Production of Gynogenetic Plants of Onion (Allium cepa L.) after Crossing with Irradiated Pollen

The possibility of producing gynogenetic plants with pollen inactivated by irradiation was explored for the onion (Allium cepa L.). Two successive experiments included the use of cytoplasmic male sterility, recessive nuclear markers in the female plants and pollen irradiation of 150 Gy. Pollinations were achieved with the aid of flies and all seeds were harvested without the use of embryo rescue. Plantlets with recessive characters could be detected and they contained haploid and diploid cells. The development of these plantlets until flowering was observed under agronomic conditions. The use of doubled haploid (DH) plants in onion breeding is discussed.     

Haploid induction from F1 hybrids between CMS shallot with Allium galanthum cytoplasm and common onion by unpollinated flower culture

The induction of haploid plants from F₁ hybrids between CMS shallot with Allium galanthum cytoplasm and common onion was examined. Starting with 535 unpollinated flowers cultured in B5 medium 25 seedlings from part henogenetic embryos were obtained of which 13 seedlings survived. Eleven seedlings were determined as haploid plants (2n = x = 8) and 2 seedlings were doubled haploid plants (2n = 2x = 16). All haploid and doubled haploid plants preserved chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) from A. galanthum. Segregation in different characters was observed among the haploid plants. The haploid and doubled haploid plants exhibited the different combinations of genes from shallot and common onion. Crossing of the doubled haploid plants with other shallot strains, common onion cultivars or related species may produce excellent F₁ hybrids for bulb production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Application of in vitro organ culture in wide-cross breeding of rapeseed

Oil radish (Raphanus sativus var. raphanistroides Makino) is resistant to drought and low temperature. In order to breed more resistant cultivars of rapeseed, the wide cross between rapeseed (Brassica napus L.) and oil radish was made. Rapeseed was not compatible with oil radish, and the frequency of hybrid plants (F₁) was very low. Moreover, the hybrid plants were sterile. In order to recover the intergeneric hybrids (F₁), the in vitro organ culture technique was applied in our experiments. The frequency of hybrid plants (F₁) was increased up to 25.55% by means of in vitro culture of pollinated ovaries. Some fertile amphidiploid hybrid plants were obtained by means of colchicine treatment of small buds obtained from cultured flower receptacle segments of hybrid plants (F₁). It is suggested that the technique of in vitro culture of pollinated ovaries and flower receptacle segments is useful in the wide-cross breeding of rapeseed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)

The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and 6-benzyladenine (6—BA) in the medium is required to induce an acceptable yield of gynogenic embryos from unfertilized ovary and flower cultures in onion. Four different exposure times of ovary and flower cultures to exogenous growth regulators (15, 30, and 45 days, and the entire culture period) were assayed. The objective was to ascertain the effect of these substances and of their period of application on the formation of gynogenic embryos and on the yield of haploids. An exposure of 15 days was sufficient for ovaries and flowers to be stimulated towards the gynogenic response, whereas, for the remaining period of 30—80 days, the pro-embryos could easily grow on a growth-regulator-free medium. In the gynogenic material obtained, phenotypic differences were visible between genetically independent lines but not between plants within each line, even when they had a different ploidy level (n or 2n). Almost all lines obtained by gynogenesis were sterile. Only a small percentage (1%) became fertile through spontaneous chromosome doubling, and these produced 2—20 seeds each, giving normal plants. The recovery of fertility occurred more often during the generations of bulbils. To exploit this natural propensity towards diploidization in this phase, different amounts and numbers of applications of colchicine were evaluated in two experiments. The treatments corresponding to 10 and 100 mg/1 of colchicine applied for 24h gave the highest number of diploid cells in root tips but no diploidization occurred in the shoot apices. Three days of colchicine treatment at 10 mg/1 produced 46% of plants completely diploid in the shoot apex. The flower fertility of these doubled haploid plants is being evaluated.     

Interspecific hybridization inBrassica juncea × Brassica hirta using embryo rescue

Summary Interspecific hybrids have been obtained in an otherwise incompatible cross betweenBrassica juncea × Brassica hirta through the in vitro culture of hybrid ovules and ovaries. The best response was observed from ovules and ovaries cultured 10–15 and 5–7 days after pollination respectively on a basal medium supplemented with indoleacetic acid, kinetin and casein hydrolysate. In some cases the basal cut end of the ovaries proliferated to form callus and shoots. The in vitro-derived hybrid seeds varied in their colour, size and shape, and the F₁ plants in the field showed a large diversity in their morphological traits. The hybrids were sterile, and had an intermediate number of chromosomes (2n=30).     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.     

Interspecific hybridization of Brassica napus and B. juncea through ovary,ovule and embryo culture

Summary Employing in vitro culture of ovaries, ovules and embryos, interspecific hybrids have been obtained amongst two important oilseed crops, Brassica napus x B. juncea and their reciprocal. The test-tube hybrid plants have been transferred to the field, and reared to maturity. The F₁ seeds obtained from the hybrid ovaries showed normal germination, and the hybrid plants exhibited a range of variation of characters.     

Genetic mapping of AFLP markers in Japanese bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis>)

Summary The first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P₁)BC₁ and (P₂)BC₁ populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P₁) and J1s-14s-20 (P₂). Based on the (P₂)BC₁ population, a linkage map of P₁ was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P₂ was constructed with the (P₁)BC₁ population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P₂ map were connected with their counterparts in P₁ map using co-dominant anchor markers, 13 SSRs and 1 CAPS.     

Optimal pollination conditions for seed set after a self-pollination,an intraspecific cross and an interspecific cross of marrow-stem kale (Brassica oleracea var. acephala)

Summary The effects of different pollination techniques, with and without emasculation and delayed pollination, were investigated to find the conditions for maximum seed set after self-pollination and intraspecific and interspecific crosses of Brassica oleracea var. acephala. The results indicated that the pollination conditions achieving maximum seed set vary with the type of pollination. After controlled self-pollination, the best seed set occurs in bud 3 to bud 10. For the intraspecific cross, the youngest flower and the oldest bud produced the largest number of developed ovules but bud pollination was productive to bud 8. The yields from these two pollination types were best when the female parent was not emasculated. In the interspecific cross with B. campestris cv. Marco the best results came from the youngest flowers and the oldest buds subjected to the standard practice of pollinating directly after emasculation. Possible reasons for these effects are discussed.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Embryo rescue and plant regeneration following interspecific crosses in the genus <Emphasis Type="Italic">Hylocereus</Emphasis> (Cactaceae)

The aim of this study was to develop an efficient methodology to rescue embryos following interspecific crosses in the genus Hylocereus. Crosses between the diploids Hylocereus polyrhizus and H. undatus in both directions were performed. Fertilized ovules carrying embryos at very early pro-embryonic stages were excised from ovaries 5 days after pollination (DAP) and placed on half-strength basal MS medium containing 680 μM glutamine, 0.55 μM α-naphthaleneacetic acid (NAA), 0.45 μM thidiazuron (TDZ) and various concentrations of sucrose. After 30 days in culture, ovules were isolated from the surrounding tissue and transferred to the same fresh medium. Significant differences were found between the main effects (cross and sucrose concentration) in ovule response, i.e., increased ovule size and callus formation. The best responses were obtained in the cross: H. polyrhizus × H. undatus; and sucrose concentration of 0.09 M. In terms of embryo conversion, polyembryony and number of regenerated plants, the highest responses were observed on the culture medium supplemented with 0.17 M sucrose in both interspecific crosses. All tested plants were found to be diploid by flow cytometric analyses. Fluorescent amplified—fragment length polymorphism (fAFLP) confirmed the hybrid origin of the regenerated plants. This study reports on the success of a three-step embryo rescue procedure for Hylocereus species. The procedure developed here provides the means for producing plants from very-early embryo stage, thus expanding the prospects for vine-cactus breeding programs.     

Gamma-irradiation of cacao (Theobroma cacao L.) pollen: Effect on pollen grain viability,germination and mitosis and on fruit set

Summary Theobroma cacao pollen fertilization capability was studied after 0, 50, 100, 200, 500 and 1000 Gy gamma-irradiation. For all irradiation doses, no alteration of pollen grain viability and in vitro germination was observed. In situ, for all doses, pollen tubes penetrated into the styles and reached the ovules 20 hours after pollination. In vitro observations of the pollen grain nuclei after 20 hours incubation showed that pollen irradiation causes inhibition of the division of the generative nucleus. Fruit survival rate 30 days after pollination decreased as irradiation doses increased from 0 to 100 Gy, and over 100 Gy no fruit set was obtained. The possibility of using irradiated pollen as a method for obtaining haploid cacao plantlets is discussed.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

Obtention of haploid embryos and plants through irradiated pollen technique in squash (Cucurbita pepo L.)

The influence of gamma ray doses (25, 50, 75, 100, 200, 300 and 400 Gy) and genotypes (Eskenderany F₁, Acceste F₁, Sakiz, Urfa Yerli) on the induction of haploid embryos obtained by irradiated pollen technique was studied in squash (Cucurbita pepo L.). Different shapes and stages of embryos were derived from seeds extracted from fruits harvested 4–5 weeks after pollination. As a result of the present study, haploid embryos and haploid plants were obtained, with haploid production strongly influenced by gamma ray doses, embryo stages and genotypes. Gamma ray doses of 25 and 50 Gy gave the highest parthenogenetical response. All of the point shape, globular shape, arrow tips and stick-shaped embryos developed into haploid plants. However, only 53.8% of torpedo and 23.1% of heart-shaped embryos gave haploid plants. In contrast, cotyledon-shaped and amorphous-shaped embryos produced only diploid plantlets. The number of embryos per 100 seeds was the highest in ‘Eskenderany F₁’ and ‘Sakiz’ genotypes. After in vitro culture, a total of 93 haploid plantlets were obtained. This revised version was published online in August 2006 with corrections to the Cover Date.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

Bud pollination and hybrid seed production in detergent-induced male sterile plants of Brassica juncea

The efficacy of a synthetic detergent (Surf Excel) as a potential chemical hybridizing agent in Brassica juncea was studied. Foliar sprays with various concentrations of the detergent caused reductions in plant height, number of branches and leaves per plant, size of leaves, anther size, pollen per flower, ovules per flower, pollen fertility, fruits per plant, fruit size, seeds per fruit, total yield per plant and 100 seed weight as compared with those of untreated plants. The style in the floral buds of plants sprayed with different concentrations of Surf Excel elongated and so the receptive stigma protruded from the buds which facilitated cross‐pollination by honey bees. The plants sprayed once with 2% Surf Excel exhibited an elongated style with a raised receptive stigma and 100% pollen sterility without causing a significant reduction in total yield.     

Hybridization in Brassica juncea × Brassica campestris through ovary culture

Summary Ovary culture has been employed for the production of interspecific hybrids of a partially compatible cross of Brassica juncea (2n=36) × Brassica campestris (2n=20). Five to seven days old ovaries cultured on White's medium supplemented with casein hydrolysate (300 mg/l) and sucrose (5%) produced more seeds than any other media tried, but seed development was better on media fortified with plant hormones. The seed yield was better in B. juncea × B. campestris than their reciprocal cross. The plants obtained from ovary-derived seeds were transferred to the field; they were intermediate in some morphological characters and chromosome number (2n=28) as compared to their parents. The flower buds generally did not open and had poorly developed anthers with mostly sterile pollen. The pod size/setting was very much reduced, but healthy seeds were obtained.     

In vitro selection for oleic and linoleic acid content in segregating populations of microspore derived embryos of Brassica napus

Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62% C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection for high oleic acid can be started already during an early stage of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphological and RAPD analysis of poplar trees of anther culture origin

Our objectives were to improve the rate of haploid plant regeneration through increasing the rate of callus initiation on the anthers and sustaining shoot regeneration frequency, and to analyze the field population of anther culture origin by morphological and molecular methods. Regarding the callus initiation, the most responsive clones were ‘N-90’(59%) in P. nigra and ‘D-29’ (75%) in P. deltoides. The rate of shoot regeneration and number of shoots/calli ranged from 4%–79% and 1–9, respectively. From the 208 rooted plants 8 haploid, 179 diploid, 4 tetraploid and 17 aneuploid plants were found. In the field population, the haploid plants could be easily identified by their retarded development and morphological characteristics (size and shape of the leaves, strong branching, etc). Several diploid plants showed depressed developmental and morphological traits similar to the haploid ones. Three traits (growth rate, leaf blade length and shape of leaf base) of the 6 different morphological characteristics measured were in correlation with the ploidy level within the poplar field population. Six primers of the 48 primers tested were able to detect polymorphism among the field plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of diploid and triploid interspecific hybrids between Lilium longiflorum and L. concolor by ovary slice culture

Ovary slice culture, after cut-style pollination, was used to develop interspecific hybrids between Lilium longiflorum and L. concolor. Reciprocal crosses between diploid cultivars (2n = 2x = 24) were carried out. On the days 30, 35, 40 and 45th after pollination (DAP), ovaries were sliced and cultured on a modified hormone-free Murashige-Skoog (M–S) medium without NH4NO3, supplemented with 6% sucrose, 50 mg/1 yeast extract and 0.25% gelrite at pH 6.3. For the L. longiflorum × L. concolor cross, ovule germination was found to be best at 30 DAP. After transfer to a M–S (half-strength) medium supplemented with 1.5% sucrose and 0.25% gelrite at pH 5.8, diploid and triploid hybrid plants were established. In contrast, ovules from the L. concolor × L. longiflorum cross did not germinate. The hybridity of the plantlets obtained was verified by karyotype and isozyme analysis. The importance of the ovary slice culture technique as a tool to develop new hybrids between incompatible lilly plants is discussed.