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.     

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.     

Influence of picloram and thidiazuron on high frequency plant regeneration in elite cultivars of wheat with long-term retention of morphogenecity using meristematic shoot segments

Meristematic shoot segments (MSSs) were used to develop an efficient and reproducible regeneration system via multiple shoot differentiation in two elite cultivars of winter wheal. Cylindrical basal shoot segments. 3 4 mm in length, were excised from seedlings derived from mature embryos that had been germinated directly on MS‐based medium containing growth regulators. These segments were further cultured on the same medium formulations. Out of 24 variations analysed, the combination of 2 mg1 4‐amino‐3.5,6‐trichloropicolinic acid (picloram) + 3 mg/1 thidiazuron (TDZ) was found to be the most effective in inducing clumps of multiple schools, buds with little callusing. Sectors of these clumps were (1) further multiplied by biweekly subculturing on 2 mg/l picloram + 2.5 mg/l TDZ leading lo highly differentiating green clusters of buds without loss of tnorphogenesis for more than 12 months so far. and without albinism, or (2) transferred to 0.1 nig I picloram + I mg/l TDZ for shoot development and elongation. The elongated shoots rooted on basal medium with 0.5 mg/l indole‐3‐butyric acid (IBA) were transferred to soil and showed normal growth and fertility. Both cultivars formed multiple schools ranging from 28 to 33 per responding MSS after 10 12 weeks.     

Influence of genotype and culture medium on callus formation and plant regeneration from immature embryos of Triticum turgidum Desf. cultivars

Twelve durum wheat cultivars were evaluated for their response to in vitro tissue culture. Zygotic immature embryos were used to induce callus formation using four different Murashige and Skoog‐based media. Each contained 9.05 μM 2,4‐dichlorophenoxy acetic acid but differed in their carbon source (sucrose or maltose) and the presence of NaCl (0 mM or 40 mM). The influence of both genotype and medium on the type and percentage of callus produced was observed. Calli were either compact and frequently embryogenic, or soft and watery. Percentages ranged from 54 to 100%, depending upon genotype and induction medium. All calli were then plated on a regeneration medium containing 20 g/l sucrose, 2.68 μM 1‐naphthaleneacetic acid and 2.22 μ 6‐benzylaminopurine. The regeneration of plantlets was higher from compact than from soft calli, with a strong dependence on genotype and type of induction medium used. MSm induction medium (30 g/l maltose) and MS40s (30 g/l sucrose plus 40 mM NaCl) were best for inducing compact calli, and gave the highest proportion of regenerated plants. The in vitro response (number of total shoots from a compact callus/number of embryos plated) was higher for immature embryos of ‘Baztan’, ‘Bradano’ and ‘Don Pedro’. These cultivars are a good starting material for experiments involving transformation of calli from zygotic immature embryos.     

Improvement of in vitro culture response of elite wheat cultivars by selecting the source spike, the scutellum size and the temperature for the induction of embryogenesis

High frequencies of regeneration are desirable characteristics of in vitro culture from elite wheat cultivars. The effect of the source spike, the scutellum size and the induction temperature on embryogenesis, regeneration and number of plants per explant were optimized in three varieties of wheat and later determined in fifteen varieties of both bread and durum wheat. The highest frequencies of embryogenesis, regeneration and plants per explant were obtained when the first spike was used as source of scutella and decreased using the scutella from the second and third spikes. Scutella from the third spike yielded half the number of plants per explant than those from the first spike. Significant differences were also found for embryogenesis, regeneration and plants per explant as affected by scutellum size. The number of plants per explant drastically decreased in commercial varieties when scutella larger than 2.0 mm were used. The optimum induction temperature was 21 and 25°C for commercial and model genotypes, respectively. However, no differences were found for the number of plants per explant as affected by the induction temperature. The in vitro culture response of twelve and four cultivars of, respectively, bread and durum wheat were then determined. In all wheat varieties tested embryogenesis, regeneration and plants per explant varied over a wide range of values. In elite wheat varieties the selection of the source spike and the scutellum size can yield high frequencies of embryogenesis and regeneration and high numbers of plants per explant.     

In vitro selection of wheat callus tolerant to high levels of salt and plant regeneration

Summary Embryogenic calli isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The effect of NaCl on the selected and unselected cell lines has been investigated. The results indicated that the relative growth rate of callus decreased as the concentration of NaCl increased in both callus lines. The selected callus line gave a higher growth weight in the presence of NaCl in the medium and was highly significant as compared with unselected callus line across medium protocols in all wheat cultivars. The dry weight of both kinds of callus lines of all wheat cultivars increased markedly with increasing NaCl concentration in most cases. The Na⁺ and Cl^- contents of both callus lines were increased with increasing salinity levels while K⁺ content was decreased. The selected callus line of each cultivar at the same salinity level produced significant amounts of Na⁺, K⁺ and Cl^- higher than the unselected callus line in most salinity levels. However, the unselected callus lines of the cultivars Giza-157 and Sakha-90 at the same salinity level produced significant amounts of K⁺ higher than the selected callus line in most salinity levels. The proline content of both kinds of callus lines for all wheat cultivars was increased with increasing salinity level. However, the selected callus line gave a significantly higher proline content than the unselected callus line in all wheat cultivars at the same Salinity level. Results from the in vitro selection for NaCl tolerance showed that the stepwise method of increasing NaCl in the medium was more effective for plant regeneration than other methods.     

Resistance to leaf rust in cultivars of bread wheat and durum wheat grown in Spain

A set of bread wheat and durum wheat cultivars adapted to Spanish conditions was tested for resistance against leaf rust caused by different pathotypes of Puccinia triticina in field trials and in growth chamber studies. Lower levels of resistance were found in durum wheat than in bread wheat. The most frequent Lr genes found in bread wheat were Lr1, Lr10, Lr13, Lr20, Lr26 and Lr28. In durum wheat, additional resistance genes that differed from the known Lr genes were identified. The level of partial resistance to leaf rust was in general low, although significant levels were identified in some bread wheat and durum wheat cultivars.     

Concentration effects of dicamba on shoot regeneration in wheat

Shoot formation and regeneration rates of calli derived from immature embryos of three spring wheat varieties were investigated to evaluate the influence on wheat regeneration of different concentrations of dicamba (0.5, 0.1 and 0.02 mg/l) in the regeneration medium. It was found that dicamba concentrations much lower (0.02 and 0.1 mg/l) than the levels reported previously (1 and 0.5 mg/l) induced significantly more shoots per callus in all three varieties, while the enhancement in regeneration rate was variety dependent. Overall, a dicamba concentration of 0.02 mg/l was more favourable for wheat regeneration.     

Identification of powdery-mildew-resistance genes in common wheat (Triticum aestivum L.). VI. Wheat cultivars grown in China

A total of 26 common wheat cultivars and advanced breeding lines grown in China were tested with a set of 11 differential powdery-mildew isolates. Seven cultivars were susceptible. Another seven cultivars showed the response pattern of resistance gene Pm2, either individually or in combination with genes Pm3d or Pm4a. Five cultivars expressed the resistance of gene Pm4b singly or in combination with Pm6. Another four cultivars exhibited the response patterns of genes Pm5, Pm6 and Pm8, respectively. Three cultivars, which included one breeding line with a pair of substituted chromosomes from Haynaldia villosa, presumably carrying the resistance gene Pm21, showed resistance-response patterns to all the isolates tested.     

Fluorescence in situ hybridization polymorphism using two repetitive DNA clones in different cultivars of wheat

Twenty‐two wheat cultivars and a wheat line were analysed with two‐colour fluorescence in situ hybridization (FISH) using the pSc119.2 and pAs1 repetitive DNA clones to detect if polymorphism could be observed in the hybridization patterns of different wheat cultivars. The FISH hybridization pattern of ‘Chinese Spring’ was compared with wheat cultivars of different origins. Differences were observed in the hybridization patterns of chromosomes 4A, 5A, 1B, 2B, 3B, 5B, 6B, 7B, 1D, 2D, 3D and 4D. Although a low level of polymorphism exists in the FISH pattern of different wheat cultivars, it is possible to identify 17 pairs of chromosomes according to their hybridization patterns with these two probes. This study will help to predict the expected variation in the FISH pattern when analysing wheat genetic stocks of different origin. It is presumed that variation in hybridization patterns are caused by chromosome structural rearrangements and by differences in the amount and location of repetitive sequences in the cultivars analysed.     

Plant regeneration from immature inflorescence callus cultures of wheat,rye and triticale

Summary Callus cultures were initiated from inflorescence explants of wheat, rye and triticale on MS medium supplemented with 2 mgl^-1 2,4-D+5% CW or 2 mgl^-1 2,4-D+0.5 mgl^-1 BA. On transfer of the cultures to medium supplemented with 15% CW+0.2 mgl^-1 NAA or 1 mgl^-1 BA+0.1 mgl^-1 IAA, shoot buds and embryoids were produced. Full fledged plantlets obtained on MS medium supplemented with NAA were transferred to the field. Cytological analysis showed the plants to be diploid. However, the regenerated plantlets were shorter, produced fewer tillers and had lower fertility compared to the control.Abbreviations BA Benzyladenine - CW coconut water - IAA indoleacetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

我国北部冬麦区小麦区域试验重复次数和试点数量的优化设计

农作物品种区域试验重复次数和试点数量的合理配置有利于提高试验的成本效率和品种选择效率。本研究分析了2010—2019年期间北部冬麦区小麦品种区域试验的重复次数和试点数量设置的合理性,依据小麦品种试验的信噪比和遗传力水平随重复次数和试点数量的变化规律,提出了重复次数和试点数量的优化设计方案。结果表明:(1)北部冬麦区小麦单点试验的遗传力平均达到0.87,需要的重复次数平均值仅为1.4,说明3次重复可以充分保证试验精确度的需求。(2)北部冬麦区水地组和旱地组小麦区域试验达到0.75的遗传力水平时,需要的试点数量分别为11个和13个,目前有效试点数量分别约为11个和8个,分别达到0.75和0.60的遗传力水平。(3)小麦品种区域试验结果对品种的审定和应用十分重要,而每年都可能有少数试验点因为各种异常情况而报废,为保证试验结果的可靠性,可按H=0.75的水平需求安排试验点数量和重复次数,即重复次数可保持当前的3次;水地组的试点数量可保持在11个左右;旱地组可将试点增加到13个;如要将遗传力提高到0.80的水平,则需约16个试点。     

Potential for effective marker-assisted selection of three quantitative trait loci conferring adult plant resistance to powdery mildew in elite wheat breeding populations

Three quantitative trait loci (QTL) associated with adult plant resistance (APR) to powdery mildew (Blumeria graminis) in wheat (Triticum aestivum) cultivar ‘Massey’ were mapped in a previous study. The three QTL were located on chromosomes 2A, 2B and 1B, and explained 50% of the total phenotypic variation. A 293 recombinant inbred line (RIL) breeding population (UJ) derived from the cross of ‘USG 3209’, a derivative of ‘Massey’, and ‘Jaypee’ was used to evaluate the potential effectiveness of marker‐assisted selection (MAS) for APR. Powdery mildew severities of the 293 UJ RILs were evaluated in 2002 (F_{5 : 6}) and 2003 (F_{6 : 7}) under natural disease pressure in the field. The 293 RILs were also evaluated for disease severity in a 2004 (F_{7 : 8}) greenhouse experiment using a composite of five different isolates of B. graminis. Selection of RILs possessing the QTL on chromosome 2A, and to a lesser extent, the one on chromosome 1B was effective in identifying powdery mildew resistance in both greenhouse and field experiments. Overall, selecting RILs with QTL on chromosomes 2A and 2B was most successful in identifying highly resistant RILs, which had mean mildew severities of 4.4% and 3.2% in 2002 and 2003 field experiments, respectively. Breeders implementing MAS programs for APR to powdery mildew via selection of RILs containing the two QTL on chromosomes 2A and 2B likely will obtain RILs having high levels of resistance in the field, however combining all three QTL may ensure greater durability.     

Effect of Mutagenic Treatments on Somaclonal Variation in Wheat (Triticum aestivum L.)

Different wheat genotypes were treated with gamma-rays, sodium azide (SA) and EMS before tissue culture and immature embryos from M₁ plants or plants shortly after exposure to gamma-rays were used to initiate callus culture. Thousands of plants were regenerated and used to investigate the effect of mutagenic treatments on the regenerated plants and somaclonal variation in the M₃R₂ and M₂R₂ generations. The results showed that mutagen-induced damage in terms of reduction in plant height, fertility and spike length were not outstanding in the regenerated plants as compared with the untreated control. In the M₃R₂ generation, only SA treatment had significantly higher frequencies of somaclonal variations than the control. Increases in the variation frequencies were observed when explant embryos were irradiated with 2.5 and 5 gy gamma-rays and the highest frequency appeared when embryos were exposed to 5 gy gamma-rays on the 5th day after anthesis. Increased variation spectra also resulted from mutagenic treatments and most of the variants recovered were unsuitable for plant improvement.     

Effect of mannitol pretreatment to improve green plant regeneration on isolated microspore culture in Triticum turgidum ssp. durum cv. 'Jennah Khetifa'

The use of doubled haploids improves the efficiency of cultivar development in many crops and can be helpful in genetic and molecular studies. The major problem with this approach is the low efficiency of green plant regeneration. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat cv. ‘Jennah Khetifa’. Tillers from donor plants were pretreated in 0.3 m mannitol and were stored at 4°C at various times: 3, 5, 6, 7, 8, 10 and 12 days. Our results showed clearly that the novel pretreatment combined mannitol 0.3 m and cold for 7 days had a strong effect on the number of embryos produced and regenerated green plants. Under this condition 13 475 mature embryos were produced from 2 693 500 microspores. Moreover, 85 green plants were obtained. High green plants regeneration frequency was recorded. As an average 11.55 green plants were produced per 100 000 microspores (about the equivalent of six plants per spike). Therefore, this study showed clearly that our results are the best ones published until now in durum wheat.     

Callus induction and plant regeneration from anther and inflorescence culture of Sorghum

Summary Twenty-five inbred lines, including grain and forage types from the USA and China, two hybrids, one Sorghum almum, and one Parasorghum (S. versicolor) were tested for their response to anther culture. Three nutrient media were effective in inducing anther calli from six cultivars (Xin White, TX 403-TSB, DDY Sommer Milo, TX 2779, Brawley, and Spur Federal) and one was effective for plant regeneration for one cultivar, Xin White. Averaged over media, callus induction frequency (number of calli per 100 anthers) was highest in cultivars Xin White and TX 403-TSB (6.7 and 3.9%, respectively). The means of cultivars for media C17-2 and Ms-t-z-2, 4.3 and 3.2%, respectively, were superior to that for medium 85D3-2 (0.1%). Expressed as an average of the six cultivars and three media the mean calli induction frequency was 2.6%; however, differential responses of genotype and medium were noted. Among the 10 regeneration media tested, medium MS-d-4 containing Murashige and Skoog basal components plus 2.0 mg/l indole-3-acetic acid (IAA) and 2.5 mg/l kinetin was the most effective for plant regeneration. Numbers of albino plants and calli developing only roots increased directly with callus-induction time, whereas the frequency of plant regeneration decreased. Regenerated plants had varied numbers of chromosomes in root tip cells: 10, 15, 20, 40, and 60. The 29 regenerated plants that reached maturity, however, were highly fertile and contained only 10 bivalents in pollen mother cells. Normal chromosome number and behavior for the regenerated plants suggest that induced calli originated from cells other than microspores. However, spontaneous chromosome doubling in microspore-derived haploids may occur. The appearance of albinos also implies that haploids may have been produced from anther culture.Joint contribution of the Dept. of Agronomy and USDA-ARS, Kansas Agricultural Experiment Station, Manhattan, KS 66506-5501, USA. Contribution no. 88-566-J.     

Genetic analysis of glume colour in common wheat cultivars from the former USSR

Genotypes for the glume colour character have been studied in 27 cultivars of common wheat (Triticum aestivum L.) originated from old landraces, and 1 specimen of T. petropavlovskyi Udacz. et Migusch. by means of analysis of the F₂ populations. The following tester lines have been used: white-glumed ‘Novosibirskaya 67’ ‘Diamant I’, and ‘Federation’, carrying the Rg1 gene alone; lines RL5405 and near-isogenic ‘Saratovskaya 29’ *5 (T. timopheevii Zhuk./T. tauschii (Coss.) Schmal.), carrying Rg2; line (1A ‘CS’ × ‘Strela’) with Rg3. The red glume colour in 21 cultivars of Triticum aestivum and in the accession of T. petropavlovskyi has been shown to be determined by the single gene Rg1, located on chromosome 1B. Five cultivars carrying the gene Rg3 for red glumes on chromosome 1A have been revealed. The cultivars ‘Zhnitsa’ and ‘Iskra’ carry the gene Rg3 alone. The red glume colour in the cultivars ‘Milturum 321’, ‘Milturum 2078’, ‘Sredneural'skaya’ is controlled by two genes, Rg1 and Rg3. In two common wheat cultivars, ‘Sarrubra’ and ‘Krasnoyarskaya 1103’ the red glume colour is determined by Rg1, inherited from local populations (‘Turka’ and ‘Kubanka’ respectively) of tetraploid wheat T. durum Desf. var. hordeiforme Host. Wide occurrence of the Rg1 gene in common wheat has been confirmed. On the contrary, none of the investigated varieties carries the gene Rg2. This revised version was published online in July 2006 with corrections to the Cover Date.     

宁麦9号与扬麦158株高及其构成因素的遗传解析

宁麦9号与扬麦158是我国长江中下游麦区的主栽品种和骨干亲本,长江中下游麦区近3年来审定品种中80%都是其衍生后代,研究其性状的遗传具重要意义。以宁麦9号与扬麦158为亲本构建的包含282个家系的重组自交系群体为材料,利用Illumina 90k芯片对群体进行基因型分析,建立高密度遗传图谱。连续3个生长季对株高及节间长度、穗长等株高构成因素进行测定,结合遗传图谱对株高及相关性状进行QTL定位,获得14个控制株高及其构成因素的稳定表达位点。通过进一步位置比对,聚焦到6个染色体区段,初步明确了各节间对株高的遗传调控机制。同时,将6个染色体区段中同源性较低的连锁标记转化为适用于高通量筛选的KASP标记,利用101份区域试验材料进行标记效应验证,结果显示聚合Qph-2D与Qph-5A.1两个位点具有较高的选择效率,继续聚合Q2A后,中选材料显著减少,可能降低选择效率;对Q2A与Q5A两个一因多效位点的选择建议以降低株高的等位变异为主;Qd1-5D可作为穗下节间(D1)的选择标记对株高展开优化选择。期望以上结果能为长江中下游麦区的小麦株高遗传改良提供帮助。     

The high and low molecular weight glutenin subunits and ω-gliadin composition of bread and durum wheats commonly grown in Portugal

A collection of 63 bread wheats (Triticum aestivum L.) and 21 durum wheats (Triticum durum Desf.) commonly grown in Portugal since 1982 were characterized for the composition of wheat storage proteins (WSP), high molecular weight glutenin subunits (HMW-GS), low molecular weight glutenin subunits (LMW-GS) and ω-gliadins. The composition of HMW-GS, LMW-GS and &-gliadins, encoded at loci Glu-1, Glu-3 and Gli-1, respectively, was revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. WSP allelic compositions of bread and durum wheat patterns were given. In the bread wheats, a total of 24, 24 and 18 patterns were observed for HMW-GS, LMW-GS and ω-gliadins, respectively. Forty-two different alleles were identified for the nine loci studied, Glu-A1 (3), Glu-B1 (7), Glu-D1 (4), Glu-A3 (5), Glu-B1 (7), Glu-D3 (2), Gli-A1 (2), Gli-B1 (8) and Gli-D1 (4). In the case of durum wheats, 19 alleles were identified: one allele at Glu-A1, two at Glu-B3, Glu-B2 and Gli-A1, three at Glu-B1, four at Glu-A3 and five at Gli-B1. For HMW-GS, LMW-GS and ω-gliadins, three, six and six different patterns were revealed, respectively. This study represents the first attempt to discriminate the bread and durum wheat varieties commonly grown in Portugal by the allelic variation of storage proteins. The database is useful for varietal identification and for plant breeders who seek to devise effective programmes aimed at improving wheat quality.