Molecular Cloning of Korrigan and Sucrose Synthase Genes and Genetic Transformation of Eucalyptus for Cellulose Enhancement

Abstract

Eucalyptus (Family: Myrtaceace) is among the fastest growing woody plant in the world and comprises of nearly 700 species distributed throughout the world. It is grown world wide due to its wide adaptability, extremely fast growing nature and most importantly excellent wood and fiber properties which make it an important source of raw material for pulp and paper industry. Cellulose, the most abundant biopolymer on earth, consists of crystalline assemblies of parallel 1,4-–linked glucan chains. Current models envision plant cellulose biosynthesis to be a three step process: (1) plasma membrane-associated sucrose synthase (SUS) which directly channel the UDP-glucose substrate to cellulose synthesizing machinery (2) coordinately expressed multiple cellulose synthase genes, organized in the form of hexagonal rosettes, polymerize glucose monomers into glucan chains while recycling liberated UDP back to SUS and (3) a membrane-associated cellulase (-1,4–glucanase; korrigan (KOR), acts as an editor of newly produced glucan chains. These two genes (namely KOR and SUS) could be important target for up regulation of the cellulose content in wood. Thus, there is a possibility for improvement of selected clones of Eucalyptus for up-regulation of cellulose content through plant genetic manipulation using these genes. Plants of elite clone (self pruning and higher biomass productivity) of E. tereticornis growing at Thapar University Campus were selected for the present study. Cultures were established from nodal explants taken from freshly coppiced shoots of elite plant of E. tereticornis (10 years old). Cultures were initiated on MS medium supplemented with 2.5 μM BA and 0.5 μM NAA. Maximum numbers of shoots per culture vessel (342) were obtained on MS supplemented with 2.5 μM BA in combination with 0.5 μM NAA, whereas shoot elongation was achieved on MS medium supplemented with 0.1 μM BA 164 and 0.5 μM NAA. Out of the three tested cytokinins i.e BA, KIN and TDZ, BA was found to be better for both shoot proliferation and elongation followed by KIN and TDZ. Average number of elongated shoots per culture vessel was significantly higher (65) when smaller shoot clump (4-5 shoots/culture) were inoculated on medium containing 0.1 μM BA along with 0.5 μM NAA as compared to larger shoot clumps (15-20 shoots per clump, 54). However, shoots proliferated per culture vessel were significantly lower (245 shoots per culture vessel) when smaller shoots clumps were cultured as compared to larger shoot clumps (342 shoots per culture vessel). Therefore, it was concluded that larger shoot clumps are better for shoot multiplication whereas, for shoot elongation smaller shoot smaller shoot were required. The effect of two light sources i.e. PAR and CFL was examined on shoot proliferation and elongation. Both, shoot proliferation rates and number of shoots elongated per culture vessel were significantly higher in cultures incubated under PAR light as compared to those inoculated under CFL. Both, chlorophyll content and osmotic potential of sap were also higher in cultures incubated under PAR light. Development of an efficient shoot regeneration protocol (through shoot organogenesis and/or somatic embryogenesis) is prerequisite for the development of an efficient genetic transformation protocol. In the present study, effect of BA along with 2,4–D or NAA was examined on shoot regeneration from leaf segments taken from microshoots. Explants showed callus formation in all combinations. However, shoot regeneration was observed in some of the combinations only. Higher percent of explants showed shoot regeneration on medium containing 2,4–D as compared to those containing NAA. On medium supplemented with 5.0 μM BA and 1.0 μM 2,4–D, a maximum of 29.8 percent explants showed shoot regeneration with an average of 14.6 shoots per explant . 165 Leaf maturity was found to influence shoot regeneration potential of leaf segments (3rd to 7th leaf from top of microshoot). Maximum percent of explants showed shoot regeneration (40.6) with 16.0 shoots per explant, when explants were taken from 5th leaf from top. This response was found to decrease from mature and younger leaves. Addition of Cefotaxime (antibiotic generally used for the elimination of Agrobacterium from cultures after co-cultivation) increased shoot regeneration potential of explants. Both percent explants showing shoot regeneration and number of shoots regenerated per explants increased with increasing concentrations of cefotaxime. Others antibiotics like carbenicillin and cephalexin inhibited shoot regeneration potential of leaf explant. The shoot regeneration potential also varied amongst different clones of E. tereticornis. Amongst the three clones tested higher number of explants showed shoots regeneration as well as callus differentiation in case of clone ‘T1’. Histological studies indicate that some of the cells in the surface layer of the callus showed intense cell division and get organized into the shoot apical meristematic zone. These meristematic zones were later organized into the shoot bud like structures, which have vascular connection with the parent tissues. These shoot buds were further observed to grow into individual shoots. Thus direct shoot regeneration was observed from these explants. Clonal uniformity of in vitro plants with that of mother plant was established using RAPD and ISSR markers. Out of 40 primers used (20 each in RAPD and ISSR), 16 RAPD and 12 ISSR primers resulted in the amplification of scorable bands. These primers gave maximum of seven and minimum of two bands. Out of the total 133 markers obtained with RAPD and ISSR, 58 markers were scored with ISSR and 75 markers were scored with RAPD primers. Size of amplified markers ranged from 200 bp 166 to 2500 bp. Similarity in banding profile of RAPD and ISSR markers in in vitro propagated plants to that of mother plant indicates the clonal nature of these palnts. The effect of different auxins i.e. NAA, IAA and IBA and strength of MS medium (Full strength, 1/2 strength and 1/4 strength) was examined on rooting of microshoots. Maximum number of shoots rooted (80.66 %) on ¼ MS medium containing 5.0 μM IBA. Maximum numbers of roots per rooted shoot (4.25) were also observed on same medium, whereas maximum root length (2.90 cm) was recorded on full strength MS medium supplemented with 5.0 μM IBA. The effect of two light sources was also examined on rooting of microshoots. Percent shoots rooted, average root length and average numbers of roots per rooted shoot were also significantly higher in cultures incubated under PAR light. In the present study, special emphasis was given to investigate the factors affecting acclimatization of plants to increase survival rate and quality of planting material. The effect of different plant growth promoting bacterial isolates namely Bacillus subtilis and Pseudomonas corrugate was tested on growth and survival of micropropagated plants. Higher survival percentage (74.6 %) was recorded for plantlets which were incubated under PAR light as compared to plantlets incubated under CFL (70.4 %). Plant survival further increased to 84 % when plants were inoculated with Bacillus subtilis followed by plantlets inoculated with Pseudomonas corrugata (80.8 %) as compared to control plants (74.6 %) where no bacteria were inoculated. For the development of genetic transformation protocol, Agrobacterium tumefaciens mediated approach was adopted. A. tumefaciens strain EHA105 harboring binary vector pBI121 was used for the development of genetic transformation protocol. Tolerance limits (sensitivity) of the leaf segments for kanamycin was determine by culturing these on shoot induction medium supplemented with different concentrations of kanamycin 167 (0, 10, 20, 30, 40,50, 70 and 100 mg/l). It was observed that the presence of kanamycin in the regeneration medium caused a drastic decline in shoot regeneration potential as compared to control (cultured on medium lacking kanamycin). The effect of various factors namely pre-culture, bacterial density, mode of injury, incubation conditions. pH, phenolics etc. on transformation of leaf explants was evaluated. In the present study; leaves, pre-cultured on medium containing 50 μM acetosyringone for 2 days and incubated under 16-h light cycle showed maximum transient GUS activity (59 %). Addition of acetosyringone (100 μM) to the cocultivation medium recorded higher transient GUS activity (62 %) in the explants. Injury of explants with the help of hypodermic needle enhanced transient GUS activity. Lower pH was found to be beneficial for T-DNA delivery. Density of bacterial suspension used for explant infection also influenced transient GUS activity. A co cultivation period of 2 days was found to be optimum. An increase in the number of explants showing GUS activity was observed, when explants were co-cultivated in 16-h light regime. Following the genetic transformation, difficulties were encountered in achieving shoot organogenesis. Therefore certain changes were made in the basal MS medium to achieve shoot organogenesis. Potassium nitrate in MS medium was completely replaced with 990 mg/l of potassium sulphate, ammonium nitrate was reduced to 391.8 mg/l and 323 mg/l of ammonium sulphate was also added to the medium. Furthermore, mesoinositol was increased to 200 mg/l. Despite optimizing most of the parameters influencing transformation were optimized, lower transformation efficiency was observed. In the present study, it took 35-40 weeks to recover transgenic shoots. The transformation efficiency varied from clone to clone, and amongst the three clones tested namely, ‘T1’, ‘CE2’ and ‘Y8’, maximum genetic transformation efficiency was recorded in case of clone ‘CE2’. 168 The kanamycin resistant shoots showed positive GUS activity. These results were confirmed by PCR amplification of DNA fragments of 750 bp specific to nptII gene and 1500 bp specific to uidA gene from transgenic shoots. Amplification specific to 16s rRNA from DNA samples isolated from transgenic shoots was not observed indicating the complete elimination of bacteria from these tissues. Isolation and cloning of korrigan (KOR) and sucrose synthase (SUS) genes was carried out using poplar (Populus deltoides) cDNA. High quality RNA from young actively growing shoots of poplar was using CTAB method. The reported sequences of KOR and SUS were retrieved from the NCBI and primers were designed to amplify partial fragments (1800 bp fragment in case of KOR and 1200 bp in case of SUS). After successful amplification, these fragments were cloned in E. coli DH5 cells and sequenced. Sequences were analyzed using BLAST and sequence analysis of both the fragments showed 99 % similarity with reported sequences from other Populus species. Subsequently, on the basis of this information, primers for full length amplification of these genes (KOR and SUS) along with overhangs of restriction enzymes BamH1 and Sac1 in case of KOR and BamH1 and Sma1 in case of SUS were designed for the directional cloning of these genes in the transformation vector pBI121. Using these primers; fragments of 1860 bp for KOR and 2400 bp for SUS were amplified. After successful amplification, these fragments were cloned in E. coli DH5_ cells and sequenced. Homology of these fragments to their respective genes was confirmed by NCBI search tool BLAST. The identification of the ORF and amino acid sequences coded by these fragments was deduced using the ORF finder program. The predicted open reading frame (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, whereas SUS gene was found to encode a protein of 805 amino acids with a molecular mass of 92.29 kDa and an isoelectric point of 6.12. In case of KOR protein, a single transmembrane domain was detected between amino acids 72–94, a feature that is similar to other known KOR proteins, whereas no transmembrane domain was detected in case of SUS protein. Both the proteins have shown the 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 and SUS belongs to sucrose synthase family of proteins, a member of UDPglycosyltransferase (GT1) super family. After theses analyses, these genes were directionally cloned into binary vector (pBI121). The ligated products were transformed into competent cells of A. tumefaciens strain EHA105 for genetic transformation of E. tereticornis clone ‘CE2’. Utilizing the protocol developed for the genetic transformation, E. tereticornis clone ‘CE2’ was transformed with 5 different transgenic lines were selected for further analysis. The putative transformed shoots were analyzed for the presence of KOR using PCR. Amplification of fragment corresponding to original cloned gene confirmed the integration of KOR gene in the host genome. qRT- PCR analysis showed over expression of mRNA specific to KOR in transformed shoots of E. tereticornis. Expression level was found to vary in different transgenic lines.