DH Lines Research Articles

Efficiency of genomic prediction of non-assessed single crosses.

An important application of genomic selection in plant breeding is predicting untested single crosses (SCs). Most investigations on the prediction efficiency were based on tested SCs using cross-validation. The main objective was to assess the prediction efficiency by correlating the predicted and true genotypic values of untested SCs (accuracy) and measuring the efficacy of identification of the best 300 untested SCs (coincidence) using simulated data. We assumed 10,000 SNPs, 400 QTLs, two groups of 70 selected DH lines, and 4900 SCs. The heritabilities for the assessed SCs were 30, 60, and 100%. The scenarios included three sampling processes of DH lines, two sampling processes of SCs for testing, two SNP densities, DH lines from distinct and the same populations, DH lines from populations with lower LD, two genetic models, three statistical models, and three statistical approaches. We derived a model for genomic prediction based on SNP average effects of substitution and dominance deviations. The prediction accuracy is not affected by the linkage phase. The prediction of untested SCs is very efficient. The accuracies and coincidences ranged from ~0.8 and 0.5 at low heritability to 0.9 and 0.7 at high heritability, respectively. We also highlight the relevance of the overall LD and demonstrate that efficient prediction of untested SCs can be achieved for crops that show no heterotic pattern, for reduced training set size (10%), for SNP density of 1 cM, and for distinct sampling processes of DH lines based on random choice of the SCs for testing.

Evaluation of neck blast resistance and agronomical performances on double haploid rice population in greenhouse and endemic field

Windarsih G, Utami DW. 2017. Evaluation of neck blast resistance and agronomical performances on double haploid rice population in greenhouse and endemic field. Nusantara Bioscience 9: 371-377. Blast disease caused by fungal Pyricularia grisea Sacc. is one of the most destructive diseases of rice in the world. The development of blast-resistant rice varieties will be essential to control this disease. This research aimed (i) to compare the resistance response to neck-blast among DH lines from double cross IR54/Parekaligolara//Bio110/Markuti and the differential varieties against three selected Indonesian blast races in greenhouse, (ii) to identify the gene(s) that caused the resistance to neck-blast based on the association between the resistance response and the genotype evaluation using molecular markers linked to Pi1, Pi33, Pib, Pir4 and Pir7 genes, and (iii) to evaluate the resistance response to leaf and neck blast on DH lines in endemic field (Sukabumi) and the agronomical performance of selected DH lines in optimum field in Ciasem of Subang, West Java, Indonesia during December 2013 to March 2014. Eleven double haploid lines from double-crossing IR54/Parekaligolara//Bio110/Markuti, the differential varieties as resistant control and the US2 variety for susceptible control were observed for neck-blast resistance response to three blast races in greenhouse and endemic field (Sukabumi), while the agronomical performances were observed in field of Ciasem-Subang. The results based on the genotyping evaluation, leaf and neck blast resistance, either in greenhouse and endemic location, and the agronomical performance in field showed that 5 selected double haploid lines had leaf and neck blast resistance and good performance on field trial. Thus they are promising for use either for further testing forwarding into releasing variety or used as donor for further blast resistant breeding activities.

Novel technologies in doubled haploid line development

Summaryhaploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.

QTL mapping for tuberous stem formation of kohlrabi (Brassica oleracea var. gongylodes L.)

The tuberous stem of kohlrabi is an important quantitative trait, which affects its yield and quality. Genetic control of this trait has not yet been unveiled. To identify the QTLs controlling stem swelling of kohlrabi, a BC1 population of 92 plants was developed from a cross of broccoli DH line GCP04 and kohlrabi var. Seine. A wide range of variation in tuberous stem diameter was observed among the mapping populations. We constructed a genetic map of nine linkage groups (LGs) with different types of markers, spanning a total length of 913.5 cM with an average marker distance of 7.55 cM. Four significant QTLs for radial enlargement of kohlrabi stem, namely, REnBo1, REnBo2, REnBo3, and REnBo4 were detected on C02, C03, C05, and C09, respectively, and accounted for the phenotypic variation of 59% for the stem diameter and 55% for the qualitative grading of tuberous stem in classes. Then, we confirmed the stability of identified QTLs using BC1S1 populations derived from the BC1 plants having heterozygous alleles at the target QTL and homozygous kohlrabi alleles at the remaining QTLs. REnBo1and REnBo2 using 128 plants of BC168S1 and 94 plants of BC143S1, respectively, and REnBo3 and REnBo4 using 152 plants of BC157S1 were detected at the same positions as the respective QTLs of the BC1 population. Confirmation of QTLs in two successive generations indicates that the QTLs are persistent. The QTLs obtained in this study could be useful in marker-assisted selection of kohlrabi breeding, and to understand the genetic mechanisms of stem swelling and storage organ development in kohlrabi and other Brassica species.

Identification of proteins related to microspore embryogenesis responsiveness in anther cultures of winter triticale (\xd7Triticosecale Wittm.)

For a better understanding of the physiological background of microspore embryogenesis (ME), the protein profile was analyzed in four winter triticale DH lines, which show extremely different embryogenic potential. The analysis were conducted with anthers at the phase of development optimal for ME induction and then after low temperature (LT, 3 weeks at 4 °C) ME-inducing tillers treatment. The sub-proteome of anthers was mapped by two-dimensional gel electrophoresis (2-DE). The protein species significantly more abundant (at least 2-fold) in responsive DH lines after LT treatment were chosen for identification by MALDI-TOF/TOF analysis. In total, 31 protein species were successfully identified as involved in the determination of microspore competence, stress response and in the regulation of ME induction. Microspore competence required sufficient energy supply and efficient system of cell protection that determine survival under prolonged LT stress treatment. LT stress was associated with increased accumulation of proteins typical for cell defence against oxidative stress (e.g., l-ascorbate peroxidase), chaperons (e.g., HSP70) and other enzymes/factors ensuring protein biosynthesis, stability and active cell divisions. Also here, effective cell defence required undisturbed energy supply. Among proteins that accumulated differentially in accordance with microspore embryogenic potential again the most important role seems to be played by the enzymes ensuring energy production and determining ability of plant stress adaptation. Two protein species (enolase, 12S storage protein), proposed earlier as candidates for markers of embryogenesis in other in vitro plant culture systems confirmed their utility for triticale anther cultures.

Chlorophyll a Fluorescence Parameters in the Evaluation of Oat DH Lines Yield Components

Chlorophyll a fluorescence can provide insight into the ability of plants to tolerate environmental conditions that can damage photosynthetic apparatus and decrease yield. The aim of the study was to determine the relationship between chlorophyll a fluorescence parameters and yield components of oat DH lines. All DH lines significantly differed in chlorophyll a fluorescence parameters and yield components. The overall performance index of PSII photochemistry (PI), showed the highest variation between DH lines, whereas the lowest had the ratio of variable to maximum fluorescence (Fv/Fm). The highest differences were observed in the number of grains per plant (21.3 to 600). Thousand-grain weight varied from 17.82 g to 41.01 g and the biomass from 8.01 g to 29.31 g. The highest negative correlations were found between Fv/Fm, Area (pool size of electron acceptors from PSII), PI and grain number per plant and biomass. Positive correlations were observed between light energy absorption (ABS/CS), grain number per plant and biomass, as well as the amount of excitation energy trapped in PSII reaction centers (TRo/CS) and biomass. Principal component analysis of chlorophyll a fluorescence parameters, together with yield components, discriminated two oat DH lines groups according to their photosynthetic efficiency and yield.

QTL analysis in Barley Across Environments in Egypt

BARLEY is one of the most important cereal crops worldwide. An advanced backcross doubled haploid mapping population was grown in four locations across Egypt (Assuit, Al Wady Al Asuity, Matrouh and Nubaria). The population included 301 BC2DH lines derived from crossing between a German elite cultivar of H. vulgare ssp. vulgare ‘Scarlett’ with an exotic accession of H. vulgare ssp. spontaneum ‘ISR42-8’. A linkage map including 371 different types of genetic markers was used to perform QTL analysis. We detected 56 putative QTLs for traits of interest. In addition, the study identified four markers with marker main and marker × environment interaction effects. The exotic alleles of those four markers could be responsible for increasing their traits across environments. Furthermore, eight markers showed pleiotropic effect across locations. Some DH lines performed better than their parents and check varieties in each environment and across four environments as well. These results might be useful in MAS for barley breeding programs in Egypt.

Transcriptome Analysis of a Female-sterile Mutant (fsm) in Chinese Cabbage (Brassica campestris ssp. pekinensis).

Female-sterile mutants are ideal materials for studying pistil development in plants. Here, we identified a female-sterile mutant fsm in Chinese cabbage. This mutant, which exhibited stable inheritance, was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and ethyl methanesulfonate mutagenesis. Compared with the wild-type line ‘FT,’ the fsm plants exhibited pistil abortion, and floral organs were also relatively smaller. Genetic analysis indicated that the phenotype of fsm is controlled by a single recessive nuclear gene. Morphological observations revealed that the presence of abnormal ovules in fsm likely influenced normal fertilization process, ultimately leading to female sterility. Comparative transcriptome analysis on the flower buds of ‘FT’ and fsm using RNA-Seq revealed a total of 1,872 differentially expressed genes (DEGs). Of these, a number of genes involved in pistil development were identified, such as PRETTY FEW SEEDS 2 (PFS2), temperature-induced lipocalin (TIL), AGAMOUS-LIKE (AGL), and HECATE (HEC). Furthermore, GO and KEGG pathway enrichment analyses of the DEGs suggested that a variety of biological processes and metabolic pathways are significantly enriched during pistil development. In addition, the expression patterns of 16 DEGs, including four pistil development-related genes and 12 floral organ development-related genes, were analyzed using qRT-PCR. A total of 31,272 single nucleotide polymorphisms were specifically detected in fsm. These results contribute to shed light on the regulatory mechanisms underlying pistil development in Chinese cabbage.

Comparative transcriptome analysis of self-incompatible flower stigmas and self-compatible bud stigmas following self-pollination in broccoli.

DH07 is a DH line of Class I S-haplotype in broccoli (Brassica oleracea var. italica), in which stigmas of flowers show self-incompatibility (SI) and stigmas of buds show self-compatibility (SC). The molecular mechanisms that lead to stigmas at different developmental stages having different responses to self-pollination are yet unknown. In the present study, comparative transcriptome profiling of the stigmas of flowers and buds before and after self-pollination was performed by RNA-sequencing using an Illumina HiSeqTM 2000. A total of 80,102,897 reads were generated for further analysis in four libraries. Comparisons of the transcriptome profiles before and after self-pollination revealed 579 differentially expressed genes (DEGs) in the stigmas of buds (SBs); of these, 431 DEGs showed increased and 148 DEGs showed decreased transcript abundance after self-pollination in SBs. There were a total of 686 DEGs between unpollinated stigmas of flowers (SFs) and pollinated SFs, among which, 517 DEGs were up regulated and 169 DEGs were down regulated. Following the self-pollination, 379 identified DEGs were common in both SBs and SFs. It was found that ARR7-like and oxysterol-binding family protein related DEGs could play key roles in SI or SC signal transduction. The results obtained in this study would form the foundation for further studies on investigating the molecular mechanisms of SI and SC in Brassica.

Identification of MFLP fingerprint for higher seed zinc accumulation in barley DH population

Selection through molecular markers for seed Zn accumulation might be an efficient complementary breeding tool in barley breeding. To develop a specific molecular markers, 150 DH lines derived from a cross between Clipper (low-Zn-accumulator) and Sahara-3771 (high-Zn-accumulator) were screened under field and glasshouse conditions. Microsatellite-anchored fragment length polymorphism (MFLP) fingerprint generated by SSR-anchor primer MF128 in combination with AFLP primer MseI-AGA (5?-GATGAGTCCTGAGTAAAGA-3?) was identified as a candidate marker for tagging seed Zn accumulation gene. The sequencing of the band showed a marker of 369 bp with the sequence of SSR anchor primer MF128 and MseI-AGA at the two ends as expected. The MFLP marker associated with higher seed Zn accumulation has potential to be converted to a simple, sequence-specific, PCR-based, low-cost marker amenable to large populations, making it potentially viable for marker-assisted selection in barley breeding. This marker might be useful in the improvement of barley productivity and nutritional quality in Zn-deficient environments.

Conversion of oat (Avena sativa L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media.

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F1 progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5–0.9 mm, 1.0–1.4 mm, and ≥1.5 mm), and transferred into 190–2 regeneration medium with different growth regulators: 0.5 mg L−1 kinetin (KIN) and 0.5 mg L−1 1-naphthaleneacetic acid (NAA); 1 mg L−1 zeatin (ZEA) and 0.5 mg L−1 NAA; or 1 mg L−1 dicamba (DIC), 1 mg L−1 picloram (PIC), and 0.5 mg L−1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0–1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L−1 NAA and 0.5 mg L−1 KIN, while the fewest germinated on medium with 1 mg L−1 DIC, 1 mg L−1 PIC, and 0.5 mg L−1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s11627-016-9788-z) contains supplementary material, which is available to authorized users.

A Major QTL, Which Is Co-located with cly1, and Two Minor QTLs Are Associated with Glume Opening Angle in Barley (Hordeum vulgare L.).

Cleistogamous and chasmogamous are two opposing phenomena for flowering in barley. Cleistogamy limits the rate of outcrossing, and increases the cost of producing hybrid barley seeds. Selecting chasmogamous lines with a large glume opening angle (GOA) is essential for the utilization of barley heterosis. In the current study, 247 DH lines derived from a cross between Yangnongpi7 and Yang0187 were used to identify and validate quantitative trait loci (QTLs) associated with the GOA in different environments using SSR markers. Three QTLs associated with barley GOA were mapped on chromosomes 2H and 7H. The major QTL QGOA-2H-2 was mapped on chromosome 2H with the flanking markers of KDH and GBM1498, explaining 63.92% of the phenotypic variation. The marker KDH was developed from the gene Cly1, which was the candidate gene for QGOA-2H-2. This new marker can be used to identify barley chasmogamous lines with a large GOA. The two minor QTLs were validated at all three locations across two seasons after removing DH lines carrying the candidate gene Cly1 of QGOA-2H-2.

Study of variation of tocochromanol and phytosterol contents in black and yellow seeds of Brassica napus L. doubled haploid populations.

In the study, an analysis of tocopherols, plastochomanol-8 and phytosterols was conducted using DH lines obtained from F1 hybrids of reciprocal crosses between yellow- and black-seeded lines. The biological material for the study consisted of two DH populations of winter oilseed rape obtained from F1 hybrids of reciprocal crosses between two DH lines: yellow- and black-seeded. Seed color was determined using a ColorFlex spectrophotometer. Fat content was determined via pulsed NMR. The levels of tocopherols, and plastochromanol-8 are analyzed using HPLC. Phytosterol contents and composition were determined by the GC method. The fat content of the black-seeded parental line was 49% and this was higher than that of the yellow-seeded parental line (44%). The fat content of DH line populations ranged from 44 to 51%. Total tocopherol content ranged from 460 to 602 mg/kg and the α-T/γ-T ratio was from 0.66 to 1.09. In parental lines H2-26 and Z-114 the total tocopherol content was 534 and 525 mg/kg, but the α-T/γ-T ratios were 0.81 and 1.21, respectively. The yellow-seeded parental line (Z-114) was characterized by a higher PC-8 content (81 mg/kg) than the H2-26 black-seeded parental line (58 mg/kg). The largest part of the total phytosterol content in seeds of both populations was β-sitosterol from 976 to 2148 mg/kg, followed by campasterol, from 636 to 1364 mg/kg, and brassicasterol from 375 to 678 mg/kg. The total tocopherol content ranged from 462 to 595 mg/kg (population HxZ) and from 460 to 602 mg/kg (population ZxH). Significantly positive correlations were observed between the seed color with α-T (r = 0.38, p < 0.01), γ-T (r = -0,34, p < 0.01) and PC-8 content (r = 0.29, p < 0.01). Correlations between the seed color with total tocopherol and total phytosterol content were not noted. Considering the range of genetic variation among doubled haploids of two populations, selected DH lines may be good parents for further breeding programs focused on increasing the amount and improving the quality of oilseed rapeseed oil. However, further studies will also be made to determine the influence of the environment on bioactive compounds in rapeseed oil. Cross direction of parental DH lines: yellow- and black-seeded has some influence, albeit not statistically significant, on the diversity of doubled haploid in their populations with regard to average fat, tocochromanol and phytosterol content.

Interactions between Glu-1 and Glu-3 loci and associations of selected molecular markers with quality traits in winter wheat (Triticum aestivum L.) DH lines

The quality of wheat depends on a large complex of genes and environmental factors. The objective of this study was to identify quantitative trait loci controlling technological quality traits and their stability across environments, and to assess the impact of interaction between alleles at loci Glu-1 and Glu-3 on grain quality. DH lines were evaluated in field experiments over a period of 4 years, and genotyped using simple sequence repeat markers. Lines were analysed for grain yield (GY), thousand grain weight (TGW), protein content (PC), starch content (SC), wet gluten content (WG), Zeleny sedimentation value (ZS), alveograph parameter W (APW), hectolitre weight (HW), and grain hardness (GH). A number of QTLs for these traits were identified in all chromosome groups. The Glu-D1 locus influenced TGW, PC, SC, WG, ZS, APW, GH, while locus Glu-B1 affected only PC, ZS, and WG. Most important marker-trait associations were found on chromosomes 1D and 5D. Significant effects of interaction between Glu-1 and Glu-3 loci on technological properties were recorded, and in all types of this interaction positive effects of Glu-D1 locus on grain quality were observed, whereas effects of Glu-B1 locus depended on alleles at Glu-3 loci. Effects of Glu-A3 and Glu-D3 loci per se were not significant, while their interaction with alleles present at other loci encoding HMW and LMW were important. These results indicate that selection of wheat genotypes with predicted good bread-making properties should be based on the allelic composition both in Glu-1 and Glu-3 loci, and confirm the predominant effect of Glu-D1d allele on technological properties of wheat grains.

The effect of light intensity on the production of oat (Avena sativaL.) doubled haploids through oat × maize crosses

Oat haploid embryos were obtained by wide crossing with maize. The effect of light intensity during the growing period of donor plants (450 and 800 µmol m−2 s−1) and in vitro cultures (20, 40, 70 and 110 µmol m−2 s−1) was examined for the induction and development of oat DH lines. Oat florets (26008) from 32 genotypes were pollinated with maize and treated with 2,4-dichlorophenoxyacetic acid. All the tested genotypes formed more haploid embryos when donor plants were grown in a greenhouse (9.4%) compared to a growth chamber (6.1%). The light intensity of 110 µmol m−2 s−1 during in vitro culture resulted in the highest percentage of embryo germination (38.9%), conversion into plants (36.4%) and DH line production (9.2%) when compared with lower light intensities (20, 40 and 70 µmol m−2 s−1). The results show that the growth conditions of the donor plant and light intensity during in vitro culture can affect the development of haploid embryos. This fact may have an impact on oat breeding programs using oat ...

An efficient shoot regeneration system andAgrobacterium-mediated transformation withcodAgene in a doubled haploid line of broccoli

Broccoli is an important vegetable crop belonging to the genus of Brassica. However, it is often affected by biotic and abiotic stresses that result in large losses in yield and quality. Genetic manipulation has opened the opportunity for germplasm improvement of broccoli. In this study, an efficient shoot regeneration and Agrobacterium-mediated transformation system was established. The optimum medium for shoot induction was selected for three types of explant including hypocotyl, petiole, and peduncle, and up to 90% regeneration frequency was obtained. The transformation procedure was developed with the EHA105 strain, harboring the PUC19 vector, along with the target gene of codA and a hygromycin-resistance gene. Several factors were optimized, including hygromycin concentration for selection and the Agrobacterium density yielding the highest transformation frequency. Among the three types of explants, peduncle explants showed the highest response to this procedure, and the highest frequency of transformation (3.4%) was obtained depending on the analysis of polymerase chain reaction and Southern blot. In conclusion, the present study introduced a reliable system for plant regeneration and genetic transformation with three types of explants for a DH line of broccoli, and peduncle explant resulted in the highest transformation frequency.

FEATURES OF SEED FORMATION IN DOUBLE HAPLOID LINES OF BROCCOLI IN DIFFERENT GENERATIONS

The biotechnological methods enable to produce the ‘pure’ lines for the short period of time that intensify the breeding program. Particularly, the anther culture is widely used to obtain double haploid lines (DH lines) in plant breeding. The technology advantage is to fast up essentially the selection of homozygous lines with different morphological traits and high level of uniformity. Contemporary breeding programs are directed at F1 hybrid development; therefore DH lines are in need to serve as the parental form for heterotic hybrid production. Nevertheless, previous data has shown that DH plants have been noticed to produce few seeds on them. To introduce the lines into breeding program for hybrid production and to multiply them, the seed formation of DH lines is worth being studied. The result of assessment of seed generation of DH lines of Broccoli that have been produced from ‘Tonus’ and convariety ‘N1’ is presented in the article There is much difference in morphological traits and both ability to recognize their own pollen and quantity of seeds in the pod among plants produced. Nine DH lines of early maturity were obtained. The differences in seed formation among these lines and between generations were revealed. It was shown the influence of genotype on seed formation in DH lines. It was also noticed the much seed formation in case of geitonogamic pollination in DH lines obtained from cultivar ‘Tonus’. The biological features of flowering in DH lines under conditions of climatic chambers have been studied.

Study of fertility and cytogenetic variability in androgenic plants (R0 and R1) of alloplasmic introgression lines of common wheat

Anther culture is one of the methods to obtain DH lines of wheat. A limitation of this method can be cytogenetic instability in plants R0, leading to a decrease in fertility or sterility. In this study, we have investigated the fertility of R0, the fertility and cytogenetic variability of R1 in alloplasmatic introgression lines of common wheat in order to develop a cytogenetically stable DH lines with introgressions from different species. Lines 311/134, 311/FL, 311/IR with the cytoplasm from H. vulgare were studied. 311/134 carries the wheat-rye 1RS.1BL and wheatwheatgrass 7DL-7Ai translocations; 311/FL has the 1RS.1BL translocation and probably introgressions from A. glaucum; and 311/IR has the wheat-rye 1RS.1BL and wheat-Ae. speltoides T2B/2S#2 translocations. Green seedlings developed in anther culture for all lines. Differences between the lines in the ability for androgenesis and in the level of fertility in R0 and R1 have been revealed. Depressed androgenesis, low fertility and high aneuploidy were observed in 311/IR. It has been proposed that the reason for this is cytogenetic instability in gametes, which is caused by Gc genes located on T2B/2S#2. 63.3 % of 311/134 and 311/FL R1 plants that were grown from low seed-set R0 plants were aneuploids. Fertile R0 regenerant plants were identified that segregated in R1 for fertility and chromosome numbers. It has been demonstrated that DH lines are best developed from highfertility R1 plants with 2n = 42 irrespective of fertility in R0.

Analysis of Yield and Yield Related Traits Variability of Winter Wheat (Triticum aestivum L.) Cv. Izolda and Double Haploid Lines

AbstractThe yield-forming potential of winter wheat is determined by several factors, namely total number of shoots per plant and total number of spikelets per spike. The field experiments were conducted during three vegetation seasons at the Plant Breeding and Acclimatization Institute – National Research Institute (PBAI–NRI), located in Radzików, Poland. The objective of this study was a comparative analysis of the structural yield-forming factor levels, which determine grain yield per spike and per plant of the DH lines and standard Izolda cultivar. Results indicate that several DH lines showed some differences in tested morphological structures of plant, yield factor levels and in grain yield per spike and per plant in comparison to standard Izolda, regardless of the year. Mean grain yield per plant of DH lines was 26.5% lower in comparison to standard Izolda only in the second year of study. It was caused by a reduction of productive tillers number. Structural yield-forming potential of DH lines was used in 38% and 59% and in case of Izolda in 47% and 61% (the second and the third year of experiment, respectively). The mean grain yield per spike of DH lines was 14.8% lower than Izolda cultivar only in third year of experiment and it was caused by about 12% lower number of grains per spike. Structural yield-forming potential of DH spikes was used in 82.4%, 85.4% and 84.9% and in case of Izolda in 83.8%, 87% and 89.5% (the first, the second and the third year of experiment, respectively). The grain yield per winter wheat plant (both DH lines and standard Izolda) was significantly correlated with the number of productive tillers per plant (r = 0.80). The grain yield per winter wheat spike (both DH lines and Izolda cultivar) was significantly and highly correlated with the number of grains per spike (r = 0.96), number of fertile spikelets per spike (r = 0.87) and the spike length (r = 0.80). Variation of spike and plant structural yield-forming factors determining grain yield levels were also analyzed. Calculated total variation coefficients values of each analyzed trait during three-year long studies were different depending on plant material – DH lines or standard Izolda. Low variation coefficients values characterized following traits (traits ranked by increasing values for DH lines and standard Izolda, respectively): total spikelets number per spike (6.6 and 6.3%), spike length (11.1 and 12.6%), fertile spikelets number per spike (13.7 and 11.7%), single grain weight (15.0 and 12.2%), shoot length (16.2 and 13.3%), grains number per spikelet (26.4 and 23.3%), total shoots number per plant (23.4 and 29.6%), grains number per spike (30.1 and 28.2%). Higher variation coefficients values were obtained for the following traits: grain yield per spike (40.0 and 35.7%), plant immature tillers number (35.8 and 42.6%), plant productive tillers number (42.2 and 43.2%), spike sterile spikelets number (46.6 and 44.7%) and number of grains per plant (58.3 and 60.5%). The highest values characterized grain yield per plant (66.9 and 60.8%).

In vitro plant regeneration from cotyledonary explants of Cucumis melo L. var. cantalupensis and genetic stability evaluation using RAPD analysis

An efficient plant regeneration protocol useful in genetic studies for important breeding lines of cantaloupe melons was developed. Three cultivars (Charentais-T, Vedrantais and Isabelle) and three doubled-haploid melon lines (NAD, DH-L2 and DH-L6) were tested for their potential to regenerate under in vitro conditions. Cotyledon explants from 6-day-old seedlings were cultured on three Murashige and Skoog based medium supplemented with different combinations of 6-benzylaminopurine (BAP) and indoleacetic acid (IAA). After 5 weeks of culture all the genotypes were able to regenerate on all media, however the regeneration frequency varied widely among the cultivars and the medium combinations used. The Charentais-T cultivar developed the highest number of shoots/explant on media containing BAP 1.0-IAA 1.0 × 10−2 mg/L (4.1) and BAP 1.2-IAA 1.2 × 10−2 mg/L (4.4) while on medium supplemented with BAP 1.3-IAA 1.3 × 10−2 mg/L the Vedrantais genotype gave the best result (2.4). The work first highlighted a good morphogenetic response of the DH lines; among all the genotypes and combination used, NAD (65.6 %) and DH-L6 (85.4 and 50.0 %) had the highest yield of regenerated plants. The results confirmed that the regeneration competence was strongly affected by genotype. Plantlets with well-developed shoot and root systems were successfully acclimatized and exhibited normal morphology and growth characteristics. In order to confirm genetic stability, a PCR-based RAPD analysis was carried out using 20 decamer-primers. No polymorphic bands were observed among all the plants revealing the genetic homogeneity of the regenerants. Furthermore, the ploidy level of regenerated plants was confirmed by flow cytometry.