An efficient Biolistic® transformation protocol for Picea abies embryogenic tissue and regeneration of transgenic plants

1999 ◽  
Vol 29 (10) ◽  
pp. 1539-1546 ◽  
Author(s):  
C Walter ◽  
L J Grace ◽  
S S Donaldson ◽  
J Moody ◽  
J E Gemmell ◽  
...  
Keyword(s):  
Picea Abies ◽  
Resistance Gene ◽  
Genetically Modified Organisms ◽  
Embryogenic Tissue ◽  
Nptii Gene ◽  
Transformation Protocol ◽  
Mature Embryos ◽  
Uida Reporter Gene ◽  
Polymerase Chain ◽  
Independent Transformation

An efficient Biolistic® transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

An efficient Biolistic® transformation protocol for Picea abies embryogenic tissue and regeneration of transgenic plants

1999 ◽  
Vol 29 (10) ◽  
pp. 1539-1546 ◽  
Author(s):  
C. Walter ◽  
L.J. Grace ◽  
S.S. Donaldson ◽  
J. Moody ◽  
J.E. Gemmell ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Picea Abies ◽  
Embryogenic Tissue ◽  
Transformation Protocol

scholarly journals Polyamines during somatic embryo development in Norway spruce (Picea abies [L.])

2009 ◽  
Vol 55 (No. 2) ◽  
pp. 75-80 ◽  
Author(s):  
J. Malá ◽  
M. Cvikrová ◽  
P. Máchová ◽  
O. Martincová
Keyword(s):  
Somatic Embryogenesis ◽  
Picea Abies ◽  
Norway Spruce ◽  
Somatic Embryos ◽  
Developmental Stages ◽  
Embryogenic Tissue ◽  
Growth Media ◽  
Mature Embryos ◽  
Proliferation Medium ◽  
Maturation Medium

Contents of free polyamines (putrescine, spermidine and spermine) were determined in different developmental stages of Norway spruce (<I>Picea abies</I> [L.] Karst.) somatic embryos by means of HPLC. Determinations were performed embryogenic tissue after 4 weeks of the growth on proliferation medium, after 2 and 5 weeks of the culturing on maturation medium, and 2 weeks after desiccation. Maturation of somatic embryos (after 5 weeks) was accompanied by increase of concentrations of putrescine (2.3 times) and spermidine (3.2 times). In comparison with above mentioned polyamines, spermine concentrations were significantly lower (4.3 times). Two weeks after desiccation, the concentrations of putrescine decreased 5.4 times and spermidine 2.2 times in comparison with mature embryos. To improve the efficiency of somatic embryogenesis of less responsive genotypes, the supplementation of growth media by polyamines is discussed.

scholarly journals Tolerance of Norway spruce (Picea abies [L.] Karst.) embryogenic tissue to penicillin, carbapenem and aminoglycoside antibiotics

2009 ◽  
Vol 55 (No. 4) ◽  
pp. 156-161 ◽  
Author(s):  
J. Malá ◽  
D. Pavingerová ◽  
H. Cvrčková ◽  
J. Bříza ◽  
J. Dostál ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Picea Abies ◽  
Norway Spruce ◽  
Aminoglycoside Antibiotic ◽  
Embryogenic Tissue ◽  
Transformation Protocol ◽  
Selective Media ◽  
Accelerated Growth ◽  
Embryogenic Tissues ◽  
Carbapenem Antibiotic

Somatic embryogenesis is conveniently utilized for the preparation of Norway spruce (<I>Picea abies</I> [L.] Karst.) transgenic clones by means of <I>Agrobacterium</I>. The establishment of successful transformation protocol requires to determine the tolerance of growing embryogenic tissue to antibiotics in culture and selective media. In 5 Norway spruce lines (genotypes) differences in the tolerance of embryogenic tissues to penicillin antibiotics (amoxicillin, carbenicillin, and ticarcillin), carbapenem antibiotic (meropenem) used for the <I>Agrobacterium</I> growth prevention, and aminoglycoside antibiotic (kanamycin) used in selective media were determined. Of the penicillin derivatives, amoxicillin was optimally tolerated in all lines and, in addition, its highest concentration accelerated growth in more rapidly growing lines. Ticarcillin was similarly tolerated but no growth acceleration was observed in any line. As regards carbenicillin, only the lowest concentration was observed to be well tolerated by all lines whereas all concentrations of meropenem were well tolerated in all lines except for slowly growing line 28, the growth of which was retarded by the concentration of 20 mg/l. The aminoglycoside antibiotic kanamycin was well tolerated by the embryonic tissue of all lines in the concentration of 10 mg/l and less in the concentration of 25 mg/l. The concentrations of 50 mg/l and 100 mg/l appeared as intolerable in all lines. Toxicity of kanamycin manifested at first in the browning and later in the growth cessation of embryogenic tissue.

Commercialization potential of somatic embryogenesis in black spruce tree improvement

1994 ◽  
Vol 70 (5) ◽  
pp. 593-598 ◽  
Author(s):  
G. W. Adams ◽  
M. G. Doiron ◽  
Y. S. Park ◽  
J. M. Bonga ◽  
P. J. Charest
Keyword(s):  
Somatic Embryogenesis ◽  
Clonal Propagation ◽  
Black Spruce ◽  
Tree Improvement ◽  
Embryogenic Tissue ◽  
Improvement Program ◽  
Mature Embryos ◽  
Somatic Plants ◽  
Number Of Individuals ◽  
Potential Tool

The somatic embryogenesis process was evaluated as a potential tool for operational vegetative propagation using individuals from families currently used in the J.D. Irving, Ltd. black spruce tree improvement program. Most families were responsive although the number of individuals within families capable of producing embryogenic tissue (ET) varied greatly (1–70%). Seventy-four percent of the ET clones produced mature embryos and most of these germinated. Greenhouse survival was initially low (11%) but improved in subsequent experiments to 45% as growing regimes were refined. Demonstration plantings of the resulting somatic plants were established at two sites in New Brunswick. A total of 206 clones were cryopreserved. The potential for integrating somatic embryogenesis techniques into tree improvement and stock production programs is discussed. Key words: tree improvement, somatic embryogenesis, clonal propagation, black spruce, biotechnology

Localization of Ds-transposon containing T-DNA inserts in the diploid transgenic potato: linkage to the R1 resistance gene against Phytophthora infestans (Mont.) de Bary

Genome ◽  
1996 ◽  
Vol 39 (2) ◽  
pp. 249-257 ◽  
Author(s):  
A. El-Kharbotly ◽  
J. M. E. Jacobs ◽  
B. te Lintel Hekkert ◽  
W. J. Stiekema ◽  
A. Pereira ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Phytophthora Infestans ◽  
Resistance Gene ◽  
Dna Sequences ◽  
Transgenic Potato ◽  
Potato Genome ◽  
Rflp Map ◽  
Polymerase Chain ◽  
R1 Gene ◽  
Map Location

The Dissociation transposable element (Ds) of maize containing NPTII was introduced into the diploid potato (Solanum tuberosum) clone J91-6400-A16 through Agrobacterium tumefaciens mediated transformation. Genomic DNA sequences flanking the T-DNAs from 312 transformants were obtained with inverse polymerase chain reaction or plasmid rescue techniques and used as probes for RFLP linkage analysis. The RFLP map location of 60 T-DNAs carrying Ds–NPTII was determined. The T-DNA distribution per chromosome and the relative distance between them appeared to be random. All 12 chromosomes have been covered with Ds-containing T-DNAs, potentially enabling tagging of any gene in the potato genome. The T-DNA insertions of two transformants, BET92-Ds-A16-259 and BET92-Ds-A16-416, were linked in repulsion to the position of the resistance gene R1 against Phytophthora infestans. After crossing BET92-Ds-A16-416 with a susceptible parent, 4 desired recombinants (Ds carrying T-DNA linked in coupling phase with the R1 gene) were discovered. These will be used for tagging the R1 gene. The efficiency of the pathway from the introduction to localization of T-DNAs is discussed. Key words : Solanum tuberosum, Phytophthora infestans, Ds element, transposon tagging, R genes, euchromatin.

scholarly journals Obtaining of transgenic alfalfa (Medicago sativa L.) and peanut (Arachis hypogaea L.) plants resistant to the herbicide Pursuit by Agrobacterium-mediated transformation

1970 ◽  
pp. 243-246
Author(s):  
S. M. Nifantova ◽  
I. K. Komarnytskyi ◽  
M. V. Kuchuk
Keyword(s):  
Gene Selection ◽  
Molecular Size ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Transformation Protocol ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Alfalfa ◽  
Arachis Hypogaea L ◽  
Selective Agents

Aim. The production of alfalfa and peanut cultivars with new properties is necessary. The purpose of this work was to develop Agrobacterium-mediated transformation protocol and to construct transgenic alfalfa and peanut plants resistant to herbicide Pursuit Methods. Genetic transformation was carried out using cocultivation of peanut and alfalfa explants with Agrobacterium tumefaciens strain GV3101 carrying genetic construct pCB004 containing mutant ahas/als gene and nptII gene. Selection was held on the solidified callus inducing medium with 50 mkg/l Pursuit. The selected callus clones were put on the regeneration medium with the same selective agents. Obtained regeneration lines were analysed using PCR-analysis. Results. 17 peanut and 14 alfalfa regeneration lines had positive signals after PCR analysis with DNA fragments of required molecular size for ahas/als and nptII genes. Conclusions. Transgenic alfalfa and peanut plants resistant to the herbicide Pursuit were obtained.Keywords: alfalfa, peanut, ahas/als gene, transformation.

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