János Pauk

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid.

Improvement of isolated microspore culture of pepper ( Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.

A study of different (CaMV 35S and mas) promoter activities and risk assessment of field use in transgenic rapeseed plants

SummaryGene fusions between the β-glucuronidase (GUS) reporter gene and the promoters of the cauliflower mosaic virus 35S RNA transcript (CaMV 35S) and the mannopine synthase (mas) genes were introduced into rapeseed varieties via Agrobacterium-mediated transformation. Fluorometric assay of β-glucuronidase activity indicated different expression patterns for the two promoters.In seedlings, the CaMV 35S promoter had maximum activity in the primary roots, while the mas promoter was most active in the cotyledons. Etiolated seedlings cultured in the dark showed reduced activity of the mas promoter.Before vernalization at the rosette stage, both promoters were more active in older plant parts than in younger ones. At this stage the highest activity was recorded in cotyledons.After the plants had bolted reduced promoter function was detected in the upper parts of the transformed plants. Both promoters were found to be functional in the majority of the studied organs of transgenic rapeseed plants, but the promoter activity varied considerably between the organs at different developmental stages.The ability of pollen to transfer the introduced genes to other varieties and related species (e.g. Brassica napus and Diplotaxus muralis) by cross-pollination was studied in greenhouse experiments, and field trials were carried out to estimate the distance for biologically-relevant gene dispersal. In artificial crossing, the introduced marker gene was transferable into other varieties of Brassica napus. In field trials, at a distance of 1 metre from the source of transgenic plants, the frequency of an outcrossing event was relatively high (10-3). Resistant individuals were found at 16 and 32 metres from the transgenic pollen donors, but the frequency of an outcrossing event dropped to 10-5.

Differential activity of the mannopine synthase and the CaMV 35S promoters during development of transgenic rapeseed plants

Abstract Fusions of the promoter of the cauliflower mosaic virus 35S RNA transcript (CaMV 35S) and the mannopine synthase ( mas ) gene to the β-glucuronidase (GUS) reporter gene have been introduced into various cultivars of Brassica napus via Agrobacterium -mediated transformation. Transgenic rapeseed plants have been also regenerated from winter cultivars (Santana, Arabella) by shoot induction from kanamycin-resistant callus tissues on the medium supplemented with AgNO 3 . Transformation was confirmed by Southern hybridization of genomic DNA from primary transformants and PCR analysis of DNA from second generation seedlings. β-glucuronidase activity analyzed by fluorometric assay or histochemical staining indicated a differential expression pattern for the two promoters. Organogenesis from in vitro cultured callus tissues was coupled with a relative increase of CaMV 35S promoter activity and reduction of mas promoter function. In seedlings, the CaMV 35S promoter had maximum activity in the primary roots, while the mas promoter was the most active in the cotyledons. Etiolated seedlings cultured in dark, showed reduced activity of the mas promoter. At rosette stage, both promoters were more active in elder plant parts than in younger ones. The highest activity values were recorded in cotyledons. After bolting, reduced promoter function was detected in upper parts of the transformed plants. Histological staining showed that the CaMV 35S promoter was active in the cortex, the phloem and the vascular cambium, while the mas promoter directed gene expression in the phloem. In conclusion, both promoters were found to be functional in majority of the studied organs of transgenic rapeseed plants, however the promoter activity varied considerably between organs and tissues at various developmental stages.

Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.)

Isolated microspore culture experiments were carried out in sweet pepper (Capsicum annuum L.) F1 hybrid genotypes. In the first experiment, four culture media (W14, B5, MS and NLN) were compared to test their effectiveness in inducing the formation of microspore-derived structures in two genotypes. The experiments revealed the superiority of B5 medium. In the second experiment, the effects of different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.2 and 0.5xa0mgxa0l−1) and kinetin (0, 0.2 and 0.5xa0mgxa0l−1) were also investigated in B5 medium with two genotypes. The effect of growth regulators were investigated on the production of microspore-derived calli and embryo-like structures (ELSs), the ratio of the two and plant regeneration (number of regenerated plantlets) in microspore culture. The histological experiments revealed the differences between the microspore-derived ELSs and calli. The most promising results were obtained on the investigated parameters in the presence of 0.1xa0mgxa0l−1 2,4-D and 0.2xa0mgxa0l−1 kinetin producing the highest number of plantlets in both genotypes tested. In the response of 11 genotypes, the androgenesis induction was successful in each sweet pepper genotypes tested using the best basic medium and growth regulators combination. In case of 11 genotypes, the number of ELSs ranged from 20 to 100/Petri dish (an average of 48.1 ELS/Petri dish), while the number of green plantlets varied from 0 to 8 plantlets/Petri dish (an average of 1.5 plantlets/Petri dish) depending on the genotype. The spontaneous rediploidization rate obtained was 25% in isolated microspore.

Phenotyping shows improved physiological traits and seed yield of transgenic wheat plants expressing the alfalfa aldose reductase under permanent drought stress

Members of the aldo–keto reductase family including aldose reductases are involved in antioxidant defense by metabolizing a wide range of lipid peroxidation-derived cytotoxic compounds. Therefore, we produced transgenic wheat genotypes over-expressing the cDNA of alfalfa aldose reductase gene. These plants consequently exhibit 1.5–4.3 times higher detoxification activity for the aldehyde substrate. Permanent drought stress was generated in the greenhouse by growing wheat plants in soil with 20xa0% water capacity. The control and stressed plants were monitored by a semi automatic phenotyping platform providing computer-controlled watering, digital and thermal imaging. Calculation of biomass values was based on the correlation (R2xa0=xa00.7556) between fresh weight and green pixel-based shoot surface area. The green biomass production by plants of the three transgenic lines was 12–26–41xa0% higherxa0than the non-transgenic plants’ grown under water limitation. Thermal imaging of stressed non-transgenic plants indicated an elevation in the leaf temperature. The thermal status of transformants was similar at both normal and suboptimal water regime. In drought, the transgenic plants used more water during the growing season. The described phenotyping platform provided a comprehensive data set demonstrating the improved physiological condition of the drought stressed transgenic wheat plants in the vegetative growth phase. In soil with reduced water capacity two transgenic genotypes showed higher seed weight per plant than the control non-transgenic one. Limitation of greenhouse-based phenotyping in analysis of yield potential is discussed.

Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.

Allele mining and haplotype discovery in barley candidate genes for drought tolerance

In the present study, allele mining was conducted on a panel of drought related candidate genes in a set of 96 barley genotypes using EcoTILLING, which is a variant of the targeting induced local lesions in genomes (TILLING) technology. Analyzing approximately 1.5 million basepairs in barley a total number of 94 verified unique haplotypes were identified in 18 amplicons designed for 9 genes. Overall, 185 single nucleotide polymorphisms (SNPs) and 46 insertions/deletions (INDELs) were detected with a mean of 1SNP/92xa0bp and 1INDEL/372xa0bp genomic sequence. Based on overlapping haplotype sequences, markers were developed for four candidate genes (HvARH1, HvSRG6, HvDRF1, HVA1), which allows distinguishing between the main haplotypes showing either differences in amino acid sequence or which have larger INDELs in the promoter region. As “proof of concept”, the HvARH1 and HvSRG6 haplotypes were tested for the level of abscisic acid-induced gene expression in subsets of genotypes belonging to different haplotype categories. An integrated database was developed to contain information about the genes, genotypes, and haplotypes analyzed in this study. The database supplies profound information about the natural variation in the tested drought related candidate genes providing a significant asset for further mapping studies dealing with this highly polygenic trait.

Effect of Induction Duration and Medium Composition on Plant Regeneration in Wheat (Triticum aestivum L.) Anther Culture

Summary The effect of induction duration, medium form and medium composition on plant regeneration from microspore-derived embryo-like structures (ELS) in anther culture of two genetically non-related spring wheat ( Triticum aestivum L.) genotypes was investigated using liquid W 14 as a basic induction medium. Induction duration had a significant effect on the regeneration capacity of ELS. The highest percentage (63 %) of regenerable ELS was obtained with 7 weeks of induction whereas the corresponding values with 6, 8 and 12 weeks were 57 %, 38 % and 9 %, respectively. Application of ficoll to the medium, changing the medium form from liquid to solid during the induction or supplementing the medium with glutamine, maltose, cellobiose or trehalose neither increased the number of ELS nor improved plant regeneration. The average number of ELS per 100 anthers decreased from 75 to 9 but the percentage of regenerable ELS increased from 53 % to 75 %, when sucrose was replaced by cellobiose. We conclude that there was an optimum time for maximum plant regeneration and that ELS should be transferred from induction to regeneration no later than 7 weeks after anther isolation.

Detection of cereal viruses in wheat ( Triticum aestivum L.) by serological and molecular methods

The reliable monitoring of field virus infections of crop species is important for both farmers and plant breeders. The aim of this study was to detect virus infections of winter wheat in the 2006/2007 season. Twelve well-known winter wheat varieties were sown on two different dates (11 th of October and 3 rd of November 2006). Leaves of two individuals from each genotype were collected on 23rd of April 2007 to detect the virus infections ( Barley stripe mosaic virus — BSMV, Barley yellow dwarf virus — BYDV-PAV, Wheat dwarf virus — WDV and Wheat streak mosaic virus — WSMV) after an extra mild autumn- and wintertime. Virus infections were detected by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). The aphid-transmitted BYDV-PAV was found frequently whereas other viruses were presented very rarely or were not detected. Forty-six per cent of the tested wheat plants proved to be infected by BYDV-PAV in ELISA, while using PCR, the virus infections with BYDV-PAV was found in 58% o...