Genetic analysis of anther culture response in wheat using aneuploid,chromosome substitution and translocation lines

Summary Marked effects of genotype on wheat anther culture response have been observed. Genetic factors have been recognised to be one of the major contributors to in vitro responses of cultured wheat tissues. In wheat anther culture, embryo induction, plant regeneration and albina/green ratio have been determined to be heritable traits. Using Chinese Spring (CS) monosomic 1D, single chromosome substitution lines of chromosome 5B or chromosome arm 5BL from Chinese Spring into six varieties, and F₁ hybrids heterozygous for the 1B chromosome structure (1BL-1BS/1BL-1RS), the anther culture response was studied: genes on CS1D chromosome and 5BL chromosome arm increases the embryo frequency; gene(s) involved in regeneration ability are located on the 1RS chromosome arm; a gene increasing albina frequency is located on Chinese Spring 5B chromosome. Our results support the fact that without gametic selection, a differential development occurred from the particular classes of microspores carrying genes for higher regeneration ability. Moreover, in some crosses, a few genes with major effects were involved in determination of anther culture response.     

Methodical improvements in rye anther culture

Summary The crucial problem in anther culture of rye (Secale cereale L.) is the very low regeneration capacity. Our study was conducted to overcome this restriction. The plant material used included a doubled haploid line (DH), two single crosses between DH Unes, and a tetraploid Secale cereale L. population. The factors carbohydrate source, post-plating temperature treatment, and gelling agent were investigated. Substantial progress was achieved by substituting maltose for sucrose. Top rates of 49 % responding anthers and 20 % green plants were obtained from one of the single crosses after a post-plating cold treatment on geirrte solidified medium. We consider our results a methodical step forward in rye anther culture.     

Chiasma distribution in rye chromosomes of diploid rye and in wheat/rye addition lines in relation to C-heterochromatin

Summary In five genetically different inbred lines of rye and in the seven Chinese Spring/Imperial wheatrye addition lines, chiasma distribution in rye chromosomes was studied with respect to the amount and position of constitutive heterochromatin (Giemsa C-bands). In all inbred lines, rye chromosomes with one primary terminal band were more frequently found as univalents than those with primary bands on both telomeres. These chromosomes were most probably 5R and/or 6R. In the addition lines a highly significant reduction in the number of arms bound by chiasmata was found for rye chromosomes 5R and 6R. Because of the similar chiasma distribution in the inbred lines and in the rye chromosomes of the addition lines, no effect of the wheat genome on the number of chiasmata in the rye chromosomes can be ascertained. However, a relationship between chiasma frequency and chromosome arm length seems to exist, since under reduced chiasma conditions the two shortest arms of the rye complement, those of chromosomes 5R and 6R, frequently fail to form a chiasma. No effect of the large blocks of constitutive heterochromatin in the telomeres of the rye chromosomes on the position of chiasmata within a bivalent could be established.This study was financially supported by the Deutsche Forschungsgemeinschaft     

Agronomic performance of lines derived from anther culture, maize pollination and single-seed descent in a spring wheat cross

Anther culture and maize hybridization are two frequently used techniques for doubled haploid production in wheat (Triticum aestivum L.). Information on the field performance of lines derived from these techniques is limited. This study was conducted to compare the performance of F_4:6 lines obtained by single-seed descent with lines obtained by anther culture and maize (Zea mays L.) pollination from the same cross of spring wheat, ’Chris’/MN 7529. Thirty-three lines derived from each of those techniques were evaluated in six environments for grain yield, protein content, test weight, heading date, kernel weight and plant height. Mean performance of the single-seed descent lines exceeded performance of the anther culture lines for grain yield, kernel weight and plant height with no apparent differences for grain protein content, test weight and heading date. No differences between trait means for the single-seed descent and maize pollination lines were found except for plant height. The best 5 lines from each method for grain yield, protein content and test weight were similar in performance except that the protein content was higher for the maize pollination lines than for the single-seed descent lines. Acceptable levels of agronomic performance could be found among lines from each method. Wide acceptance of the doubled haploid technique for pure line production in breeding programs may, however, be limited by the often poor efficiency of doubled haploid line production, resulting in smaller population sizes for selection of desirable traits in comparison to the single-seed descent method. Received: 31 July 1998 / Accepted: 28 November 1998     

Alloplasmic wheat - Elymus ciliaris chromosome addition lines.

Alloplasmic euploid wheat with the cytoplasm of Elymus ciliaris (2n = 4x = 28, ScScYcYc) is male sterile and has reduced vigor. However, alloplasmic plants with E. ciliaris chromosomes 1Sc or 1Yc marked by gliadin genes Gli-Sc1 and Gli-Ycl, respectively, are vigorous and fertile. The Rf genes on 1Sc and 1Yc are named Rf-Sc1 and Rf-Yc1. Two chromosome translocations involving 1Yc were isolated. The first involved the short arm of 1Yc translocated to the short arm of wheat chromosome 3B. The second involved the short arm of 1Yc translocated to the short arm of a chromosome, designated L, of E. ciliaris. The second line also has another E. ciliaris chromosome designated A and lacks wheat chromosome 6A. This line is resistant to Puccinia recondita. The relationship between fertility restoration and nucleolar organizing regions is discussed. Key words : Triticum aestivum, Elymus ciliaris, chromosome addition, Rf genes, nucleolar organizing regions.     

Cytodifferentiation and transformation of embryogenic callus lines derived from anther culture of wheat

Three types of callus tissues established from anther culture of eleven doubled haploid (DH) lines of wheat (Triticum aestivum L.) were evaluated for their ability in enhancing friable embryogenic (Type II) culture differentiation and genetic transformation. Differences between types of callus inocula were highly significant (P < 0.001), suggesting that the quality of the initial callus explant is of profound importance in encouraging the proliferation of Type II cultures. Other factors found to be crucial included weekly subculture of friable embryogenic callus tissues on a maintenance medium containing 30 microM dicamba and a predominance of amino-acid nitrogen supplement. Transfer and integration of the beta-glucuronidase gene was also affected by the type of inoculum when suitable embryogenic cell cultures were transformed using silicon carbide whiskers and high velocity microprojectiles. Expression of the hygromycin phosphotransferase selectable marker gene sequence was confirmed in all the stably transformed cell lines maintained on selection media containing lethal levels of hygromycin. Comparatively, there were differences in the frequency of regenerable, transgenic clonal segments between whisker-treated and microprojectile bombarded tissues mainly as a result of the fact that cultures vortexed with whiskers were more capable of post-treatment cell proliferation and embryo differentiation than those bombarded with cDNA-coated microprojectiles. Conditions for obtaining these results are outlined and discussed in relation to the suitability of the two transformation strategies for producing transgenic cell aggregates of wheat.     

Solid-stemmed spring wheat cultivars give better androgenic response than hollow-stemmed cultivars in anther culture

Solid-stemmed spring wheat cultivars (Triticum aestivum L.) are resistant to the stem sawfly (Cephus cinctus Nort.) and lodging. Anthers of 24 spring wheat cultivars with varying content of pith in the stem were used in the experiment. All were classified into three groups: solid, medium–solid and hollow stems. There was considerable influence of the cultivar on callus formation and green plant regeneration. The highest efficiency of green plant regeneration (24%) was observed for the solid-stemmed AC Abbey cultivar. There was no regeneration from the explants of four cultivars: CLTR 7027, Alentejano, Marquis and Bombona. Principal component analysis showed no differences between the cases under observation (callus induction and green plant regeneration) in their response to pre-treatment temperatures (4 and 8°C). The examination of the effects of various auxin types in the induction medium on callus formation and green plant regeneration revealed that the strongest stimulation of these processes was observed in the C17 medium with 2,4-D and dicamba. The efficiency of callus formation and green plant regeneration was greater in solid-stemmed cultivars than in hollow-stemmed cultivars.     

Callus induction and organogenesis in wheat/Aegilops longissima chromosome addition lines

The influence of individual Aegilops longissima addition chromosomes on callus induction and organogenesis of in vitro cultivated immature embryos of wheat was studied. Immature 14-day-old embryos of seven Chinese Spring lines with added chromosomes of Aegilops longissima were in vitro cultivated. Although relatively high number of calli were formed in embryo cultures of all lines studied, the different genotypes expressed variation in respect to their response to in vitro cultivation. The number of initiated calli of line D, containing chromosome l l arms 7S L and 4S L was relatively low. The lines showed substantial differences in respect to the number of initiated morphogenic calli and regenerants recovered. The highest number of morphogenic calli and regenerants l l l were obtained in cultures of lines containing the chromosomes 2S , 6S and to less extend 3S.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.     

Reaction of alien chromosome substitution and addition lines of hard red spring wheat to common root rot and black point.

Resistance to common root rot and black point, caused by Cochliobolus sativus, was evaluated in alien chromosome substitution and addition lines of the cultivars 'Cadet' and 'Rescue'. Substitution of chromosome 5B in 'Rescue' with 5Ag from Agropyron elongatum decreased root rot susceptibility to a level intermediate between that in the susceptible 'Rescue' and the resistant 'Cadet'. The substitution of 'Rescue' chromosome 5A or 5D with 5Ag, or the addition of 5Ag to 'Rescue' complement had no consistent effect on root rot susceptibility. The root rot resistance of 'Cadet' was unaffected by substitution of chromosomes 5A, 5B, or 5D with 5Ag, or the addition of 5Ag. This indicates that the susceptible allele of the gene Crr is the primary determinant in the reaction of wheat to common root rot. Black point resistance in the susceptible cultivar 'Rescue' was significantly increased by substitution of chromosome 5B with 5Ag, or the addition of 5Ag. No corresponding effect was demonstrated for black point incidence in the moderately resistant cultivar 'Cadet' with substitution of chromosome 5Ag for 5B, or the addition of 5Ag. Chromosome 5Ag apparently carries one or more genes conferring resistance to black point. The identity of these lines was confirmed by restriction fragment length polymorphism analysis using group 5 chromosome arm specific probes. This extends the use of these molecular probes to the Agropyron genome.     

Homoeology of rye chromosome arms to wheat

Summary Cytological markers such as diagnostic C-bands, telocentrics, and translocations were used to identify the arms of rye chromosomes associated with wheat chromosomes at metaphase I in ph1b mutant wheat × rye hybrids. Arm homoeologies of rye chromosomes to wheat were established from the results of metaphase I pairing combined with available data on the chromosomal location of homoeoloci series in wheat and rye. Only arms 1RS, 1RL, 2RL, 3RS, and 5RS showed normal homoeologous relationships to wheat. The remaining arms of rye appeared to be involved in chromosome rearrangements that occurred during the evolution of the genus Secale. We conclude that a pericentric inversion in chromosome 4R, a reciprocal translocation between 3RL and 6RL, and a multiple translocation involving 4RL, 5RL, 6RS, and 7RS are present in rye relative to wheat.     

Genotypic features of morphogenesis in spring barley anther culture

The variability of characteristics in haploid production was assessed depending on the growth conditions for donor plants, ear pretreatment technique, and nutrient medium composition. The ranks of genotypes included in the experiment remained unchanged in terms of their in vitro ability toward androgenesis, which evidences the significant contribution of a genetic component to the general variability and demonstrates the need to investigate how genetic control of androgenesis works.     

GISH analysis of disomic Brassica napus-Crambe abyssinica chromosome addition lines produced by microspore culture from monosomic addition lines

Two Brassica napus--Crambe abyssinica monosomic addition lines (2n=39, AACC plus a single chromosome from C. abyssinca) were obtained from the F₂ progeny of the asymmetric somatic hybrid. The alien chromosome from C. abyssinca in the addition line was clearly distinguished by genomic in situ hybridization (GISH). Twenty-seven microspore-derived plants from the addition lines were obtained. Fourteen seedlings were determined to be diploid plants (2n=38) arising from spontaneous chromosome doubling, while 13 seedlings were confirmed as haploid plants. Doubled haploid plants produced after treatment with colchicine and two disomic chromosome addition lines (2n=40, AACC plus a single pair of homologous chromosomes from C. abyssinca) could again be identified by GISH analysis. The lines are potentially useful for molecular genetic analysis of novel C. abyssinica genes or alleles contributing to traits relevant for oilseed rape (B. napus) breeding.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

Dissection of rye chromosome 1R in common wheat

Rye chromosome 1R contains many agronomically useful genes. Physical dissection of chromosome 1R into segments would be useful in mapping 1R-specific DNA markers and in assembling DNA clones into contig maps. We applied the gametocidal system to produce rearranged 1R chromosomes of Imperial rye (1R(i)) added to common wheat. We identified rearranged 1R(i) chromosomes and established 55 1R(i) dissection lines of common wheat carrying a single rearranged 1R(i) chromosome. Fifty-two of the rearranged 1R(i) chromosomes had single breakpoints and three had double breakpoints. The 58 breakpoints were distributed in the short arm excluding the satellite (12 breakpoints), in the satellite (4), in the long arm (28), and in the centromere (14). Out of the 55 lines, nine were homozygous for the rearranged 1R(i) chromosomes, and the remaining lines were hemizygous. We developed 26 PCR-based EST markers that were specific to the 1R(i) chromosome, and nine of them amplified 1R(i) arm-specific PCR products without restriction-enzyme digestion. Using the nine EST markers and two previously reported 1R-specific markers, we characterized the 55 1R(i) dissection lines, and also proved that we can select critical progeny plants carrying specific rearranged 1R(i) chromosomes by PCR, without cytological screening, in 48 out of the 55 hemizygous dissection lines.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.