Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

Induction of haploid and diploid plants though in vitro anther culture of haploid wheat (n=3x=21)

Summary Five haploid plants of wheat were used for anther culture. Embryos were formed and six plants were regenerated. Of these, two were haploid (n=3x=21) and two diploid (2n=6x=42). The two diploids derived from the anthers of the same haploid wheat plant gave seeds, but the fertility was reduced in one of them showing, abnormalities at meiosis.     

Temporal changes in floral nectar production, reabsorption, and composition associated with dichogamy in annual caraway (Carum carvi; Apiaceae)

The dynamics of nectar production were studied in perfect florets of two varieties (Karzo, Moran) of annual caraway (Carum carvi L., Apiaceae). Florets were protandrous and strongly dichogamous, lasting 7-15 d but producing nectar from the stylopodia for 4-12 d, in an interrupted fashion. Nectar secretion began during a floret's phase of stamen elongation and anther dehiscence. After reabsorption of uncollected nectar, at which point nectary surfaces were completely dry, the two styles elongated and a second bout of secretion commenced during the female phase, up to 5 d later, when a floret became receptive to pollination. During the male and female phases, respectively, 0.392 ± 0.064 μL and 1.083 ± 0.261 μL of nectar of similar solute concentration (844 mg/mL) was produced per ten florets. On a daily basis, florets yielded 1.5-fold more nectar in the female than during the male phase. First-time nectar removal throughout the female phase did not match the sum of nectar quantities from male and female phases combined, suggesting that under natural conditions, any uncollected male-phase nectar, once reabsorbed, is not made available to visitors of the same florets when in the female phase. Nectar-sugar composition differed between bouts of secretion; it was hexose-rich (59.6% fructose, 26.9% glucose, 13.6% sucrose) initially, but hexose-dominant (70.2, 26.8, 3.1) during the female phase. A 5.7-fold difference in mean nectar production per floret occurred among plants.     

Gametic selection in anther culture of rice (Oryza sauva L.)

Summary The segregation and recombination of heterozygous isozyme markers have been monitored in anther culture derivatives (i.e., six nonmorphogenic microspore-derived callus [NMC] populations and two anther culture plant [ACP] populations) and F₂ plants generated from six F₁ hybrids of rice, including five japonica upland/improved indica tropical hybrids. The alleles in excess at some loci displaying skewed segregations in the F₂s were consistently overrepresented in the NMC populations. These alleles were also generally found to be overabundant in the two ACP populations except for certain loci that contrastingly segregated in a 11 ratio. Additional distortions were found to be specific to AC derivatives indicating the existence of in vitro gametic selection. Overall, however, the gametic selection in the ACP materials was neutral with regard to the indica and japonica differentiation. Estimates of linkages between markers borne by chromosome 6 using AC-derivative data were consistent with those noted in the F₂s and with current knowledge of the isozyme locus linkage map. Given the average neutrality of gametic selection and the consistency of linkage relationships in the ACPs, their further use as rice molecular mapping and gene tagging populations can be investigated with confidence.Joint contribution with Research Institute for Tropical Food Crops, Department of the International Centre of Agronomical Research for Development (IRAT-CIRAD), 45bis avenue de la Belle Gabrielle, F-94736 Nogent s/Marne Cédex, France and International Rice Research Institute (IRRI), P.O.Box 933, Manila, Philippines     

Genetic studies of corn (Zea mays L.) anther culture response

Summary Anthers of two maize (Zea mays L.) inbred lines, DBTS (P₁) and B73 (P₂), their F₁, F₂ and first backcross generations — F₁ x DBTS (B₁), and F₁ x B73 (B₂) — were float cultured in YP medium to study the inheritance of corn anther culturability using generation mean analysis. Significant effects of generation were observed for the three traits measured: anther response (%), frequency of embryos (%) and anther productivity. Variation among the generations was similar for anther response and frequency of embryos: no significant differences were found among the P₁, F₁, F₂ and B₁ means, but the means of P₂ and B₂ were significantly lower than those of the other generations. For anther productivity, the F₂ generation tended to have a slightly higher tendency for multiple embryo formation. A simple additive-dominance model was adequate in explaining the inheritance of anther response and frequency of embryos, but digenic epistasis (additive x dominance) was involved in the inheritance of anther productivity. Additive genetic variance was higher than non-additive genetic variance for all the traits; however, only environmental variance was significant. Narrow-sense heritability estimates were 65% and 75% for anther response and frequency of embryos, respectively. Significant inter-plant variation was observed within generations, even for the inbred line DBTS, but isozymic analysis involving five enzyme loci did not reveal any genotypic variability within the inbred lines DBTS and B73.     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Genetic studies of rice (Oryza sativa L.) anther culture response

Anthers of two rice (Oryza sativa L.) varieties, Lunhui 422 (P₁) and Jinzao 5 (P₂), their F₁, F₂ and first backcross generation-F₁ x Lunhui 422 (B₁), and F₁ x Jinzao 5 (B₂)-were cultured in L8 medium to study the inheritance of rice anther culturability using generation mean analysis. Significant effects of generation were observed for the four traits measured: anther response (%), frequency of callus induction (%), frequency of callus differentiation (%) and culture efficiency (%). Variation among the generations was similar for all traits: significant differences were found among six generations and the means of the P₂ and B₂ were significantly lower than those of the other generations. The frequency of callus differentiation showed a nonsignificant difference among the P₁, F₁, F₂ and B₁ generations which had slightly high values than the P₂ and B₂. Additive genetic variance (V_A) was higher than non-additive genetic variance (V_D) for anther response and frequency of callus induction. However, A_V was lower than V_D for frequence of callus differentiation and culture efficiency, V_A was significant for all traits except for the culture efficiency, and V_D was nonsignificant for all traits except for the frequency of callus differentiation. On the other hand, environmental variation (V_E) was significant for the 4 traits. Narrow-sense heritability estimates were 95.52%, 82.19% and 13.54% for anther response, frequency of callus induction and culture efficiency, respectively.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Substitution analysis of callus induction and plant regeneration from anther culture in wheat (Triticum aestivum L.)

The genetic determination of callus induction, total plant regeneration and green plant regeneration from anther culture were studied using a Chinese Spring/Cheyenne substitution series. All the three characteristics were found to be polygenically determined, but their inheritance was independent from one another. The 7A and 18 chromosomes had a considerable effect on callus induction. In the case of total plant regeneration the most influential chromosome as the 3A while the 2D chromosome showed a definite influence on green plant regeneration. The interaction between the genetic background of the recipient plant and the substituted chromosome plays an important role in the manifestation of the studied features.     

Nuclear genes affecting albinism in wheat (Triticum aestivum L.) anther culture

Summary Inheritance of the ability to respond in wheat anther culture was studied from 6×2 reciprocal crosses between six varieties with high and two varieties with low capacity for green plant formation and their parents, replicated in two environments. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 78.4% and 85.4% of total variation, respectively, while smaller genetic effects were indicated for regeneration. Nuclear genes could explain almost all the genotype effects in this material. Embryo formation showed heterosis over high parent for 5 of the 12 hybrids, while percentages of green plants from the hybrids were intermediate to the parents. General Combining Ability (GCA) could explain 78.8% of the variation for embryo formation among the hybrids, whereas differences in percentage of green plants were dominated by Specific Combining Ability (SCA), accounting for 67.9% of hybrid variation. A positive correlation (r=0.81^**) was observed between the genetic capacity for regeneration and green plant formation. Analysis of covariance indicated that effects causing GCA for green plant formation were mainly responsible for this correlation. A regression model with two parallel lines divided the six parent lines with high green plant formation into three groups with respect to their reactions with the two testers. The results are discussed with regard to possible involvement of two sets of nuclear genes affecting the percentage of green plants obtained in wheat anther culture: one set consisting of mainly additive effects affecting green plant percentage through an initial effect on regeneration ability, and another set of two or a few more major genes with dominance or epistatic effects uncorrelated with regeneration.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

Direct regeneration of haploid or doubled haploid plantlets in cucumber (Cucumis sativus L.) through ovary culture

This paper reports a simple and effective method of directly producing cucumber plants through unfertilized ovary culture. The paper involves an experiment investigating factorial interactions between TDZ treatment, cold pretreatment, genotypes of cucumber, to improve plant induction. Cold pretreatment was effective in stimulating the ovary. The results showed that cold pretreatment for 4 days, TDZ for 0.06 mg·L^?1, the interaction with genotype can be used as an effective strategy to improve the efficiency of gynogenesis. The plant regeneration induction rate was highest (79.3%). In addition, we observed the process of cucumber megasporogenesis and plant regeneration. The plants obtained from ovary culture of cucumber were identified as diploid or haploid by flow cytometry, consistent with the results of chromosome counting. The diploid plants were further identified as pure doubled haploid using simple sequence repeats (SSR). The doubling treatment we used was one of the simplest and most effective methods, completed in a short time (1 h) with a doubling rate of 75%. The acclimation rate for the surviving was 70%. This work provides a basis for promoting haploid breeding in cucumber.

     

Anther culture and haploid plant regeneration in purple coneflower (Echinacea purpurea L.)

Anthers containing microspores at the uninucleate stage were excised from capitula of field grown plants of purple coneflower, Echinacea purpurea, and cultured on medium conducive to callus growth. In callus induction cultures, N6 basal medium was more effective than Murashige and Skoog (MS), and a combination of benzyladenine (BA) at 2.22 μM with naphthaleneacetic acid (NAA) at 0.054 μM was more effective than 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.52, 9.05 and 13.57 μM. Callus induction rate as high as 85.8% was achieved, and no statistically significant differences were found among cultures with various densities of 20, 40, 60, 80 and 100 anthers per bottle each filled with 40-ml medium. Shoots were regenerated from calluses on MS medium containing 2.22 μM BA and various concentrations of NAA, with the highest regeneration rate of 95.24% obtained when 0.27 μM NAA was applied. Although a large portion of the␣regenerated shoots had prominent symptom of vitrification, some normal shoots could be easily rooted on MS medium containing 0.054 μM NAA. Thirty regenerated plants were randomly selected and 19 of them were confirmed to be haploid by observation of chromosome number of root-tip cells.     

Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

Summary Several factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory. Published with approval of the Director of Arkansas Agricultural Experimental Station.     

Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH doubled haploid - LS Linsmaier and Skoog basal medium - BAP benzylaminopurine - GLM Generalized Linear Model - SAS Statistical Analysis System     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.