Spontaneous Doubled Haploids Research Articles

IN VITRO DIPLOIDIZATION OF HAPLOID PLANTLET FROM ANTHER CULTURE OF EGGPLANT (Solanum melongena L.) USING PENDIMETHALIN

The availability of anther culture methods for producing pure lines in a doubled-haploid (DH) plant form makes it possible to accelerate the development of hybrid varieties in eggplant. Previous studies have developed an efficient eggplant anther culture method. However, the formation of spontaneous DH plants through this method is relatively low, ranging from 25%–30%. Recently several studies reported that pendimethalin effectively duplicates the chromosome numbers of several plant species in vitro. This study aimed to determine the pendimethalin effect on the diploidization of eggplant haploid plantlets from anther culture. The study compared three concentrations of antimitotic pendimethalin: 100 μM, 300 μM, and 1000 μM in three incubation durations: two, four, and six days. The results showed the treatment with a pendimethalin concentration of 300 μM incubated for two days gave the highest level of plantlet diploidization at 75.0%. In vitro, chromosomal duplication treatment using pendimethalin with different concentrations and duration of incubation time affected the plantlet survival and growth of eggplant haploid plantlets. The increase in pendimethalin concentration and incubation duration inhibited the physiology and growth of plantlets and affected alteration in the ploidy level of eggplant haploid plantlets.

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding.

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the '1009' genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.

Generation of doubled haploids in cauliflower

Hybrids of cauliflower are in high demand world over due to their high yield potential, earliness, better quality, better resistance to biotic and abiotic stresses. Conventionally, hybrids are developed from the intercrossing of two diverse inbred parental lines which are developed through continuous inbreeding for 8–10 generations and still don't attain complete homozygosity. Doubled haploid technology on the other hand generate completely homozygous inbred lines in a single step. Therefore, a study was undertaken at Punjab Agricultural University, Ludhiana, to develop a protocol for the development of doubled haploid lines in cauliflower. The anthers were excised from the flower buds of different genotypes viz. Jyoti, Pusa Sharad, Kartiki, CAUMH-2, CAUMH-10, LS-2, LS-3, and LS-5 followed by their culture on five different callus induction media compositions. Genotypes differed significantly in the ability to induce callus which was maximum in Jyoti followed by LS-2. Different media compositions also varied significantly in callus induction efficiency which was maximum on MS media+1.5 mg/L 2,4-D +1.0 mg/L NAA. Maximum shoot regeneration was recorded in genotype Kartiki followed by LS-2 when cultured on MS media+3.0 mg/L BAP+2.0 mg/L Kin. The regenerated shoots thus obtained were rooted on ½ MS media +1.0 mg/L IBA. Ploidy analysis of root tips revealed that 22.2% of the regenerated plantlets were haploids, 27.8% were spontaneous doubled haploids, 16.7% were tetraploids and remaining 33.3% were mixoploids.

Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Genetic variation of doubled haploids derived from anther culture of M1 red rice plants

Abstract. Nurmansyah, Setyadi AH, Fatumi NC, Fatmawati Y, Wulandari RA, Purwantoro A. 2021. Genetic variation of doubled haploids derived from anther culture of M1 red rice plants. Biodiversitas 22: 4923-4929. The doubled haploid (DH) technology when integrated with induced mutation could accelerate development of local rice cultivars with several desirable traits. Anthers from the first generation of mutant (M1) plants were utilized for DH production. However, the effectiveness of inducing and fixing mutation requires detailed evaluation with the help of molecular techniques for its accuracy and reliability than that of morphological or biochemical assessments. The objective of this research was to develop and detect genetic variation of DH plants derived from anther culture of M1 plants. Seeds of local red rice cultivar, Cempo Abang, were treated with four gamma-ray doses (0, 100, 200, 300 Gy) and planted to produce the M1 plants. Panicles at the booting stage of the M1 plants were collected and used as donor anthers. The present study suggested that gamma irradiation treatments indirectly could increase callus formation. However, it also reduced the number of callus producing plantlets due to higher frequency of brown calli. Eleven spontaneous DH plants obtained in the study along with parental plants were assessed to determine the occurrence of genetic variation using six RAPD primers. The RAPD primers generated 51 bands, of which 34 alleles or 66.7% were polymorphic with an average of 5.6 polymorphic alleles per primer. The genetic similarity among parent and 11 DH lines based on Jaccard’s similarity index ranged from 0.622 to 0.902. The DNA polymorphism among the DH plants demonstrated the effect of gamma irradiation to create genetic variation. Therefore, this method could be used as an alternative for rice breeding programs especially to develop preferred traits in the local rice cultivars.

Effect of incubation temperature and sucrose concentration on callus induction and plant regeneration in cauliflower (Brassica oleracea var. botrytis)

Androgenic and plant regeneration potential from antherculture were investigated in cauliflower genotypes underdifferent incubation temperatures, sucrose concentrationsand regeneration media compositions. Anthers werecultured on MS medium supplemented with 1.5 mg/L 2,4-Dand 1.0 mg/L NAA growth hormone with four differenttreatments of sucrose i.e. 3%, 5%, 10% and 13%. Maximumandrogenesis has been observed with 10% sucroseconcentration. Anthers of all the varieties were incubatedunder three different temperatures i.e. 25°C, 32°C and 35°C.Among these incubation temperatures, higher androgenesiswas observed at 32°C incubation temperature. Callus werecultured on five shoot regeneration media compositions,among which maximum regeneration has been observed onMS media supplemented with 5.0 mg/L BAP and 1.0 mg/LNAA followed by MS media supplemented with 6.0 mg/LBAP and 1.0 mg/L NAA. Maximum root regeneration hasbeen observed in ½ MS medium supplemented with 1.0 mg/L IBA growth hormone followed by ½ MS mediumsupplemented with 0.4 mg/L IBA. In cytological examination,20.3% of the regenerated plants were found to be haploidwhile 29.8% were spontaneous doubled haploids.

In situ Parthenogenetic Doubled Haploid Production in Melon "Piel de Sapo" for Breeding Purposes.

Doubled haploids in cucurbit species are produced through in situ parthenogenesis via pollination with irradiated pollen for further use as parental lines for hybrid F1 production. In this study, seven genotypes of melon “Piel de Sapo” were appraised for agronomic traits and pathogen resistances to evaluate its commercial value and used as donor plant material for the parthenogenetic process. Then, in situ parthenogenetic capacity of melon “Piel de Sapo” germplasm was evaluated and optimized. Several steps of the parthenogenetic process were assessed in this study such as melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of parthenogenetic embryos and plantlets, in vitro and in vivo chromosome doubling with colchicine or oryzalin and fruit set of doubled haploid lines. Parthenogenetic efficiencies of “Piel de Sapo” genotypes showed a high genotypic dependency during the whole process. Three different methods were assayed for parthenogenetic embryo detection: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was four times faster than one-by-one method and jeopardized eight times less parthenogenetic embryo obtention than liquid medium. One third of melon fruits set after pollination with irradiated pollen contained at least one parthenogenetic embryo. The 50.94% of the embryos rescued did not develop into plantlets because failed to germinate or plantlet died at the first stages of development because of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of the 26 parthenogenetic plantlets obtained was: 73.08% haploid, 23.08% spontaneous doubled haploid and 3.84% mixoploid. Two in vitro chromosome doubling methods, with colchicine or oryzalin, were compared with a third in vivo colchicine method. In vivo immersion of apical meristems in a colchicine solution for 2 h showed the highest results of plant survival, 57.33%, and chromosome doubling, 9.30% mixoploids and 20.93% doubled haploids. Fruit set and seed recovery of doubled haploids lines was achieved. In this study, doubled haploid lines were produced from seven donor genotypes of melon “Piel de Sapo,” however, further improvements are need in order to increase the parthenogenetic efficiency.

In vitro androgenesis: spontaneous vs. artificial genome doubling and characterization of regenerants.

Androgenesis has become the most frequently chosen method of doubled haploid (DH) production in major crops. Theoretically, plantlets derived from in vitro cultured microspore encompass halfof the normal chromosome number of donor plants and thus, considered to be haploid. However, depending on species/genotype and the method of haploid production, either via anther or isolated microspore culture, different ratios of spontaneous DHs and diploid (2n) or even polyploid plants originating from somatic tissues or unreduced gametes may also arise in the cultures. Adopting the method of haploid identification, anti-microtubular agent for restoring fertility, and discriminating spontaneous DHs from undesired heterozygote plants will substantially affect the success of androgenesis in breeding programs. The recent advances in the last 2decades have made it possible to characterize the in vitro regenerants efficiently either prior to genome duplication or using in breeding programs. The herein described approachesand antimicotubular agents are, therefore, expected to improve the efficiency of DH-based breeding pipeline through the in vitro androgenesis.

Improvement of anther cultures conditions using the Taguchi method in three cereal crops

Background: Plant tissue cultures have the potential to reprogram the development of microspores from normal gametophytic to sporophytic pathway resulting in the formation of androgenic embryos. The efficiency of this process depends on the genotype, media composition and external conditions. However, this process frequently results in the regeneration of albino instead of green plants. Successful regeneration of green plants is affected by the concentration of copper sulfate (CuSO 4 ) and silver nitrate (AgNO 3 ) and the length of induction step. In this study, we aimed at concurrent optimization of these three factors in barley ( Hordeum vulgare L.), wheat ( Triticum aestivum L.), and triticale (x Triticosecale spp. Wittmack ex A. Camus 1927) using the Taguchi method. We evaluated uniform donor plants under varying experimental conditions of in vitro anther culture using the Taguchi approach, and verified the optimized conditions. Results: Optimization of the regeneration conditions resulted in an increase in the number of green regenerants compared with the control. Statistic Taguchi method for optimization of the in vitro tissue culture plant regeneration via anther cultures allowed reduction of the number of experimental designs from 27 needed if full factorial analysis is used to 9. With the increase in the number of green regenerants, the number of spontaneous doubled haploids decreased. Moreover, in barley and triticale, the number of albino regenerants was reduced. Conclusion: The statistic Taguchi approach could be successfully used for various factors (here components of induction media, time of incubation on induction media) at a one time, that may impact on cereals anther cultures to improve the regeneration efficiency. Normal 0 false false false PL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:PL;}

Utilization of in Vitro Anther Culture in Spelt Wheat Breeding.

The efficiency of in vitro anther culture was screened in a full diallel population of four spelt wheat genotypes and ten F1 hybrids. Genotype dependency was observed based on the data of embryo-like structures (ELS), green-, albino plantlets. In the diallel population and ten F1 hybrids, the green plantlets production ranged from 13.75 to 85.00 and from 6.30 to 51.00, respectively. The anther culture-derived plants of F1 hybrids were grown up in the nursery. At the harvest, 436 spontaneous doubled haploid (DH) plants were identified among the 1535 anther culture-derived transplanted and grown up individual plants. The mean of spontaneous rediploidization was 28.4% which ranged from 9.76% to 54.24%. In two consecutive years, the agronomic values of ‘Tonkoly.pop1’ advanced line were compared with seven DH lines of ‘Tonkoly.pop1’ in the nursery. The DH lines achieved competitive values in comparison with ‘Tonkoly.pop1’ advanced line based on the 11 measured parameters (heading date, plant height, yield, hardness, width and length of seed, TKW, hulling yield, flour yield, protein and wet gluten content). These observations presage the efficient utilization of anther culture in spelt wheat breeding.

Male flower induction significantly affects androgenesis in cucumber (Cucumis sativus L.)

ABSTRACTMale flower induction and androgenesis in three genotypes of cucumber (Cucumis sativus), two Beth alpha F1 hybrids (BT1 and BT2, gynoecious) and Dastgerdi (DTG, monoecious), were evaluated. Induction of male flowers in gynoecious cucumber cultivars was assessed by single and combinatorial spraying of AgNO3 (3 mM), GA3 (1.5 mM) and CoCl2 (3 mM) after the first true leaf emergence. After 6 weeks from the beginning of treatments, the average numbers of male flowers induced by spraying AgNO3, GA3, and CoCl2 were 42, 14.5, and 0, respectively. Androgenesis was performed in two gynoecious cultivars, in which the donor plants were treated with AgNO3 (3 mM) or GA3 (1.5 mM) and the DTG cultivar without treatment. Among the three cultivars, the DTG showed the highest percentage of embryogenic calli formation (62.2%) and number of embryos per anther (1.81). In the gynoecious cultivars, the percentage of embryogenic calli formation (27.1%) and number of embryos per anther (0.23) in the anthers of GA3 treated plants were significantly higher than those treated by AgNO3. Cytological and SSR marker analysis of regenerants showed that 16 plants were spontaneous doubled haploid, 2 plants were diploid and one plant was tetraploid.

Line × tester analyses for anther culture response of bread wheat (Triticum aestivum L.)

The aim of this study was to investigate the gene effects that play a role in the inheritance of the anther culture response of the F1 and F2 populations obtained from the hybridization between the seven doubled haploid (DH) wheat line and four bread wheat cultivar using line × tester method as well as, to determine suitable parents and promising hybrids. Eleven parents and their twenty-eight hybrid combinations were examined for androgenic properties related to anther culture response such as the number of callus, green, albino, haploid, spontaneous DH and fertile plants. The obtained data were evaluated by the combining ability analysis calculated using the line × tester method and by estimating the heritability degrees. According to the results, for all traits examined, the non-additive gene effects were found as significant and narrow sense heritability at a low level. Significant combining ability effects have been identified for some parents and hybrids regarding traits examined. DH19, DH22, and Harmankaya-99 have been identified as suitable parents to develop the androgenic traits, while crosses formed between them have been determined as promising. A total of 444 plants (142 F1, 302 F2) were transferred to the greenhouse from the plants obtained from the anther culture, and 205 (68 in F1, 137 in F2) were fertile. Ninety-three of the fertile plants were obtained from the parents. The seeds of 112 DH plants obtained from hybrids were replicated to be used in breeding programs.

High Frequency of Doubled Haploid Plant Production in Spelt Wheat

AbstractThis is the first study to report an efficient anther culture (AC) method for spelt wheat, which has an increasing importance not only in applied research but also in organic farming and changing nutritional standards. In this study, an efficient AC protocol has been described for ‘GK Fehér’ spelt wheat. The number of AC-derived embryo-like structures (ELS) was 62.2/100 anthers, from which we were able to regenerate 30.6 green plantlets per 100 anthers. The percentage of green plantlets production was 89.0% among the regenerated plantlets, while the phenomenon of albinism was restricted (3.8/100 anthers). Altogether, from AC of ‘GK Fehér’ 306 green plantlets were producedin vitroand 241 plants were acclimatized to the greenhouse conditions. Based on ploidy level analyses, 83 spontaneous doubled haploid (DH) plants were produced (8.3 DH plants/100 anthers), so the percentage of spontaneous rediploidization was 34.4%. The spontaneous DH plants produced fertile spikes, while a few seeds were harvested from seven partially fertile plants.

High-purity Seed Production of Doubled Haploid Chinese Cabbage [Brassica rapa L. ssp. pekinensis (Lour.)] Through Microspore Culture

The purpose of this study was to produce doubled haploid red color heading type Chinese cabbage using isolated microspore culture. Genotypic differences in embryogenic response and regenerative ability of microspore-derived embryos to plants were observed. A high rate of plant regeneration from microspore-derived embryos was achieved by an improved protocol involving replacement of culture media and adjustment of heat shock temperature. More than 60% of regenerated plants were spontaneous doubled haploids. Haploids were characterized by short and malformed stamen and few viable pollen grains as compared to spontaneous doubled haploids and dihaploids. The seeds harvested from the 142 spontaneous doubled haploid plants were designated as H1; 25 seeds in each population was grown and characterized. The H1 generation has been shown to display considerable phenotypic variation and high purity. These spontaneous doubled haploids may be directly exploited as new varieties in a red colored Chinese cabbage breeding program.

High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.)

We developed an efficient culture system for producing cotyledonary embryos from isolated microspores of hot pepper (Capsicum annuum L.) and analyzed the ploidy levels of regenerated plants. Three culture protocols were studied: liquid, double-layer, and two-step culture. In the double-layer culture, cotyledonary embryos were produced more efficiently when the same medium composition was used for the liquid upper-layer and the solid under-layer. The two-step culture system, in which microspores were first incubated on liquid medium and then subcultured on double-layer medium, was most effective for producing cotyledonary embryos. Cotyledonary embryos were produced more efficiently when the isolated microspores were cultured in liquid medium for 1 week in 60 × 15-mm plates at a density of 8–10 × 104/mL and microspore suspensions from two liquid culture plates were combined into a single 100 × 20-mm plate containing solid medium, and the culture was continued for an additional 3 weeks. When cotyledonary embryos obtained from this two-step culture were transplanted into regeneration medium, more than 95 % developed into plants. Only 31 of the 190 analyzed plants (16.3 %) generated by this method were spontaneous doubled haploids. This two-step culture system outperforms all previously reported culture protocols for isolated microspores of hot pepper, and appears to be a promising tool for the production of haploid plants for hot pepper breeding.

A new microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials, expresses auxin and efficiently regenerates doubled-haploid plants in Brassica napus

BackgroundMicrospore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.ResultsWe have developed a new in vitro system at lower temperature (18°C) to efficiently induce microspore embryogenesis throughout two different developmental pathways: one involving the formation of suspensor-like structures (52.4%) and another producing multicellular embryos without suspensor (13.1%); additionally, a small proportion of non-responsive microspores followed a gametophytic-like development (34.4%) leading to mature pollen. The suspensor-like pathway followed at 18°C involved the establishment of asymmetric identities from the first microspore division and an early polarity leading to different cell fates, suspensor and embryo development, which were formed by cells with different organizations and endogenous auxin distribution, similar to zygotic embryogenesis. In addition, a new strategy for germination of microspore derived embryos was developed for achieving more than 90% conversion of embryos to plantlets, with a predominance of spontaneous doubled haploids plants.ConclusionThe present work reveals a novel mechanism for efficient microspore embryogenesis induction in B. napus using continuous low temperature treatment. Results indicated that low temperature applied for longer periods favours an embryogenesis pathway whose first division originates asymmetric cell identities, early polarity establishment and the formation of suspensor-like structures, mimicking zygotic embryogenesis. This new in vitro system provides a convenient tool to analyze in situ the mechanisms underlying different developmental pathways during the microspore reprogramming, breaking or not the cellular symmetry, the establishment of polarity and the developmental embryo patterning, which further produce mature embryos and plants.

Efficiency of SSR markers for determining the origin of melon plantlets derived through unfertilized ovary culture

The effects of temperature pre-treatment, thidiazuron, naphthaleneacetic acid, and 6-benzylaminopurine on in vitro gynogenic plant production from un-pollinated melon (Cucumis melo L.) ovaries were investigated. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. The temperature pre-treatment (4°C) for 4 days increased embryo formation frequency (63.3%) significantly. Addition of thidiazuron (0.04 and 0.02 mg/l) in the induction medium significantly increased the number of responding ovaries (46.6%, 65.83%), respectively. The maximum number of plantlet regeneration (22.5%) was achieved by culturing the ovary derived embryos on Murashigue and Skoog medium (MS medium) supplement with 0.6 mg/l 6-benzylaminopurine. Spontaneous doubled haploids originated directly through embryogenesis were subjected to genetic analysis using SSR molecular marker with 23 primers pair for homozygosity. SSR markers with microsatellite CMGA172, confirmed that the alleles in the parental material were also present in the gynogenic plantlets, but amplified only two alleles as compared to four alleles of the heterozygous parent material at same locus. Therefore these regenerated plantlets were consider homozygous and produced through a process of gametophytic embryogenesis.

Effectiveness of combining resistance to Thielaviopsis basicola and Tomato spotted wilt virus in haploid tobacco genotypes.

Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F1 hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F1 hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.

Regeneration of oat androgenic plants in relation to induction media and culture conditions of embryo-like structures

The effect of C17 and W14 induction media on the formation of embryo-like structures (ELS) from F3 generation of nine hexaploid oat hybrids was investigated in the study. In all genotypes, the highest number of ELS (0.6 - 12.1/100 anthers) was obtained on C17 medium. The efficiency of plant regeneration on medium 190-2 was tested, in relation to different ELS culture conditions. The highest rate of green plants per 100 ELS (3.3 - 42.4) was produced by incubation at 22<sup>o</sup>C in the dark for the first two weeks. Among 36 green regenerants, 28 (77.8%) were haploid and 8 (22.2%) were spontaneous doubled haploids, fully fertile. After colchicine treatment of haploid plants, 19 were partially fertile and set from 1 to 15 seed per panicle.

Study of androgenesis and spontaneous chromosome doubling in barley ( Hordeum vulgare L.) genotypes using isolated microspore culture

This research aimed to study the androgenesis and spontaneous chromosome doubling of five barley genotypes using an isolatedin vitromicrospore culture technique, involving a completely randomized design (CRD) with three replications. Statistical analysis of embryogenesis and cytogenetic results showed that genotype had a significant effect on haploid embryogenesis (P<0.01) and on spontaneous chromosome doubling (P<0.05). The genotype Igri was found to have the highest potential to produce haploid embryos (1577 embryos from 100 anthers), followed by the genotypes Boyer/Rojo, Afzal/Turkman/Kavir, Ashar/Hebo and Agrigashar/Matico with 369, 304, 278 and 150 embryos from 100 anthers, respectively. The highest percentage of spontaneous chromosome doubling (76%) was observed for the genotype which had the lowest embryogenesis (Agrigashar/Matico) and the lowest (65%) for the genotype with the highest androgenic capacity (Igri). Microspore embryogenesis also showed comparatively higher genotypic (109.2) and phenotypic (109.5) coefficients of variation, heritability (99.62) and genetic advance (1206.77), indicating the pre-dominance of additive gene action in the control of this character in the material studied. Estimates of genetic parameters (PCV, GCV and heritability) for microspore embryogenesis were higher than for spontaneous doubled haploids. These results indicated that selection for androgenic capacity would be more effective than for spontaneous doubled haploids. The findings showed a negative relationship (r= −0.68) between embryogenesis and spontaneous chromosome doubling in the barley genotypes studied. All the large embryos used had high regenerability and good plantlet formation.