Plant regeneration through callus initiation from mature embryo of Triticum

The behaviour of diverse Triticum genotypes in the tissue culture response of mature embryo callus was compared, and factors affecting tissue culture response were studied in this paper. Significant differences were detected in callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency when mature embryos of 31 plants of different Triticum species were compared. These were the main wheat cultivars of the Chinese northern winter‐type wheat region and breeding lines (Triticum aestivum L.), durum wheat (Triticum durum Desf.), cultivable emmer wheat (Triticumdicoccum Schuble) and the common wheat progenitors Triticum dicoccoides and Triticum aegilopides. The genotype dependency was particularly high in tissue culture of mature embryos of these Triticum genotypes. The efficiency of induction, differentiation and regeneration of mature embryos callus was high in genotypes selected out. Mature embryo‐derived callus of HB341, TS021, SN2618, T. dicoccum, HB188, and T9817 showed better tissue culture response than the other genotypes. Plantlets can be regenerated from mature embryo‐derived callus of 31 genotypes, saving on growth facility resources and time required for the collection of other explants, and providing a solid basis for the genetic transformation and molecular plant breeding of Triticum plants.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes

Seven genotypes of winter durum wheat (Triticum durum Desf.) were cultured to establish an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of immature and mature embryo cultures. Immature embryos were aseptically dissected from seeds and placed, with the scutellum upwards, in dishes containing Murashige and Skoog's (MS) mineral salts and 2mg 2,4- dichlorophenoxyacetic acid (2,4-D) per litre. Calli and regenerated plants were maintained on 2,4-D-free medium. Mature embryos were moved slightly on the imbibed seeds. For callus formation, the seeds with moved embryos were placed, furrow downwards, in dishes containing 8 mg 2,4-D per litre. The developed calli and regenerated plants were maintained on the MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Variability was observed among the wheat genotypes tested for various culture responses in both explant cultures. Callus induction rate and regeneration capacity of callus were independent of each other. Mature embryos have a low frequency of callus induction but a high regeneration capacity. Considering availability, rapidity and reliability, this form of mature embryo culture can be used as an alternative method for immature embryo culture.     

Embryo-rescue in the genus Tulipa L.; successful direct transfer of T. kaufmanniana Regel germplasm into T. gesneriana L.

Summary Embryo-rescue was studied as a means to overcome post-fertilization barriers in interspecific crosses in the genus Tulipa. With compatible T. gesneriana L. cultivar crosses, ovule culture was found to be superior to isolated embryo culture. Complete plantlet formation was possible from an embryo size of about 0.5 mm onwards.In the interspecific cross T. gesneriana x T. kaufmanniana, which is hampered by embryo breakdown, successful rescue of abortive embryos was demonstrated. Optimal embryo-rescue was achieved in cultures started seven to nine weeks after pollination. With cultures initiated at a later time, the rate of success decreased. A low number of germinative seeds were obtained after normal ripening of the seed pods, but by using ovule culture the efficiency of seedling formation could be increased dramatically.The ovule culture procedure will allow novel crosses and will offer new possibilities for the introduction of desirable genes into tulip cultivars.     

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.     

Interspecific hybridization in Cucumis L. II. The crossability of species,an investigation of in vivo pollen tube growth and seed set

Summary The crossability of 12 Cucumis species of African and Asiatic origin was studied in a diallel cross, in order to find ways to realise the cross between the common cucumber (C. sativus L.) and its wild relatives which carry resistances against diseases and pests.Self-pollinations and cross-pollinations within species gave normal pollen tube growth and seet set. The different accessions within a species, as a rule reacted alike in interspecific crosses. In crosses between African species different crossing patterns were found, viz. bilateral congruity, bilateral incongruity and unilateral incongruity. Within C. sativus all accessions intercrossed freely, except one, which displayed unilateral incongruity.Good seed was harvested from several crosses and in some cases embryo culture was needed for further development of seeds. No good seeds were obtained from any cross between a species of the African group and C. sativus L.     

Embryo rescue in plants—a review

Summary The plant breeders usually rescue inherently weak, immature or hybrid embryos to prevent degeneration. The successful production of plants from the cultured embryos largely depends upon the maturation stage and the composition of the medium. Abortion of embryos at one or the other stage of development is a characteristic feature of distant hybridization. For the first time successful embryo culture to obtain an interspecific cross between Linum perenne × L. austriacum was demonstrated by Laibach (1925, 1929). Since then several refinements have been made in embryo culture/embryo rescue techniques which have been a popular approach for raising hybrids from a number of incompatible crosses. Currently embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining plants from inherently weak embryos, obtaining haploid plants as well as for shortening the breeding cycle.     

Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is an important warm season turf and pasture grass. In vitro tissue culture of St Augustinegrass could serve as an important mean for its improvement through genetic transformation as well as induced somaclonal variation. To optimize tissue culture conditions for plant regeneration of St Augustinegrass, tissue culture responses of 11 explant tissues and four callus induction/subculture media have been examined. Embryogenic calli with regeneration potential were observed on cultures of early immature embryo [3 days after pollination (DAP)], immature embryo (7–14 DAP), and shoot base of young seedlings. The addition of benzyladenine (BA) in the callus induction/subculture medium enhances callus regeneration ability and does not harm callus induction for immature embryos. The best response came from 7 to 14 DAP immature embryo on MS medium containing 1 mg/l 2,4‐dichlorophenoxyacetic acid and 0.5 mg/l BA. The callus induction and regeneration rates were 97.7% and 47.6% respectively. However, BA supplement reduced callus formation and failed to enhance regeneration for young leaf bases. Scanning electron microscopy revealed that plant regeneration of St Augustinegrass is via somatic embryogenesis.     

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora x A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.Abbreviations DAP days after pollination - MS medium medium according to Murashige & Skoog (1962)     

Crossability and embryo rescue enhancement in wide crosses between wheat and three Agropyron species

Summary Soft winter wheat lines were crossed with Agropyron intermedium, A. elongatum and A. trichophorum using pollen from single plants of Agropyron spp. to pollinate wheat spikes. Not only species but also individual plants within varieties of Agropyron species differed in percent seed set with a wheat genotype. In two arrays of crosses between two phenotypically different plants of A. elongatum and nine wheat lines, one Agropyron plant gave higher seed set (overall=27.1%) than the other (overall=3.7%). The differences were significant in seven of the nine cross combinations. Results are consistent with the hypothesis that these two plants differ in their crossability as pollen parents with wheat, and suggest the possibility of occurrence of crossability genes in wheatgrasses. The success rate of hybrid embryo rescue was higher (87.5%) with cold treatment (4°C) than without cold treatment (75.0%) of excised embryos on culture media. Results underscore the significance of genotype of the alien species for crossing with low crossable wheats, and of the physical factors for improving embryo rescue in wide crosses.Contribution No. 11,825, Purdue Agric. Exp. Stn., West Lafayette, Indiana 47907, USA. The research was supported in part by Public Varieties of Indiana.     

Shortening the seed-to-seed cycle in artichoke breeding by embryo culture

Globe artichoke breeders have two important problems: lengthy seed to seed cycle and seed‐borne diseases caused by fungal pathogens. Both problems can be solved by embryo rescue. The objective of this work was to determine the best moment to isolate and culture the embryos and to determine the optimal in vitro culture conditions for their development. For that purpose embryos were collected from five cultivars in different postpollination stages and transferred into two different culture media. Growth regulators present in the medium resulted in poor root induction, but did induce a better shoot per explant development. Data have shown that 20 days is the optimal time for embryo rescue and that a medium without growth regulators would be more suitable for embryo development.     

Osmoregulation in birdseed millet under conditions of water stress II. Variation in F3 lines of Setaria italica and its relationship to plant morphology and yield

We report, for the first time, there generation of four homozygous lines in sweet cherry (Prunus avium L.) byin situ parthenogenesis followed by embryo and cotyledon culture. The sweet cherry cultivar ‘Altenburger’ was pollinated with marked pollen irradiated by γ-rays at doses ranging from 250 to1200 Gy. Pollination with such irradiated pollen affected fruit set and the quality of the embryos, and induced the formation of parthenogenic embryos. The immature embryos extracted from the stones, 35 or 75days after pollination, were cultivatedin vitro in an embryo or cotyledon culture. Although flow cytometrical analysis demonstrated the diploid level for all regenerants, four lines could be characterized as homozygous using isoenzyme analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific hybridization between Vicia faba (L.) and Vicia narbonensis (L.): Early pod growth and embryo-sac development

Summary Fertilized embryo-sac development and pod growth was studied in one Vicia faba cultivar, one Vicia narbonensis population and their reciprocal crosses. The initial development of endosperm and embryo was at least four days faster in V. narbonensis than in V. faba. Pods and ovules developed also faster in V. narbonensis than in V. faba. The growth rate of the hybrid pods followed the growth rate of the mother species, but was slower than that of the pods from selfed flowers. In the cross V. narbonensis × V. faba the ovules stopped growing 9 days after pollination, while in the reciprocal cross they stopped growing 15 days after pollination. Hybrid embryo-sacs from V. faba × V. narbonensis were aborted before they reached the stage of 256 endosperm nuclei or 200 embryo cells. Selfed V. faba embryo-sacs reached this stage in less than 9 days after pollination. In the reciprocal cross the embryo-sacs were aborted before they reached the stage of 128 endosperm nuclei or 80 embryo cells. Selfed V. narbonensis embryo-sacs reached this stage at the 4th day after pollination. Given that at these stages the embryo has less than 200 cells it was concluded that an in-ovule embryo culture technique should be developed to obtain hybrid plants.     

Studies of the Genetic Relationship between Anther Culture and Somatic Tissue Culture Abilities in Wheat

A population of thirty-eight doubled haploid lines, developed from the F₁ between two wheat parents differing in anther culture and somatic tissue culture responses, ‘was used to examine the genetical control of responses to these in vitro systems. During anther culture genetic variation between lines was exhibited for frequencies of callus induction., embryo production and embryo regeneration rates. In addition the relative frequencies of green and albino plants was shown to be genotype dependent. However, there was no correlation, between the frequencies of embryo production and the regeneration rate of those embryos suggesting an independent genetic control of these two components. Transgressive segregation for performance was observed for all components indicating that at least two genes are involved in the response of each, and lines for improved performance, combining high ernoryo production rates and good regeneration capacity were identified. No genetic variation for frequencies of callus induction from immature embryos was observed in this cross. However, genetic variation for the regeneration frequencies of plants was observed. Lines with an improved tissue culture response over the two parents were identified. There was no correlation between the performance of lines in anther culture and somatic tissue culture, indicating separate genetical control, and lines with alternative levels of response to the two systems were identified.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

Parental effects on crossability,embryo differentiation and plantlet recovery in wheat x rye hybrids

Summary One hexaploid wheat cultivar (Triticum aestivem) and two tetraploid wheat lines (T. durum) were crossed with seventeen inbred lines of rye (Secale cereale). Seed set, degree of hybrid embryo differentiation at the time of excision for in vitro culture and recovery of amphihaploid plantlets from various embryo categories were studied. Degree of embryo differentiation was predominantly determined by maternal wheats, paternal rye genotypes appearing to be of minor importance. T. aestivum x rye hybrid embryos were superior to those produced from T. durum for degree of differentiation. The proportion of plantlets developing from differentiated embryos was high for all wheat parents, whereas undifferentiated embryos were mostly unsuitable for plantlet production. The results revealed that cross-incompatibility in hexaploid wheat x rye crosses was due to failure of fertilization, while in tetraploid wheat x rye crosses it was caused by lack of embryo differentiation. Correlation analyses showed that seed set provided a criterion to predict the amphihaploid plantlets to be expected from a particular wheat x rye combination.     

Evaluation of androgenic competence through anther culture in common eggplant and related species

Anther culture is a convenient technique to obtain androgenic haploid and doubled haploid (DH) plants. In common eggplant (Solanum melongena), this technique has been used to develop DH pure lines for producing uniform F1 hybrid seed of some commercial varieties. However, a comprehensive study of the variation of this useful trait among different materials of common eggplant and related species is still lacking. In this work, we studied the androgenic response of 12 accessions of common eggplant and related materials from the primary (eggplant complex) and secondary genepools. We cultured anthers of all the accessions under the same experimental conditions, and studied their competence to produce calli, embryos and plants, as well as the quality and origin of the embryos produced. In our conditions, anthers of 11 out of the 12 accessions produced somatic calli, whereas only 5 also produced microspore-derived embryos, with variable results in terms of embryo quality and of frequency of embryo induction and plant germination. Embryos of responding accessions were initially haploid, and reached the DH status, verified with SSR markers, after a defined period of culture. In addition to other aspects common to many androgenesis-responsive species, our results allowed us to extract conclusions particular to common eggplant and relatives, including the difficulty for finding sources of androgenic competence out of S. melongena, the reduced impact of calli in the production of non-DH individuals, and the need to avoid the occurrence of severe anatomical and functional problems in the apex of most embryos, which seriously reduces their germinative success.     

Interspecific hybridization in the genus Gossypium through embryo culture

Summary With embryo rescue and culture techniques interspecific hybrids have been obtained amongst diploid cultivated (Gossypium arboreum, G. herbaceum) and wild species (G. stocksii, G. anomalum) of cotton. The early abscission of the young bolls was prevented by repeated application of growth regulators followed by the culture of immature hybrid embryos (15 days after pollination). The best growth and development were obtained on modified Murashige and Skoog's medium with supplements. The plants were stouter when the cultures were first incubated in the dark for 15–25 days, followed by exposure to light. The hybrid plants transferred to soil developed further and matured, and were more or less intermediate between their respective parents.     

A genetic linkage map of <Emphasis Type="Italic">Populus adenopoda</Emphasis> Maxim. × <Emphasis Type="Italic">P. alba</Emphasis> L. hybrid based on SSR and SRAP markers

Populus adenopoda Maxim. and P. alba L. [section Populus (aspen), genus Populus] are two tree species of ecological and economic value. To date, no high-density genetic maps are available for these two species. In this study, 1100 interspecific hybrids were obtained by controlled crossing and embryo culture. Simple sequence repeat (SSR) and sequence-related amplified polymorphisms (SRAP) were used to genotype 189 F₁ individuals. The genetic linkage map of P. adenopoda × P. alba generated from this study includes 212 markers (192 SSRs and 50 SRAPs) and consists of 26 linkage groups spanning 2178.5 cM, with an average distance of 11.7 cM between markers. This is the first SSR- and SRAP-containing genetic linkage map for aspen. The SSRs on the map will serve both as bridges for comparison with the poplar maps published to date and as a direct link to the Populus genomic sequence. Future studies focusing on the data presented here should enhance the density and precision of the map for identifying and localizing quantitative trait loci and promote genomic research on the genus.     

Somatic hybridization as a tool for tomato breeding

Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.Abbreviations cpDNA chloroplast DNA - mtDNA mitochondrial DNA