Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). II. Evaluation of first- and second-cycle doubled haploids

Summary First- and second-cycle doubled haploids of burley tobacco (Nicotiana tabacum L.) were examined to ascertain the significance of source genotype on the performance of anther-derived material and to determine the magnitude of changes from the haploid-production and chromosome-doubling stages. The anther-derived lines evaluated in this study did not exhibit siginficant yield reductions. However, significant interactions between the cycle comparison and the source genotypes indicated that source genotype can affect the degree of vigor reduction observed. Significant variation was also observed among the second-cycle doubled haploids examined in this study. Partitioning of this variation indicates that most of it is due to alteration present at the haploid-production step instead of the chromosome-doubling step. In general, the variation present in the second-cycle doubled haploids was distributed around the values observed for the source first-cycle doubled haploids. Even though unexpected variation is often observed in anther-derived materials, these results suggest that reduced vigor is not intrinsically associated with androgenetic lines of burley tobacco. The most plausible explanations for the variation and vigor losses in anther-derived lines propose that natural phenomena, given the opportunity for expression via anther culture, are responsible. Spontaneous variants present in the gametic pool of inbreds or first-cycle doubled haploids offer one possible explanation for the performance observed in anther-derived lines of tobacco.Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091. This research was supported, in part, by a grant from R. J. Reynolds Tobacco Co. This paper (no. 85-3-29) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Variation among androgenic and gynogenic doubled haploids of tobacco (Nicotiana Tabacum L.)

Summary Agronomic field performance traits and leaf chemical characteristics of gynogenic doubled haploids (GDH) and androgenic double haploids (ADH) were compared with selfed lines of a burley tobacco cultivar to determine which type of doubled haploids was superior. Three sets of ADH and GDH lines performed similar to their selfed lines, but one set each of ADH and GDH lines had reduced yield, plant height, leaf number, stalk diameter, and leaf length in comparison to the respective selfed lines of KY 17.Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. This paper (No. 87-3-287) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

Addition of Colchicine to Wheat Anther Culture Media to Increase Doubled Haploid Plant Production

Chromosome doubling is critical for obtaining doubled-haploid plants from wheat (Triticum aestivum L.) anther culture. The most common doubling method applies colchicine to the plant. However, colchicine is phytotoxic and can induce a high frequency of plant death. In this experiment, anthers from two wheat genotypes (“Pavon 76” and ‘Centurk’) were placed on nine embryoid initiation media having three sugar sources (maltose, sucrose, and maltose + glucose) with three colchicine concentrations (0.0, 0.1, and 0.2 g · l^-1). Wheat starch was used as a gelling agent. After three days, the anthers were washed and moved to fresh media without colchicine. Increasing the colchicine concentration decreased the number of embryoids produced from 77.4 embryoids/100 anthers to 29.9 embryoids/100 anthers, but did not significantly affect the frequency of plant regeneration (0.49 green plants/embryoid to 0.40 green plants/embryoid), and increased the frequency of doubled-haploid plants (19.0 doubled-haploid plants/100 green plants to 72.3 doubled-haploid plants/100 green plants). Considering the total number of doubled-haploid plants produced, low levels of colchicine added to the initiation media were very effective.     

开发实用的染色体加倍体系构建成烟草DH群体

通过对4种染色体加倍方法(烟草浸花药法、浸花培苗法,叶片再生法及浸腋芽法等)的比较研究表明：以4g／L浓度的秋水仙碱浸泡花药法加倍率最高达47．5％～75％,其次为叶片再生法(30％～36．67％)和浸苗法(16．67％～32．35％),而浸腋芽法较低(17．78％)。前两种方法加倍率虽高,但有较高畸形苗比率,叶片再生法工序较繁琐。作者认为烟草加倍单倍体产生,应以采用浸苗法为主。在使用秋水仙碱浸苗加倍时,添加DMSO可明显促进秋水仙碱的加倍效率,且促进作用随时间延长而提高：从高效、快捷和节约等原则考虑,我们开发了烟草有效的染色体加倍体系,以4g／L的秋水仙碱 20g／LDMSO溶液浸苗48h的效果最好。对两个组合烟草单倍体苗浸12h以上,其染色体加倍效率达到对照的2．30-2．93倍。本试验用较低浓度秋水仙碱添加DMSO有利于节约实验费用。通过完善染色体加倍技术程序已构建成2个DH群体,供基因定位研究。     

Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum)

Summary Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25° C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l glutamine and solidified by gelrite (4g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15 E m^–2 S^–1) at 26°C at 16h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28°C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regenerated and fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparisons between doubled haploid lines produced by anther culture,the Hordeum bulbosum-method and lines produced by single seed descent in barley crosses

Summary Possible negative effects of tissue culture on qualitative and quantitative characters have been investigated in three crosses of spring barley, using doubled haploid lines produced by anther culture (AC, with maltose as a carbon source) or the Hordeum bulbosum-method (HB). In one cross inbred lines produced by single seed descent (SSD) have also been included. Quantitative characters were investigated in a 2-year field experiment at one location. The results show that although the methods in the majority of cases gave similar results, the genotypic arrays produced were not identical. Different markers deviated in the various cross/method combinations. The ranking of methods as well as the frequencies of lines transgressive for grain yield differed between crosses. Thus no consistently negative impact of anther culture in barley has been found, and, although not identical, the various methods may be considered equivalent.Abbreviations AC Anther Culture method - SSD Single Seed Descent method - HB Hordeum bulbosum method     

Climatic adaptation in subterranean clover populations

Summary Long-term persistence and, hence, agronomic success as a pasture of the annual species subterranean clover depend primarily on seed yield and seed survival over seasons. In natural populations, plant characteristics influencing seed setting and formation of seed reserves in the soil are expected to be adjusted to the prevailing environmental conditions of the sites of origin. Knowledge on plant/environment relationships may provide information on adaptive strategies of persistence, and guidelines for selecting adapted varieties to specific conditions. On pure lines from a number of populations such relationships were assessed for flowering time, seed yield, burr fertility, individual seed weight, initial hardseededness, and rate of hardseededness breakdown over summer. Flowering time decreased on decreasing annual rainfall, i.e., on shortening the growing season, as adaptive response to the need of producing adequate seed before the onset of the dry season. Individual seed weight decreased on decreasing rainfall, and increasing temperatures. Hard-seed maintenance over summer was higher in populations from hot and dry environments, where the marked effect of temperature on hardseededness breakdown exerts a strong selective pressure. Within-population variation, assessed on flowering time, was particularly wide, with early genotypes occurring even in populations from long-season environments. The adaptive relevance of maintaining high levels of within-population polymorphism to cope with unpredictable climatic fluctuations is discussed. Number of constituent lines as a measure of the population structure, and intra-population variation were both influenced by altitude and rainfall, tending to decrease as the climatic selective pressure becomes severe, under both low-rainfall, hot conditions and high-elevation, cold-prone environments.     

Agronomic Performance of Wheat Doubled-Haploid Lines Derived from Cultivars by Anther Culture

To determine the level of gametoclonal variation among doubled-haploid lines (DHLs) of Triticum aestivum L. developed using anther culture techniques and its effect on agronomic performance, 70 anther culture-derived DHLs of ‘Kitt’ were compared for 7 agronomic traits to 50 single-seed descent-derived lines (SSDLs) of ‘Kitt’ and to the cultivar ‘Kitt’. In a second experiment, 26 DHLs of ‘Chris’ were compared for 7 agronomic traits to 29 SSDLs of ‘Chris’ and to the cultivar ‘Chris’. Each experiment was grown as a randomized complete block design with three replications in three environments. For ‘Kitt’, the DHLs averaged significantly lower grain yields than the comparable SSDLs. For ‘Chris’, the DHLs averaged lower, but not significantly lower, grain yield than the SSDLs. In both ‘Kitt’ and ‘Chris’, the genetic component of variance for yield of the DHLs was significantly larger than that of the SSDLs indicating the presence of gametoclonal variation. The lower average grain yield of the DHLs was explained by a larger group of low-yielding DHLs than was present in the SSDLs. Six ‘Kitt’ DHLs and 3 ‘Chris’ DHLs were lower yielding than the lowest yielding SSDL, respectively. Elite DHLs and SSDLs were similar for mean grain yield performance. Though the DHLs and SSDLs were significantly different for some yield components, the affected yield component changed with the cultivar and there was no consistent effect. Significant genotype × environment interactions were detected for some traits which were caused by changes in the magnitude of differences, rather than reversals in ranking, indicating that low yielding DHLs could be culled on the basis of visual selection or single-environment testing. Hence, gametoclonal variation was induced by the anther culture techniques used in this study, tended to be deleterious for grain yield, and was sensitive to the growing environment. However, as the DHLs and SSDLs had similar expected population means based upon expected gains from selection, this gametoclonal variation should not be a major hindrance to wheat breeding.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

The effect of the ‘Golden Promise’ dwarfing gene on partial resistance to leaf rust and powdery mildew in spring barley

Summary F₂-plant progenies, derived from seven crosses between susceptible erectoides breeding lines and conventional, nutans breeding lines with partial resistance to leaf rust incited by Puccinia hordei Otth., derived from Vada x Cebada Capa, were examined in a field and a glasshouse experiment. In the field experiment, the nutans plant progenies generally were more resistant to leaf rust and powdery mildew than the erectoides progenies. In the glasshouse experiment, the components of partial resistance to leaf rust of six erectoides and six nutans progenies derived from one cross were studied. The nutans progenies generally had lower infection frequencies than erectoides progenies with a similar infection level in the field experiment. The variation for leaf rust and powdery mildew infection in the field was similar for the nutans and erectoides progenies. This suggests that erectoides lines with a fairly high level of partial resistance to leaf rust and powdery mildew can be selected from these populations.     

The relationship between powdery mildew (Sphaerotheca fuliginea) resistance and leaf chlorosis sensitivity in cucumber (Cucumis sativus) studied in single seed descent lines

Summary The genetic relation between powdery mildew resistance and sensitivity for leaf chlorosis of glasshouse cucumber was investigated. The powdery mildew resistant, leaf chlorosis sensitive hybrid variety Profito was crossed with the powdery mildew susceptible, non chlorosis sensitive hybrid variety Corona. Forty four F₆ inbred lines of this cross, produced by single seed descent, were tested for powdery mildew resistance (PMR) and leaf chlorosis sensitivity (LCS). PMR and LCS were positively correlated (r=0.59). One or more of the PMR genes probably causes LCS as a pleiotropic effect or is closely linked to LCS, but also other factors, not genetically linked to PMR can cause LCS. Five F₆ lines combined a significantly higher level of PMR than Corona with a significantly lower LCS than Profito. Three of these lines could hardly be distinguished from Corona for the level of LCS.Abbreviations LCS leaf chlorosis sensitivity - PMR powdery mildew resistance - SSD Single Seed Descent     

Peroxidase isozyme differences in tall fescue cultivars and allopolyploid accessions

Summary The use of isoelectric focusing for the isozymic identification of cultivars and wild forms in tall fescue (Festuca arundinacea Schreb.) and determination of ploidy effects was investigated. Peroxidase zymograms from allopolyploid accessions (4x, 6x, 8x, and 10x, x=7) and randomly sampled plants of the cultivars Kenhy, Kentucky 31, and Missouri 96, were compared for band number, position, and staining intensity. There was little isozymic variation among plants in the tetraploid and hexaploid, but considerably more in the octoploid and decaploid members of the series. Yet no significant effects of ploidy on isozyme complexity or band number were observed. There were no consistent differences in band number, position, or frequencies among the three cultivars in spite of their diverse parentage. Isoclectric focusing can be used successfully in tall fescue, but the limited variability of the peroxidases encourages investigation of other isozyme systems in the future.Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 8627. Research supported by USDA-SEA Competitive Research Grant 5901-0410-9-0366-0.     

Molecular mapping of QTLs for non-parasitic leaf spot resistance and comparison of half-sib DH populations in spring barley

Quantitative trait loci (QTLs) for resistance against non-parasitic leaf spots (NPLS) were first characterized in a spring barley double haploid population derived from the cross IPZ 24727/Barke (Behn et al., 2004). The aim of the present study was to identify QTLs for NPLS resistance in the half-sibling DH population IPZ 24727/Krona and to compare them with the QTLs of the population IPZ 24727/Barke. An anther culture-derived doubled haploid population of 536 DH lines was developed from the cross IPZ 24727 (resistant)/Krona (susceptible). Field trials were performed over two years in two replications, scoring NPLS and agronomic traits that might interact with NPLS. A molecular linkage map of 1035 cM was constructed based on AFLPs, SSRs and the mlo marker. QTL analyses for NPLS identified three QTLs that accounted for 30% of the phenotypic variation. For comparison of the QTLs from each DH population, a consensus map was generated comprising 277 markers with a length of 1199 cM. In both populations, the QTLs for NPLS mapped to chromosomes 1H, 4H and 7H. A common QTL with a great effect in both populations and over all environments was localized at the mlo locus on chromosome 4H, indicating that the mlo powdery mildew resistance locus has a considerable effect on NPLS susceptibility. The steps necessary to validate the QTLs and to improve the NPLS resistance by breeding were discussed.     

The quantitative relationship between telomeric heterochromatin and secalin synthesis in Secale cereale L.

Summary The relationship between the amount of prolamine (secalin) synthesized and the amount of telomeric heterochromatin in ten inbred lines of rye (Secale cereale L.) cv. Self Fertile Spring was investigated. There was no association between the total amount of secalin synthesized and the amount of telomeric heterochromatin per haploid genome. However, a positive relationship was found between total secalin synthesis and the amount of telomeric heterochromatin on the short arm of chromosome 7 (7RS). Lines synthesizing high levels of secalin were found to have approximately 60% less telomeric heterochromatin on chromosome arm 7RS than lines synthesizing low levels of secalin. There was no positive relationship between secalin synthesis and the amount of telomeric heterochromatin present on any other telomere.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

Colchicine-induced heritable variation in cell size and chloroplast number in the leaf cells of inbred ryegrasses (Lolium perenne,L. multiflorum)

Summary A 3 hr period of treatment with a 0.2% aqueous solution of colchicine was given to one week old seedlings of 3 inbred lines of perennial ryegrass (Lolium perenne) and 4 lines of Italian ryegrass (L. multiflorum). The surviving plants developed as mixoploids and were self-pollinated. In the following generation (CT1) the seeds were grown without further treatment, and were screened cytologically at the seedling stage to separate out diploids from tetraploids. Only the diploids were kept, and in this CT1 generation comparisons were then made between controls (2x) and colchicine-treated diploids (C2x) for leaf mesophyll cell size and chloroplast number variation. Heritable differences were found such that the cell plan areas and/or the numbers of chloroplasts per cell were significantly greater in the C2x compared with the 2x treatment in 3 and 5 out of the 7 lines. In each case there was enhancement of chloroplast numbers independently of the variation in cell size.     

Comparison of fertility between the F1, F2 and anther derived lines in the crosses of indica/japonica and japonica/indica in rice (Oryza sativa L.)

Summary Response of anthers in in vitro culture was examined in the indica-japonica hybrids of rice (Oryza sativa L.). Significant genotypic differences were observed for callus induction and regeneration among the different interracial hybrids of indica-japonica races. Induction frequency of haploids ranged from 57.7 to 72.9 per cent and doubled haploid androgenic lines ranged from 27.1 to 42.3 per cent in the anther culture of the different hybrids. The indica-japonica hybrids recorded partial pollen grain and spikelet fertility in F1 (29.9 to 41.5% and 19.4 to 48.7% respectively) as well as in F2 (42.7 to 50.6% and 37.1 to 54.4% respectively). In contrast, the androgenic doubled haploid lines recorded significant increase and the pollen grain and spikelet fertility was 76.3 and 78.6 per cent respecitively. The results suggested that the sterility barriers for realising genetic recombinants and fixation of fertile homozygous lines in indica-japonica hybridization programme could be overcome through F1 anther culture technique.Abbreviations BAP Benzyl Amino Purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - MS Murashige & Skoog medium - IAA Indole Acetic Acid