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.     

Regeneration potential of CIMMYT durum wheat and triticale varieties from immature embryos

Twenty‐five durum wheat elite advanced lines and released varieties, and five triticale varieties were evaluated for their ability to produce embryogenic callus using three different media. For callus initiation and maintenance there were basal Murashige and Skoog (MS) medium containing double strains of macroelements and 2.5 mg/l 2,4D (DW1), basal MS medium containing 2.0 mg/l 2,4D (DW2), or basal MS medium supplemented with 1.0 mg/l 2,4 D and coconut milk (DW3). Plant regeneration was achieved on basal MS medium with indoleacetic acid and 6‐benzylaminopurine, and plants rooted on MS with 1‐naphthale‐neacetic acid. DW3 medium proved better than the other media tested for embryogenic callus initiation and maintenance. Regeneration rates varied widely with both genotype and initiation medium, with values ranging from no regeneration to 100% regeneration; the plantlets produced per embryo ranged from five to 20. Fourteen of the durum wheat genotypes showed 63–100% regeneration from DW3 callus formation medium, four lines from DW1 medium, and two lines from DW2. Four of the triticale varieties had regeneration of 48–100% from DW3 medium. After six subcultures, over a 6‐month period, genotypes lost their ability to regenerate plants. Only 10 lines retained some plant regeneration potential but regeneration was at reduced levels. Successful regeneration of durum wheat and triticale varieties will be used as an integral part of the transformation process.     

The Influence of Dicamba on Somatic Embryogenesis and Frequency of Plant Regeneration from Cultured Immature Embryos of Wheat (Triticum aestivum L.)

An investigation was made to discover the influence of dicamba on the somatic embryogenesis of winter wheat cultivars-. Immature embryos of Triticum aestivum cv, ‘Sage’, ‘Caribo’ and ‘Kanzler’ were cultured, on modified N6-medium with the addition of 1 mg/13,6 dichlor-2-methoxy benzoe acid (dicamba). The young embryos were placed with the embryo axis on to the medium. Under this condition the scutella of the embryos at different stage of development produced compact calli and embryoids which regenerated plants with a high frequency (70 %) four to: six weeks later. The results suggest that dicamba could be of value in the induction of somatic embryogenesis.     

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.     

Callusing and Regeneration of Plantlets via Somatic Embryogenesis from Inflorescence Cultures of Triticum aestivum L.: Role of Genotype and Long-Term Retention of Morphogenic Potential

In the present investigation we have succeeded in obtaining a high frequency of regeneration of plantlets via somatic embryogenesis from callus derived from immature inflorescence explains of Triticum aestivum var, ‘Sonalika’. The explants were cultured on MS medium supplemented with 2,4-D, casein hydrolysate and coconut milk. A large number of embryoids germinated, to form plantlets on the medium when 2, 4-D was omitted altogether or provided at low concentration. Plantlets were transferred to soil under natural environmental conditions and were shown to have the normal chromosome number of 2n = 6x = 42. Experiments with nineteen other varieties show that there is a marked effect of genotype both on initiation of callusing as well as on regeneration. So far ‘Sonahka’ has proved to be the most responsive among varieties tested by us. With callus of the variety ‘Sonalika.’, we also conducted an investigation on long-term retention of regenerative potential. During Song-term culture, for about 12 months, the morphogenic potential gradually diminished and was finally lost, but the regeneration potential could be restored by subculturing at very short intervals.     

Large-scale production of wheat and triticale double haploids through the use of a single-anther culture method

A total of 257 parental wheat and 38 triticale lines were used for anther culture. On average, 2.1 green wheat haploids were obtained per spike. This response occurred irrespective of the origin of the material (Germany, France, Sweden or UK) and 5 years of testing. Triticale responded with 5.3 green haploids per spike. Using the criterion that one parental line should give at least one green haploid per spike in the screening experiment, green haploids were produced from 88 out of 91 F₁ wheat breeding combinations and from each of 21 F₁ and F₂ triticale breeding combinations. An average of 4.7 green plants were obtained per spike from the wheat production programme, while the triticale programme gave an average of 6.2 green plants per spike. A single medium supplemented with different hormones for anthers and embryos was used for culture of both crops.     

Comparison of callus culture with embryo culture at different times of embryo rescue for primary triticale production

Summary Callus culture of immature wheat-rye hybrid embryos was compared with embryo culture in two experiments. Embryos were rescued from field grown mother plants at two day intervals 13–21 days after pollination and plated for 1) callus culture on Murashige and Skoog medium (MS) supplemented with 2 mg/12,4-dichlorophenoxy acetic acid followed by plant regeneration on hormone free MS medium with half strength mineral salts, 2) embryo culture on Taira and Larter medium (TL). Observations were made on embryo size and condition at time of rescue (Experiment 1) and embryo development directly into plants (embryo culture) or through embryogenesis (callus culture). Fewer 19 and 21 day old embryos developed into plants from callus culture than from embryo culture in Experiment 1. Callus culture was more efficient than embryo culture in promoting plant recovery from 17 day old embryos in Experiment 2. The number of plants per embryo was significantly higher from callus culture than from embryo culture. In both experiments callus culture promoted embryogenesis in more embryos than developed in embryo culture. Embryo rescue 15–17 days after pollination was optimal in both experiments.     

Somatic Embryogenesis and Plant Regeneration from Tritordeum

Regeneration of plants by somatic embryogenesis from immature embryos of hexaploid tritordeum (AABBH^chH^ch, amphiploid Hordeum chilense×Triticum turgidum conv. durum) and durum wheat (Triticum tergidum) was induced on MS medium supplemented with different 2.4-D concentrations. Well-defined embryoids were formed with a high frequency on the scutellar callus from 1 or 2 weeks onwards and plantlets were developed from them. In the best cases from one single explant more than 100 plants could be obtained. Plants were also regenerated by somatic embryogenesis from inflorescences of Hordeum chilense×Triticum turgiditm conv. durum hybrid and its respective hexa-amphiploid. With regard to callus induction and regenerative ability, evident differences between hexa- and octoploid (H. chilense×T. aestivum) tritordeum were found, the latter showing a very low response.     

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.     

Vernalization of immature embryos of winter wheat genotypes

Summary The effect of direct vernalization of immature embryos on flowering was studied in six winter wheat genotypes. Fourteen-, 17-, and 20-day-old embryos were excised and vernalized for 0–6 weeks on synthetic medium during a conditioning period. Percent germination of embryos was high (overall 96.1%), and free from genotypic effects. Genotypes differed for flowering in response to cold treatment of excised embryos. Embryo vernalization was as effective as or more than conventional vernalization (control, seedling vernalization for 6 weeks). Seventeen-day-old embryos were the most responsive to vernalization. With a 5-week vernalization of 17-day-old embryos, the percentage of plants anthesed was higher than those from 14-and 20-day-old embryos. For 17-day-old embryos vernalized for 5 weeks, the mean number of days from culture to anthesis was less than that of 6 week vernalization, less than that of 14- and 20-day-old embryos, and less than controls.Purdue Univ., Agronomy Dept., W. Lafayette, IN 47907, USA.     

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

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

Inheritance of Karnal bunt-free trait in bread wheat

A Karnal bunt (KB)‐free wheat stock (‘KBRL22’) obtained from a cross of two resistant lines (‘HD29’ and ‘W485’) was used as a donor to introgress the KB‐free trait into ‘PBW343’(an ‘Attila’ sib), the most widely grown wheat cultivar in India. The number of KB‐free and KB‐affected plants in BC ₁, BC₂, BC₃ and BC₄ as well the F₂ was recorded after artificial inoculations. The segregation pattern in these generations clearly indicated two independently segregating, dominant genes which jointly confer the KB‐free attribute. The importance of the KB‐free line generated in this experiment is discussed.     

冬小麦遗传再生体系及根癌农杆菌介导的转化研究

摘要:以冬小麦品种临优145的幼胚为外植体,消毒后用解剖刀挑取幼胚,盾片朝上接种于MS+2,4-D 2 mg/L+肌醇100 mg/L＋MES 400 mg/L＋CH 100 mg/L[7]＋40g/L麦芽糖＋8g/L琼脂的培养基中诱导愈伤组织, 每两周继代一次,将诱导出的淡黄色、颗粒状Ⅱ型胚性愈伤组织接种于分化培养基（MS+ZT 1 mg/L+IAA 1mg/L+ MES 400 mg/L＋CH 100 mg/L+麦芽糖40g/L+gelrite 2.6g/L,PH5.8）上培养2－3周,然后转接到再生培养基(1/10MS+NAA 0.5mg/L+KT 0.5mg/L+蔗糖30g/L+ gelrite 2.6g/L,PH5.8)中进行再生成苗。接种1229块幼胚,得到987块愈伤组织, Hyg抗性愈伤组织123块,抗性再生植株34株。在建立了再生体系的基础上,用根癌农杆菌介导法将GUS基因导入幼胚愈伤组织,抗性植株的PCR检测呈阳性。X－gluc染色表明, 少部分愈伤组织出现肉眼可见的蓝色晕斑,说明GUS基因已经在小麦幼胚愈伤组织中表达。     

Plant tissue culture of Secale: A review

Summary Plant tissue culture of rye employs different parts of plant bodies originating from various stages of rye ontogenesis. For the culture initiation mainly diploid and after that tetraploid forms were used, however, haploids and triploids were subject of investigation. The development of plant regeneration via somatic embryogenesis or organogenesis required the using of numerous basal media with various plant growth hormones. Haploidization appeared to be the most difficult problem and only interspecies hybridization helped to overcome this problem. Wide sexual intergenera and interspecies hybridization in cereals require the development of proembryo and immature embryo culture system. Using the nurse culture basing on immature endosperm resulted in getting fully formed rye plants. There is no progress in somatic cell genetic manipulation of rye because of lack of plant regeneration system in mesophyll green leaf and suspension protoplast cultures.Abbreviations 2, 4, 5, Cl₃POP... 2, 4, 5-Cl₃-phenoxypropionic acid, dicamba...-3, 6-dichloroasinic acid - GA₃... gibberellic acid - BA... benzyladenine - IBA... 3-indolebutric acid - IAA... indoleacetic acid - NAA... 1-naphtylacetic acid - 2, 4-D... 2, 4-dichlorophenoxyacetic acid - 2, 4, 5-T... 2, 4, 5-trichloroacetic acid - p-CPA... parachlorophenoxyacetic acid - CM... coconut milk - CW... deproteined coconut milk - Kin Kinetin     

Complexity of the Gli-A3 locus in bread wheat

Two bread wheat cultivars, ‘Ariana 8’ and ‘Cajeme 71’, and 129 F₂, grains from the cross between them were analysed for gliadin composition. Two monodimensional (A-PAGE and SDS-PAGE) and two different two-dimensional (SDS-PAGE x SDS-PAGE and A-PAGE x SDS-PAGE) electrophoretic methods were used. Parents differed at the Gli-Al locus, detected by A-PAGE. The SDS-PAGE of the aqueous ethanol extractable protein under nonreduced conditions showed two bands of ‘Ariana 8’ and one of ‘Cajeme 71’, encoded by genes located 22 cM from the Gli-Al locus, and therefore, located at the Gli-A3 locus. This locus has been considered to contain genes coding for ω-gliadins alone. The two-dimensional maps of the parents showed that one band from ‘Ariana 8’ was an ω-gliadin, but the other two bands, one from each parent, were γ-gliadins. Results obtained indicated that GH-A3, like Gli-Al, is a complex locus coding for both ω-and γ-gliadins.     

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.     

Genetic basis of seedling-resistance to leaf rust in bread wheat 'Thatcher'

The bread wheat cultivar ‘Thatcher’ is documented to carry the gene Lr22b for adult‐plant resistance to leaf rust. Seedling‐resistance to leaf rust caused by Puccinia triticina in the bread wheat cultivar ‘Thatcher’, the background parent of the near‐isogenic lines for leaf rust resistance genes in wheat, is rare and no published information could be found on its genetic basis. The F₂ and F₃ analysis of the cross ‘Agra Local’ (susceptible) × ‘Thatcher’ showed that an apparently incompletely dominant gene conditioned seedling‐resistance in ‘Thatcher’ to the three ‘Thatcher’‐avirulent Indian leaf rust pathotypes – 0R8, 0R8‐1 and 0R9. Test of allelism revealed that this gene (temporarily designated LrKr1) was derived from ‘Kanred’, one of the parents of ‘Thatcher’. Absence of any susceptible F₂ segregants in a ‘Thatcher’ × ‘Marquis’ cross confirmed that an additional gene (temporarily designated LrMq1) derived from ‘Marquis’, another parent of ‘Thatcher’, was effective against pathotype 0R9 alone. These two genes as well as a second gene in ‘Kanred’ (temporarily designated LrKr2), which was effective against all the three pathotypes, but has not been inherited by ‘Thatcher’, seem to be novel, undocumented leaf rust resistance genes.     

Seed storage protein composition of Hellenic bread wheat cultivars

The allelic diversity in seed storage proteins of 25 bread wheat cultivars grown in Hellas was investigated. In total, 15–20 seeds per cultivar were used for the determination of the alleles present at the loci coding for high‐molecular‐weight glutenin subunits and gliadins. For this purpose, acid polyacrylamide gel electrophoresis for gliadins and SDS‐electrophoresis for glutenins were employed. Analysis of the electrophoretic patterns revealed that intravarietal selections obtained from the cultivar ‘Nestos’, together with the cultivar ‘Eurydice’ which was selected from the cultivar ‘Nestos’, were identical to their original cultivar and to the cultivar ‘Dodoni’. The cultivars ‘Penios’, ‘Siette Cerros E’, ‘Gorgona’ and ‘Louros’, although recorded to differ in descent, were found to be identical at all the loci examined. Finally, it was revealed that four of the Hellenic cultivars carry the wheat‐rye 1BL/1RS translocation. These data could be beneficial for a better understanding of the existing differences in quality and stress‐resistance between the cultivars examined.     

Identification and characterization of quantitative trait loci related to lodging resistance and associated traits in bread wheat

Lodging is a major constraint to increasing yield in many crops, but is of particular importance in the small‐grained cereals. This study investigated the genetic control of lodging and component traits in wheat through the detection of underlying quantitative trait loci (QTL), The analysis was based on the identification of genomic regions which affect various traits related to lodging resistance in a population of 96‐doubled haploid lines of the cross ‘Milan’בCatbird’, mapped using 126‐microsatellite markers. Although major genes related to plant height (Rht genes) were responsible for increasing lodging resistance in this cross, several other traits independent of plant height were shown to be important such as fool and shoot traits, and various components of plant yield. Yield components such as grain number and weight were shown to be an indicator of plant susceptibility to lodging. QTL for lodging and associated traits were found on chromosomes IB, ID. 2B. 2D. 4B, 4D. 6D and 7D. QTL for yield and associated traits were identified on chromosomes IB, ID. 2A. 2B. 2D. 4D and 6A,     

The HMW Glutenin Subunit and Gliadin Compositions of German-Grown Wheat Varieties and their Relationship with Bread-Making Quality

An in vitro system for the initiation of somatic embryogenesis and plant regeneration of spring- and winter-type rye has been developed. The optimal developmental stage of immature embryos to be used as explants is the late stage of spherical coleoptile when the tissue turns milky in colour and the coleoptile is enlarged but spherical. The type and concentration of auxin in the induction medium is of importance for obtaining high efficiency of somatic embryogenesis. CC-medium high 30 μM Dicamba resulted in well developed somatic embryos in large umbers. Positive effects on the efficiency, intensity and speed of development of somatic embryos have been observed by the addition of coconut water to CC-medium, replacing agar by agarose and culturing the explants in low light intensity. Following a “step by step” optimization, culture conditions have been defined giving rise to a 90—100% efficiency of somatic embryogenesis and a high number of regenerated plants.