Efficient in vitro plant regeneration from shoot apices and gene transfer by particle bombardment in Jatropha curcas

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5–6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm−3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.

[1]  Daniel Lord,et al.  Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch , 2004, Plant Cell, Tissue and Organ Culture.

[2]  K. Ripley,et al.  Micropropagation of Euphorbia Lathyris L. , 2004, Plant Cell, Tissue and Organ Culture.

[3]  M. Jordan,et al.  Regeneration of Pouteria lucuma (Sapotaceae) plants in vitro , 1992, Plant Cell, Tissue and Organ Culture.

[4]  Mariam B. Sticklen,et al.  Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops , 2005, In Vitro Cellular & Developmental Biology - Plant.

[5]  E. Rech.  Morphological Factors Influencing Recovery of Transgenic Bean Plants (Phaseolus vulgaris L.) of a Carioca Cultivar , 1997, International Journal of Plant Sciences.

[6]  P. Toivonen,et al.  Regeneration of plantlets from in vitro cultured cotyledons of white spruce (Picea glauca (Moench) Voss) , 1988, Plant Cell Reports.

[7]  N. Albarello,et al.  Micropropagation of Rollinia mucosa (JACQ.) baill , 2001, In Vitro Cellular & Developmental Biology - Plant.

[8]  M. Sujatha,et al.  Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.) , 1998, Plant Cell Reports.

[9]  M. Ramesh,et al.  Transient expression of -glucuronidase gene in indica and japonica rice (Oryza sativa L.) callus cultures after different stages of co-bombardment , 2005 .

[10]  P. Ponmurugan,et al.  In vitro Multiple Shoot Proliferation and Plant Regeneration of Vanilla planifolia Andr. - A Commercial Spicy Orchid , 2006 .

[11]  P. Mukherjee,et al.  Somatic embryogenesis in Jatropha curcas Linn., an important biofuel plant , 2007, Plant Biotechnology Reports.

[12]  B. M. Johri,et al.  Morphogenesis in mature endosperm cultures of Jatropha panduraefolia , 1974 .

[13]  Sushil Kumar,et al.  Genetic variability and divergence studies in seed traits and oil content of Jatropha (Jatropha curcas L.) accessions , 2007 .

[14]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[15]  M. Sujatha,et al.  Rapid plant regeneration from various explants of Jatropha integerrima , 1993, Plant Cell, Tissue and Organ Culture.

[16]  P. Chand,et al.  High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.) , 2000 .

[17]  B. M. Johri,et al.  Morphogenesis in endosperm cultures , 1973 .

[18]  J. Hawker,et al.  In vitro Propagation of Latex-producing Plants , 1982 .

[19]  S. Peyachoknagul,et al.  High efficiency of stable genetic transformation in Dendrobium via microprojectile bombardment , 2007, Biologia Plantarum.

[20]  H. Raheman,et al.  Biodiesel production from jatropha oil (Jatropha curcas) with high free fatty acids: An optimized process , 2007 .

[21]  P. Bregitzer,et al.  Inheritance and Expression of Transgenes in Barley , 2003 .

[22]  S. D. Purohit,et al.  Micropropagation of Achras sapota through enhanced axillary branching , 1998 .

[23]  V. Srivastava,et al.  Accelerated production of transgenic wheat (Triticum aestivum L.) plants , 1996, Plant Cell Reports.

[24]  M. Sujatha,et al.  Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic Jatropha curcas L. , 2005, Plant Growth Regulation.

[25]  P. Lemaux,et al.  Increased Chromosomal Variation in Transgenic versus Nontransgenic Barley (Hordeum vulgare L.) Plants , 2000 .

[26]  Shiping Zhang,et al.  Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells , 1996, Plant Cell Reports.

[27]  K. Kartha,et al.  Effect of Growth Regulators on Plant Regeneration from Shoot Apical Meristems of Cassava (Manihot esculenta Crantz) and on the Culture of Internodes in vitro , 1979 .

[28]  M. Bevan,et al.  GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. , 1987, The EMBO journal.

[29]  R. Schilperoort,et al.  Plant Molecular Biology Manual , 1995, Springer Netherlands.

[30]  R. Islam,et al.  Regeneration of plantlets from in vitro cultured cotyledons of Aegle marmelos Corr. (Rutaceae) , 1994 .

[31]  C. Fauquet,et al.  Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis , 1997, Plant Cell Reports.

[32]  M. Sujatha,et al.  Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas , 1996, Plant Cell, Tissue and Organ Culture.

[33]  N. Geetha,et al.  High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeonpea (Cajanus cajan l.) , 1998 .

[34]  W. Crosby,et al.  A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants. , 1991, Gene.

[35]  R. Vallejos,et al.  Hygromycin resistance as an efficient selectable marker for wheat stable transformation , 1996, Plant Cell Reports.

[36]  I. Potrykus,et al.  Transient expression of visible marker genes in meristem cells of wheat embryos after ballistic micro-targeting , 1993, Planta.

[37]  P. Srivastava.  In vitro Induction of Triploid Roots and Shoots from Mature Endosperm of Jatropha panduraefolia , 1971 .

[38]  N. Saleh,et al.  Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) using the biolistic device , 1997 .

[39]  R. Twyman,et al.  Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns , 2000, Transgenic Research.

[40]  L. Xiao,et al.  Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment , 1997, Plant Cell Reports.

[41]  N. S. Shekhawat,et al.  Factors affecting in vitro clonal propagation of Prosopis cineraria , 1993, Plant Growth Regulation.

[42]  K. Kamo,et al.  Tissue specificity and expression level of gusA under rolD, mannopine synthase and translation elongation factor 1 subunit a promoters in transgenic Gladiolus plants , 1999, Plant Cell Reports.

[43]  S. Rogers,et al.  Extraction of DNA from plant tissues , 1989 .

[44]  N. Jayabalan,et al.  An efficient micropropagation system for Eclipta, alba—A valuable medicinal herb , 2005, In Vitro Cellular & Developmental Biology - Plant.

[45]  S. Xing,et al.  GENETIC TRANSFORMATION AND HYBRIDIZATION Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus , 2008 .

[46]  G. Galili,et al.  Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures , 1992, Molecular and General Genetics MGG.

[47]  S. Muthukrishnan,et al.  Generation of low copy number and stably expressing transgenic creeping bentgrass plants using minimal gene cassette bombardment , 2008, Biologia Plantarum.

[48]  A. Ganapathi,et al.  In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes , 2002, Agroforestry Systems.

[49]  J. Heller.  Physic nut: Jatropha curcas L. , 1996 .

[50]  P. Christou.  Genetic transformation of crop plants using microprojectile bombardment , 1992 .