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

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.

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.

scholarly journals Norway spruce (Picea abies) genetic transformation with modified Cry3A gene of Bacillus thuringiensis.

2013 ◽  
Vol 60 (3) ◽  
Author(s):  
Jindřich Bříza ◽  
Daniela Pavingerová ◽  
Josef Vlasák ◽  
Hana Niedermeierová
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Bacillus Thuringiensis ◽  
Picea Abies ◽  
Norway Spruce ◽  
Somatic Embryos ◽  
Marker Gene ◽  
Selection Marker ◽  
Embryogenic Tissue ◽  
35S Promoter ◽  
Rt Pcr

Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.

scholarly journals Somatic embryogenesis of selected spruce species (Picea abies, P. omorika, P. pungens 'Glauca' and P. breweriana)

2011 ◽  
Vol 77 (3) ◽  
pp. 189-199 ◽  
Author(s):  
Teresa Hazubska-Przybył ◽  
Krystyna Bojarczyk
Keyword(s):  
Somatic Embryogenesis ◽  
Picea Abies ◽  
Sucrose Concentration ◽  
Medium Composition ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Embryogenic Cell ◽  
Nursery Production ◽  
Embryogenic Cell Lines

Somatic embryogenesis was studied in four spruce species (<em>Picea abies</em>, <em>P. omorika</em>, <em>P. pungens</em> 'Glauca' and <em>P. brewenana</em>) to determine if this method can be used for in vitro propagation of coniferous trees. The highest frequency of initiation of embryogenic tissue was obtained when mature zygotic embryos were used as explants. It ranged then from 10.8% (<em>P. brewenana</em>) to 23.75% (<em>P. omorika</em> and <em>P. pungens</em> 'Glauca'). The frequency of embryogenic tissue initiation was strongly affected by medium composition, i.e. addition of appropriate auxins (2,4-D, NAA, Picloram) and sucrose concentration (10-20 g<sup>-1</sup>"1). A lower frequency was obtained in <em>Picea omorika</em> (10%) when megagametophytes (endosperms with immature zygotic embryos) were used as explants. No emryogenic tissue was produced from hypocotyls, cotyledons and needles. A satisfactory frequency was achieved with the use of somatic embryos of <em>Picea abies</em> (30%). The proliferation of embryogenic cell lines of spruces was affected by medium type. The experiments resulted in production of somatic plantlets of <em>P. abies</em> and <em>P. omorika</em>. This enables the application of this method of spruce micropropagation for genetic and breeding research or for nursery production.

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 Standardized Genetic Transformation Protocol for Chrysanthemum cv. ‘Jinba’ with TERMINAL FLOWER 1 Homolog CmTFL1a

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 860
Author(s):  
Saba Haider ◽  
Yaohui Gao ◽  
Yike Gao
Keyword(s):  
Transgenic Plants ◽  
Genetic Transformation ◽  
Model Organism ◽  
Plant Morphology ◽  
Pcr Analysis ◽  
Transformation System ◽  
Transformation Protocol ◽  
Genetic Transformation System ◽  
Chrysanthemum X Morifolium

Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis. The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.

scholarly journals Organogenesis and Sonication-Assisted Agrobacterium-Mediated Transformation of Poplar Roots

2021 ◽  
Author(s):  
Thais Salete Giovanella ◽  
Juliana Degenhardt ◽  
Laudiane Bruna Zanella ◽  
João Carlos Bespalhok Filho
Keyword(s):  
Plant Regeneration ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Transformation Efficiency ◽  
Populus Alba ◽  
Quantitative Real Time Pcr ◽  
Regeneration Rate ◽  
Transformation Protocol ◽  
Cultivation Period ◽  
Kanamycin Concentration

Abstract Functional genomics along with genetic transformation and plant regeneration are used to identify genes of interest, comprising essential tools to obtain plants with desired characteristics. In this study, we described an organogenesis and a genetic transformation protocol for Populus tremula x Populus alba clone 717-1B4 using roots. In the organogenesis experiments the PGRs zeatin, BA and kinetin at different concentrations were evaluated. The effect of explant age was evaluated and at 30 days “old” roots (6 months old) showed a higher regeneration rate when compared to “young” roots (2 months old). In the genetic transformation experiments kanamycin concentration, the use of sonication (SAAT), co-cultivation period and explant age were evaluated. Sonication positively affected transformation, while co-cultivation time did not interfere. Regarding explant age, no statistical differences were observed. Quantitative real-time PCR (qPCR) data showed that among the six transgenic plants evaluated, two presented two copies and the others only one copy of the gene. In this study, efficient protocols of organogenesis and genetic transformation for poplar roots are presented with a transformation efficiency of 58%.

Transgenic Miscanthus lutarioriparius that co-expresses the Cry 2Aa# and Bar genes

2019 ◽  
Vol 99 (6) ◽  
pp. 841-851
Author(s):  
Yanan Xia ◽  
Jun Xu ◽  
Junyi Duan ◽  
Qingbo Liu ◽  
Hongmei Huang ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Herbicide Tolerance ◽  
Transformation Method ◽  
Energy Crop ◽  
Embryogenic Calli ◽  
Transformation Protocol ◽  
Agrobacterium Mediated Transformation ◽  
Domestic Species ◽  
Miscanthus Lutarioriparius

Miscanthus lutarioriparius, a domestic species of China, belongs to the genus of perennial rhizomatous Miscanthus and is a promising energy crop for biomass production. To establish an Agrobacterium tumefaciens transformation protocol and obtain transgenic plants co-expressing the Cry 2Aa# and Bar genes, an Agrobacterium-mediated transformation method for M. lutarioriparius was developed in this study. To inhibit tissue browning, optimized callus induction and regeneration media were developed. Embryogenic calli were efficiently induced from immature inflorescences on Murashige & Skoog (MS) medium containing 0.2 g L−1 citric acid. Agrobacterium tumefaciens EHA105 carrying pC3300 with the Cry 2Aa# and Bar genes were introduced into embryogenic calli. Six transgenic lines were selected by Basta screening and confirmed by genomic PCR. The herbicide tolerance of M. lutarioriparius transgenic plants was verified by spraying with glufosinate and expression of the Cry 2Aa# protein by ELISA. Therefore, an Agrobacterium-mediated transformation protocol for M. lutarioriparius was established and the co-expression of herbicide tolerance and Cry 2Aa# was successfully introduced with the system.

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