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.     

In vitro selection of tomato transformants at the immature embryo stage

Summary A method is described for selecting resistant transformed tomato genotypes in vitro at the stage of immature embryos. The utilization of HLH nutrient medium with the selective agent kanamycin is proposed. Normal development of seedlings from immature embryos, which do not form callus, is a good and true indicator for isolation of resistant genotypes. The immature embryos do not germinate and develop on MS selective medium.     

Embryo rescue in Rosa hybrida L.

Summary Two crosses between Rosa hybrida L. cultivars, that fail to produce progenies by conventional means, were carried out. In one of them, total hip abscision occured seven weeks after pollination; in the other one, only 2% of the fertilized hips remained on the plants after eleven weeks. Four- and five-week-old fertilized ovules were isolated in the first cross and six-week-old embryos in the second. The ovules were cultured on six media which differed in their mineral salt and sucrose concentrations while the embryos were cultured on only one of these media and eventually cold treated for one month at 4° C before being placed in a culture room set at 23° C. The embryos that had been isolated in ovulo when they were still not visible under a binocular lens developed atypically. The embryos isolated in ovulo when they were heart-shaped and on average 0.27 mm long performed in ovulo germination on some media and/or enlargement on all of them after two weeks; after another two week culture, once isolated from the ovule, all of them germinated. The cultured isolated embryos, that were exposed to cold, enlarged and germinated more rapidly when placed at 23° C than those not exposed to cold. Furthermore, the plantlets resulting from untreated embryo germination were characterized by large cotyledons which were only partially green. These results are discussed in regard to embryogenesis, precocious germination and dormancy.     

In vitro screening of strawberry plants for cold resistance

Formation of embryo autonomy of strawberry, plant regeneration fro membryo components, plant freezing conditions in vitro and the possibility to differentiate objectively genotypes by freezing them in vitro and in vivo were studied to create strawberry screening technology in vitro for cold resistance. It was established that autonomy of strawberry embryos manifests itself not earlier than on 14–16^th day after pollination and full autonomy is reached on 20–22^nd day. Plants regenerated from 26 days old embryos grew most intensively. At the highest rate strawberry plants regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons x on the medium with 1.0 BA and 0.5 NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was -8 to 12 ^°C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo, provided hardening proceeded not less than 21 days. The correlation between cold resistance in vitro and in vivo reached 0.93. Domination of cold resistance manifested itself in strawberry seedlings from various crossing combinations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interspecific hybridization between Cicer arietinum and C. pinnatifidum

An interspecific hybrid between Cicer arietinum cv. GL 769 and a wild species C. pinnatifidum was obtained after emasculation, pollination and application of growth regulators. Ovules were cultured and embryos were later dissected to obtain hybrid plants. These plants were albinos and morphologically resembled C. pinnatifidum. Shrivelled seeds were also obtained in 2% of the crosses, which on germination gave rise to albino plants. These plants did not survive beyond 20 days. The hybrid nature of these plants was confirmed by esterase isozyme studies. Hybrid shoots obtained from germinating embryos were cultured on modified ML-6 medium with BAP 2 mg/1, IAA 0.5 mg/1, where they turned green after 3–4 weeks. Transmission electron microscopy (TEM) studies on leaf sections from green hybrid shoots showed an improvement in the chloroplast structure, with better organized grana.     

New interspecific hybrids in the genus Agropyron

Summary Fifteen species from the genus Agropyron were crossed together. Fourteen of the crosses did not produce fruits. Twenty crosses produced varying numbers of caryopses. Of the fruits produced, 22% contained no embryos. For a variety of reasons it was possible to obtain only 2 plants from the remaining 140 embryos. The crosses which yielded viable plants were between A. trachycaulum cv. Primar (2n=28) and A. intermedium cv. Chief (2n=42) as well as between A. trachycaulum from Lethbridge (2n=28) and A. desertorum cv. Nordan (2n=28). The somatic chromosome numbers for the hybrids are 37 and 28. respectively. Studies of vegetative plant character are presented.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. I. Production of F1 hybrids through embryo rescue

Summary Triazine resistant Brassica napus ssp. oleifera and ssp. rapifera were hybridized to cultivars of B. oleracea ssp. italica, ssp. botrytis, ssp. capitata and ssp. fimbriata. The interspecific embryos did not survive in vivo but could be rescued in vitro using a culture medium developed by Monnier (1973). The embryos did not grow directly into normal plants but were successfully regenerated using the protocol developed by Keller (1984). Hybridization efficiency ranged from 0 to 2.64 hybrids per pollination. Interspecific embryo abortion may be related to abnormal endosperm development.     

Characterization of a green non‐dormant sunflower (Helianthus annuus) mutant NDG

A sunflower (Helianthus annuus L.) mutant was observed in the progeny of a cross between the sunflower cultivar ‘HA 89’ and an amphiploid of a Helianthus divaricatus L. × P21 cross that exhibited loss of dormancy induction in the developing embryo. Seeds of this mutant frequently germinate on the head about 40 days after pollination. The cotyledons of this mutant remain green, whereas some other non‐dormant mutants exhibit loss of pigmentation. The objectives of this investigation were to compare levels and activities of abscisic acid (ABA), a plant hormone that induces dormancy in developing embryos, in the non‐dormant green mutant (NDG) and ‘HA 89’ from which NDG was derived. Immunoassays showed that abscisic acid was present in NDG and the levels were not significantly different from those in ‘HA 89’. Exposure of excised NDG mutant embryos to 40 μm abscisic acid failed to prevent germination, suggesting that non‐dormancy could result from impairment in ABA receptors or from a defect in other proteins participating in the subsequent signalling pathway that normally induces dormancy.     

The evaluation of superior Hordeum bulbosum L. genotypes for use in a doubled haploid barley breeding programme

Summary Eight Hordeum bulbosum selections were produced from a cross between Cb 2920/4 and Cb 2929/1, two genotypes widely used in doubled haploid breeding programmes. The selections were hybridized with barley to evaluate their ability to produce high proportions of well-differentiated haploid embryos compared with Cb 2929/1 as control. We report here an initial small-scale investigation followed by a larger-scale test in two different environments to assess seed setting, haploid embryo differentiation rates and VB hybrid formation. These VB embryos contain both parental sets of chromosomes and occur more frequently in the glasshouse during the winter. Two of the eight selections were identified as combining the desirable characteristics of both parents, namely high seed setting on cv. Vada which is partially incompatible with H. bulbosum, large numbers of well-differentiated haploid embryos and a low incidence of VB hybrids. The selections are available for release to interested research workers and plant breedersAbbreviations DH doubled haploid - VB a hybrid from H. vulgare x H. bulbosum which contains both parental sets of chromosomes     

Plant Regeneration from in vitro Cultured Anthers of Black Mustard (Brassica nigra Koch)

Anthers of Brassica nigra, excised from fresh as well as cold-pretreated (3 days at 3 ± 2°C) buds cultivated on modified B₅ medium (Gamborg et al. 1968) containing sucrose level varying from 2 % to 10 %, along with 1O^?6M BAP (benzylaminopurine) and 9 × 10^?6M 2,4-D (2,4-dichlorophenoxyacetic acid), developed calli and/or embryos. The latter response was observed only in anthers reared on media containing 6 % or higher levels of sucrose. On media containing two or four per cent sucrose, the anthers produced calli, exclusively. The growth of embryos was inhibited or else they started callusing if left on the media containing higher levels of sucrose. However, on transfer to MS medium (Murashige and Skoog 1962), containing 2 % sucrose, embryos started callusing and subsequently a few secondary embryos differentiated. Such embryos were sub-cultured on MS + 5 × 10^?6M BAP + 2 % sucrose, wherein numerous shoots developed from embryos. The shoots were rooted by transferring to a medium containing 5 × 10^?6M NAA (naphthalene acetic acid). Within two months of culture, some of these plants started flowering in vitro.     

Enhanced pollen grain embryogenesis and plant regeneration in anther cultures of Brassica juncea cv. PR-45

Summary Pollen grain embryogenesis in anther cultures of Brassica juncea cv. PR-45 was considerably enhanced by treating the donor plants with 4 mll^-1 (v/v) of ethrel or delayed sowing of the donor plants, the latter treatment being superior. The anthers derived from plants sown about two months after the normal sowing period showed 18% androgenesis as compared to 3.5% in the control.Pollen grain embryos normally showed very poor germination (10%) on B₅ or B₅ containing GA₃. However, ABA or cold treatment promoted normal germination of these embryos. Exposure of the embryos to 4°C for 6 days, which proved to be the best treatment, induced 66% germination of the embryos.     

Doubled haploids of Coffea canephora: development,fertility and agronomic characteristics

Summary Doubled haploids (DH) of Coffea canephora Pierre were developed using haploid embryos which occur spontaneously in association with polyembryony. The frequencies of polyembryonic seeds and haploid embryos varied according to the parental genotypes. However, production of a large number of DH seemed possible from all genotypes. More than 750 DHs produced from various genotypes were grown under field conditions and evaluated for different characters of agronomic importance. Approximately half of DH genotypes did not survive, suggesting a strong, negative effect of homozygosity. Inbreeding depression is particularly severe on general vigor and reproductive aspects. For several characters studied such as leaf shape, leaf rust resistance and hundred bean weight, considerable genetic variations were observed within and between groups of DHs constituted by the DHs produced from the same clone. Despite their low vigor and reduced fertility, the DHs of C. canephora offer new possibilities in genetic research and coffee breeding.     

Genetic analysis of in vitro wheat somatic embryogenesis

Summary A spring wheat genotype which produces somatic embryos in vitro, after short and long-term culture, was tested for its ability to sexually transmit this embryogenic trait. Reciprocal crosses were performed between a embryogenic line and a nonembryogenic variety.Immature embryos were cultured on Murashige and Skoog medium plus 2 mg/l 2,4-dichlorophenoxyacetic acid, gelled with 5.5 g/l agarose. Somatic embryogenesis was not expressed in the F₁'s. In contrast, from several hundred immature embryos of the F₂ generation of one cross, 10.7% and 1.6% expressed somatic embryogenesis in short and long-term cultures respectively. These percentages of embryogenic: non-embryogenic fits a model of a few complementary genes. The embryogenic capacity of the F₂ genotypes depends on the presence of recessive alleles at these gene loci. The long-term wheat somatic embryogenesis capacity requires a more complex mechanism than the short-term one.Abbreviations CS Chinese Spring - Aq Aquila - E Embryogenic - NE Nonembryogenic - SC Subculture     

In-vitro Fertilization of Ovules of Some Species of Brassicaceae*

The method of in-vitro fertilization of ovules can be successfully applied to various species of Brassincaceae. Mature embryos and plants were obtained after in-vitro pollinating the ovules of Arabis caucasica, Brassica napus, B. oleracea var. sabellica (kale), B. oleracea var. italica (broccoli), Diplotaxis tenuifolia, Moricandia arvensis and Sisymbrium Loeselli. In the case of Sinapis alba fertilization and embryo development did not occur. The same method has been successfully used for obtaining hybrid immature embryos at different stages of development from crosses between B. napus X D. tenuifolia, B. napus X M. arvensis, B. oleracea var. italica X D. tenuifolia, D. tenuifolia x B. napus, D. tenuifolia. X M. arvensis and D. tenuifolia X S. Loeselli. The present findings show that in-vitro pollination of ovules of various species of Brassicaceae makes it possible to (a) perform the whole process of fertilization and embryogenesis; and (b) obtain intergeneric hybrid embryos.     

In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis

Embryo development in vivo has been studied in four Citrus aurantium L. polyembryonic genotypes. Seeds were collected 65, 85, 105, 125 and 220 days after pollination (DAP). None of the immature seeds harvested 65 and 85 DAP contained visible embryos. A single embryo at a more advanced developmental stage was observed in the central position at the micropylar apex of the embryo sac in about 74% of seeds harvested at 105 DAP, while at 125 and 220 DAP the majority of seeds had two or more embryos at the same developmental stage crowded together. Restriction fragment length polymorphism (RFLP) analysis of lowand high-copy-number nuclear DNA was used to distinguish zygotic from nucellar seedlings. Analysis of plantlets derived from in vitro culture of the bigger embryos, located in the central position at the micropylar apex of the embryo sac of seeds harvested at 105 DAP, established that the frequencies of zygotic embryos ranged from 82 to 88%. Media for immature embryo germination in vitro were based on the nutrients and vitamins of Murashige and Skoog (MS) and Murashige and Tucker (MT) media supplemented with various concentrations of sucrose and growth regulators. A total of 76% of globular stage embryos (<0.3mm) germinated on MT medium containing 150 mM sucrose and 14.4 μM gibberellic acid. Heart stage embryos (0.3-0.8 mm) germinated at 95% on MT medium supplemented with 150 mM sucrose and 2.9 μM gibberellic acid. The addition of 500 mg/l malt extract to MS medium increased the germination of early cotyledon stage (0.8-2.0mm) embryos to 98%. The optimum sucrose concentration for embryo rescue was 150 mM for the three embryo developmental stages. The ability to form plants in vitro strongly increased with increasing embryo developmental stage.     

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.     

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.     

Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos

Summary Experiments upon in vitro culture of immature durum wheat embryos, harvested at different growth stages, were made in two consecutive years. Callus formation and plantlet regeneration were obtained. The ability to form callus and the degree of morphogenetic processes varied with the different hormonal treatments used and with the age of the embryos. In the first year the best response for callus growth was observed with 2,4-D 2 mg l^-1 plus adenine 50 mg l^-1 or 2,4-D 5 mg l^-1 alone in the more mature embryos (15 and 20 days after anthesis). On the contrary, NAA 5 mg l^-1 had a greater shoot regeneration effect. In the next year, at all 2,4-D concentrations and for the two different ages of the embryos tested, all embryos formed callus. Regeneration of plantlets was obtained in higher percentage in calli originated from the more developed embryos. The effect of changed media upon plantlet regeneration was studied after callus transplant.Investigation by cytophotometry and chromosome counts on different calli showed, practically in all cells, a diploid condition. A histological analysis demonstrated embryogenic somatic characteristics in many samples of callus. The pattern of organogenesis seemed to be via adventitious bud formation but structures resembling embryoids were also observed in the callus.     

The Effectiveness of Vernalization of Immature Embryos of Winter Wheat var. Grana as Related to Age and Exogenous Phytohormones

Starting from the 10th day after pollination, immature embryos of winter wheat var. Grana were isolated and then vernalized for 4 to 8 weeks on Murashige and Skoog nutrients containing BAP (0.5 or 2 mg/dm³), NAA (0.5 or 2 mg/dm³), or GA₃ (5 or 20 mg/dm³). Vernalized seedlings were cultivated in a greenhouse and the number of days to the heading of individual plants as well as the percentage of plants capable of generative development were estimated. The lower limit of size for 50 % survival of embryos strongly depended on the phytohormone used: from 0.9 mm in control, 1.1 mm in nutrient containing BAP, 1.2 mm for NAA, up to 1.7 mm in nutrient with GA₃. Exogenous GA₃ was lethal for embryos younger than 18 days but induced elongation of older embryos. Embryos isolated 2.5 to 4 weeks after pollination showed minimal requirements for the length of vernalization. BAP increased the percentage of heading plants originated from the youngest embryos. GA₃ improved partial vernalization, strongly increasing the percentage of heading plants, but did not change the time from the end of vernalization to heading. It has been postulated that GA₃ increases number of plants capable of overcoming the threshold of induction of generative development but does not accelerate the flowering process. Hormonized plants showed no deformation of generative development. As the effectiveness of vernalization increased, the heading of plants was faster but they were shorter, forming spikes with a smaller number of spikelets and producing fever lateral shoots. The very young embryos probably have in reserve sufficient amounts of auxins and gibberellins and therefore exogenous GA₃ decreases their viability or even exerts a deleterious effect. However, as the embryos' ageing, gibberellin starvation develops. This being especially pronounced during vernalization. The de novo synthesis or activation of gibberellins takes place during the second stage of vernalization. This is why exogenous hormone improves the effectiveness of partial vernalization, though it is not possible to substitute by gibberellins the vernalization requirements of immature embryos.     

Intraspecific Variation in Mean Endosperm Cell Cycle Time in Vicia faba (L.) and Interspecific Hybridization with Vicia narbonensis (L.)

The rate of early embryo-sac development was studied in seven faba bean (Vicia faba L.) cultivars grown in a controlled environment. The embryo-sac stage was determined from the number of endosperm nuclei per embryo-sac and the number of embryo cells during the first 12 days after pollination. Differences in early embryo-sac development were observed between the genotypes studied. In addition, five different V. faba×V. narbonensis crosses and reciprocals were made using genotypes with small differences in mean endosperm cell cycle time and genotypes with large differences. The percentage of success (pod set) in V. faba×V. narbonensis crosses ranged between 9 % and 59 % while in the reciprocals it ranged from 12 % up to 30 %. However, cytological studies showed that the high percentage of success (pod set) observed in the cross A-107 × A-202 was not associated with a higher percentage of interspecific hybrid embryos. The results indicate that genotypes of the two species with smaller differences in mean endosperm cell cycle time result neither in a higher percentage pod set nor in bigger hybrid embryos.