Castor (Ricinus communis L.) is an important plant for production of industrial oil. The systemat... more Castor (Ricinus communis L.) is an important plant for production of industrial oil. The systematic evaluation of the molecular diversity encompassed in castor inbreds or parental lines offers an efficient means of exploiting the heterosis in castor as well as for management of biodiversity. Two DNA-based molecular marker techniques, viz., random amplified polymorphism DNA (RAPD) and inter simple sequence repeat (ISSR), were used to assess the genetic diversity in castor genotypes. Out of the 200 RAPD and 21 ISSR primers screened, a total of 35 polymorphic primers (30 RAPDs and 5 ISSRs), were used in this study. Amplification of genomic DNA of 22 genotypes, using RAPD analysis, yielded 256 fragments, of which 205 were polymorphic, with an average of 6.83 polymorphic fragments per primer. Number of amplified fragments with RAPD primers ranged from 6 to 12, with the size of amplicons ranging from 160 to 3000 bp in size. The polymorphism ranged from 27.2 to 100.0, with an average of 80.2%. The 5 ISSR primers produced 47 bands across 22 genotypes, of which 32 were polymorphic, with an average of 6.4 polymorphic fragments per primer. The number of amplified bands varied from 8 to 13, with size of amplicons ranging from 240 to 2700 bp. The percentage of polymorphism using ISSR primers ranged from 33.3 to 100.0, with an average of 68.1%. The Mantel test between the two Jaccard's similarity matrices gave r = 0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within the groups was not similar when RAPD and ISSR derived dendrograms were compared, whereas, the pattern of clustering of the genotypes remained akin in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.53 to 0.91, 0.55 to 1.00, and 0.51 to 0.93 with RAPD, ISSR, and combined dendrogram, respectively. Knowledge on the genetic diversity of castor can be used to future breeding programs for increased oil production to meet the ever increasing demand of castor oil for industrial uses as well as for biodiesel production.▶ Random amplified polymorphism DNA (RAPD) and inter simple sequence repeat (ISSR) markers can be aptly used to assess the genetic diversity in castor genotypes. ▶ Both ISSR and RAPD markers exhibited ample genetic polymorphism among the various genotypes of castor used in the study. ▶ The level of polymorphism revealed by RAPD and ISSR, indicate presence of abundant genetic diversity among the various castor genotypes used in the study.
RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay we... more RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.
The host range specificity ofAgrobacterium with five tea cultivars and an unrelated species (Arte... more The host range specificity ofAgrobacterium with five tea cultivars and an unrelated species (Artemisia parviflora) having extreme surface characteristics was evaluated in the present study. The degree ofAgrobacterium infection in the five cultivars of tea was affected by leaf wetness, micro-morphology and surface chemistry. Wettable leaf surfaces of TV1, Upasi-9 andKangra jat showed higher rate (75%) ofAgrobacterium infection compared to Upasi-10 and ST-449, whereas non-wettable leaves ofA. parviflora showed minimum (25%) infection. This indicated that the leaves with glabrous surface having lower 8 (larger surface area covered by water droplet), higher phenol and wax content were more suitable forAgrobacterium infection. Caffeine fraction of tea promotedAgrobacterium infection even in leaves poor in wax (Upasi-10), whereas caffeine-free wax inhibited bothAgrobacterium growth and infection. Thus, study suggests the importance of leaf surface features in influencing theAgrobacterium infection in tea leaf explants. Our study also provides a basis for the screening of a clone/cultivar of a particular species most suitable forAgrobacterium infection the first step inAgrobacterium-mediated genetic transformation.
Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel pl... more Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.▶ Toxic and non-toxic varieties of Jatropha curcas are chemically and genetically different. ▶ An efficient method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed. ▶ TDZ is most potent cytokinin for high-frequency plant regeneration.
Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential ... more Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.
Jatropha curcas, the energy plant has attained great attention in recent years because of its bio... more Jatropha curcas, the energy plant has attained great attention in recent years because of its biodiesel production potential; however, oil and deoiled cakes are toxic. A non-toxic variety of J. curcas is reported from Mexico. A simple and efficient protocol has been developed for plant regeneration using cotyledonary petiole explants of non-toxic variety of J. curcas. The percentage of induction of shoot buds (59.11%), and the number of shoot buds (5.01) per explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 2.27 μM thidiazuron (TDZ). These induced shoot buds multiplied when subcultured on MS medium supplemented with 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BAP), and 5.5 μM α-naphthaleneacetic acid (NAA) for 4 weeks and subsequent elongation achieved on MS medium supplemented with 2.25 μM BAP and 8.5 μM indole-3-acetic acid (IAA). Shoots more than 2 cm long were harvested and cultured on MS medium containing different concentrations and combinations of IBA, IAA, NAA, and 0.25 mg L−1 activated charcoal, and 19.91% rooting was achieved in 15 μM IBA, 5.7 μM IAA, and 16.5 μM NAA after 4 weeks with more than 90% survival rate.
An efficient and reproducible method for the regeneration of Jatropha curcas plants has been deve... more An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L−1 activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L−1 activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.
Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl expla... more Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L−1 TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca2+ concentrations to 0.22 g L−1 in the germination medium increased the number of responding explants.Induced shoot buds, upon transfer to MS medium containing 2 mg L−1 Kn (Kinetin) and 1 mg L−1 BAP (6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS medium supplemented with 1.5 mg L−1 IAA (indole-3-acetic acid) and 0.5 mg L−1 BAP and 3.01–3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant differences were observed. The elongated shoot could be rooted by pulse treatment on half-strength MS medium supplemented with 2% sucrose, 3 mg L−1 IBA (indole-3-butyric acid), 1 mg L−1 IAA, 1 mg L−1 NAA (α-naphthalene acetic acid) and subsequent transfer on 0.25 mg L−1 activated charcoal medium. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes.► Efficient and reproducible in vitro propagation protocol in Jatropha curcas via hypocotyls explants was developed. ► Regeneration was found to be genotype dependent. ► In vitro source of explants led to higher regeneration efficiency as compared to ex vitro source. ► Number of shoot buds induced were affected by the explants position on seedling axis. ► Age of seedling also influenced the regeneration potential which decreased with increase in age. ► Transient decrease of Ca2+ concentrations in germination medium increased percentage response.
A simple, high frequency, and reproducible method for plant regeneration through direct organogen... more A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l−1 activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.
Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential ... more Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explants. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), β-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explants, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD600 = 0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 μM acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 μM thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 μg ml−1 hygromycin. Selected shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 μM BA and 8.5 μM indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 μM indole-3-butyric acid (IBA), 5.7 μM IAA, 5.5 μM NAA, and 0.25 mg l−1 activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was achieved for leaf explants using this protocol.
Castor (Ricinus communis L.) is an important plant for production of industrial oil. The systemat... more Castor (Ricinus communis L.) is an important plant for production of industrial oil. The systematic evaluation of the molecular diversity encompassed in castor inbreds or parental lines offers an efficient means of exploiting the heterosis in castor as well as for management of biodiversity. Two DNA-based molecular marker techniques, viz., random amplified polymorphism DNA (RAPD) and inter simple sequence repeat (ISSR), were used to assess the genetic diversity in castor genotypes. Out of the 200 RAPD and 21 ISSR primers screened, a total of 35 polymorphic primers (30 RAPDs and 5 ISSRs), were used in this study. Amplification of genomic DNA of 22 genotypes, using RAPD analysis, yielded 256 fragments, of which 205 were polymorphic, with an average of 6.83 polymorphic fragments per primer. Number of amplified fragments with RAPD primers ranged from 6 to 12, with the size of amplicons ranging from 160 to 3000 bp in size. The polymorphism ranged from 27.2 to 100.0, with an average of 80.2%. The 5 ISSR primers produced 47 bands across 22 genotypes, of which 32 were polymorphic, with an average of 6.4 polymorphic fragments per primer. The number of amplified bands varied from 8 to 13, with size of amplicons ranging from 240 to 2700 bp. The percentage of polymorphism using ISSR primers ranged from 33.3 to 100.0, with an average of 68.1%. The Mantel test between the two Jaccard's similarity matrices gave r = 0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within the groups was not similar when RAPD and ISSR derived dendrograms were compared, whereas, the pattern of clustering of the genotypes remained akin in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.53 to 0.91, 0.55 to 1.00, and 0.51 to 0.93 with RAPD, ISSR, and combined dendrogram, respectively. Knowledge on the genetic diversity of castor can be used to future breeding programs for increased oil production to meet the ever increasing demand of castor oil for industrial uses as well as for biodiesel production.▶ Random amplified polymorphism DNA (RAPD) and inter simple sequence repeat (ISSR) markers can be aptly used to assess the genetic diversity in castor genotypes. ▶ Both ISSR and RAPD markers exhibited ample genetic polymorphism among the various genotypes of castor used in the study. ▶ The level of polymorphism revealed by RAPD and ISSR, indicate presence of abundant genetic diversity among the various castor genotypes used in the study.
RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay we... more RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.
The host range specificity ofAgrobacterium with five tea cultivars and an unrelated species (Arte... more The host range specificity ofAgrobacterium with five tea cultivars and an unrelated species (Artemisia parviflora) having extreme surface characteristics was evaluated in the present study. The degree ofAgrobacterium infection in the five cultivars of tea was affected by leaf wetness, micro-morphology and surface chemistry. Wettable leaf surfaces of TV1, Upasi-9 andKangra jat showed higher rate (75%) ofAgrobacterium infection compared to Upasi-10 and ST-449, whereas non-wettable leaves ofA. parviflora showed minimum (25%) infection. This indicated that the leaves with glabrous surface having lower 8 (larger surface area covered by water droplet), higher phenol and wax content were more suitable forAgrobacterium infection. Caffeine fraction of tea promotedAgrobacterium infection even in leaves poor in wax (Upasi-10), whereas caffeine-free wax inhibited bothAgrobacterium growth and infection. Thus, study suggests the importance of leaf surface features in influencing theAgrobacterium infection in tea leaf explants. Our study also provides a basis for the screening of a clone/cultivar of a particular species most suitable forAgrobacterium infection the first step inAgrobacterium-mediated genetic transformation.
Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel pl... more Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.▶ Toxic and non-toxic varieties of Jatropha curcas are chemically and genetically different. ▶ An efficient method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed. ▶ TDZ is most potent cytokinin for high-frequency plant regeneration.
Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential ... more Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.
Jatropha curcas, the energy plant has attained great attention in recent years because of its bio... more Jatropha curcas, the energy plant has attained great attention in recent years because of its biodiesel production potential; however, oil and deoiled cakes are toxic. A non-toxic variety of J. curcas is reported from Mexico. A simple and efficient protocol has been developed for plant regeneration using cotyledonary petiole explants of non-toxic variety of J. curcas. The percentage of induction of shoot buds (59.11%), and the number of shoot buds (5.01) per explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 2.27 μM thidiazuron (TDZ). These induced shoot buds multiplied when subcultured on MS medium supplemented with 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BAP), and 5.5 μM α-naphthaleneacetic acid (NAA) for 4 weeks and subsequent elongation achieved on MS medium supplemented with 2.25 μM BAP and 8.5 μM indole-3-acetic acid (IAA). Shoots more than 2 cm long were harvested and cultured on MS medium containing different concentrations and combinations of IBA, IAA, NAA, and 0.25 mg L−1 activated charcoal, and 19.91% rooting was achieved in 15 μM IBA, 5.7 μM IAA, and 16.5 μM NAA after 4 weeks with more than 90% survival rate.
An efficient and reproducible method for the regeneration of Jatropha curcas plants has been deve... more An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L−1 activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L−1 activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.
Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl expla... more Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L−1 TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca2+ concentrations to 0.22 g L−1 in the germination medium increased the number of responding explants.Induced shoot buds, upon transfer to MS medium containing 2 mg L−1 Kn (Kinetin) and 1 mg L−1 BAP (6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS medium supplemented with 1.5 mg L−1 IAA (indole-3-acetic acid) and 0.5 mg L−1 BAP and 3.01–3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant differences were observed. The elongated shoot could be rooted by pulse treatment on half-strength MS medium supplemented with 2% sucrose, 3 mg L−1 IBA (indole-3-butyric acid), 1 mg L−1 IAA, 1 mg L−1 NAA (α-naphthalene acetic acid) and subsequent transfer on 0.25 mg L−1 activated charcoal medium. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes.► Efficient and reproducible in vitro propagation protocol in Jatropha curcas via hypocotyls explants was developed. ► Regeneration was found to be genotype dependent. ► In vitro source of explants led to higher regeneration efficiency as compared to ex vitro source. ► Number of shoot buds induced were affected by the explants position on seedling axis. ► Age of seedling also influenced the regeneration potential which decreased with increase in age. ► Transient decrease of Ca2+ concentrations in germination medium increased percentage response.
A simple, high frequency, and reproducible method for plant regeneration through direct organogen... more A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l−1 activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.
Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential ... more Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explants. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), β-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explants, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD600 = 0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 μM acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 μM thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 μg ml−1 hygromycin. Selected shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 μM BA and 8.5 μM indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 μM indole-3-butyric acid (IBA), 5.7 μM IAA, 5.5 μM NAA, and 0.25 mg l−1 activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was achieved for leaf explants using this protocol.
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