Description of specific and original methodologies in LIH

In Lithuanian Institute of Horticulture (LSDI) DNA extraction, search for PCR and molecular markers is performed when implementing various projects and tasks. When implementing Lithuanian priority science field project “Plant Adaptation and its Control Using Biotechnology Measures” (ABIOTECHA) supported by Lithuanian State Science and Studies Foundation (contract no. C-01/2006) (head dr. Aušra Ražanskienė, Institute of Biotechnology), the following biotechnology works were performed:

  1. Creation of forms of currants resistant to reversion virus (BCRV), using post-transcription repression of gene activity and scFv expression. DNA of various species of currants was extracted for controlling purpose.
  2. Identification of genes resistant against strawberry phytophtorosis (Phytophthora fragaria) Rpf, and construction of a vector transmitting Vf genes with various promoters as well as investigation of expression in transformed plants in vitro. DNA of different species of strawberries (14) and apple-trees (21) was extracted.
  3. Investigation and modification of genetic factors, determining resistance against cold, in various orchard and model plants, DNA of strawberries (4 species), quince (3 clones), sweet cherries and cherries (6 species) as extracted.
  4. Expression of Rol B gene, controlled with the help of different promoters, in ligneous plants. DNA of quinces (3 clones) as extracted.
  5. Cloning of genes, controlling the evolution cycle of the plant (blossoming induction, formation of generative organs, cropping), creation of vectors. DNA of thale cress (Arabidopsis thaliana) was extracted.

Currently implemented Industrial Biotechnology Development in Lithuania in 2007 – 2010 program project “Resistance against Cold and Regulation of it Using Biotechnology Measures” (RESISTANCE AGAINST COLD) (head dr. Rytis Rugienius, Lithuanian Institute of Horticulture) works are also related to the extraction of DNA. DNA is extracted from strawberries (Fragaria ananassa), quince (Cydonia oblonga), rape (Brassica napus), and maiden grass (Miscanthus sp).

Implementing another project of the same program “Fruity Plants – Producers of Natural Anthocyanins” (ANTHOCYANINS) (head dr. Tadeušas Šikšninas) DNA of black currants (Ribes nigrum) was extracted, it was planned to extract the DNA of black chokeberry (Aronia melanocarpa), black lace elderberry (Sambucus nigra) and other plants.

Despite the above mentioned plants in other and previous projects the DNA of Percian cumins (Carum carvi), peonies (Paeonia sp.) as well as other plants was extracted.

This work describes extraction of genomic DNA from apple-trees in accordance with modified protocol of Doyle and Doyle (1990). For extraction of DNA and further Vf1 gene synthesis with the help of PCR the following species of Malus domestica were used: “Freedom”, “Prima”, “OR33T90”, “Štaris”, “Aldas”, “Skaistis” and “Rudenis”. For construction of the vector with Vf1 gene the common DNA of apple-tree species “Freedom” as used. The common DNA was extracted from the leave tissue homogenized in liquid nitrogen (0.2 g) using the modified CTAB method (Doyle and Doyle, 1990). This method was applied for the orchard plants used in the work (apple-trees, also sweet cherries, quince and strawberries). Extracting DNA from cumins, it was gained contaminated with phenol compounds, therefore, it was necessary to perform additional cleaning; using filters with glass dust (Eppendorf et al.).

Scientists from Lithuanian Institute of Horticulture used RNA extraction mainly seeking afterwards to synthesize the copies DNA. cDNA is used for the cloning of plants and the viral genes inside them, for identifying viruses, for investigating gene expression. The most important problem, when extracting vegetable RNA – its quick fragmentation due to the impact of R-Nasis present in the air and other places. The work (at least the initial stages) is performed in the laminary box.

The work also provides information about the authors of the above mentioned methodologies, workers of the Lithuanian Institute of Horticulture: D. Gelvonauskienė, B. Gelvonauskis and R. Rugienius.

Seeking to extract the fragments of the virus of double blossoms of black currants (project ABIOTECHA) RNA extraction as performed using Qiagen RNAeasy Plant Mini Kit set. Approximately 100 mg of tissue is homogenized in liquid nitrogen with the help of the pestle and pistil, afterwards the work is performed as shown within the recommendations of the manufacturer of the set. RNA extracted from 100 mg of tissue was re-suspended in 50 µl of water; concentration determined using spectrophotometer – approximately 0.1 mg/ ml.

PolyA+ RNA was extracted from the common RNA using Qiagen Oligotex mRNA mini Kit set following the recommendations of the manufacturer. From 10 µg of the common RNA received polyA+ RNA is suspended in 20 µl of water, the concentration of the received RNA is about 5 µg/ ml.

Polymerase chain reaction (PCR) is a method allowing receiving a large amount of specific DNA fragments in vitro from a small amount of complex compound. Two primers are necessary for the implementation of polymerase chain reaction, which would restrict the investigated sequence and are directed at 5’->3’. The primers hybridise with opposite DNA chains and having added DNA polymerase the synthesis reaction takes place on both of the chains of the initial DNA, restricted by the primers. PCR is a very sensitive method, used for solving most of the molecular biology, diagnostics, and population genetics problems. PCR method is based on reproduction of enzyme DNA fragments, using oligonucleotide primers. These primers with the help of polymerase allow reproducing certain DNA sequences. At the very beginning DNA chains are distinguished under high temperatures. Later on the temperature is decreased and the primer attaches to the complementary DNA fragment. Afterwards polymerisation reaction takes place and complementary chains are produced. These cycles are repeated several times. For the development of the method a significant moment was the usage of thermostable polymerase from thermophilic bacterium Thermus aquaticus (taq DNA poolymerase).

PCR of the Lithuanian Institute of Horticulture (LSDI) was used in most of the above mentioned projects. Samples of PCR methodologies performed by the Lithuanian Institute of Horticulture (LSDI):

For the construction of the vectors, transmitting Vf genes with various promoters (project ABIOTECHA), during the amplification of DNA fragments of Vf1 gene pairs of primers were used: VF1ABF1 and VF1ABR1 (at 61 ºC temperature), VF1BCF1 and VFBCR1 (at 61 °C temperature), VF1SPF and VF1SPR (at 47 °C temperature) (Annex no. 3). Primers selected according to the sequence AJ297739 of Gene Bank (NCBI), publicised on the internet http://www.ncbi.nlm.nih.gov/entrez/. PCR: DNA denaturation was performed at 94 ºC temperature within 4 min, later 35 cycles at 94 ºC temperature for 1 min 45 s, depending on the primers: 47 ºC, 61 ºC 1 min 45 s, 72 ºC 2 min 30 s, at the end – 72 ºC for 6 min. 1.25 units of polymerase mixture High Fidelity PCR Enzyme Mix* (“Fermentas”) were used for the PCR reaction. The reaction was performed, at 1.5 mM MgCl2, 1 µM primers and 0.2 mM dNTP. Amplified Vf1 gene fragments of the expected size, affected by XbaI and HindIII restriction endonucleases (“Fermentas”) and purified using 1 % agarose gel, using Cyclo-Pure Gel Extraction Kit from Agarose Slides set (“Amresco”). The purified fragments of Vf1 gene were cloned into the plasmid pUC57R.

For the extraction of strawberry resistance to phytophorosis gene Rpf1 marker using PCR method (project ABIOTECHA), polymorphous primer OPO16 offered by Van de Weg (1997) was used. The common DNA was extracted from the species of strawberries and hybrid clone 005001-2 (“Selen” x “Tristar”), “Anapolis”, “Redgauntlet”, 940101 (“Guardian” x “Pegasus”)‚ “Elsanta”‚ “Selen”‚ “Tristar” plants, using CTAB method (Doyle and Doyle, 1990). The first cycle of PCR was performed 95 ºC 4 min, later on 30 cycles 95 ºC 1 min, 35 ºC 1 min, 72 ºC 1.30 min. 1 unit of Taq DNA polymerase (“Fermentas”) was used for the PCR reaction, reaction carried out at 1.5 mM MgSO4, 0.2 mM dNTP, 1µM of primer OPO16. After amplification, having performed DNA electrophoresis, it was found out that the electrophoregrams of DNA of resistant and non-resistant species differ by one DNA fragment. This DNA fragment/ marker of the species “Elsanta” was extracted from the gel using “AMRESCO” set in accordance with the recommendations provided by the manufacturer. The extracted fragment was afterwards cloned into plasmid pTZ57 using the cloning set InsT/AcloneTM PCR Product Cloning Kit, (“Fermentas”) in accordance with the recommendations of the producer. This DNA fragment (450 bp) was sequenced in the Sequencing Centre of Institute of Biotechnology (BTI). Primers specific to the sequence of the marker (SCAR) were selected. PCR was used just like using polymorphous primer, except for the fact that the temperature of attachment of the primers was 51 ºC.

Investigation and modification of genetic factors, controlling resistance to cold in various orchard and model plants (project ABIOTECHA). Extraction of common RNA of winter wheat “Duoniai” and thale cress was performed using the set Eppendorf Perfect RNA, Eukaryotic, Mini (“Grida”), following the recommendations of the manufacturer. RNA of wheat and thale cress was extracted from the plants, incubated at the temperature of +4 °C from 4 to 40 days.

COR 15 and COR47 gene cDNA synthesised using the set RevertAid™ H Minus First Strand cDNA Synthesis Kit (“Fermentas”), applying the methodology provided by the manufacturer and primers CHT46F1, COR15F3 and COR47F3.

For PCR reaction dedicated to amplification of cDNA 40 µl of reaction mixture as used, it comprised 2.5 units of PCR polymerase mixture (High Fidelity Enzyme Mix, “Fermentas”, Lithuania), 0.8 µg cDNA, 1.5 mM MgCl2, 1 µM primers: COR15F3 and COR15R3, COR47F3 and COR47R5 (Annex no. 1), 0.2 mM dNTP. Polymerase chain reaction conditions: 95 ºC 4 min, 94 ºC 1 min 30 s, 61 ºC 1 min 20 s, 72 ºC 3 min, 72 ºC 6 min (35 cycles).

Amplified fragments of the investigated genes, purified from agarose gel, using Cyclo-Pure Gel Extraction Kit from Agarose Slides set (“Amresco”). Isolated DNA fragments with COR15 and COR47 genes were cloned into pUC57 plasmid. Transformed bacteria were selected growing them in LB media with ampicillin, IPTG and X-Gal. Fragment was sequenced in Sequencing Centre (Institute of Biotechnology, Lithuania).

Extraction of DNA plasmids, restriction, transformation was performed according to the described methods (Maniatis et al., 1982; Sambrook et al., 1989).

Primers were selected according to the sequences publicised on the internet http://www.tigr.org/tdb/e2k1/ath1/ath1.shtml by the Gene Bank.

The expression of rolB gene, controlled by different promoters, in ligneous plants (project ABIOTECHA). For the extraction of rolB gene Rhizobium rhizogenes stem pRiA4 was used bought from ATCC-LGC Promochem. Rhizobium rhizogenes total DNA was extracted using the set “Genomic DNA Purification Kit” (“Fermentas”), following the recommendations provided by the manufacturer. Extracting rolB gene the first cycle of the polymerase chain was performed 95 oC 5 min, 58 oC 1 min, 72 oC 2.5 min, afterwards 30 cycles 95 oC 1 min, 58 oC 1 min, 72 oC 2.5 min. 1 unit of Taq DNA polymerase (“Fermentas”) was used for PCR reaction, the reaction was performed with 1.5 mM MgSO4, 0.2 mM dNTP, 1 µM of primers. The following primers were used: rolB5pro, rolB3ter1, rolB3ter2, rolB5kod and rolB3kod. XbaI cloning target was inserted into all the primers.

Seeking to ensure, whether transformation actually took place in the regenerants after of cultivation of explants with A. tumefaciens, DNA of the regenerated plants was extracted and PCR was performed, using rolB characteristic primers ROLB661F and ROLB661R. The common DNA was extracted from quince plants grown in vitro, using CTAB method (Doyle and Doyle, 1990). First cycle of PCR was performed 95 ºC 4 min, afterwards 30 cycles 95 ºC 1 min, 49 ºC 1 min, 72 ºC 1.30 min. 1 unit of Taq DNA polymerase (“Fermentas”) was used for PCR reaction, the reaction was performed at 1.5 mM MgSO4, 0.2 mM dNTP, 1 µM of primers.

For the cloning* (project ABIOTECHA) of genes, controlling plant evolution cycle (blossoming induction, formation of generative organs, cropping) the common DNA of thale cress (Arabidopsis thaliana) was extracted from the plants kept in the daylight, using CTAB method (Doyle and Doyle, 1990). For the synthesis of VRN2 gene cDNA, RNA was extracted from plants incubated for 20 days at +8 °C temperature. For PHYA and PHYB gene cDNA synthesis, RNA was extracted from the plants kept in daylight, 660 nm together with 735 nm, 660 nm wave length light. Extraction of the common RNA of thale cress was performed using Eppendorf Perfect RNA, Eukaryotic, Mini (“Grida”) set in accordance with the recommendations provided by the manufacturer.

When performing the screening of apple-tree species in accordance with the presence of Vf gene (Gelvonauskienė et al, 2005), different species of the apple-trees were used for the investigation: “Noris”, “Papirovka” – sensitive to scabs, “Antonovka”, “Katja”, “Tellissaare” and “Auksis” – resistant, “Prima”, “Štaris”, “Aldas”, “Skaistis”, “Rudenis” – immune to scabs. DNA extraction from the apple-trees was performed according to Delaport (1983). 1 unit of Taq DNA polymerase was used for PCR (MBI “Fermentas”, Lithuania), 1.5 mM MgSO4, 0.2 mM dNTP and 1µM of each oligonucleotide primer. DNA denaturation took place in the following way: 95 ºC 4 min, afterwards 35 cycles – 94 ºC 1.15 min, 47 ºC 1.15 min and 72 ºC 2 min.

The results showed that the species “Prima”, “Aldas”, “Skaistis” and “Rudenis”, which have immunity against scabs, 1 - 5 Venturia ineaqualis races, have DNA fragment of 500 bp size, characteristic to Vfa1 gene (see Figure). Immune species “Štaris” did not have this fragment. Nevertheless, this fragment was characteristic to the species “Tellissare”, which has a polygenous mechanism of resistance against the scabs. These fragments were not identified in DNA of the apple-tree species “Noris” and “Papirovka”, which are sensitive to scabs, just like in the species, which have polygenous resistance (“Katja” and “Auksis”).

Part of the data on application of molecular markers is provided in the section about PCR

For the construction of the vectors, transmitting Vf genes with various promoters, on the basis of the sequences provided in the gene bank (http://www.ncbi.nlm.nih.gov), primers were synthesised, which were characteristic to Vf1 gene sequence. Using these primers, applying PCR method, DNA fragments – Vf1 gene markers were synthesised, which obviously proved the presence or no presence of this gene in the investigated species of the apple-trees (Gelvonauskienė et al, 2005).

For the extraction of strawberry resistance to phytophtoresis gene Rpf1 marker, applying PCR method, using polymorphous primer OPO16, fragments of DNA of the strawberries were synthesized, one of which (430 bp) was related with strawberry resistance against phytophtoresis (Phytophthora fragariae) gene Rpf1 (Van de Weg, 1997). Nevertheless, this marker is difficult to identify among other DNA fragments. That is characteristic to most of the polymorphous primers, which are used for RAPD analysis. Therefore, we decided to synthesize the primers characteristic to the DNA sequence of this marker, application of which would allow synthesizing during PCR only one DNA fragment, which would be related to Rpf1, but easily identified. For this purpose this DNA fragment/ marker of the species “Elsanta” was extracted from the gel using “Amresco” set in accordance with the recommendations provided by the manufacturer. The extracted fragment afterwards was cloned into the plasmid pTZ57, using the cloning set InsT/AcloneTM PCR Product Cloning Kit (“Fermentas”) following the recommendations of the manufacturer. The cloned DNA fragment (450 bp) was sequenced in the Sequencing center of the Institute of Biotechnology (BTI). Primers unique to the sequence of the marker (SCAR) were selected (see the table). Using this marker it was easy to identify the species of the strawberries which had or did not have Rpf1 gene (Figure 4) (Rugienius et al, 2006).

When implementing the project together with agricultural subject: identification of molecular DNA markers for identifying Lithuanian species of sweet cherries, the common DNA of Lithuanian and foreign species of sweet cherries was extracted, polymerase chain reaction (PCR) were carried out, DNA fragments were amplified using electrophoresis. Polymorphism of S alleles was investigated in 13 Lithuanian sweet cherry species of different ripening period and in 12 sweet cherry species - standards. Polymorphism of simple sequence repeats (SSR) was investigated in Lithuanian species of sweet cherries.

The results of the project will be used by scientists of genetics, breeding, when analysing the origins of the species present in the working collections and their relations, the gardeners, when selecting the composition of the species within sweet cherry sections, seeking to ensure cross-compatibility and stable cropping, owners of the licences of the species, reproducers of seedlings, exporters, seeking to defend their rights.

Several factors are important for successful usage of the protoplast culture: nature of the initial material, its growth conditions, enzyme influence conditions and the composition of nutritional media. Protoplasts can be extracted from various types of tissues, but most often the potassium culture, the leaves or cell suspension are used. According to the methodology prepared by us, protoplasts are extracted from half vitrified in vitro grown strawberry plant leaves. In such a way it is possible to receive sufficient amount of vital protoplasts. Species and plant growing conditions before the extraction are very important factors for the extraction of protoplasts. Another factor is the composition and the concentration of the enzymes destroying the membrane of the cell. The concentrations of the enzymes offered in the methodology allow receiving optimal amount of the protoplasts. Increase of the concentration accelerates extraction of the protoplasts, but decreases the vitality thereof. The vitality of protoplasts and efficiency of membrane destruction can be obviously evaluated by painting them using fluorescence paint. Sufficient amount of vital protoplasts is a significant factor of successful somatic hybridization.