Anther Culture Response in Perennial Ryegrass (Lolium perenne L.)

20 diploid clones from 7 varieties, and 10 tetraploid clones from 3 varieties of Lolium perenne were tested in replicated anther culture experiments. Embryos or calluses, were obtained from all clones, and plants were regenerated from all clones except one. The total yield of plants (albino and green plants) ranged from 0 to 61 plants per 100 cultured anthers among genotypes, and there was a general tendency for tetraploic genotypes to be more responsive. Viable green plants were obtained from 5 diploid and 7 tetraploid clones representing 2 and 3 varieties, respectively. Their origin from reduced pollen was confirmed by a haploid chromosome number in some regenerants and by homozygosity for isozyme loci in spontaneously chromosome doubled plants produced from heterozygous diploid donor plants. A large number of the plants were successfully established in the soil. For most donor genotypes, green plants were rare exceptions, but two diploic clones consistently produced 2.3 and 3.8 green plants per 100 cultured anthers, respectively. Estimates of variance components from replicates with greenhouse and field-grown donor plants showed that genotypes accounted for about 73 per cent of the total variation in yield of embryos/calluses, while only 14—15 per cent of the total variation was due to replicates. Hence at present, emphasis should be placed on die selection of high-response genotypes in material of high agronomic potential.     

Ploidy Manipulation and Introgression of Resistance to Alternaria Helianthi from Wild Hexaploid Helianthus Species to Cultivated Sunflower (H. annuus L.) Aided by Anther Culture

The present investigation discusses the scope for transferring of resistance to leaf spot disease incited by Alternaria helianthi from two hexaploid wild species (H. tuberosus and H. resinosus) to diploid cultivated sunflower. Interspecific hybrids produced between sunflower and these two hexaploid species were partially fertile with tetraploid chromosome status. Backcrosses of these interspecific hybrids with cultivated sunflower resulted in the formation of sterile triploid plants. To overcome the problem of sterility and facilitate backcrosses with cultivated sunflower, anther culture of the tetraploid interspecific hybrids was carried out to bring down their chromosome number to diploid status. Anthers from both interspecific hybrids were cultured on basal Murashige and Skoog media supplemented with varying concentrations of organics and the growth regulators benzyladenine and naphthaleneacetic acid. Anthers of interspecific hybrids involving H. resinosus responded well and regenerated through an embryogenic route at a frequency of 98.7%. But in interspecific hybrids with H. tuberosus, anthers formed callus and subsequently regenerated shoots through an organogenic pathway. DNA ploidy analysis of anther culture plants of interspecific hybrids derived from H. tuberosus crosses was carried out to identify plants with desired diploid status. In vitro screening of parents, interspecific hybrids and anther culture plantlets against A. helianthi showed resistance in 68.5% of the anther culture plants of interspecific hybrids from H. tuberosus and in 24.3% of the plants derived from interspecific hybrids involving H. resinosus.     

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 2006 with corrections to the Cover Date.     

Components of Response in Barley Anther Culture

Anther culture response with 17 widely-grown varieties and one model variety of barley was studied with one replication from field-grown donor plants and one replication from a growth-chamber. Plants were regenerated from all 18 varieties and green plants were obtained from 16 of them. On average, 1.6 green plants were obtained per 100 cultured anthers from all the material. Estimated variance components for the formation of embryos/callus from the anthers were dominated by the effects of the genotypes and interactions between plant material and environments which together accounted for 60.1 and 17.0 % of the total variation respectively, while environments were nonsignificant for this character. Plant regeneration from embryos/callus were not significantly influenced by either genotype or environments. Components of variance for green plant formation were dominated by the effects of the genotypes, accounting for 73.2 % of the total variation, and a smaller effect from environments accounting for 11.2% of the total variation. Main effects from genotypes on the percentage of green regenerants divided 7 varieties into two distinct groups, indicating that major genetic factors were involved. The genetic basis for green plant regeneration seems different from that governing embryo formation. The results are discussed with respect to the possible prediction of anther culture response for new barley hybrids, as a means for directing the use of barley anther culture towards material that responds well.     

Monitoring genetic fidelity vs somaclonal variation in Norway spruce (Picea abies) somatic embryogenesis by RAPD analysis

Summary Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation (eg. in many forest trees), or in the regeneration of genetically transformed plants. Random amplified polymorphic DNA (RAPD) was used to analyse somatic embryos and plants regenerated from embryogenic cell lines in Norway spruce, Picea abies (L.) Karst. RAPD facilitated the identification of clones, as material from the same cell lines shared identical patterns of amplified fragments, whereas regenerants from different cell lines were easily distinguishable by their respective patterns. For comparisons with explant donor genotypes, cell lines were initiated from cotyledons. Some of the seedlings that had parts of their cotyledons removed were grown on as control plants. Somatic embryos regenerated from cotyledon cell lines showed no aberrations in RAPD banding patterns with respect to donor plants. We conclude that gross somaclonal variation is absent in our plant regeneration system.Abbreviations ESM embryogenic suspensor mass - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - (2,4-dichlorophenoxy)acetic acid 2,4-D - 1-naphthaleneacetic acid NAA     

Genetic Variability for Haploid Production in Crosses Between Tetraploid and Hexaploid Wheats with Maize

Crosses were made between 14 wheat genotypes (11 tetraploid, 3 hexaploid) and a single Fl hybrid of maize that was used as the male parent. The experimental design consisted of randomized blocks with three replications. Plants were grown under controlled greenhouse conditions (day length 16 h and temperature 25 °C/15 °C, day/night). To enhance embryo survival, 2, 4-D treatment (10 mg/1) was applied to spikes 24 h after pollination with maize. Embryos were recovered from all tetraploid and hexaploid wheats at a rate of 2.09 to 26.76 per 100 pollinated florets. Haploid and doubled haploid plants were obtained from all hexaploid genotypes (T. aestivum) and from 5 of 11 tetraploid genotypes (T. turgidum var.). The most important point of these experiments was the ability to produce haploid plants from tetraploid wheat for two reasons: firstly, anther culture cannot be applied in tetraploid wheat (T. turgidum var.) due to the inefficiency of embryo formation and the high proportion of albino plants. Secondly, to date, crosses between tetraploid wheat and maize have resulted in embryo formation, but not in haploid plants.     

Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Summary Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of Mickylee were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.     

Analysis of protoplast-derived plants of Saintpaulia ionantha H. Wendl

Of 3272 plants regenerated from protoplasts of 10 Saintpaulia ionantha genotypes, 98.4% survived transfer to the greenhouse. The frequency of regenerants with chlorophyll deficiencies, i.e. variegated leaves or albinos, was low (1.5%). There was a higher number of polyploid, in most cases tetraploid plants, regenerated from protoplasts (16%) which were identified by their altered morphology. Measurements of stomatal length and counting the number of chloroplasts per guard cell also allowed a clear differentiation between diploid and polyploid plants. The classification was confirmed by DNA content determination using flow cytometry. Mechanisms leading to polyploidization included spontaneous protoplast fusion as well as chromosome doubling during callus growth and shoot regeneration. Two genotypes with instabilities in flower colour showed completely altered flower colours in plants regenerated from protoplasts as well as in plants regenerated on leaf explants in vitro.     

The Response of Anther Culture in a Genetically Wide Material of Winter Wheat (Triticum aestivum L.)

In a screening experiment with 215 different winter cultivars of Triticum aestivum L. for response in anther culture, pollen embryos or callus were obtained from 200 different cultivars and green plants from 93 different cultivars. On average, from the whole material, 1.3 green plants were obtained per 100 anthers cultured. Variance components estimated from replicates with selected parts of the plant material indicated that for the formation of pollen embryos from anthers, interactions between genotypes and replications were dominating, accounting for 45 to 50 per cent of the variation. Main effects from genotypes were less prominent, accounting for 25 to 30 per cent of the variation, and the replications showed relatively little effect, accounting, for about 4 to 12 per cent of total variation. Regeneration of plants from pollen embryos or callus was not significantly influenced by either genotype or replicates. The frequencies of plants regenerated being green, however, were influenced from both genotype and environments contributing 42.5 and 34.9 per cent of the total variation respectively, while only minor interactions between genotype and environments were indicated for this character.     

Plants regenerated from embryo cultures of an apomictic clone of Kentucky bluegrass (Poa pratensis L. ‘Baron’) are not apomictic in origin

Plants were regenerated from tissue cultures of embryos dissected from seeds that were harvested from a self-pollinated clonal selection of Kentucky bluegrass (Poa pratensis L.) ‘Baron’, an apomictic cultivar. Plants were regenerated from 35 embryo-derived callus cultures of the 3280 embryos that were plated. Flow-cytometric (FCM) and RAPD-marker analyses were performed to determine if regenerants were or were not apomictic in origin. Fifteen regenerants with a 3c DNA content were classified as arising from 2n + n aberrant embryos, which was a higher frequency than expected, based on a chi-square analysis. Of the remaining 20 regenerants with a 2c DNA content, a chi-square test showed that all could have arisen from n + n sexually-derived embryos, based on the observed segregation of n + n regenerants, which fit the expected 3:1 ratio of dominant:recessive RAPD-marker phenotypes. The apparent lack of regenerants of apomictic origin, and implications for genetic transformation and breeding of Kentucky bluegrass are discussed.     

Effects of D-genome chromosomes on crossability of hexaploid triticale (X Triticosecale Wittmack) with maize

With the aim of producing polyhaploids of hexaploid triticale, 20 genotypes from a CIMMYT breeding programme and eight D-genome chromosome substitution lines of ‘Rhino’ were crossed with maize. In crosses between 20 triticale genotypes and maize, 15 lines produced embryos. Frequencies of embryo formation ranged from 0.0 to 5.4%, with an average of 1.1%. From a total of 200 pollinated spikes, 62 plants were regenerated. Most regenerated plants were polyhaploids with 21 chromosomes, and few aneuhaploids with 22 chromosomes were found. In crosses of triticale substitution lines with maize, all the lines produced embryos, while ‘Rhino’ produced no embryos at all. Higher frequencies of embryo formation were obtained in substitution lines with chromosomes 2D and 4D. These results suggest that D-genome chromosomes in a triticale genetic background have the effect of increasing the frequency of polyhaploid production in triticale x maize crosses.     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

Genotypes of Perennial Ryegrass (Lolium perenne L.) with High Anther-Culture Response through Hybridization

Anther-culture response in Lolium perenne was studied in the hybrid populations from three responsive diploid parent clones to obtain genotypes with superior anther-culture response through hybridization. Genetic variation for anther-culture response was significant both within and between the four hybrid families and their parents. A reciprocal effect was indicated for embryo formation and regeneration percentages for one of the cross combinations. Among 55 hybrid clones tested in two environments, 6 clones were identified with an anther-culture response level superior to their parents. While the parents produced one green plant or less per 100 anthers, these superior clones produced from 11 to 59 green plants/100 anthers, mainly as the result of improved embryo formation and improved percentages of regenerated plants being green. A significant correlation (r = 0.397^**) was observed between embryo formation and percentage of green plants. The results are discussed with regard to the possible transfer of anther-culture response to non-responsive breeding material through hybridization.     

Genetic analysis for haploid-regenerations responses of the hexaploid-wheat anther cultures

Genetic variability in response to anther culture was investigated in 49 winter hexaploid wheats, comprising 33 pure lines (F₁₀) derived from a composite cross programme and their 16 parental genotypes. All genotypes were grown in a randomized block design with three replications in a controlled greenhouse. The number of embryoids and total plant regeneration per 100 anthers, as well as the numbers of green and albino plants regenerated per 100 embryoids, were measured. Significant genetic variability was observed among the 49 genotypes for all the traits studied. All traits showed high heritability. Among the genotypes compared, DC²30N and 1BPT-40 gave the best results for the production of embryoids and IBPT-78 had the highest value for the production of green plants. The genotype IBPT-34 developed a large number of albino plants, and it should be useful as a parent in studies to determine the genetic control of albino plants in wheat.     

Evidence for genetic control and media effect on haploid regeneration in the anther culture of hexaploid wheat (Triticum aestivum L.)

Crosses were made between seven hexaploid wheat genotypes. Twenty-one F₁ hybrids and their parents were grown in a greenhouse with 16 h day/8 h night at 25°C and 15°C, respectively. The experiment was a complete randomized block design with three replications. Each replication consisted of one pot with three plants. Anther culture was performed in two different induction media (CHB and W14) and androgenetic traits were studied. Statistical analysis was carried out separately for each induction medium. Genetic variation was highly significant for androgenetic traits and the best parent (IBPT 19) produced 68 embryos and 9.3 green plants per 100 anthers in CHB medium. Genetic components were affected by induction media and some components were significant in one medium and non significant in the other. General combining ability (GCA) was significant for all androgenetic traits, except for albino plant regeneration in both media and total plant regeneration in CHB medium, whereas specific combining ability (SCA) was not significant for the traits studied. Narrow sense heritability was high for embryo induction frequency and green and total plant regeneration. All our results indicate that androgenetic parameters can be improved in hexaploid wheat by genetic means.     

Analysis of Chinese Spring regenerants obtained from short- and long-term wheat somatic embryogenesis

Summary Somatic embryogenesis was initiated from immature embryos on Murashige-Skoog (MS) medium plus 2 mg.l^-1 2,4-dichlorophenoxyacetic acid, 2% sucrose and 0.6% agarose. Somatic embryos were isolated and regenerated into whole green plants on MS medium devoid of 2,4-D. These regenerants were previously demonstrated to differ in their mitochondrial DNA organization. In order to estimate their characteristics three progenies of short-term culture regenerants and three progenies of long-term culture regenerants were analyzed and compared to the parental line. These somaclones obtained from the wheat variety Chinese Spring were evaluated for variation of 13 agronomic and morphological quantitative characters in comparison to the parental line. Significant variation was observed for plant height, spike length, main tiller diameter, between the somaclones regenerated from long-term culture and their parent. Differences were observed to increase with the duration of culture, leading to a significant modification of the structure of the plants. Several changes occurred during the somatic tissue cultures, but to a lesser extent than has previously been described in the literature.     

Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM

Isolated microspore cultures from two doubled haploid (DH) lines of wheat, Triticum aestivum L., were used to develop an in vitro chromosome-doubling protocol. During the initial 24 h or 48 h of culture the microspores were treated with either of the two antimicrotubule herbicides trifluralin or amiprophos-methyl (APM) in concentrations ranging from 0.1 μM to 10μM. Untreated control cultures yielded 209 embryos per 100000 microspores, which is the equivalent of one spike. Among the regenerated plantlets 67% were green, and 15% of the flowering plants were spontaneously chromosome doubled. Treatments with both the herbicides had a significant effect on chromosome doubling, measured as the percentage of fertile regenerants. With the best combination of treatment duration (48 h) and herbicide concentration (10/μM) the percentage of fertile plants among regenerants could be increased up to 74% with APM and up to 65% with trifluralin. The largest numbers of DH plants per spike could be obtained with herbicide concentrations at 1–3 μM. Treatments with either herbicide at these concentrations resulted in an estimated average between the two genotypes of 27 DH plants per 100 000 microspores. These results demonstrate the high potential of APM and trifluralin as chromosome-doubling agents in isolated microspore cultures. The in vitro treatment integrated into tissue culture procedures will constitute an efficient method for chromosome doubling in future wheat breeding     

The origin of clones among androgenic regenerants of hexaploid triticale

Doubled haploids (DH) have become a standard tool in breeding and genetic studies of many crops and in most cases androgenesis is the only available route of their production. It has been recently observed that some populations of DH lines obtained via androgenesis contain high proportions of clones. This seriously reduces the efficiency of breeding and may jeopardize genetic studies. This study was designed to determine at which stage of androgenesis these clones are created, using samples set aside during routine production of DH lines in breeding of hexaploid triticale. The fate of each androgenic structure was carefully followed through the entire regeneration process, and all obtained plants were genotyped using DNA markers. Overall, 189 plants were regenerated forming 33 families, each originating from a single original androgenic structure (callus, polyembryos). In ca. 80 % of cases all members of a family were genetically identical. However, in about 20 % of cases the families of regenerants were genetically heterogeneous, showing that not all androgenic structures originate from single microspores. The evidence shown here demonstrates that retention of single plants from each original structure guarantees the production of only unique genotypes but it reduces the total output of plants. If maximum output is desired, multiple regenerants from single callus can be retained but must be genotyped using at least 10 polymorphic markers to identify clones.     

Genetic stability at nuclear and plastid DNA level in regenerated plants of <Emphasis Type="Italic">Solanum</Emphasis> species and hybrids

In this work we detected the extent of variability at nuclear and cytoplasmic DNA level of regenerated plants belonging to Solanum genotypes with a different genetic background and somatic chromosome number. As for the nuclear characterization, a total of 66 (18.5%) polymorphic bands were scored using 13 ISSR primers on 45 randomly selected regenerants. Our results show that the regenerants obtained from clone cmm 1T and, at lower level, those from cph 1C are unstable under in vitro conditions or rather more prone to in vitro-induced stress leading to somaclonal variation than the other genotypes used. Two types of changes were observed: disappearance of parental ISSR fragments, termed “loss”; appearance of novel ISSR fragments, termed “gain”. The most frequent event occurring in the regenerants was the loss of fragments (41 bands). Regenerated plants were analyzed with seven plastid universal primers to determine the cytoplasmic composition at chloroplast level. All cpDNA primer pairs tested produced amplicons of the same size in all genotypes analyzed and no polymorphic fragments were observed with any universal primers used. Our results show that under in vitro culture conditions genotype affects the integrity of the genome. In addition, the absence of polymorphism at plastid level confirms the greater genetic stability of cytoplasmic DNA.