In vitro plants that are free of pathogens are crucial for biotechnological breeding methods. The present study investigates the effects of sterilization with sodium hypochlorite (NaClO) and the ...addition of Plant Preservative Mixture (PPMTM) to the growth medium on pathogen elimination, germination, and seedling development of the winter wheat (Triticum aestivum L.) variety Mara. The sterilization treatments differed in the duration of seed sterilization in 4% NaClO and the PPM concentration added to the growth medium. Pathogenic fungi of the genera Aspergillus, Cladosporium, Penicillium, and Bipolaris were completely eliminated when the seeds were sterilized in NaClO and placed on growth media with the addition of PPM. Extending the duration of the sterilization treatment with NaClO to 50 min reduced Fusarium contamination, while the interaction between the 50 min sterilization treatment with NaClO and the addition of PPM to the growth medium reduced Alternaria contamination. Our results suggest that PPM could complement sterilization procedures with NaClO in the introduction of highly infected wheat seeds in vitro. Seed germination was not affected by sterilization with NaClO or by the addition of PPM. However, PPM at a concentration of 4 mL L−1 had a negative effect on seedling development.
Drought negatively affects plants by altering morphological, physiological and metabolic processes and ultimately reducing yields. Garlic (
L.), an important member of the Alliaceae family, is also ...sensitive to drought and maximizing the yield of garlic bulbs is largely dependent on water availability. The objective of this study was to determine the effects of drought stress on morphological and physiological characteristics, as well as on phenolic, sugar, inulin and free amino acid content and antioxidant activity in two Croatian garlic ecotypes, 'Istarski crveni' (IC) and Istarski bijeli (IB). Drought was induced by using polyethylene glycol 8000 (PEG) solution (-0.6 MPa) starting 21 days after clove planting and lasted for 20 days. Drought reduced plant height, number of leaves and plant weight, but increased root length in both ecotypes compared to the control treatment. Among the physiological parameters, significant differences were observed between the two ecotypes studied in the spectral characteristics of the leaves, namely reflection in red, green and blue, VAL, values of the vegetation indices related to the chlorophyll content (CHI, GI), and the anthocyanin content (ARI). Ecotype IC showed higher antioxidant activity in the control treatment due to higher total phenolic content (TPC), but under drought conditions higher DPPH radical scavenging activity was determined in ecotype IB and higher values of FRAP in IC. Sucrose and glucose generally decreased under drought, while inulin increased in IB but decreased in IC. Total free amino acid content increased under drought in both ecotypes. In conclusion, drought tolerance of IB might be associated with increased accumulation of inulin and higher levels of amino acids, especially those shown to contribute to drought resistance. In IC, drought tolerance is associated with an increase in some amino acid compounds and better root growth in depth, probably due to a more efficient translocation of sucrose to the underground part of the plant.
Viruses are responsible for more than 50% of annual potato tuber yield losses and cause great economic damage. The traditional Croatian potato cultivar ‘Brinjak’ is important for local growers ...because of its economically profitable production and as a gene pool for future breeding programs. However, the full genetic potential of the cultivar cannot be exploited due to virus infection. In this study, we attempted to eliminate potato virus M (PVM) and potato virus S (PVS) from potato cultivar ‘Brinjak’ and to evaluate the effects on physiological parameters and yield. Shoot apices were isolated from PVM + PVS-infected sprouts and cultivated for six weeks on MS medium with the addition of 50 or 100 mg L−1 ribavirin. The surviving shoot apices were micropropagated. The in vitro post-eradication period lasted 200 days. DAS-ELISA and RT-PCR were performed on R0 and R1 plants 90 days after acclimatization to determine the sanitary status of the plants. Chlorophyll fluorescence and multispectral imaging were performed on the R0 plants at the same time. The success of PVS elimination was 33% at both ribavirin concentrations. However, neither concentration was successful in eliminating PVM. Plants with mixed infection (PVM + PVS) had more severe disease symptoms compared to PVM-infected plants, affecting photochemistry and multispectral parameters and, consequently, yield. PVM + PVS plants had significantly lower number and weight of tubers per plant and lower average tuber weight than plants with single PVM infection in most of the generations studied. The results indicate a strong negative impact of PVS in mixed infections with PVM and show the importance of its elimination from potato plants.
Amelanchier alnifolia, a deciduous shrub or small tree with edible berry-like fruit, is gaining importance as a commercial fruit crop. The application of micropropagation could complement ...conventional propagation methods of A. alnifolia. The present study aimed to investigate how two light treatments—a mix of red and blue LED lights with dominant wavelengths of 457 and 658 nm (RB LED) and conventional fluorescent lamps emitting light at broad wavelengths of 400-700 nm (FL)—in combination with three cytokinins (CK) belonging to either substituted adenines (6-benzylaminopurine, BAP; meta-Topolin, mT) or to phenylurea cytokinins (thidiazurone, TDZ) affected the multiplication rate, chlorophyll content, and stomata formation in in vitro culture of A. alnifolia axillary shoots. The two light sources proved equally effective in producing axillary shoots, but FL favorably influenced the elongation and chlorophyll content. On the other hand, RB LED lights triggered a significantly higher stomata number of A. alnifolia plantlets in vitro compared with those developed under FL. Presented results reveal a negative ratio between the stomata number and chlorophyll content in response to different cytokinins: TDZ induced the highest number of stomata while BAP significantly increased the chlorophyll content. As the largest number of axillary shoots per explant was achieved on the medium with addition of TDZ, it could be beneficial to use TDZ for multiplication, whereas the use of BAP could be advantageous in the last passage of multiplication before rooting.
Caper (Capparis orientalis Veill.) is a species rich in bioactive compounds, with positive effects on human health. It has a great adaptability to harsh environments and an exceptional ability to ...extract water from dry soils. In Croatia, the caper grows as a wild plant, and its cultivation is insignificant, which is probably due to propagation difficulties. Micropropagation could be a solution for this. The aim of this study was to investigate the success of the micropropagation, in vitro rooting, and acclimatization of Capparis orientalis Veill. Shoot proliferation was tested in a Murashige and Skoog (MS) medium, with sucrose or glucose, and in 13 treatments, presenting the combined effect of different cytokinins and their concentrations. The success of rooting was examined in relation to the impact of various auxins, durations of rooting, and carry-over effects. A better proliferation was achieved when sucrose was used. The highest number (18) of shoots/explants was obtained in the medium supplemented with 0.6 mg·L−1 meta-topolin, while the rooting was equally efficient in the media supplemented with 2 mg·L−1 of indole-3-acetic acid or indole-3-butyric acid, or in hormone-free rooting medium. A prolonged time in the media increased the rooting efficiency, while the carry-over effect had no influence. The acclimatization rate reached 66%. Additional efforts should be made to find out how to speed upthe rooting and enhance the acclimatization rate of caper grown in Croatia.
We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy ...and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L⁻¹ benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The present study was conducted to establish a protocol for the regeneration of virus-free garlic plants through somatic embryogenesis of two Croatian garlic ecotypes. Basal parts of cloves from ...mother plants were cultured on a full Murashige and Skoog (MS) or modified MS medium (¼ of KNO3 and NH4NO3 and 2xMgSO4) containing 0.1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg L−1 2,4-D + 0.5 mg L−1 kinetin (Kin) and representing four different treatments. Plants were regenerated in MS medium containing 0.1 mg L−1 2,4-D and rooted in a medium containing 0.05 mg L−1 1-naphthaleneacetic acid (NAA) + 0.005 mg L−1 6-(γ,γ-dimethylallylamino)purine (2iP). The presence of viruses (i.e., sanitary status) of the mother plants and regenerants was checked by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The mother plants were infected with onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In addition, the presence of garlic common latent virus (GCLV) was confirmed in four mother plants. Embryogenic callus developed in all four treatments with success ranging from 55% to 81% depending on treatment and ecotype. Plant conversion was significantly higher in somatic embryos developed in media containing 0.1 mg L−1 2,4-D than those developed in media containing 1 mg L−1 2,4-D + 0.5 mg L−1 Kin. Virus elimination success ranged from 13.3% up to 62.5% depending on garlic ecotype and treatment. The overall rate of virus elimination by somatic embryogenesis for both treatments and ecotypes were 20.7%, 22.9%, and 30.5% for OYDV, GCLV, and LYSV, respectively. Based on these results, somatic embryogenesis has been shown to be equally or more successful in eliminating garlic viruses compared to other in vitro methods.
'Topaz' is a modern Czech apple cultivar well accepted by consumers and scab-resistant, providing reasons for the significant spreadof cv. 'Topaz' in European orchards, especially in the organic ...fruit production industry. Growing the apple trees on their own rootsprovides some advantages in comparison with grafted trees. Micropropagation is the method of choice for plantlet production for thispurpose as well as for the establishment of healthy mother stock trees as a source of scions. The efficiency of axillary shoot proliferationwas examined on four media differing in plant growth regulators and their concentrations, and from three explant types: intact ordecapitated and defoliated microshoots placed vertically and one-nodal segments placed horizontally. All media consisted of Quoirin and Lepoivre (QL) macroelements and Murashige and Skoog (MS) microelements. Furthermore, rooting efficiency on six different media/treatments was analyzed. Media with 1 mg/L 6-benzylaminopurine (BA) or BA (0.5 mg/L) + 1.5 mg/L kinetin (Kin) produced similarnumber of microshoots per inoculated one (2.5 and 2.4, respectively). Medium with 1 mg/L thidiazuron (TDZ) produced significantlyhigher number of shoots (3.6) but they were fasciated. Three different explant types also produced similar numbers of microshoots.High rooting efficiency (68.7%), a high number of roots per shoot (6.6) and the best quality of shoots were obtained in rooting mediumcontaining 2 mg/L of indole-3-butyric acid (IBA). An efficient method of shoot proliferation was established, and, since rooting was themost critical step, an efficient procedure for rooting apple cv. 'Topaz' was established.
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IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, UL, UM, UPUK
•We bring new method for synthesis of grapevine intraspecific chimeras based on Meristematic Bulk Tissue.•Microsatellite (SSR) markers were successfully applied for detection of synthetic ...chimeras.•This opens for the first time the possibility of Synthetic Chimeral Breeding application for Grapevine.
Chimeral breeding is an alternative breeding method for vegetatively propagated species which can combine desirable traits from different cultivars. Main problem of its application is related to the difficulties in development of synthetic chimeras in most species. Grapevine is one of the most important crops in the world with significantly reduced varietal diversity used in production accompanied by difficult acceptance of new cultivars developed by existing breeding methods. Main objective of this study was to investigate the possibility of grapevine intraspecific chimeras development using in vitro method based on meristematic bulk tissue grafting. Meristematic bulk tissue was successfully developed from two cultivars (Cabernet sauvignon and Babic). After successful grafting and regeneration, 118 plants were obtained. Detection of chimeral plants was performed using combination of phenotyping and genotyping. Phenotyping was based on visual detection of cultivar specific characteristics displayed by young plants. Six microsatellites markers loci were evaluated for their efficiency in detection and quantification of different share of DNA belonging to two cultivars in mixed DNA samples. VVS2 locus was selected and used in genetic characterization of plants obtained from tissue culture. Chimeral status was confirmed in case of seven plants and different share of two cultivars within was detected chimeras. Described method shows promising results due to high regeneration power of meristematic bulk tissue and successful and precise detection of chimeral plants.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The present study was undertaken to evaluate efficiency of callogenesis and regeneration by somatic embryogenesis of the subendemic Iris species Iris illyrica from Croatia and to select highly ...regenerative donor plants/genotypes. Leaf base segments were used as explants. Callogenesis and somatic embryogenesis were induced on MS media supplemented with: (1) 4.52 micromole 2,4-dichlorophenoxyacetic acid (2,4-D) + 4.83 micromole 1-naphthaleneacetic acid (NAA) + 0.46 micromole kinetin (Kin); (2) 4.52 micromole 2,4-D + 4.6 micromole Kin; (3) 13.4 micromole NAA + 2.3 micromole Kin. Transfer of embryogenic calli onto hormone-free medium enabled the development of mature somatic embryos. Frequency of callogenesis was influenced by the donor plant. Among 15 donor plants tested, 3 of them exhibited high regeneration capability and produced 87 regenerants. Seven morphological traits were observed in order to assess phenotypic variability of flowering regenerants. Regenerants with higher values of fall and standard width and length show potential for further breeding of new varieties of Illyrian iris. Flowering and non-flowering regenerants had the same ploidy level as donor plants. Also, the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 12.936 ± 0.038 pg).
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