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Title: Regeneration and genetic transformation of Populus deltoides Bartr. ex Marsh. for cellulose enhancement
Authors: Sharma, Saloni
Supervisor: Reddy, M Sudhakara
Kumar, Anil
Keywords: Populus deltoides;Shoot organogenesis, Somatic embryogenesis, Cellulose enhancement, BAP, IAA, Sucrose synthase,;Korrigan;Southern blotting, qRT-PCR
Issue Date: 23-Aug-2022
Abstract: Wood is the primary raw material for the production of pulp and paper. The Indian pulp and paper sector has made considerable progress during the last decades and characterized one of the key industries in India with a turnover of about 6.5 billion euros. India is considered the fastest-growing paper market globally. Populus spp. is gaining attention because of its faster growth and multipurpose wood. Species of Populus and their hybrids are grown for various uses such as plywood, timber, paper, fuelwood, and bioenergy production. Because of its higher commercial value, there is a need to adopt strategies for developing productive clones of Populus. Populus deltoides, a native of North America, is a widely planted species in India. For establishment of micropropagation protocol Populus deltoides elite clones G48 and L34 were selected for the present study based on their higher biomass productivity and wood quality. Further based on the regeneration and transformation efficiencies P. deltoides clone G48 was used to enhance the cellulose content to increase its economic value. A successful micropropagation and regeneration protocol was established. Agrobacterium tumefaciens mediated transformation protocol was optimized for cloning of two essential genes, Korrigan and sucrose synthase, directly involved in cellulose biosynthesis. Southern blot analysis was used to confirm the stable integration of transgene and check the copy number of the gene, which was further confirmed by qRT-PCR. The genes were successfully cloned, and their expression pattern was studied. This study has established a highly efficient regeneration and transformation protocols. The direct shoot organogenesis was achieved from leaf explant of two commercially important clones of Populus deltoides on MS medium enriched with 15 mg/L adenine sulphate, 5 mg/L Ascorbic acid, 250 mg/L (NH4)2 SO4 (referred to as PD1 medium) supplemented with 2.5 µM each of 6-benzylaminopurine and indole-3-acetic acid. Higher shoot organogenic potential was recorded from the explants of clone G48 as compared to clone L34. The age of leaf explant also affected the shoot organogenic potential, and maximum shoot organogenesis was recorded in case of 5th leaf from the top of microshoot. Histological studies revealed altered cell division resulting in the formation of meristematic pockets after 5 days of culture, these meristematic pockets grew into dome protuberances by 10th day. Organized shoots were visible after 15 days of culture. A clear three phases of shoot organogenesis viz induction (0–4 days), initiation and organization (4–10 days) and growth (11–16 days onwards) were observed. Marked variation in the activity of enzymes such as catalase, peroxidase, polyphenol oxidase and acid phosphatase were observed during these phases. The activity of these enzymes was found to increase in cultures grown on the medium resulting in shoot organogenesis during shoot development (after 7 days of culture). Different hormones reported to induce somatic embryogenesis were tested by varying their concentrations to induce somatic embryos. A combination of 2,4-D and BAP was used at various concentrations. Among them a concentration of 2.5 µM BAP and 5.0 µM 2,4-D showed highest frequency of nodular compact callus induction. These calli did not show any regeneration thereafter. Subsequently various concentrations of NAA were tested and it was found that 25 µM NAA showed induction of direct embryogenesis from leaf explants. We also tested a combination of NAA and BAP at different concentrations for induction of somatic embryogenesis. Combination of NAA and BAP gave the best results for induction of somatic embryogenesis. The highest rate of somatic embryogenesis was observed at the combination of 5 µM each of NAA and BAP. Histological studies was performed to confirm these embryo-like structures. The formation of spherical appearance proved the first key feature of somatic embryo development, followed by the cotyledonary initials represented by a precise heart-shaped stage. This heart-shaped embryo progressed toward a late-heart shaped or torpedo-shaped embryo that achieved well-developed and mature cotyledons. In the present study sucrose synthase (PdeSuSy) and korrigan gene (PdeKor) were cloned, characterized and overexpressed in Populus deltoides Bartr. ex Marsh. (Clone G48). The cloned PdeKor gene had an 1860 bp open reading frame (ORF). The predicted ORF of KOR gene was found to encode a protein of 619 amino acids with a calculated molecular mass 169 of 68.45 kDa and an isoelectric point of 8.92. In KOR protein, a single transmembrane domain was detected between amino acids 72–94, a feature that is similar to other known KOR proteins. Kor protein showed presence of glycosylations and phosphorylation sites in their sequences which indicate that both proteins undergo post translational modifications for proper functioning. The predicted KOR belongs to glycosyl hydrolase family 9 (GH9) of proteins. PdeSuSy consisted of an open reading frame of 2412 bp encoding a protein of 803 amino acids. Sequence analysis PdeSuSy showed glycosylation and phosphorylation sites with no transmembrane domain. The PdeSuSy gene was mobilized into binary vector pBI121 under CaMV 35S promoter with a specifically designed strategy due to the presence of multiple restriction sites of the vector in the gene. Transformation of G48 clone of P. deltoides was carried out using leaf as explants. These explants were highly sensitive to kanamycin and tolerated only up to 10 g/ml. The transformants were selected on PD medium (modified MS) containing 10 g/ml kanamycin and 500 g/ml cefotaxime. Transgenic shoot lines overexpressing PdeSuSy were recovered from infected explants on a selection medium supplemented with 2.5 M each of 6-benzyl adenine and indole-3-acetic acid. Since PdeSuSy and PdeKor are present in P. deltoides, to confirm the stable transformation of recombinant vector in the regenerated plant, we conducted the PCR of selection marker, that is, neomycin specific gene. DNA isolated from the leaves of putative transgenic and untransformed shoots was used for PCR amplification of DNA fragments specific to the nptll gene present in the pBI121 vector. The amplified PCR product was to confirm stable integration of transformed genes into the cells. A fragment of about 750bp specific to nptII was amplified. Plasmid DNA of pBI121 was used as a positive control, and leaf explant from untransformed shoots of P. deltoides were the source of DNA for negative control. Overexpression of PdeSuSy and PdeKor gene in transgenic lines of P. deltoides was studied using real-time PCR. PdeSuSy and PdeKor expression was studied by amplifying the genes from randomly excised elongated shoots from each line and was compared to untransformed shoots. We observed a 2–3-fold increase in expression of PdeKor and 3–4 folds increase in PdeSuSy gene in all the transformed lines. Southern blotting was performed to confirm the presence of the gene of interest in the genomic DNA of P. deltoides and to rule out the pitfalls of potential false positives. In case of PdeKor the results from Southern blotting showed a copy number of 1–3 gene copies in different transgenic lines. PdeSuSy results showed a copy number of 1–2 gene copies in different transgenic lines. The results for quantitative PCR are either similar or showed higher copy number than southern blot. The presence of T-DNA tandem repeats might be the cause of variation in copy number. The determination of cellulose content was done according to a standardized protocol (Updegraff, 1969). PdeKor showed no increase in cellulose content. Transgenic Line 2 in case of PdeSuSy transformants showed a 4% increase in cellulose content in comparison to control.
Description: Ph.D. Thesis
Appears in Collections:Doctoral Theses@DBT

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