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Developing transgenic Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland.

Joshi M, Jha A, Mishra A, Jha B - PLoS ONE (2013)

Bottom Line: Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis.Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl.Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

View Article: PubMed Central - PubMed

Affiliation: Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India. bjha@csmcri.org

ABSTRACT
Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

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Genetic transformation of J. curcas via microprojectile bombardment.(A) Transient gus expression on embryo axis after 24 h of bombardment. Selection of transformants (B) during 1st round (8 days old), (C) during 3rd round (60 days old) on hygromycin. Regeneration of transformants; (D) multiple shoot induction, (E) multiple shoot regeneration (120 days old), (F) rooting (200 days old), (G) hardening of transgenic plant in plastic jar (250 days old). GUS assay of (H) non transformed leaf and (I) leaf of transgenic plant.
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pone-0071136-g002: Genetic transformation of J. curcas via microprojectile bombardment.(A) Transient gus expression on embryo axis after 24 h of bombardment. Selection of transformants (B) during 1st round (8 days old), (C) during 3rd round (60 days old) on hygromycin. Regeneration of transformants; (D) multiple shoot induction, (E) multiple shoot regeneration (120 days old), (F) rooting (200 days old), (G) hardening of transgenic plant in plastic jar (250 days old). GUS assay of (H) non transformed leaf and (I) leaf of transgenic plant.

Mentions: Five days pre-cultured embryo axes were bombarded at optimized parameters, i.e. 1 µm microcarrier size, 1100 and 1350 psi He pressure, with target distances of 9 and 12 cm, respectively [12]. After bombardment, 3–5 transformed embryos per shot per plate were selected randomly after 24 h and transient gus expression was assessed. Most of the explants showed discrete blue spots or regions (Figure 2a). Bombarded embryo axes were regenerated into transgenic plants after hygromycin selection (Figure 2b–f) as reported previously [12]. Putative transgenic plants with rooted shoots were transplanted into pots for hardening and acclimatization (Figure 2g and Table S1). Regenerated young leaves from putative transformed plants were assayed for constitutive expression of the gus gene using non-transformed leaves as control (Figure 2h–i).


Developing transgenic Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland.

Joshi M, Jha A, Mishra A, Jha B - PLoS ONE (2013)

Genetic transformation of J. curcas via microprojectile bombardment.(A) Transient gus expression on embryo axis after 24 h of bombardment. Selection of transformants (B) during 1st round (8 days old), (C) during 3rd round (60 days old) on hygromycin. Regeneration of transformants; (D) multiple shoot induction, (E) multiple shoot regeneration (120 days old), (F) rooting (200 days old), (G) hardening of transgenic plant in plastic jar (250 days old). GUS assay of (H) non transformed leaf and (I) leaf of transgenic plant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733712&req=5

pone-0071136-g002: Genetic transformation of J. curcas via microprojectile bombardment.(A) Transient gus expression on embryo axis after 24 h of bombardment. Selection of transformants (B) during 1st round (8 days old), (C) during 3rd round (60 days old) on hygromycin. Regeneration of transformants; (D) multiple shoot induction, (E) multiple shoot regeneration (120 days old), (F) rooting (200 days old), (G) hardening of transgenic plant in plastic jar (250 days old). GUS assay of (H) non transformed leaf and (I) leaf of transgenic plant.
Mentions: Five days pre-cultured embryo axes were bombarded at optimized parameters, i.e. 1 µm microcarrier size, 1100 and 1350 psi He pressure, with target distances of 9 and 12 cm, respectively [12]. After bombardment, 3–5 transformed embryos per shot per plate were selected randomly after 24 h and transient gus expression was assessed. Most of the explants showed discrete blue spots or regions (Figure 2a). Bombarded embryo axes were regenerated into transgenic plants after hygromycin selection (Figure 2b–f) as reported previously [12]. Putative transgenic plants with rooted shoots were transplanted into pots for hardening and acclimatization (Figure 2g and Table S1). Regenerated young leaves from putative transformed plants were assayed for constitutive expression of the gus gene using non-transformed leaves as control (Figure 2h–i).

Bottom Line: Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis.Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl.Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

View Article: PubMed Central - PubMed

Affiliation: Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India. bjha@csmcri.org

ABSTRACT
Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

Show MeSH
Related in: MedlinePlus