-2007-03-M

Name of the Major Advisor

Dr.(Mrs.) Rajinder Kaur

Year of Completion of Degree

2008

Title of the thesis

 “Studies on characterization of pecan [Carya illinoensis (Wang) K. koch] germplasm using RAPD markers”

Abstract

The present studies on characterization of pecan [Carya illinoensis (Wang) K. koch] germplasm using RAPD markers. Genomic DNA was isolated from young and green leaves of 32 pecan accessions by using CTAB method (Doyle and Doyle, 1987) with modifications. Genetic variation was studied using 25 random decamer primers. Of these only 13 produced polymorphism. Total number of bands amplified were 75, out of which all were polymorphic and 22 bands examined as unique bands. The amplified fragments were ranged from 2000bp to 7000bp and all pecan genotypes examined can be clearly discriminated and identified using 13 primers selected in this study. The average PIC value of 13 primers were 0.78. Similarity matrix was constructed by using Jaccard;s Coefficient and it ranged from 0.00 to 0.55. Maximum similarity matrix was obtained between Kanza and Chickasaw. Dendrogram of all pecan accessions. Cophenetic correlation measures the accuracy with which dendrogram represents the similarity matrix constructed from the RAPD data was 0.91. From the data obtained in this study it can be concluded that polymorphism can be easily scored and used for studying diversity between pecan genotypes.

Name of the student

Shweta Pathania

Admission Number

H-2004-10-M

Name of the Major Advisor

Dr. (Mrs.) Manju Modgil

Year of Completion of Degree

2006

Title of the thesis

Plant regeneration studies and assessment of genetic variation among regenerants of apple rootstock MM111

Abstract

The present studies were aimed to develop technique for high frequency regeneration in clonal apple rootstock MM111 through leaves as explants and to assess the genetic variability among regenerants using RAPD-PCR. Callus was induced on high auxin containing medium and organogenesis was obtained from secondary callus obtained on 2 mg/l NAA and 0.5 mg/l BA after about 30 days in the shoot induction medium. Both direct and indirect regeneration was obtained on MS medium with BA (2, 3, 4 mg/l) and NAA 1 mg/l) in light as well as in dark but relatively higher regeneration was found in explants grown in light. The maximum regeneration (33.76 %) was obtained in 3 mg/l BA with 1 mg/l NAA with 1.67 shoots per explant and 1.5 – 2.0 cm shoot length. The regeneration percentage increased to 43.33 % with 0.6 mg/l TDZ and 0.5 mg/l NAA with 3 shoots per explant and maximum shoot length of 3.0 – 3.5 cm. The potential of regeneration in leaves increased when exposed to growth regulators for a brief period of time. The same combination i.e. 0.6 mg/l TDZ and 0.5 mg/l NAA resulted in maximum regeneration percentage of about 49 % with maximum of 6 shoots per explant and maximum shoot length of 3 cm, in comparatively less time period. 0.2 mg/l TDZ with 1 mg/l IBA resulted in regeneration but with low frequency. Regenerated shoots were multiplied and rooted. The rooted regenerants were hardened. DNA was isolated from field grown apple rootstock MM111 and 5 regenerants, which were subjected to RAPD-PCR analysis using 10 primers initially. Out of total 10, only 6 primers yielded 19 scorable bands of which 7 were polymorphic and 12 monomorphic with 36.8 per cent polymorphism. Each primer produced a unique set of amplification products ranging in size from about 300 – 1000 bp. Thus, regeneration from leaf proved to be an effective method to create variability which could be valuable for crop improvement.

Name of the student

Yashveer Singh Verma

Admission Number

H-2006-24-M

Name of the Major Advisor

Dr. (Mrs.) Kamlesh Kanwar

Year of Completion of Degree

2008

Title of the thesis

In vitro regeneration and genetic transformation in    Terminalia chebula Retz.

Abstract

The present investigation entitled ‘In vitro regeneration and genetic transformation in Terminalia chebula Retz.” was conducted to obtain complete plantlets through indirect regeneration from juvenile explants. Seed kernels obtained after excising the dried seed were inoculated on MS basal medium after surface sterilization and maximum 75.00 per cent germination was recorded. In vitro raised seedlings of Terminalia chebula were used as a source of juvenile explants (hypocotyl and cotyledon). Maximum callus was induced from juvenile explants on MS basal medium containing 1.0 mg/l 2, 4-D after about 30 days of inoculation, while hypocotyl showing 90.00 per cent and cotyledon 75.00 per cent callus induction. Shoot regeneration was recorded from cotyledonary callus on shoot induction medium comprising BAP in combination with NAA. The maximum shoot regeneration (36.67) was obtained in 1.5 mg/l BAP with 0.10 mg/l NAA with three shoots per explant and 1.56 cm average shoot length. Regenerated shoots were further transferred to ½ strength MS medium containing activated charcoal for root induction. Best rooting (43.75 %) was reported in ½ strength MS medium with 0.5% activated charcoal with 2.2 number of roots per shoot and 2.15 cm average root length. Transgenic callus was produced through Agrobacterium tumefaciens mediated genetic transformation carrying gus and npt-II gene from cotyledonary explants. 72 hours of co-cultivation preceded by 72 hours of pre-conditioning was found best for callus induction. PCR studies were carried out to evaluate control and putative transgenic samples. Out of three putative transgenic callus samples two showed the amplified band of integrated gus gene which is at par with the band of positive control (Agrobacterium tumefaciens LBA 4404) , confirming the integration of gus gene.

Name of the student

Neha Sharma

Admission Number

H-2007-09-M

Name of the Major Advisor

Dr S. V. Bhardwaj 

Year of Completion of Degree

2009

Title of the thesis

Studies on genomics and proteomics of a potyvirus infecting summer squash (Cucurbita pepo L.) in H.P.

Abstract

                Summer Squash (Cucurbita pepo L.) is one of the important vegetable crops of family Cucurbitaceae. Zucchini yellow mosaic virus (ZYMV; Family: Potyviridae, Genus: Potyvirus) causes great losses to both crop and ornamental  cucurbit crop production. In the present study, after detection using ELISA, molecular characterization of ZYMV (at genomic and proteomic level) infecting summer squash was carried out. A cDNA of approximately 700bp was amplified from infected tissue with the help of primers specific for potyvirus group. The PCR amplified product was sequenced and analyzed. The sequence of partial coat protein of 154 nucleotides of Una (Indian) isolate of zucchini yellow mosaic virus (ZYMV) was determined and translated to proteins. Later, the sequence was submitted to NCBI and has got accession no. GU144796 with protein id ACZ36948. In BLASTN analysis, nucleotide test sequence showed 91% homology with D13914 (sequence from USA), whereas, protein test sequence was 75.9% homologous in BLASTP analysis with a number of protein sequences present in the database. The alignment score of test sequence with 67 other isolates of ZYMV retrieved from NCBI database was highest for USA among varied countries and lowest for China in case of nucleotides and Korea in case of proteins. Phylogenetic analysis revealed 91% similarity of the test virus sequence with a USA ZYMV CP (D13914) and 75.9% similarity of the partial polyprotein sequence with that of Japan (BAE75935). Conserved domain of the test virus was found to show homology with the potyvirus coat protein domain alignment collection (pfam00767). Computational restriction digestion revealed that 22 different restriction enzymes restrict present isolate of ZYMV. Secondary structures for polyprotein of the test virus was predicted which inferred dominance of alpha (α) helix in the protein sequence.

Name of the student

Tripti Baheti

Admission Number

H-2007-18-M

Name of the Major Advisor

Dr  S. K. Sharma

Year of Completion of Degree

2009

Title of the thesis

“Plant regeneration from callus and cell suspension culture of carnation  Dianthus caryophyllus)”

Abstract

                The present investigation entitled “ Plant regeneration from callus and cell suspension culture of carnation (Dianthus caryophyllus).”, the regeneration protocols were standardized using ex vitro derived leaf explants. The sterilized explants were inoculated on MS medium supplemented with different concentrations of growth regulators for callus induction. The highest percent callus induction and growth was achieved with MS medium supplemented with 0.5 mg l‑1 2, 4-D + 0.5 mg l-1 NAA. The compact and regenerative calli obtained on MS medium supplemented with 0.5 mg l-1 TDZ + 1 mg l-1 NAA was found most suitable for regeneration. The highest number of calli producing shoots and average number of shoots per callus was recorded on MS medium supplemented with 2 mg l-1 zeatin + 1 mg l-1 IAA. The calli derived from leaf explants cultured in media containing 0.5 mg l-1 2, 4-D + 0.5 mg l-1 NAA were highly friable, had poor regeneration potential and were selected for cell suspension studies. Cell suspensions were initiated with optimal quantity of callus (1.25-1.50 g) per 20 ml in liquid MS medium containing 0.5 mg l-1 2, 4-D + 0.5 mg l-1 NAA and subcultured weekly to fresh medium to obtain maximum cell density. The cultures were allowed to grow into microcolonies in liquid medium and subsequently into calli on solid MS medium. Calli differentiated into shoots on MS medium supplemented with 2 mg l-1 zeatin + I mg l-1 IAA. The maximum average number of roots per shoot and average root length was obtained on MS medium supplemented with 2.0 mg l-1 IBA and 0.2% activated charcoal. Plantlets were obtained from in vitro derived shoots with 32-56% survival after 30 days of their transfer to pots.

Name of the student

Deepka Sharma

Admission Number

H-2007-02-M

Name of the Major Advisor

Dr (Mrs.) Rajinder Kaur

Year of Completion of Degree

2009

Title of the thesis

Studies on relatedness among cultivars and selected clones of almond [Prunus dulcis Miller (D. A. Webb)] using RAPD markers

Abstract

                In the present studies on relatedness among cultivars and selected clones of almond [Prunus dulcis Miller (D. A. Webb)] using RAPD markers, DNA from fresh, young and healthy leaves of 32 almond accessions was isolated using CTAB method (Doyle and Doyle, 1987) with some modifications. Out of forty random decamer primers, only sixteen were able to amplify genomic DNA. These sixteen random decamer primers gave 87 polymorphic  bands, 14 unique bands sized 100 bp to 1500 bp. The PIC value (Polymorphic Information Content) for all the sixteen primers was calculated and average was found to be 0.684. The primer S073 gave highest PIC value (0.8687) and lowest PIC value (0.2551) was obtained with  primer S081. From this it was found that primer S073 was most informative. Similarity matrix using Jaccard’s coefficient was constructed and it ranged from 0.000 to 0.667. Maximum similarity was found  between ‘V.1’ and ‘Tree No. 104’. Dendrogram based on UPGMA method divided the accessions into three main clusters ‘A’, ‘B’ ,‘C’ and one singlet (Nauni Selection) was obtained at 0.02 similarity index value. Cophenetic correlation was found to be 0.89. From all the data obtained it can be concluded that polymorphism can be easily scored and used for studying relatedness almond genotypes using RAPD markers.

Name of the student

Anish Sharma

Admission Number

H-2007-02-M

Name of the Major Advisor

Dr (Mrs.) Rajinder Kaur

Year of Completion of Degree

2009

Title of the thesis

Studies on relatedness among cultivars and selected clones of almond [Prunus dulcis Miller (D. A. Webb)] using RAPD markers

Abstract

                In the present studies on relatedness among cultivars and selected clones of almond [Prunus dulcis Miller (D. A. Webb)] using RAPD markers, DNA from fresh, young and healthy leaves of 32 almond accessions was isolated using CTAB method (Doyle and Doyle, 1987) with some modifications. Out of forty random decamer primers, only sixteen were able to amplify genomic DNA. These sixteen random decamer primers gave 87 polymorphic  bands, 14 unique bands sized 100 bp to 1500 bp. The PIC value (Polymorphic Information Content) for all the sixteen primers was calculated and average was found to be 0.684. The primer S073 gave highest PIC value (0.8687) and lowest PIC value (0.2551) was obtained with  primer S081. From this it was found that primer S073 was most informative. Similarity matrix using Jaccard’s coefficient was constructed and it ranged from 0.000 to 0.667. Maximum similarity was found  between ‘V.1’ and ‘Tree No. 104’. Dendrogram based on UPGMA method divided the accessions into three main clusters ‘A’, ‘B’ ,‘C’ and one singlet (Nauni Selection) was obtained at 0.02 similarity index value. Cophenetic correlation was found to be 0.89. From all the data obtained it can be concluded that polymorphism can be easily scored and used for studying relatedness almond genotypes using RAPD markers.

Name of the student

Nguyen Thanh Binh

Admission Number

H-2001-10-M

Name of the Major Advisor

Dr.S.V.Bhardwaj

Year of Completion of Degree

05.12.2003

Title of the thesis

Development of virus resistant cell lines of bell pepper (Capsicum annum L. cv. California wonder) through in vitro techniques

Abstract

Virus diseases of bell pepper constitute an important factor contributing to low yields and reduced fruit quality rendering the growing of pepper uneconomical. Since, no effective control measure against virus diseases are known, some of the in vitro approaches introduced during past few decades, have proved very efficient in retrieving virus free plants. The present investigations are also an effort towards obtaining virus resistant cell lines through in vitro techniques using green islands. The plant growing in the field were indexed for presence or absence of virus(es). Explants for callus induction and proliferation were taken from fully expanded mature leaves showing clear mosaic symptoms and then indexed. The presence of only Potato Y Potyvirus (PVY) in Capsicum annuum L. var. California Wonder was recorded in the field samples as well as cultures. The explants i. e. leaf discs were sterilized in Bavistin (0.2%) for 5 minutes, followed by a treatment with sodium hypochlorite (20%) in the presence of 1-2 drops of Tween- 20 for 10 minutes. Nodular, hard and green coloured callus was obtained on MS medium supplemented with BAP (0.5-2.0 mg/l) alone or in combination with NAA (0.1-1.0 mg/l), whereas, loose, friable, creamish and brown coloured callus was obtained on MS medium supplemented with 2, 4-D (0.5-2.0 mg/l). The best medium for multiplication and maintenance of cali was worked out to be MS medium supplemented with 1.0 mg/l of BAP in combination with 0.5 mg/l of NAA. However, shoot regeneration could not be obtained in the presence of BAP (0.5mg/l) alone or in combination with various auxins like NAA (0.5mg/l), IAA (0.5 mg/l) and IBA (0.5 mg/l). Among the eighty calli derived from dark green island tissues of infected leaves only eighteen tested negative against Potato Y Poyuvirus antisera. The virus free and/or resistant cell lines obtained under present investigations need further studies to obtain virus free and/or resistant plants.

Name of the student

Ruchika Sharma

Admission Number

H-2008-18-M

Name of the Major Advisor

Dr. (Mrs.) Amarjit K. Nath

Title of the thesis

“Studies on antioxidant activity in fruits of crab apple”

Abstract

Crab apple (Malus baccata) fruits of Himalayan region were analyzed for total antioxidant activity, phenol content, ascorbic acid content and antioxidant enzyme activities viz., superoxide dismutase, catalase, peroxidase and ascorbate peroxidase. The results obtained were compared with fruits of commercial apple cultivar Red Delicious (Malus domestica ‘Red Delicious’). Superoxide dismutase enzyme was purified and characterized from pulp extract of crab apple fruits by ammonium sulphate precipitation, gel filtration chromatography (Sephadex G-100) and ion exchange chromatography (DEAE-Sephadex). On DEAE-Sephadex, three isoforms of superoxide dismutase (SOD) were eluted with water, 0.1M and 0.2 M KCl gradients, thereby indicating different charges on the isoforms. .The isoforms, superoxide dismutase enzyme-1, superoxide dismutase enzyme-2 and superoxide dismutase enzyme-3 were purified to 14.1, 8.8 and 10 fold with 16.6, 14.0 and 15.4 per cent recovery, respectively. The molecular weight of purified isoforms of superoxide dismutase enzyme-1, superoxide dismutase enzyme-2 and superoxide dismutase enzyme-3 were found to be 15,848; 17,378 and 20,892 daltons, respectively, as revealed by SDS-PAGE. Isoforms of superoxide dismutase enzyme were found to be heat stable up to 900C. All the three isoforms had pH optima of 7.8. The activity of all the three isoforms of superoxide dismutase enzyme was found to increase upon incubation with increasing concentrations of zinc (Zn2+) ions.              

                Superoxide dismutase is a powerful free radical scavenger which has been clinically shown to protect the brain, skin, heart, spinal cord from ischemic injury and for treatment of the disorders associated with free radical damage and long-term exposure to toxins like cigarette smoke. Keeping in view the role of superoxide dismutase enzyme in stress tolerance in plants, there is a need to identify novel genes of this enzyme from native germplasm.

Name of the student

Monika Gupta

Admission Number

H-2007-04-D

Name of the Major Advisor

Dr (Mrs.) Poonam Shirkot

Title of the thesis

“Isolation & characterization of Thermus aquaticus and production of Taq DNA polymerse enzyme”

Abstract

Thermophiles are the organisms which are adapted to live at high temperatures. The enzymes from thermophiles find a number of commercial applications because of their thermostability and thermoactivity. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes. In keeping with this view, hot water springs of Himachal Pradesh could serve as a good source for new thermophilic microorganisms with novel industrially important properties. The aim of this research was therefore the isolation of Thermus aquaticus and production of Taq DNA polymerase enzyme.

Eight hot water springs viz., Manikaran, Vashisht, Khirganga, Tattapani, Jeory and Rajgarh were purposely selected for the present studies. Ten samples from each hot water springs were collected. The pH and temperature of the eight thermal springs were recorded and ranged from 4.1-6.8 and 23^oC-105^oC respectively. The chloride, sulphate, total hardness, calcium hardness and magnesium content ranged from 198.0-2673.0, 12.0-70.0, 105.0-698.0, 33.66-258.0, 84.0-544.0 and 1.46-55.16 mg/l respectively. Forty two bacterial isolates were isolated from the selected eight hot water springs of Himachal Pradesh using different media. All the bacterial isolates were studied for various morphological characters and on the basis of morphological characterization, 20 isolates were screened out. The selected 20 isolates were further investigated for biochemical characters and five isolates of Thermus i.e, TMA₅, TMA₇, TVB₈, TK₁₀ & TT₅ were selected for further enzymatic studies. The selected five bacterial isolates were screened for the intracellular production of Taq DNA polymerase enzyme. The maximum (0.03 U/mg protein) Taq polymerase was released by thermophilic bacterial isolate TMA₅. Optimization of culture conditions for enzyme production was carried out. The intracellularly released Taq polymerase from thermophilic bacterial isolate TMA5 was partially purified by ammonium sulhate precipitation followed by gel permeation chromatography and SDS-PAGE. The Taq polymerase assay of the partially purified enzyme fraction revealed that although percent recovery of enzyme was low but it resulted in increasing the purity of Taq DNA polymerase by 1.67 folds.

                Genomic DNA was isolated from the selected isolate TMA₅. PCR of the isolated DNA was carried out using genus specific primers for 16S rDNA gene and amplification of the DNA was carried out using primers. Sequencing of the PCR product was done using similar primers. Sequence of the TMA₅ isolate so obtained was found to be 1325 bp. BLASTN analysis showed 95-98% homology of the query sequence with other isolates of Thermus aquaticus. A total of 17 Thermus aquaticus sequences were mined and these sequences were used to compare the 16S rDNA sequence of the test isolate TMA₅. Alignment score was highest for Thermus aquaticus strain YT-1 16S ribosomal RNA, partial sequence (NR_025900.1). Phylogenetic analysis based on nucleotide sequences using NJ method was achieved via phylip 3.68 and EXOME^TM HORIZON.

Name of the student

Ashish Verma

Admission Number

H-2008-03-M

Name of the Major Advisor

Dr. (Mrs.) Rajinder Kaur

Title of the thesis

Studies on molecular characterization of some Prunus rootstocks

Abstract

 In the present studies on molecular characterization and fingerprinting of some Prunus rootstocks, DNA from fresh, young and healthy leaves of 18 Prunus rootstocks was isolated following CTAB method (Doyle and Doyle, 1987) with some modifications. Out of a total of 20 SSR primers used, only 16 were able to amplify the genomic DNA .Eight microsatellite primers were found to be monomorphic and eight were found to be polymorphic. The eight polymorphic primers gave a total of 13 polymorphic bands out of which only five were found to be unique for rootstocks ‘Cadaman’, ‘GF 677’, ‘Wild apricot’, ‘Manicot’ and ‘Gisela 5’. Similarity matrix using NTSYSpc ver. 2.02h and dissimilarity matrix using DARwin5 ver. 5.0.155 CIRAD was constructed and it ranged from 0.533 to 1.00 and 0.00 to 0.34, respectively. The rootstocks ‘Wild peach’ and ‘Paja’ and ‘GF 31’ and ‘Julior’ were found to be identical. Dendrogram based on UPGMA method divided the rootstocks into two broad clusters ‘A’ and ‘D’ and the rooted tree based on Neighbour-Joining method of DARwin5 ver. 5.0.155 CIRAD also divided the rootstocks as per the results of the dendrogram. The PIC value for all the eight polymorphic primers was calculated and the mean PIC value was found to be 0.38. The primer IISRS-8 gave highest PIC value (0.67) and the lowest PIC value (0.09) was obtained with primer IISRS-9. Polymorphism was found to be 43.33%. From all the data obtained, it can be concluded that more number of informative primers would be incorporated in the study to molecular characterize and fingerprint these rootstocks.

Name of the student

Monika Kashyap

Admission Number

H-2008-13-M

Name of the Major Advisor

Dr (Mrs) Kamlesh Kanwar

Title of the thesis

“Studies on cell culture for genetic transformation in Punica granatum L.”

Abstract

 The present investigation aims at “Studies on cell culture for genetic transformation in Punica granatum L.”. Genetic transformation experiment was conducted with cotyledonary explants excised from in vitro raised seedlings of Punica granatum L. cv. Kandhari Kabuli. Explants were preconditioned on standardized callus induction medium (solid MS medium supplemented with 4.0 mg/l BA and 3.0 mg/l NAA) for 48 hours followed by 48 hours of co-cultivation with Agrobacterium tumefaciens. Friable putative transgenic were used for initiation of cell suspension culture. Cells of various shapes such as oval, elongated, columnar, elliptical and isodiametric were observed in the suspension culture. Cells proliferated best on selective MS medium supplemented with 4.0 mg/l BA + 3.0 mg/l NAA and 50 mg/l kanamycin along with 500 mg/l cefotaxime with average cell doubling time of 5 days. Combination of a cytokinin (BA) and an auxin (NAA) was found to be better for cell proliferation as compared with the combination of two cytokinins (BA and TDZ). Growth curve of cells in suspension culture consisted of lag phase (5-6 days), exponential phase (7 days) and stationary phase (4-5 days) followed by cell death. Plating efficiency of 0.7 % was obtained by plating of cell aggregates (5-6 cells) on selective MS medium supplemented with 4.0 mg/l BA and 3.0 mg/l NAA utilizing modified Bergman technique. Putative transgenic macrocalli did not show shoot regeneration. Positive PCR results were observed in transgenic calli obtained through cell suspension culture. The protocol, thus offer an opportunity to produce the putative transformed tissues/ plantlets with single cell origin.

Name of the student

Mahak Tufchi

Admission Number

H-2007-06-M

Name of the Major Advisor

Dr (Mrs) Kamlesh Kanwar

Title of the thesis

“In vitro genetic transformation through shoot bud in Punica granatum L. cv. Kandhari Kabuli by Agrobacterium tumefaciens”

Abstract

The present investigation aims at “In vitro genetic transformation through shoot bud in Punica granatum L.cv. Kandhari Kabuli by Agrobacterium tumefaciens.” Shoot buds/nodal segments were taken as explant from the juvenile newly emerging current shoots from 8 years old field grown tree. Sterilization of the shoot buds was done by 0.2% bavistin (3 min) and 0.5% sodium hypochlorite (10 min). Establishment of shoot buds was carried out on MS medium supplemented with 2.0 mg l^-1 BAP that resulted in nearly about 52.77 per cent bud break. The multiplication of the microshoots was carried out on multiplication medium supplemented with 2.0 mg l^-1 BAP + 0.5 mg l^-1 Kn + 0.5 mg l^-1 GA₃. Highest in vitro rooting of the microshoots was obtained on half strength MS medium supplemented with 0.05% activated charcoal that showed 70.8 % rooting. Effect of subculturing on the number of microshoots, number of roots and root length was also observed. Hardening of the plantlets was carried out in cocopeat and sand in 1:3 ratio with 26.66 per cent survival of plants. By using enhanced axillary branching the in vitro raised shoots with shoot bud as explant were taken for genetic transformation with Agrobacterium tumefaciens carrying cry1Ab gene and npt II (neomycin phosphotransferase gene). Effect of pre conditioning duration and co-cultivation duration on the frequency of transformation was studied. Maximum transformation frequency (3.33%) was obtained at 48 hours of pre conditioning and 48 hours of co-cultivation with 0.1mM Acetosyringone. Successful genetic transformation in the transformed shoots was confirmed by PCR analysis and one out of four putative transformants showed positive result.
 

Name of the student

Karuna Dhiman

Admission Number

H-2007-05-M

Name of the Major Advisor

Dr S.K. Sharma

Title of the thesis

Plant regeneration and Agrobacterium-mediated gene transfer studies in broccoli (Brassica oleracea L. var. italica) tissues.

Abstract

An efficient protocol for in vitro plant regeneration and genetic transformation has been developed in broccoli (Brassica oleracea L. var. italica cv. Solan green head) tissues. Hypocotyl and cotyledon were used as explants for in vitro plant regeneration studies in broccoli. The explants were excised from sterile germinated seedlings and placed on shoot induction medium. The hypocotyl  and cotyledon explants showed high frequency of shoot regeneration (62.50% and 60.00 %) on MS medium supplemented with 2.5 mg/l BAP and 2.0 mg/l NAA and MS medium supplemented with 3.5 mg/l Kn and 0.1 mg/l NAA respectively. MS medium supplemented with 0.2 mg/l NAA was found to be best for root regeneration (90.00%). The broccoli plantlets were able to regenerate plantlets within 6-7 weeks. Regenerated plantlets were successfully acclimatized. The high frequency regeneration system served as an excellent tool for the establishment of an efficient transformation method for broccoli. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β- glucuronidase [uid A (gus)] gene in binary vector (pBI 121) system along with kanamycin  resistance gene (npt-II) for selection in both bacteria and plant was used for co- cultivation experiment  to transfer uid A (gus) and npt-II genes in broccoli. After co-cultivation only the transformed cells were able to grow on selective shoot regeneration medium (50mg/lt Kanamycin and 500mg/lt cefotaxime) whereas non-transformed cells/explants died on the selective medium. Transformation experiment could be scored as early as 3-4 weeks after selection. The effect of pre-culturing and co-cultivation was studied. Explants pre-cultured for 72 hours prior to co-cultivation for 48 hours resulted in improved transformation frequency. Putative transformed calli and shoot from hypocotyl explants obtained, which were able to grow on the selective medium containing 50mg/lt Kanamycin. The presence of npt-II and uid A (gus) was confirmed by Polymerase chain reaction using designed primers. Out of 4 putative transformed calli and 1 shoot, 2 calli and a shoot have shown the amplification of npt-II and uid A (gus) genes. Expression of uid A (gus) gene was analyzed in these transformed calli by GUS assay and two transformed calli which showed presence of npt-II and uid A (gus) genes were also GUS positive. A protocol for plant regeneration and genetic transformation in broccoli tissues has been standardized.

Name of the student

Anish Kumar Sharma

Admission Number

H-2008-04-M

Name of the Major Advisor

Dr S.V. Bhardwaj

Title of the thesis

Development of mass multiplication protocol for Asparagus adscendens Roxb.

Abstract

The present investigations entitled ‘‘Development of mass multiplication protocol for Asparagus adscendens Roxb.” were conducted to obtain plantlets through direct regeneration from juvenile nodal segments and rhizome explants from mature plants. Nodal segments obtained after excising spines and cladodes while tuberous roots from rhizome explants were carefully removed using scalpel blade without damaging the explants. Nodal segments were surface sterilized with 0.525% NaOCl for 15 minutes followed 0.2% bavistin for 15 minutes and finally 0.1% HgCl₂ for 1.5 minutes and resulting in 91.33% survival. The rhizome explants were sterilized using 3% NaOCl for 2 minutes followed by 2% bavistin + 2% Mancozeb for 20 minutes and finally 0.1% HgCl₂ for 2 minutes achieving 88.3% survival of explants. Maximum establishment of cultures from both explants was obtained on MS medium supplemented with 0.2 mg/l BAP and 0.2 mg/l Kn. In nodal explants maximum shoot proliferation rate (5.50) on MS medium containing 0.05 mg/l NAA and 0.3 mg/l Kn was obtained, while in rhizome explants multiplication rate of 3.50 was obtained on MS medium with 0.2 mg/l BAP and 0.2 mg/l Kn after 8 weeks on multiplication medium. Regenerated shoots of appropriate size (3-4cm) were transferred to 14 different media combinations tried for root induction but failed to get any success which can attributed due to genetic variation in the concerned species. On the basis of this study it is concluded that a reliable protocol for in vitro shoot multiplication of Asparagus adscendens has been developed which can be used for conservation of this endangered plant species.

Name of the student

Adil Ahmad Kamal

Admission Number

H-2006-01-M

Name of the Major Advisor

Dr (Mrs). A. K. Nath

Title of the thesis

“Studies on characterization of edible Rubus species using RAPD markers”

Abstract

The present studies on characterisation of Rubus species were carried out by using RAPD markers. Genomic DNA was isolated from young and green leaves of 10 Rubus species (Rubus moluccanus, Rubus assamensis, Rubus hypargyrus, Rubus nepalensis, Rubus treutleri, Rubus ulmifolius, Rubus paniculatus, Rubus laciocarpus, Rubus opulifolius and Rubus ellipticus) by using CTAB method (Doyle and Doyle, 1987) with modifications. Genetic variation was studied using 30 random decamer primers. Of these only 22 produced polymorphism. Total number of bands amplified were 115, out of which 86 were polymorphic and 16 were specific RAPD markers. The amplified fragments were ranged from 1006 bp to 6039 bp and percentage of total polymorphic bands was 75. All Rubus species examined can be clearly discriminated and identified using the 22 primers selected in this study. Similarity matrix was constructed by using Dice coefficient and it ranged from 0.62 to 0.83. Low similarity value was obtained between Rubus assamensis and Rubus hypargyrus and high between Rubus paniculatus and Rubus laciocarpus. Dendrogram was constructed by using UPGMA method for the clustering of all Rubus species. All the Rubus species grouped together except Rubus assamensis, which formed another cluster. From the data obtained in this study it can be concluded that polymorphism can be easily scored and used for studying  diversity between Rubus species. The specific RAPD marker obtained could be used for identification of Rubus species.

Name of the student

Shweta Pathania

Admission Number

H-2004-10-M

Name of the Major Advisor

Dr. (Mrs.) Manju Modgil

Title of the thesis

Plant regeneration studies and assessment of genetic
variation among regenerants of apple rootstock MM111

Abstract

The present studies were aimed to develop technique for high frequency regeneration in clonal apple rootstock MM111 through leaves as explants and to assess the genetic variability among regenerants using RAPD-PCR. Callus was induced on high auxin containing medium and organogenesis was obtained from secondary callus obtained on 2 mg/l NAA and 0.5 mg/l BA after about 30 days in the shoot induction medium. Both direct and indirect regeneration was obtained on MS medium with BA (2, 3, 4 mg/l) and NAA 1 mg/l) in light as well as in dark but relatively higher regeneration was found in explants grown in light. The maximum regeneration (33.76 %) was obtained in 3 mg/l BA with 1 mg/l NAA with 1.67 shoots per explant and 1.5 – 2.0 cm shoot length. The regeneration percentage increased to 43.33 % with 0.6 mg/l TDZ and 0.5 mg/l NAA with 3 shoots per explant and maximum shoot length of 3.0 – 3.5 cm. The potential of regeneration in leaves increased when exposed to growth regulators for a brief period of time. The same combination i.e. 0.6 mg/l TDZ and 0.5 mg/l NAA resulted in maximum regeneration percentage of about 49 % with maximum of 6 shoots per explant and maximum shoot length of 3 cm, in comparatively less time period. 0.2 mg/l TDZ with 1 mg/l IBA resulted in regeneration but with low frequency. Regenerated shoots were multiplied and rooted. The rooted regenerants were hardened. DNA was isolated from field grown apple rootstock MM111 and 5 regenerants, which were subjected to RAPD-PCR analysis using 10 primers initially. Out of total 10, only 6 primers yielded 19 scorable bands of which 7 were polymorphic and 12 monomorphic with 36.8 per cent polymorphism. Each primer produced a unique set of amplification products ranging in size from about 300 – 1000 bp. Thus, regeneration from leaf proved to be an effective method to create variability which could be valuable for crop improvement.

Name of the student

Neha

Admission Number

H-2005-10-M

Name of the Major Advisor

Dr. (Mrs.) Manju Modgil

Title of the thesis

In vitro screening of apple rootstock M7 regenerants against Dematophora necatrix Hartig 

Abstract

Soil borne pathogens cause important damages to plant health. One of the most important diseases of apple is white root rot caused by Dematophora necatrix Hartig.  Presently, the disease is being managed by drenching chemicals deep in the soil, which is not only cumbersome and expensive, but also disturbs the soil ecology. Therefore, a study was conducted with the objective to develop a technique for in vitro screening of apple rootstocks M7 regenerants against Dematophora necatrix culture filtrate.  Direct organogenesis has been obtained from in vitro leaf explants on MS medium supplemented with BA (4.0, 4.5 mg/l) and IAA (1.0, 1.5 mg/l) in dark.  The highest regeneration percentage (59.15) was obtained on 4.0 mg/l BA with      1.0 mg/l IAA with 18-19 number of shoots after 6 weeks.  Both direct and indirect regeneration was obtained on combinations of TDZ (0.2, 0.4, 0.6, 0.8, 1.0 mg/l) with NAA (0.5, 1.0 mg/l) or IAA (1.0 mg/l) or IBA (1.0 mg/l).  These regenerants after multiplication were exposed to selective medium with different concentrations of fungal culture filtrate for the selection of putative resistant shoots.  Fourty per cent of shoots survived on 70 per cent FCF while no shoot could survive on 72.5 per cent FCF. The regenerated shoots which were tolerant to 70 per cent culture filtrate were further exposed to three selection cycles. The selected/tolerant and unselected regenerants were rooted and hardened for pathogenecity test, dose of inoculum was standardized by inoculating unselected regenerants with different doses of inoculums. Tolerant regenerants were similarly tested with standardized dose of inoculum with positive and negative control. Finally, five resistant lines were obtained.

Name of the student

Parul Sharma

Admission Number

H-2004-7-M

Name of the Major Advisor

Dr. (Mrs.) Manju Modgil

Title of the thesis

Micropropagation of apple rootstock Malling 26

Abstract

The present studies were made to develop a workable micropropagation technique of apple rootstock M26 through terminal/axillary buds as explants taken from field trees. During establishment of explants, highest survival percentage (18.82 %) was recorded during summer and spring seasons. Surface sterilization with HgCl₂ for 3 minutes was found to be the best as it gave maximum number of uncontaminated buds (37.54 %) as well as the highest bud break. MS medium supplemented with BA (1.0 mg/l), IBA (0.1 mg/l) and GA₃ (0.5 mg/l) resulted in maximum bud break and survival. Better multiplication was found on MS medium supplemented with 0.5 mg/l BA, 0.5 mg/l Kin and 0.05 mg/l IBA, which showed 4 – 9 fold multiplication with 0.5 – 4.0 cm long shoots. Maximum rooted microshoots (98 %) was observed in 0.3 mg/l IBA supplemented with 162 mg/l phloroglucinol in two step method. Rooted plantlets (without callus) showed 85-90 per cent success in potting mixture during hardening. Plantlets with intermediate callus were difficult to establish in pots. DNA was isolated from field grown apple rootstock M26 and 20 micropropagated shoots and subjected to RAPD-PCR. Of the 10 primers used, 4 yielded 12 scorable bands. Among them, 10 were monomorphic and 2 were polymorphic with 16.6 per cent polymorphism. Tissue cultured plants showed maximum genetic similarity among themselves and with the parent.

Name of the student

Mohit Soni

Admission Number

H-2003-08-M 

Name of the Major Advisor

Dr. (Mrs.) Manju Modgil

Title of the thesis

Studies on in vitro propagation of apple                                          rootstock, Merton 793

Abstract

The present studies were made to develop a workable micropropagation technique of apple rootstock M793 through terminal/axillary buds as explants taken from field grown trees.  During establishment of explants, highest survival percentage (77.53 – 84.89 %) was recorded during summer and spring seasons due to less contamination and less exudation of phenols.  The explants of size 0.9 – 1.7 cm were found to be the most appropriate for maximum explant survival i.e. 52.27 – 55.5 %.  Surface sterilization with HgCl₂ for 3 minutes was found to be the best as it gave maximum number of uncontaminated green buds (87 %) as well as highest bud break.  Out of two media (MS and WPM) attempted for explant establishment, former was found more suitable.  MS medium supplemented with BAP (0.5 – 1.0 mg/l) and IBA (0.1 mg/l) gave maximum bud break and survival.  Better multiplication was found on MS medium supplemented with 0.5 mg/l BA and 0.01 mg/l IBA, which gave 3-4 fold multiplication with 2 – 3.5 cm long shoots without callus.  All the three types of explants i.e. shoot tip, nodal cuttings and cluster of 2 – 3 small shoots were suitable for multiplication of shoots as well as for making longer shoots which can be used for rooting.  Maximum rooted microshoots (70 %) was observed in 10 days dark treatment in IBA containing LM.  Rooted plantlets without callus showed 85 – 90 % success in potting mixture during hardening.  Plantlets with intermediate callus were difficult to establish in pots.

Name of the student

Richa Sood

Admission Number

H-2007-16M

Name of the Major Advisor

Dr. (Mrs) A. K. Nath

Title of the thesis

Biochemical characterization to assess genetic diversity in wild Pomegranate of H.P           

Abstract

The present investigation on “Biochemical characterization to assess genetic diversity in wild pomegranate of H.P” was carried out using randomly amplified polymorphic DNA (RAPD) markers. On the basis of morphological studies collection Shoghi-2 of Shoghi site was seen to possess maximum petiole length and vitamin C content in aril juice. Genomic DNA was isolated from young and green leaves of 24 collections of six sites of wild pomegranate using CTAB method (Doyle and Doyle,1987) with slight modifications. Genetic variation was studied using 25 random decamer primers,  out of these only 19 primers produced polymorphism. Total number of bands amplified were 142, out of which 116 were polymorphic and 19 were specific RAPD markers. The amplified fragments ranged in size from 178-3895 bp and percentage of total polymorphism band was 70.All the 24 collections of the six sites were distinguished with the combination of 19 primers selected in this study. Similarity matrix was constructed using Dice and Jaccard coefficient. It ranged from 0.42 -0.91 (Jaccard coefficient) and 0.60-0.92 (Dice coefficient). Low similarity value was obtained between Rajgarh-3 and Kandaghat-2 and high similarity was between Badiyal-2 and Shoghi-4. Dendrogram was constructed by using UPGMA method for the clustering for all the collections of wild pomegranate. All the collections were grouped together except for Rajgarh-3, which formed another cluster. Relationship between individual site and its collections were not clear from the dendrogram, for this purpose individual dendrogram between single site and its collections was constructed. From the data obtained in this study it can be concluded that RAPD studies can be useful in breeding programmes allowing the identification of different collections and assessing the genetic similarity among different collections of wild pomegranate which would facilitate their use as identified genetic stock in future breeding programmes.

Name of the student

Richa Taank

Admission Number

H-2007-15-M

Name of the Major Advisor

Dr. (Mrs) A. K. Nath

Title of the thesis

“Studies on a-amylase inhibitor
(a-AI) in some grain legumes”

Abstract

Eighteen different grain legume cultivars of Himalayan region were analyzed for a-amylase inhibitor activity. a-Amylase unit inhibited per gram seed weight was maximum in Hans cultivar of Phaseolus vulgaris L. a-Amylase inhibitor from seeds of Hans cultivar of Phaseolus vulgaris L. was purified using ammonium sulphate precipitation, gel filtration chromatography (Sephadex G-200) and ion exchange chromatography (DEAE-Sephadex). The inhibitor was purified to homogeneity as judged by native PAGE with 34.42 fold purification and 69.35 per cent recovery. The purified inhibitor had a molecular weight of 16,218 daltons and was found to be monomer as revealed by SDS-PAGE. It was found to be heat stable upto 30⁰C-40⁰C and had two pH optima of 5.0 and 6.9. Nature of inhibition was found to be of non competitive type as determined by Lineweaver burk plot. The purified inhibitor was found to have inhibitory activity against a-amylases extracted from larvae of Callosobruchus chinensis, Tribolium castaneum and mid gut of Corcyra cephalonica. Purified a-amylase inhibitor was also found to inhibit human salivary a-amylase.

Name of the student

Sonia Kumari

Admission Number

H-2008-19-M

Name of the Major Advisor

Dr. D.K. Srivastava

Title of the thesis

Studies on genetic transformation in brinjal (Solanum  melongena L.)       

Abstract

Genetic transformation studies were carried out to standardize a protocol for Agrobacterium –mediated gene transfer in brinjal (Solanum melongena L. cv. Pusa Purple long) with npt-II and gus genes. Plant regeneration studies were carried out using two types of explants viz. hypocotyl and cotyledon which were used from 10-12 days old in vitro grown seedlings. For shoot regeneration from hypocotyl and cotyledon explants the best shoot regeneration medium was found to be MS + 2.5 mg/l BAP + 0.5 mg/l IAA and MS + 2.5mg/l Kn + 0.4 mg/l IAA, respectively. The cotyledon explants showed high frequency of shoot regeneration (77.46%)  as compare to hypocotyl explants (57.17%). MS medium supplemented with 0.10 mg/l IAA was found best for root regeneration from in vitro developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of hypocotyl tissues and to select transgenic shoots during genetic transformation experiment. Kanamycin concentration as low as 20 mg/l was toxic to the explants (hypocotyl) on selective shoot regeneration medium. Agrobacterium tumefaciens strain 4404, carrying binary vector pBI 121 containing npt-II and gus genes under the control of NOS and CaMV35s promoters was used for genetic transformation studies. Preincubation of 72 hrs and co-cultivation of 48 hrs was found optimum as it gave maximum transgenic shoot regeneration on selective medium. The transformation efficiency was low in cotyledon explants  (2.20%)  and hypocotyl explants (6.8%) on  the selective shoot regeneration medium. Root regeneration from putative transgenic shoots was obtained on selective root regeneration medium. The putative transformants were randomly selected for the amplification of gus and npt-II genes with specific designed primers and out of 8 randomly selected putative transgenic plantlets 5 have shown the amplification of gus and npt-II genes there by indicating the presence/integration of gus and npt-II genes into the genome of transgenic brinjal. The expression of gus gene was studied by using biochemical and histochemical techniques of GUS assay. The gus gene was expressed in the PCR positive transgenic plantlets of brinjal. A protocol for genetic transformation in brinjal with npt-II and gus genes has been standardized.

Name of the student

Madhvi Soni

Admission Number

H-08-12-M

Name of the Major Advisor

Dr (Mrs.) Rajinder Kaur

Title of the thesis

Studies on in vitro propagation and conservation of Viola pilosa Blume

Abstract

A protocol for in vitro propagation and conservation was developed for Viola pilosa, a  valuable medicinal plant The sterilized explants (buds) cultured on MS medium supplemented with 1 mg/l BA, 1 mg/l TDZ and 0.50 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoot on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found out to be the best medium for in vitro shoot multiplication. Also effect of different types of solidifying agents on the growth of in vitro multiplying shoots was studied and out of three different solidifying agents viz. agar agar, agarose and gelrite gellan gum, the gelrite gellan gum containing medium was found out to be the best medium for in vitro shoot multiplication. Callus was obtained as a by product from shoots on MS medium supplemented with 1 mg/l BA, 1 mg/l TDZ and 0.50 mg/l GA₃ and this callus was further multiplied on medium supplemented with 1.5 mg/l NAA. The shoot regeneration was obtained from the callus on MS basal medium supplemented with 0.1% activated charcoal. 100%  root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA.

In vitro conservation was successfully demonstrated by using two different approaches namely slow growth at low temperature and cryopreservation following vitrification. In slow growth at low temperature studies the in vitro shoots incubated at 10^oC,  though showed 100% retrieval, however, shoots became etiolated, while the shoots incubated at 4^oC showed retrieval of 85.7% with no morphological variation during incubation. In cryopreservation - vitrification studies the vitrified shoot buds gave maximum retrieval of 41.66% when they were precooled at 4^oC while only 16.66% vitrified shoots were retrieved from those precooled at 10^oC. In vitro formed plantlets were hardened and transferred to soil with 55.55% survival.

Name of the student

Nidhi Jindal

Admission Number

H-08-14-M

Name of the Major Advisor

  Dr S.K. Sharma

Title of the thesis

Studies on in vitro selection and regeneration of Chrysanthemum(Dendranthema grandiflorum Tzvelev) cv. ‘Snow Ball’ resistant to the culture filtrate of Fusarium  oxysporum f.sp. chrysanthemi

Abstract

The reproducible protocol was optimized for in vitro selection and regeneration of chrysanthemum (Dendranthema grandiflorum Tzvelev) cv. ‘Snow Ball’ resistant to the culture filtrate of Fusarium oxysporum f.sp .chrysanthemi. The callus was induced on the medium (MS basal medium + 10 mg/l kinetin + 1 mg/l NAA). Callus was subjected to different concentrations of culture filtrate of Fusarium oxysporum f.sp. chrysanthemi where selective dose to culture filtrate was found to be 8 percent on which 4.54 percent cell survival of chrysanthemum variety ‘Snow Ball’ was recorded. The best medium for callus differentiation and shoot bud regeneration was (MS basal + 0.2 mg/l BA + 0.1 mg/l NAA + 1 mg/l GA₃) on which 66.67 percent calli regenerating shoots was recorded. Percent survival of the shoots regenerated from selected calli when tested in vitro with spore suspension recorded was 20 percent.

Name of the student

Rashmi Ranade

Admission Number

H-2007-14-M

Name of the Major Advisor

Dr (Mrs) Kamlesh Kanwar

Title of the thesis

In vitro cell line selection and plant regeneration of carnation cv. Master against Fusarium oxysporum f. sp. dianthi

Abstract

The present investigation aims at ‘In vitro cell line selection and plant regeneration of carnation cv. Master against Fusarium oxysporum f. sp. dianthi’. Micropropagation of carnation was standardized through direct regeneration of adventitious shoot buds and indirect regeneration from callus using leaf as explant. The best callus induction medium obtained for the induction of callus from leaf explants was MS medium supplemented with 1.0 mg/l TDZ and 0.2 mg/l NAA. The best medium for callus differentiation and shoot regeneration was MS medium supplemented with 1.0 mg/l TDZ and 1.5 mg/l TIBA, MS medium supplemented with 1.0 mg/l TDZ and 0.5 mg/l TIBA was best for induction of adventitious shoot buds in leaf explants. Fresh weight of callus increased progressively upto third subculture passage while number of shoots and shoot length increased significantly with progressive subculturing upto third subculture passage. Microshoots were rooted on MS medium supplemented with 0.2 per cent activated charcoal. In vitro flowering was observed in callus and microshoot cultures. Cell line selection was done by using fungal culture filtrate of Fusarium oxysporum f. sp. dianthi as a selective agent. Selection and isolation of cell lines was done using calli and plantlets were regenerated from cell lines selected at 12.5% of fungal culture filtrate and the selected microshoots were rooted on the rooting medium. Rooted control as well as selected plantlets were hardened eventually. RAPD-PCR evaluation was done for the comparison of control and selected plants which revealed presence of one polymorphic band in the putative resistant plant samples. The selected plant showed resistance development to Fusarium oxysporum f. sp. dianthi in in vivo testing.

Name of the student

Neelam Prabha Negi

Admission Number

H-2007-10-M

Name of the Major Advisor

Dr. (Mrs.) Manju Modgil

Title of the thesis

Studies on induction of rooting in in vitro grown shoots of apple clonal rootstock Merton 793

Abstract

An efficient and reliable procedure for rooting and hardening of in vitro raised shoots of apple rootstock, Merton 793 has been developed. 66.78 per cent rooting was obtained with half strength MS medium supplemented with 0.1 mg/l NAA but the roots were thick and with profuse callus. 0.2% activated charcoal was used to suppress the callus but rooting efficiency reduced. Successful rooting was related to exposure of shoots to NAA supplemented liquid medium for few days and transferred to solid medium without NAA. Reduction of MS salts (1/3 and ¼) and sucrose (20 and 15 g/l) in root elongation medium showed decreased rooting in comparison to ½ MS with 25 g/l sucrose. Among various substrates tested, agar was found the best among sand, perlite and tapioca pearls. There was no rooting in sand and perlite while, less rooting in tapioca pearls. Rooted plantlets of about 2-3 cm height were subsequently transferred to peat-sand for hardening. 70 per cent plantlets established when roots were induced without callus. On the other hand, 80 per cent of the plantlets established successfully when partial in vitro root initiation was carried out in liquid medium and ex vitro root elongation and hardening in peat-sand. For direct rooting, around 55 per cent plant establishment was observed when shoots were dipped for 10 mins in higher concentration of NAA solution and planted in portrays containing peat-sand mixture. Partial in vitro rooting or direct rooting of shoots of M793 may be recommended for commercial propagation programme.

Name of the student

  Shweta Sen

Admission Number

H-2006-03-D

Name of the Major Advisor

Dr. S.K. Sharma

Title of the thesis

“Agrobacterium mediated transfer of rice chitinase gene for fungus resistance in chrysanthemum (Dendranthema grandiflorum Tzvelev cv. Snow Ball).”

Abstract

An investigation was conducted to standardize the protocols for fungus resistance gene (chi 11) transfer studies in chrysanthemum cultivar Snow Ball using Agrobacterium mediated gene transfer technique. Agrobacterium tumefaciens strain containing a binary vector pCAMBAR:chi 11 having a marker genes (hpt and bar) and fungus resistance (chi11) gene was used for genetic transformation studies. Plant regeneration studies were carried out using two regeneration systems viz. through callus cultures and direct adventitious shoot regeneration from leaf and internode explant. The regeneration protocols were standardized which were utilized during genetic transformation experiments. Hygromycin, cefotaxime and kanamycin sensitivity experiments were conducted to study the effect of antibiotics on relative growth of tissues of chrysanthemum and hygromycin, cefotaxime and kanamycin concentration of 10 mg l^-1, 300 mg l^-1 and 10 mg l^-1, respectively was optimized for selection of transformed tissues. The explants were dipped in the Agrobacterium suspension having 10⁸ cells/ml (OD-0.521at 540 nm) and 20 and 15 min dipping duration was optimum for leaf and internode explants, respectively. The highest transformation frequency with leaf and internode explant was achieved with their pre-conditioning for 48 h followed by 96 h co-cultivation on selective medium inducing callus (10 mg l^-1 kinetin + 0.5 mg l^-1 NAA + 10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime and 0.5 mg l^-1 BA + 0.5 mg l^-1NAA +10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime, respectively). On the other hand, highest transformation frequency from explants inducing direct adventitious shoots was achieved with 48 h pre-conditioning followed by 96 hr co-cultivation on selective medium (2.0 mg l^-1 BA + 0.5 mg l^-1 NAA + 10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime for leaf explant  and 2.0 mg l^-1 BA + 0.25 mg l^-1 NAA + 10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime for internode explant). The transformed calli  from leaf explant was differentiated on selective medium containing 0.5 mg l^-1 BA + 0.1 mg l^-1 IAA + 10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime, wheras, the transformed calli  from internode explant was differentiated on selective medium containing 0.5 mg l^-1 BA + 0.25 mg l^-1 IAA + 10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime. The putative transgenic shoots were multiplied on selective medium (0.5 mg l^-1 BA + 0.1 mg l^-1 IAA + 1 mg l^-1  GA₃ + 10 mg l^-1 hygromycin + 300 mg l^-1 cefotaxime) by repeated subculture to remove the escapes. The shoots were rooted on selective medium (1/2 MS + 0.2 mg l^-1 IBA + 5 mg l^-1 hygromycin) and acclimatized with 60-90% success.  The putative transformed shoots were analyzed by PCR for the presence of chi11 gene with specific primers and by southern blot analysis for the integration of chi11 gene in plant genome. Only 7 plants out of 20 randomly chosen hardened plants showed the presence of chi11 gene in PCR analysis. PCR confirmed plants were further analyzed for the integration of chi11 gene in the genome of chrysanthemum plant and only 2 out of 7 PCR positive showed the integration of gene in southern hybridization experiment. The 2 plants were multiplied and hardened  and were analysed for the expression of chi11 gene through bioassays which were carried out by spraying Septoria obesa  spore suspension on  the transformed plants kept under warm and humid conditions. Preliminary studies with the spore suspension of Septoria obesa indicated a significant expression of chitinase  (chi11) gene in the transformed plants with no development of symptoms on the transformed plants in comparison to control plants.

Name of the student

Ranjana Gupta

Admission Number

H-2005-04-D

Name of the Major Advisor

Dr S. V. Bhardwaj

Title of the thesis

Effect of Asparagus adscendens Roxb. on target receptor interactions

Abstract

            Asparagus adscendens Roxb. is a important medicinal plant having capability to be used as a drug to control AIDS. Based on this information the antiviral protein from this plant was isolated from leaf samples and purified to homogeneity by chromatography on CM-Sepharose. The molecular weight of antiviral protein was found to be 53,000 daltons. It exhibited antiviral activity against Potato Virus Y in its test host Datura metel. Presence of PVY was determined in induced resistant Tomato plants at the respective observations times by bioassay and ELISA. Asparagus antiviral protein mixed with PVY virus strongly decrease the number of local lesions on leaves of Datura metel. Total RNA was extracted from the leaves of Asparagus adscendens Roxb. and messenger RNA (mRNA was separated out using mRNA as a template, complementary DNA (cDNA) was synthesized and amplified by reverse transcription coupled PCR using gene specific primers. A product of 854bp was selected based on the size of AAVP of nucleotide was translated and its homology with already reported sequences of antiviral proteins was analyzed. After elution the product was purified and cloned into pGEM Easy vector and mobilized into E. coli strain, JM 109 and sequenced.  Phylogenetic analysis based on nucleotide sequence using Maximum Parsimony method inferred that AAVP has been found in the same cluster as that of Saponaria officinalis. Predictions for various restriction enzymes and PCR primers were carried out. Alpha helix secondary structure was predicted to dominate in the AVP sequence. Homology model of AAVP was built using EXOME HORIZON software and validated using Ramachandran plot.

Name of the student

Bandna Kumari

Admission Number

H-2008-07-M

Name of the Major Advisor

Dr (Mrs.) Amarjit K. Nath

Title of the thesis

Studies on α -Amylase inhibitor in local collections of Colocasia

Abstract

Corms of nine local collections of Colocasia of Himachal Pradesh were analysed for α–amylase inhibitor activity. Colocasia collections (C2) of Bhota village (Hamirpur district, H.P.) was found to contain maximum α–amylase unit inhibited per gram corm fresh weight  The α–amylase inhibitor from corms of Colocasia collections (C2) was purified to 17.21 folds with 61.61 percent recovery  using ammonium sulphate precipitation, gel filtration chromatography (Sephadex G-200) and ion exchange chromatography (DEAE-Sephadex). A single band of the purified inhibitor was obtained by Native–PAGE. SDS-PAGE revealed the purified inhibitor to be a monomer with molecular weight of 13,900 daltons. The nature of inhibition was found to be of non-competitive type as determined by Lineweaver-Burk plot and Dixon’s plot and the Ki value was 0.54 nmole.  The inhibitor was found to be most stable at 30°C and retained 81.50 percent activity at 70°C temperature. Inhibitor was found to have one pH optima of 6.9. The purified inhibitor was found to have inhibitory activity against α–amylases extracted from larvae of Callosobruchus chinensis, Tribolium castaneum, Corcyra cephalonica and  midgut α-amylase of Spodoptera littoralis. 100 percent larval mortality of Corcyra cephalonica was observed after 7 days when they were fed on wheat flour mixed with 0.0036 % (w/w) of purified inhibitor. Purified α–amylase inhibitor was also found to inhibit the activity of human salivary α–amylase. It also had inhibitory activity against potato α–amylases and reduction in sugar content of treated potato slices was observed. The purified inhibitor was found to be glycoprotein.

Name of the student

Pooja Verma

Admission Number

H- 2007- 13- M

Name of the Major Advisor

Dr. S. V. Bhardwaj

Title of the thesis

Molecular characterization of a coat protein gene of Potato virus Y infecting tomato in HP

Abstract

Potato virus Y is one of the most economically important and damaging species of family potyviridae. It stands at top of the ladder bringing down the economic returns below threshold level in tomato. Samples (1-6) showing prominent virus like symptoms were collected from six different locations of Solan and Shimla districts. PVY was seems to be is prevalent in Shimla district of H.P. as confirmed by serological detection of Shimla isolates using DAS- ELISA. cDNA of the virus nucleic acid was synthesized and PCR amplification gave amplicon of approximately 600bp when specific designed primers for PVY^O CP gene were used, which proved the presence of PVY^O in samples. Molecular characterization was performed by sequencing of CP region of the viral genome. The sequence of Kufri isolate has a chain of 549 nucleotides and translated nucleotide sequence has 103 amino acids. This sequence was submitted to Genebank and has been assigned Accession no. GQ891044. In Kumarsain isolate, amplicon was found to be 465 nucleotides long. The two isolates shared 94-96% similarity at amino acid level. Studies on coat protein gene sequence of different strain of PVY depicted that the test isolate PVY-Kufri (GQ891044) shared 3-98% and 5-100% homology at nucleotide and amino acid levels respectively with 107 other previously reported PVY strains of the world. The sequence alignment analysis revealed high ongoing evolutionary rate of CP gene of the virus. Three motifs were form in multiple sequence alignment of CP amino acid sequences. Phylogenetic trees were constructed by NJ and MP methods. Close phylogeny of test isolate of Kufri with China and Japan suggests the origin of test strain of PVY in either of these two countries or vice-versa.

Name of the student

Bhawna Saxena

Admission Number

H-2006-01-D

Name of the Major Advisor

Dr.(Mrs.) Rajinder Kaur

Title of the thesis

Identification of quantitative trait loci for resistance to Xanthomonas campestris pv. campestris  in Brassica oleracea var. capitata

Abstract

The present investigation on Brassica oleracea var. capitata was carried out with the objective to identify quantitative trait loci for resistance to Xanthomonas campestris pv. campestris by employing RAPD and SSR markers. Work was also done to identify genetics of resistance to blackrot in cabbage. F₂ population raised from a cross between January King Sel Improved (resistant parent) and Golden Acre (susceptible parent), was used as mapping population. 200ml of bacterial suspension containing 6.02 X 10⁶ bacterial cells per ml of suspension was used to screen 200 F₂ plants by scissor cut method and spraying through a sprayer. 110 resistant and 90 susceptible plants were recorded. The goodness of fit of observed ratio (9:7) was tested by Chi-square test with χ² value as 0.0625, hence, showing polygenic dominant control of resistance. Polymorphism was studied in seven cultivars of cabbage using 35 SSR and 20 RAPD primers. Results analyzed in form of dendrogram, scatter-plot and dissimilarity studies showed that the choice of parental lines i.e. January King Sel Improved as resistant parent and Golden Acre as susceptible parent, is most appropriate as both are at a good distance from each other. Phenotypic data for 200 F₂ individuals was recorded for symptom score and percentage severity towards blackrot reaction. Genotypic data was recorded on the basis of survey polymorphism in 200 F₂ individuals with 80 RAPD and 120 SSR primers. After polymorphism survey and segregation distortion, genotypic data of 2 RAPD and 30 SSR markers were retained for further analysis. Both phenotypic and genotypic data were used to construct a linkage map using software MAPMAKER ver. 2.0. A total of five linkage groups were constructed, spanning a distance of 1,538 cM with average distance between loci as 48.06 cM. A total of four QTL (XccDs1-1, XccDs2-1, XccDs3-1 and XccDs3-2) were identified for symptom score and four (XccDi1-1, XccDi3-1, XccDi3-2 and XccDi5-1) for logarithm of percentage severity. The effect of each QTL was ranging from 9.4% to 31.8%.

Name of the student

Anshu Sharma

Admission Number

H-08-01-M

Name of the Major Advisor

Dr  S K Sharma

Title of the thesis

“In vitro selection and regeneration of hybrid lily against the culture filtrate of Fusarium oxysporum f.sp. lilii”

Abstract

The reproducible protocol was optimized for in vitro selection and regeneration of hybrid lily against culture filtrate of Fusarium oxysporum f.sp. lilii. Callus initiation was recorded on medium AS₁₉ (MS basal medium +2 mg/l NAA +1.5 mg/l BA, 5% sucrose when incubated under dark condition. Calli were subjected to different concentrations of culture filtrate of Fusarium oxysporum f. sp. lilii where selective dose of culture filtrate was found to be  15 per cent on which 6.6 per cent cell survival of lilium cv. ‘Casa Blanca’ was recorded. The best medium for bulblet regeneration was ASR₄ (MS basal medium + 2mg/l NAA+1.5 mg/l BA and 3% sucrose) on which 75.0 per cent calli regenerating bulblets were recorded. Per cent survival of the bulblets regenerating from the selected calli when tested in vitro with spore suspension was 10 per cent respectively.

Name of the student

Era Vaidya

Admission Number

H-2008-09-M

Name of the Major Advisor

Dr. (Mrs.) Rajinder Kaur

Title of the thesis

Studies on datamining for development of dbEST – SSRs for    genotype identification in cauliflower (Brassica oleracea var. botrytis)

Abstract

The present investigation on cauliflower were carried out to develop EST – SSR markers from the EST database and to characterize different genotypes using genomic and dbEST – SSRs. EST sequences belonging to Brassica oleracea var. botrytis, Brassica oleracea var. capitata, Brassica oleracea var. italica, Brassica oleracea var. gemmifera and Arabdopsis thaliana, available on the EST database on the NCBI website (www.ncbi.nlm.nih.gov/nucest) were screened for SSR motifs using two programs i.e. MISA and SSRIT. 29 cauliflower, 165 Arabidopsis and 194 broccoli EST sequences were detected to contain SSRs and out of these, 16 sequences (two of cauliflower, 10 of broccoli and four of Arabidopsis thaliana) were selected for primer designing using PRIMER3 software. For polymorphism studies using 18 genomic SSR and 16dbEST – SSR primers, DNA from fresh, young leaves of 20 genotypes of cauliflower was isolated by CTAB method (Doyle and Doyle, 1987). Out of the 18 genomic SSRs, 16 gave amplification, eight of which were found to be polymorphic, revealing 65.85% polymorphism among the cauliflower genotypes. On the other hand, 13 out of 16 dbEST – SSRs amplified the genomic DNA, 11 primers being polymorphic, revealing 52.35% polymorphism. PIC (Polymorphism Information Content) value for all the primers was calculated and was found to range from 0.09 – 0.80 for genomic SSRs and from 0.09 – 0.60 for dbEST – SSRs. Similarity matrices and dendrograms were generated using NTSys ver.2.02h and dissimilarity matrices and rooted trees were generated using DARwin5 ver.5.0.155, for polymorphism data for both the primer sets. The dendrograms and rooted trees generated using UPGMA and Neighbour Joining algorithms, respectively, generated for both the SSR marker sets, divided the cauliflower genotypes into two main clusters, while genotype ‘US Agri Seeds’ was singled out from rest of the genotypes. Seven unique markers were given by three genomic SSRs and a single unique marker by one EST – SSR. These unique makers could identify three cauliflower genotypes.

Name of the student

Nisha

Admission Number

H-07-12-M

Name of the Major Advisor

Dr. D.K. Srivastava

Title of the thesis

Molecular characterization of cabbage (Brassica oleracea  L.  var. capitata.) using RAPD.

Abstract

            The present studies on ‘Molecular characterization of cabbage (Brassica oleracea var. capitata) genotypes using RAPD was carried out. The aim of this study was to investigate genetic relationship among different genotypes of cabbage. Sixteen cabbage genotypes namely, Golden acre, Best of all, BC-79, Giddeon, Green kid, Cabbage Mangla, General Cabbage, Darl Cabbage, EC-30191, AC-204, No-4, Green Emperor, Green Europium,KGAT-3, Pusa Drum Head and  No-29 were selected for the study. A total of 17 primers were tried to generate RAPD profile, out of these reproducible patterns were obtained with 7 primers. A total of 22 bands were obtained out of which  all were polymorphic  and 2 were unique bands. The percentage of total polymorphism was 100%. Similarity index was computed based on Jacquard coefficient and used for cluster analysis based on UPGMA. RAPD technology could be useful for identification of different accessions as well as accessing the genetic similarity among different accessions of cabbage. RAPD analysis could also be useful in certification scheme for releasing certified plant material.

Name of the studnt

Geetika Gambhir

Admission Number

H-2007-04-M

Name of the Major Advisor

Dr S. K. Sharma    

Title of the thesis

“In vitro production of bulblets in Lilium by anther and ovary cultre”

Abstract

In the present investigation entitled “in vitro production of bulblets in Lilium by anther and ovary culture”, the sterilized explants (anther and ovary) were inoculated on MS medium supplemented with different concentrations of growth regulators. The number of days for bulblet initiation decreased significantly when MS medium was supplemented with growth regulators in case of ovary, whereas anther failed to produce any bulblets. The highest per cent of explant forming bulblets and maximum number of bulblets were observed on MS medium supplemented with  2.0 mg/l NAA + 1.5 mg/l BA. The highest  average fresh weight per bulblet obtained when MS medium was supplemented with 2.0 mg/l NAA + 1.5 mg/l BA. The maximum per cent bulblet (100%) forming and maximum roots per bulblet (7.83) was observed with 0.5 mg/l NAA. The highest root length (20.60mm) was observed on the same concentration. Chromosome counts of root tips of regenerated plantlets indicated no change in ploidy.

Name of the student

Pardeep Pathania

Admission Number

H-2008-15-M

Name of the Major Advisor

Dr (Mrs.) Manju Modgil

Title of the thesis

Genetic identification of apple genotypes by using molecular markers

Abstract

Random amplified polymorphic DNA (RAPDs) and simple sequence repeats (SSR) were used to test the genetic variability and relatedness among 20 cultivars of apple. 28 RAPD and 5 SSR primers were used to amplify the DNAs of all 20 cultivars. RAPD generated 107 polymorphic bands with 65.6% polymorphism while SSR generated 15 polymorphic alleles with 71.4% polymorphism. The average PIC value was 0.70 for RAPD and 0.64 for SSR and the PIC value was the highest in OPA-16(RAPD) and IISRS-3; IISRS-18(SSR) primers. RAPD and SSR revealed different genetic similarities among 20 apple cultivars. The highest similarity was detected between ‘Fuji’ and ‘Fuji Rekaki’ (0.859) followed by ‘Thanedar early flowering’ and ‘Fuji’ by RAPD while between ‘Galaxy’ and ‘Jonadel’ (1.000) followed by ‘Neomi’ with ‘Arlet’, ‘Fuji’ and ‘Royal Gala’; ‘Fuji Rekaki’ with ‘Thanedar early flowering’ and ‘Reinette-Du-Canada’ by SSR. The dendrogram generated from RAPD data grouped ‘Fuji’ and ‘Fuji Rekaki’ in one cluster and ‘Jonadel’ and ‘Jonagold’ in other cluster, while dendrogram derived from SSR data grouped ‘Jonadel’ and ‘Jonagold’ in one cluster and ‘Arlet’ and ‘Royal Gala’ in other cluster, which is generally in accordance with the recorded pedigree information.

Name of the student

Ayesh Gaur

Admission Number

H-2008-06-M

Name of the Major Advisor

Dr. D.K. Srivastava

Title of the thesis

Studies on genetic fidelity of tissue culture raised plants of Himalayan poplar (Populus ciliata wall.)  

Abstract

The present investigation was undertaken with an objective of enhancing the frequency of plant regeneration in Himalayan poplar (Populus ciliata wall.) and assessing their genetic fidelity. A high efficiency plant regeneration protocol has been developed from leaf and petiole explants in Himalayan poplar (Populus ciliata wall.). The explants were excised, surface sterilized and cultured on shoot induction medium. A high efficiency of shoot regeneration was observed in leaf (80.00%) and petiole (85.70%) explants on MS medium supplemented with 0.024mg/l TDZ and 79.7 mg/l adenine and on MS medium supplemented with 0.004 mg/l TDZ and 79.7 mg/l adenine respectively. Root regeneration from in vitro developed shoots was observed on MS medium containing 0.10mg/l IAA. A protocol for high frequency plant regeneration has been standardized. Genomic DNA was isolated from leaves of randomly selected 20 in vitro raised plantlets of Himalayan poplar using CTAB method with some modifications. The quantified DNA was then subjected to PCR and a total of 25 primers were used for genetic fidelity studies. Among the 25 primers initially screened, 20 produced clear and scorable amplification products. A total of 94 fragments were amplified by 20 random primers out of which 80 were found to be monomorphic and a high degree of monomorphism (89.46%) was observed among in vitro raised plantlets of Himalayan poplar. On an average, 4.44 amplified fragments were observed per primer. Thus the technique of RAPD-PCR was found to be reliable to assess the genetic fidelity of tissue culture raised plantlets of Himalayan poplar (Populus ciliata wall.).