Retention of relict satellite DNA sequences in Anemone (Ranunculaceae)

Satellite DNAis a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNAis an important element in genome organization and evolution in plants. Here we study the presence, physical distribution and abundance of the satellite DNAfamily AhTR1 in Anemone. Twenty-two Anemone accessions were analyzed by PCR to assess the presence of AhTR1, while fluorescence in situ hybridization and Southern hybridization were used to determine the abundance and genomic distribution of AhTR1. The AhTR1 repeat unit was PCR-amplified only in eight phylogenetically related European Anemone taxa of the Anemone section. FISH signal with AhTR1 probe was visible only in A. hortensis and A. pavonina, showing localization of AhTR1 in the regions of interstitial heterochromatin in both species. The absence of a FISH signal in the six other taxa as well as weak signal after Southern hybridization suggest that in these species AhTR1 family appears as relict sequences. Thus, the data presented here support the »library hypothesis« for AhTR1 satellite evolution in Anemone. Similar species-specific satellite DNAprofiles in A. hortensis and A. pavonina support the treatment of A. hortensis and A. pavonina as one species, i.e. A. hortensis s.l

Indukcija multiplih izdanaka i regeneracija biljaka vrste Betula pendula in vitro

Shoot development and plant regeneration through axillary bud culture of the European birch (Betula pendula Roth.) were successfully achieved. The modified ACM medium supplemented with 2.2 μM BA was used as initial medium. Both adventitious and axillary shoot development was induced in 2-year-old genotype explants, while in 4- year-old genotype cultures only axillary shoots developed. The establishment of multiple shoot culture was genotype depending. It was found that ACM nutrition medium, supplemented with 2.2 μM BA, was more effective for shoot multiplication than WPM and BTM. Microcuttings were easily rooted on ACM medium supplemented with 1.0 μM IBA. The complete plants obtained were successfully transferred to soil.Razvitak multiplih izdanaka i regeneracija biljaka dobivena je kulturom aksilarnih pupova obične breze (Betula pendula Roth.). Za indukciju izdanaka korištena je modificirana hranidbena podloga ACM uz dodatak 2,2 μM BA. Kod dvogodišnjih genotipova za razliku od četverogodišnjih, inducirani su osim aksilarnih i adventivni izdanci. Mogućnost uspostavljanja kulture multiplih izdanaka ovisila je o genotipu. Utvrđeno je da je podloga ACM s 2,2 μM BA u odnosu na WPM i BTM s istom koncentracijom citokinina bila pogodnija za umnažanje izdanaka. Izdanci su uspješno zakorjenjivani na ACM hranidbenoj podlozi uz dodatak 1,0 μM IBA. Kompletne biljke uspješno su se prilagodile na vanjske uvjete uzgoja

Allium-test — odgovor na djelovanje cijanazina

The effects of cyanazine on mitosis in Allium ce pa L. can be grouped as mitotic and chromosomal effects. Mitotic effects induced by delay in spindle formation were produced immediately after 3 hours treatments in all the concentrations. The mitodepressive activities persisted in two highest concentrations (10-2 M and 10-3 M) only, whereas in solution of cyanazine intermediate concentrations (10-4 — 10-8 M) mitotic activity increased even more than in the control. Chromosomal effects such as laggards, bridges, fragments, fragmentations and micronuclei were noticed after a 3-hours treatment but they were more intensively after 24- or 120-hours treatments in all the concentrations. Stickiness was very frequent and accompanied all the instabilities. Comparison of these two effects shows that mitotic activity was parallel to the frequency of total changes in higher and intermediate concentrations. Recovery from the cyanazine effects, after one day in water, caused normalization of mitotic activity, but a higher frequency of chromosome aberration continued in all the treated materials, depending on the concentration. We can conclude that there i no reversibility of cyanazine effects.Upotrebom Allium-testa ispitan je citogenetski odgovor na djelovanje triazinskog herbicida cijanazina u raznim vremenskim intervalima (3, 24 i 120 sati) kao i u otopinama raznih koncentracija (od 10-2 do 10-8 M), te nakon 24 sata oporavka u vodovodnoj vodi. U svrhu dobivanja traženog odgovora u tretiranom i kontrolnom materijalu, tj. u stanicama meristema korijena isklijalih lukovica crvenog luka (Allium čepa L.) vršena je analiza različitih tipova citogenetskih promjena, kao i komparacija mitotske aktivnosti i učestalosti sveukupnih kromosomskih aberacija. Utvrđeno je da u višim (10-2, 10-3 i 10-4) i srednjim (10-5 i 10-6 M) koncentracijama mitotsku aktivnost paralelno prati učestalost sveukupnih kromosomskih poremećaja, dok je u nižim koncentracijama (10-7 i 10-8 M) mitotska aktivnost bila slična kontroli. Mitodepresivno djelovanje cijanazina zadržalo se samo u najvišim koncentracijama, a u otopinama srednjih koncentracija njegovo djelovanje bilo je stimulativno i mitoza je bila učestalija od kontrole. Oštećenja genetičkog materijala, evidentirana kromosomskim aberacijama, javljala su se u otopinama svih koncentracija, kao i u svim vremenskim periodima, a također su se ovisno o koncentracijama održala i nakon 24 sata oporavka u vodovodnoj vodi. Rezultati provedenog Allium-testa pokazuju da cijanazin nesumljivo inducira oštećenja genetičkog materijala te da dio citogenetičkog učinka može nadživjeti eliminacijsku staničnu selekciju pošto se oštećenje vjerojatno ugradilo u molekulu DNA test organizma luka (Allium cepa L.)

Karyotypes of Allium senescens L. ssp. montanum (Fries) Holub populations from the Mt. Biokovo region

The karyotypes of three Allium senescens ssp. montanum (Fries) Holub populations from the Mt Biokovo region were investigated. This species has been poorly cytogenetically analysed, and with respect to Croatian populations such investigations have never been carried out. The chromosome number, morphology and distribution of heterochromatin were investigated. The number, position and activity of nucleolar organising regions (NORs) were also analysed. In all three populations, only plants with a tetraploid (2n = 4x = 32) chromosome complement were found. The chromosome complement consisted of seven groups of four metacentric and one group of four submetacentric chromosomes bearing the satellite on the short arm. Chromosomes exhibit four types of Giemsa C-bands: telomeric, intercalary, centromeric bands and those located on satellites. Eight chromosomes with the characteristic banding pattern were distinguished. Silver staining revealed a maximum of four nucleoli that corresponded to the maximum number of active NORs (Ag-NORs) located terminally on the short arm of four submetacentric chromosomes. The results obtained suggest that A. senescens ssp. montanum from the Mt Biokovo region is an autotetraploid species

Usporedba intraplazmidne rekombinacije u bakterijama Agrobacterium tumefaciens i Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism.U ovom smo radu konstruirali plazmid koji nam je omogućio da usporedimo intraplazmidnu rekombinaciju u bakterijama Agrobacterium tumefaciens i Escherichia coli. Plazmid sadržava dvije istosmjerno ponovljene kopije gena odgovornog za rezistenciju na spektinomicin, pri čemu jednoj kopiji nedostaje 5\u27, a drugoj 3\u27 kraj gena, a međusobno su homologne u duljini od 570 pb. Osim toga, DNA koja se nalazi između ove dvije istosmjerno ponovljene sekvencije sadržava gen koji daje rezistenciju na antibiotik ampicilin. Homolognom rekombinacijom između nepotpunih gena za rezistenciju na spektinomicin nastaje funkcionalni gen, odgovoran za pojavu rezistencije. Pritom može doći do delecije gena za rezistenciju na ampicilin ili njegovog umnožavanja, zajedno s nepotpunim genima za otpornost na spektinomicin. Ovaj eksperimentalni sustav omogućio nam je da po prvi put pratimo pojavu delecija i amplifikacija tijekom intraplazmidne rekombinacije u bakteriji A. tumefaciens. Pokazali smo da delecije i amplifikacije u bakterijama Agrobacterium tumefaciens i Escherichia coli nastaju prvenstveno RecA-neovisnim mehanizmom. Osim toga, ustanovili smo da se delecije i amplifikacije pojavljuju s podjednakom učestalošću, što upućuje na to da je mehanizam oba rekombinacijska događaja sličan

The Repetitive DNA Composition in the Natural Pesticide Producer Tanacetum cinerariifolium: Interindividual Variation of Subtelomeric Tandem Repeats

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), a plant species endemic to the east Adriatic coast, is used worldwide for production of the organic insecticide, pyrethrin. Most studies concerning Dalmatian pyrethrum have focused on its morphological and biochemical traits relevant for breeding. However, little is known about the chromosomal evolution and genome organization of this species. Our study aims are to identify, classify, and characterize repetitive DNA in the T. cinerariifolium genome using clustering analysis of a low coverage genomic dataset. Repetitive DNA represents about 71.63% of the genome. T. cinerariifolium exhibits linked 5S and 35S rDNA configuration (L-type). FISH reveals amplification of interstitial telomeric repeats (ITRs) in T. cinerariifolium. Of the three newly identified satellite DNA families, TcSAT1 and TcSAT2 are located subterminally on most of T. cinerariifolium chromosomes, while TcSAT3 family is located intercalary within the longer arm of two chromosome pairs. FISH reveals high levels of polymorphism of the TcSAT1 and TcSAT2 sites by comparative screening of 28 individuals. TcSAT2 is more variable than TcSAT1 regarding the number and position of FISH signals. Altogether, our data highlights the dynamic nature of DNA sequences associated with subtelomeres in T. cinerariifolium and suggests that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes

Kromosomske promjene u klijanaca crnog bora izazvane regulatorima rastenja

Cytogenetic effects caused by two synthetic auxins: 1-naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA) as well as by synthetic cytokinin benzyladenine (BA), on root tip cells of ten-day-old European black pine (Pinus nigra Arn.) seedlings were observed. The results showed that auxins induced stronger cytogenetic effects than cytokinin. After treatment with auxins the mitotic activity and the corresponding number of chromosomal aberrations increased in parallel to the concentrations used. Both auxins, NAA and IBA, induced almost the same types of nuclear changes such as spindle failure, chromosome fragmentation, laggards and polyploidy. NAA induced a higher frequency of chromosomal changes than IBA. Also, the recovery period after the application of NAA was much slower as compared to that of IBA. Cytokinin BA showed the opposite effect. After treatment with the highest concentration the mitotic activity was very similar to the control, but after the recovery period the mitotic index increased significantly. This was also the case with a number of aberrant cells.Istraženo je djelovanje dva sintetska auksina: 1-naftalenoctene kiseline (NAA) i indolil-3-maslačne kiseline (IBA) i sintetskog citokinina benziladenina (BA) na meristemske stanice vrška korjenčića 10 dana starih klijanaca crnog bora (Pinus nigra Arn.). Auksini imaju snažnije citogenetsko djelovanje na genom meristemskih stanica od citokinina. Nakon tretmana auksinima mitotska aktivnost stanica je porasla paralelno s upotrijebljenom koncentracijom. Broj aberantnih stanica također se povećavao s koncentracijom auksina. Oba auksina induciraju gotovo iste tipove promjena na razini diobenog vretena i na razini kromosoma. Sintetski citokinin BA je pokazao suprotno djelovanje. Nakon tretmana s najvećom koncentracijom mitotska aktivnost bila je vrlo slična onoj u kontrolnom uzorku. Međutim nakon oporavka značajno je porasla, a također je porastao i broj kromosomskih aberacija

Indukcija multiplih izdanaka i regeneracija biljaka vrste Betula pendula in vitro

Shoot development and plant regeneration through axillary bud culture of the European birch (Betula pendula Roth.) were successfully achieved. The modified ACM medium supplemented with 2.2 μM BA was used as initial medium. Both adventitious and axillary shoot development was induced in 2-year-old genotype explants, while in 4- year-old genotype cultures only axillary shoots developed. The establishment of multiple shoot culture was genotype depending. It was found that ACM nutrition medium, supplemented with 2.2 μM BA, was more effective for shoot multiplication than WPM and BTM. Microcuttings were easily rooted on ACM medium supplemented with 1.0 μM IBA. The complete plants obtained were successfully transferred to soil.Razvitak multiplih izdanaka i regeneracija biljaka dobivena je kulturom aksilarnih pupova obične breze (Betula pendula Roth.). Za indukciju izdanaka korištena je modificirana hranidbena podloga ACM uz dodatak 2,2 μM BA. Kod dvogodišnjih genotipova za razliku od četverogodišnjih, inducirani su osim aksilarnih i adventivni izdanci. Mogućnost uspostavljanja kulture multiplih izdanaka ovisila je o genotipu. Utvrđeno je da je podloga ACM s 2,2 μM BA u odnosu na WPM i BTM s istom koncentracijom citokinina bila pogodnija za umnažanje izdanaka. Izdanci su uspješno zakorjenjivani na ACM hranidbenoj podlozi uz dodatak 1,0 μM IBA. Kompletne biljke uspješno su se prilagodile na vanjske uvjete uzgoja

Utjecaj svjetlosti na proizvodnju betanina s pomoću transformiranih stanica šećerne repe

The objective of this work is to transform sugar beet (Beta vulgaris L. var. altissima) cells using Agrobacterium tumefaciens, a wild octopine strain B6S3, and to study metabolic changes associated with the transformation. From the primary tumours on leaf fragments, two tumour lines were established: one pale green and the other red-violet in colour. The red-violet pigment was identified by means of high-performance liquid chromatography as betanin. Betanin production is strongly light-dependent. To enhance the betanin yield, nutrient media with different carbohydrates like sucrose, a combination of glucose and fructose, or only glucose or only fructose were tested. The selection of carbohydrate affected betanin production and yield. It was observed that the production of betanin per g of dry mass was 20–40 % higher on simple carbohydrates, especially fructose, than on sucrose or the combination of glucose and fructose. However, due to higher biomass production on the medium with sucrose, the highest total yield of betanin was obtained in the presence of sucrose. The most suitable time for possible betanin extraction was between days 7 and 14 for tissue grown on sucrose. The tumour line seems to be promising as an alternative source of betanin as well as a model to study sugar-regulated genes involved in tissue morphology control.Svrha je rada bila transformirati stanice šećerne repe (Beta vulgaris L. var. altissima) bakterijom divljeg soja B6S3 Agrobacterium tumefaciens, te proučavati metaboličke promjene nakon transformacije. Od primarnih tumora koji su se razvili na zaraženim listovima, uspostavljene su dvije tumorske linije: svijetlozelena i crveno-ljubičasta. Pigment iz crvene linije identificiran je visokotlačnom tekućinskom kromatografijom kao betanin. Proizvodnja betanina ovisila je o svjetlosti. Da bi se pojačao prinos betanina, ispitana je hranidbena podloga s različitim šećerima, i to: saharozom, glukozom i fruktozom te samo s glukozom ili samo s fruktozom. Proizvodnja i prinos betanina ovisili su o dodanom šećeru. Proizvodnja betanina po gramu suhe tvari bila je 20-40 % viša na monosaharidima, posebice fruktozi, nego na saharozi ili kombinaciji glukoze i fruktoze. Međutim, zbog obilnije biomase, najveći prinos betanina ostvaren je na podlozi sa saharozom. Najpovoljnije vrijeme za iskorištavanje betanina je između prvog i drugog tjedna supkulture na saharozi. Zaključno, dobivena tumorska linija prikladan je alternativni izvor pigmenta betanina, a i model za istraživanje gena koji uz pomoć šećera regulira morfologiju biljnih tkiva