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Leucojum aestivum L. is an important medicinal plant which produces Amaryllidaceae alkaloids, especially galanthamine and lycorine. Research is currently exploring the possibility of producing these alkaloids using biotechnological methods, including in vitro cultures. The biosynthesis of alkaloids may be affected by the types and concentrations of carbohydrate sources used in the medium. In the present investigation we performed such studies on in vitro cultures of L. aestivum with a view to obtaining plant material of good quality, characterized, in particular, by a high content of valuable Amaryllidaceae alkaloids. Methods We examined the effects of various types of carbohydrate sources-sucrose, glucose, fructose and maltose-at different concentrations (30, 60 and 90 g/L)-on the quality of L. aestivum plants grown in the RITA® bioreactor. The plants' quality was assessed by their biomass increments, as well by as analysing photosynthetic pigments, endogenous sugar, phenolics and Amaryllidaceae alkaloid content. We also investigated the effect of sugars on the activity of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Results The highest biomass increments were observed in plants cultivated in the medium containing 90 g/L sucrose. The highest CAT activity was noted in cultures growing in the medium supplemented with 90 g/L maltose, while the highest POD activity was observed in the presence of 90 g/L fructose and 60 g/L maltose. No differences in SOD activity were observed. Moreover, the sugars did not affect the contents of chlorophyll a and carotenoids, whereas the highest amount of chlorophyll b was recorded in plants growing in the medium with 60 g/L maltose. No statistically significant differences were observed in the contents of endogenous sugars and phenolics in any in vitro conditions. However, the addition of sugar had a decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distribution of alkaloids occurred in plants cultured in the medium containing 60 g/L sucrose. Six Amaryllidaceae alkaloids were detected in the plant tissue. The addition of 30 g/L fructose in the medium resulted in the accumulation of five alkaloids, including ismine, which was not identified in other analysed tissues. The highest concentration of galanthamine was observed in plants cultured in the presence of 30 g/L fructose and 60 g/L sucrose (39.2 and 37.5 μg/g of dry weight (DW), respectively). The plants grown in the medium containing 60 g/L sucrose exhibited the highest lycorine content (1048 μg/g of DW). Conclusions The type and concentration of sugar used in the medium have an essential influence on the biosynthesis of Amaryllidaceae alkaloids in L. aestivum plants cultured in a RITA® bioreactor. The results point to an interesting approach for commercial production of galanthamine and lycorine. [ABSTRACT FROM AUTHOR]

Background. The oat x maize addition (OMA) lines are used for mapping of the maize genome, the studies of centromere-specific histone (CENH3), gene expression, meiotic chromosome behavior and also for introducing maize C4 photosynthetic system to oat. The aim of our study was the identification and molecular-cytogenetic characterization of oat x maize hybrids. Methods. Oat DH lines and oat x maize hybrids were obtained using the wide crossing of Avena sativa L. with Zea mays L. The plants identified as having a Grande- 1 retrotransposon fragment, which produced seeds, were used for genomic in situ hybridization (GISH). Results. A total of 138 oat lines obtained by crossing of 2,314 oat plants from 80 genotypes with maize cv. Waza were tested for the presence of maize chromosomes. The presence of maize chromatin was indicated in 66 lines by amplification of the PCR product (500 bp) generated using primers specific for the maize retrotransposon Grande-1. Genomic in situ hybridization (GISH) detected whole maize chromosomes in eight lines (40%). All of the analyzed plants possessed full complement of oat chromosomes. The number of maize chromosomes differed between the OMA lines. Four OMA lines possessed two maize chromosomes similar in size, three OMA--one maize chromosome, and one OMA--four maize chromosomes. In most of the lines, the detected chromosomes were labeled uniformly. The presence of six 45S rDNA loci was detected in oat chromosomes, but none of the added maize chromosomes in any of the lines carried 45S rDNA locus. Twenty of the analyzed lines did not possess whole maize chromosomes, but the introgression of maize chromatin in the oat chromosomes. Five of 66 hybrids were shorter in height, grassy type without panicles. Twenty-seven OMA lines were fertile and produced seeds ranging in number from 1-102 (in total 613). Sixty-three fertile DH lines, out of 72 which did not have an addition of maize chromosomes or chromatin, produced seeds in the range of 1-343 (in total 3,758). Obtained DH and OMA lines were fertile and produced seeds. Discussion. In wide hybridization of oat with maize, the complete or incomplete chromosomes elimination of maize occur. Hybrids of oat and maize had a complete set of oat chromosomes without maize chromosomes, and a complete set of oat chromosomes with one to four retained maize chromosomes. [ABSTRACT FROM AUTHOR]

Background. Melatonin (MEL) is a signaling molecule in plants that affects developmental processes during vegetative and reproductive growth. Investigations have proved that exogenously applied MEL also has the potential to improve seed germination and plant development. Methods. In the present study, seeds of stevia, a species with a very low germination rate, were germinated on an agar gel (AG) containing MEL at various concentrations (5, 20, 100, and 500 µM) in light. Seeds germinated on AG without MEL were used as controls. For the first 24 or 48 h of germination, the seeds were maintained in darkness as a pre-incubation step. Some seeds were not exposed to this pre-incubation step. Results. At concentrations of 20 and 5 µM, MEL significantly improved germination, but only in seeds pre-incubated in darkness for 24 h (p < 0.001). At concentrations of 100 and 500 µM, MEL had an inhibitory effect on germination, regardless of the pre-incubation time. Melatonin also affected plantlet properties. At a concentration of 20 µM, MEL increased plantlet fresh weight and leaf numbers. At a concentration of 5 µM, it promoted plantlet height. Regarding root development, the most favorable MEL concentration was 500 µM. Biochemical analysis revealed that MEL promoted higher pigment concentrations but hampered superoxide dismutase activity. On the other hand, the concentrations of sugars and phenolics, as well as the activities of catalase and peroxidase, increased at a MEL concentration of 500 µM. Discussion. The results suggest that MEL can improve germination of positively photoblastic stevia seeds and that it can play a role in plantlet development. However, the effects observed in the present study depended on the quantity of MEL that was applied. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the influence of aqueous extracts from the leaves of peppermint (Mentha ×piperita L.) on the seedlings and plants of common sunflower (Helianthus annuus L.). Different concentration of peppermint leaves extract: 1, 3, 5, 10, 15% was used to examine the growth (length), fresh and dry mass of common sunflower seedlings and plants watered peppermint extracts in phase of growth. We measured the length of seedlings and separated organs of plant: root, hypocotyl, epicotyl, petioles of the first row and remainder of the shoot. Additionally, we determined fresh and dry mass of these organs and cotyledons, leaves of the first order and remainder of the leaves. Depending on the concentration the aqueous extracts showed a stimulatory (low extract concentration) or inhibitory (high extract concentration) effect on growth, fresh and dry mass of common sunflower seedlings and plants. [ABSTRACT FROM AUTHOR]

The QTLs controlling germination and early seedling growth were mapped using seeds acquired from mapping population and parental lines of Chinese Spring and SQ1 grown under water-limited conditions, severe drought (SDr) and well-watered plants (C). Germination ability was determined by performing a standard germination test based on the quantification of the germination percentage (GP24) of seeds incubated for 24 h at 25°C in the dark. Early seedling growth was evaluated on the basis of the length of the root and leaf at the 6th day of the experiment. QTLs were identified by composite interval mapping method using Windows QTLCartographer 2.5 software. For the traits studied, a total of thirty eight additive QTLs were identified. Seventeen QTLs were mapped in C on chromosomes: 1A, 2A, 7A, 1B, 2B, 3B, 4B, 5B, 6B, 7B, 2D, 3D, 4D and 6D, while twenty one QTLs were identified in SDr on chromosomes: 1A, 2A, 5A, 2B, 3B, 4B, 5B, 6B, 7B, 3D, 5D and 6D. Most of the QTLs for GP and early leaf growth parameters were clustered on chromosome 4B (associated with the Rht-B1 marker) both in C and SDr plants. The results indicate the complex and polygenic nature of germination. [ABSTRACT FROM AUTHOR]

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer's disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro- o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM). [ABSTRACT FROM AUTHOR]

The aim of the study was to optimize the method of oat haploid production by pollination with maize. Seventeen oat genotypes were used in the experiment. Various factors influencing the growth and development of ovaries and embryo production were investigated: genotype, time of pollination, growth regulators and time of their application. Emasculated before anthesis, oat florets were pollinated with maize pollen after 0, 1 or 2 days. Next, one of two auxins analogues (2,4-D or dicamba) were applied to oat pistils. These auxins had no significant influence on the number of enlarged ovaries and embryos. The time of application of these growth regulators had a significant influence on embryo production. Haploid embryos were obtained from all used genotypes, although the frequency of enlarged ovaries and obtained embryos did not differ markedly between the genotypes. On average, 85% of ovaries were enlarged and 11.7% of them produced haploid embryos. Depending on the regeneration medium, 24-41% of embryos were germinated, of which 12% had developed into green plants. A strong significant difference in the number of germinating embryos and haploid plants was observed between the kind of regenerating medium used. There were no albino plants and all the obtained plants were haploid. [ABSTRACT FROM AUTHOR]

Haploidization is one of the methods of creating new cultivars that allows to shorten the production time of new cultivars. The aim of this study was to increase the efficiency of oat haploid embryos germination obtained by the wide crossing method. The experiments were performed on 40 oat (Avena sativa L.) F1 progeny derived from Strzelce Plant Breeding Ltd. Oat emasculation was done 8 weeks after sowing. Plants were pollinated with maize and treated with a 2,4-dichlorophenoxyacetic acid (2,4-D). Three weeks after pollination, enlarged ovaries were collected and surface sterilized. Next embryos were isolated and placed on four regenerating media: (1) - 190-2 (Wang and Hu, 1984) with 0.5 mg dm-3 kinetin, 0.5 mg dm-3 1-naphthaleneacetic acid (NAA), 9% maltose, 0.6% agar and pH 6.0, (2) - medium (1) with 50 mg dm-3 cysteine, (3) - medium (1) with 10% v/v coconut water and (4) - medium (1) with 50 mg dm-3 casein hydrolysate. Induction and regeneration of embryos were performed at 16 h photoperiod and light intensity of 130 μmol (photons) m2 s-1 and at 20/17 °C (day/night). Germinated embryos were transferred to MS medium (Murashige and Skoog, 1962) with 1.5% sucrose, solidified with 0.6% agar and pH 5.8. The obtained haploid plants were acclimated to ex vitro conditions. Well-rooted haploid plants were treated with colchicine in order to double the number of chromosomes. Ploidy level of plants was evaluated using a MACS Quant flow cytometer (MACS Quant, prod. Miltenyi Biotec). Of the 40 oats (F1) genotypes, 974 panicles (26960 florets) were emasculated. Seven hundred forty five haploid embryos were obtained, while 211 germinated. The highest number of haploid embryos germinated on medium (1) - 0.40%, and the lowest on medium (4) with casein hydrolysate - 0.09%. On medium (2) with cysteine and on medium (3) with coconut water the percentage of germinating embryos was similar (0.24% and 0.20%, respectively). Addition of cysteine, coconut water and casein hydrolysate inhibited embryos germination. As a result of haploid plants acclimatization to natural conditions and chromosome doubling, 67 doubled haploid (DH) lines were obtained. The number of embryos and regenerated plants depended on the genotype. The embryos were induced in 39 of 40 examined genotypes. The embryos of eight genotypes: 5.8514, STH5.8529, STH4.403/1, STH5.451/1, 5.8422, STH5.8528, 5.8430 and STH5.5046 did not develop into haploid plants. DH lines were obtained from 28 genotypes. The highest number of embryos per emasculated florets (over 5%) was obtained from the genotypes 5.8508, 5.8525 and STH5.8518/1. The most DH lines generated 5.8507, STH5.8504/1 and STH5.8518/1 genotypes. On average, of all tested genotypes, 2.69% of embryos, 0.76% of germinating embryos, 0.43% of haploid plants and 0.24% of DH lines per one hundred emasculated florets were obtained. [ABSTRACT FROM AUTHOR]

A doubled haploid population of 94 lines from the Chinese Spring × SQ1 wheat cross (CSDH) was used to evaluate additive and epistatic gene action effects on total phenolic content, grain yield of the main stem, grain number per plant, thousand grain weight, and dry weight per plant at harvest based on phenotypic and genotypic observations of CSDH lines. These traits were evaluated under moderate and severe drought stress and compared with well-watered plants. Plants were grown in pots in an open-sided greenhouse. Genetic parameters, such as additive and epistatic effects, affecting total phenolic content, were estimated for eight year-by-drought combinations. Twenty-one markers showed a significant additive effect on total phenolic content in all eight year-by-drought combinations. These markers were located on chromosomes: 1A, 1B, 2A, 2B, 2D, 3A, 3B, 3D, 4A, and 4D. A region on 4AL with a stable QTL controlling the phenolic content, confirmed by various statistical methods is particularly noteworthy. In all years and treatments, three markers significantly linked to QTLs have been identified for both phenols and yield. Thirteen markers were coincident with candidate genes. Our results indicated the importance of both additive and epistatic gene effects on total phenolic content in eight year-by-drought combinations. [ABSTRACT FROM AUTHOR]

Wide crossing of cereals with maize is often used to produce doubled haploids (DH). Usually in this method total elimination of maize chromosomes are commonly observed in most cereals, but oat can retain one of more maize chromosomes. The aim of the study was identification, morphological characterization and yield components analysis of the OMA (oat-maize addition) lines. In order to identify maize chromosomes retained in oat genome, oat genomic DNA was isolated from the flag leaf. DNA samples were analyzed by PCR method with using 5'-AAA GAC CTC ACG AAA GGC CCA AGG-3' and 5'-AAA TGG TTC ATG CCG ATT GCA CG-3' primers to detect the presence of maize chromatin. When the whole chromosomes or fragments of maize chromosomes were retained in oat genome, genomic DNA of 500 bp (specific maize retrotransposon fragment, Grande I) was amplified, and detected by electrophoresis. All OMA lines with detected Grande I were analyzed with genomic in situ hybridization (GISH) in order to detect and visualize maize chromosomes. To determine which maize chromosome was present in the OMA lines, DNA amplification using SSR-PCR with primers specific for maize chromosomes was performed. Of all the examined oat lines, in 47 OMA lines retrotransposon Grande I was detected. Twenty-six fertile OMA lines were analyzed with GISH. The whole maize chromosomes as well as a banding pattern probably colocalised with 25S rDNA sites in examined lines were found. Additional hybridization signals found in 2-3 pairs of chromosomes (depending on the analyzed line) might correspond 5S rDNA sequences (in the oat genome these sequences are located on 4 pairs of chromosomes). However, it can not be excluded that these signals came from other maize DNA sequences that have been incorporated into oat chromosomes. Studies based on SSR-PCR enabled the detection of 4, 5 and 6 maize chromosomes in three OMA lines. There were no significant morphological differences between OMA and DH lines. The obtained lines were varied mainly in number of grains and thousand grain weight. [ABSTRACT FROM AUTHOR]

Wide hybridization is one of the haploid-inducing techniques that can accelerate the breeding process. Obtaining new cultivars is crucial to solve the problem of the constantly growing world population and global increase in demand for food, feed and renewable energy under changing environmental conditions. Here, we present a detailed protocol for obtaining oat (Avena sativa L.) doubled haploids (DHs) by pollination with maize (Zea mays L.). After fertilization, not only oat homozygotes, but also oat × maize hybrid zygotes can be formed, and during early embryo development, maize chromosomes are preferentially eliminated, which ultimately results in haploid plant formation. This chapter describes a method to produce oat DHs by crossing oat with maize, covering all steps from crossings to haploid plant regeneration and chromosome doubling., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Production of doubled haploids (DHs) by androgenesis is a promising and convenient alternative to traditionally used breeding techniques. Low response of anther culture and strong genotype dependency in the development of embryo-like structures (ELS) was reported for oat (Avena sativa L.). Total homozygosity has been reached in one generation. This chapter describes a step-by-step protocol that can be useful for androgenesis studies and oat DH line production through anther culture., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Leucojum aestivum L. is an important medicinal plant which produces Amaryllidaceae alkaloids, especially galanthamine and lycorine. Research is currently exploring the possibility of producing these alkaloids using biotechnological methods, including in vitro cultures. The biosynthesis of alkaloids may be affected by the types and concentrations of carbohydrate sources used in the medium. In the present investigation we performed such studies on in vitro cultures of L. aestivum with a view to obtaining plant material of good quality, characterized, in particular, by a high content of valuable Amaryllidaceae alkaloids., Methods: We examined the effects of various types of carbohydrate sources-sucrose, glucose, fructose and maltose-at different concentrations (30, 60 and 90 g/L)-on the quality of L. aestivum plants grown in the RITA ® bioreactor. The plants' quality was assessed by their biomass increments, as well by as analysing photosynthetic pigments, endogenous sugar, phenolics and Amaryllidaceae alkaloid content. We also investigated the effect of sugars on the activity of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD)., Results: The highest biomass increments were observed in plants cultivated in the medium containing 90 g/L sucrose. The highest CAT activity was noted in cultures growing in the medium supplemented with 90 g/L maltose, while the highest POD activity was observed in the presence of 90 g/L fructose and 60 g/L maltose. No differences in SOD activity were observed. Moreover, the sugars did not affect the contents of chlorophyll a and carotenoids, whereas the highest amount of chlorophyll b was recorded in plants growing in the medium with 60 g/L maltose. No statistically significant differences were observed in the contents of endogenous sugars and phenolics in any in vitro conditions. However, the addition of sugar had a decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distribution of alkaloids occurred in plants cultured in the medium containing 60 g/L sucrose. Six Amaryllidaceae alkaloids were detected in the plant tissue. The addition of 30 g/L fructose in the medium resulted in the accumulation of five alkaloids, including ismine, which was not identified in other analysed tissues. The highest concentration of galanthamine was observed in plants cultured in the presence of 30 g/L fructose and 60 g/L sucrose (39.2 and 37.5 µg/g of dry weight (DW), respectively). The plants grown in the medium containing 60 g/L sucrose exhibited the highest lycorine content (1048 µg/g of DW)., Conclusions: The type and concentration of sugar used in the medium have an essential influence on the biosynthesis of Amaryllidaceae alkaloids in L. aestivum plants cultured in a RITA ® bioreactor. The results point to an interesting approach for commercial production of galanthamine and lycorine., Competing Interests: The authors declare that they have no competing interests., (© 2020 Ptak et al.)