An Improved In Vitro Culture Procedure for Embryoids Derived from Isolated Microspores of Rape (Brassica napus L.)

An in vitro culture technique was developed that allowed the effective culturing of embryos derived from isolated microspores of rape (Brassica napus L.). The method consists of the use of microspores from buds at well-defined development stages, the use of a double-layer culture medium containing activated charcoal and the incubation of embryoids in culture flasks on a shaker. These culture modifications, together with the growing of embryoids on solid medium with high agar concentration, resulted in a high frequency of embryoids growing directly into plants.     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

Effect of Sugars in Wheat Anther Culture Media

Sugars are critical components in bread wheat (Triticum aestivum L.) anther culture media for successful somatic embryo initiation and plant regeneration. In this experiment, anthers from three wheat genotypes were cultured on a modified Liang's 85D12 initiation medium with seven sugar combinations (I-sugars: galactose, mannose, maltose, fructose, sucrose, glucose, maltose + glucose) at 0.26 M, and 2,4-dichlorophenoxyacetic acid (2 mg/L), 1-naphthalene acetic acid (1 mg/L), and glutamine (254 mg/L). Wheat starch (5 % W/V), a potential source of sugars, was used as the medium gelling agent. No previous research has studied the effect of different sugars with wheat starch. A split-plot experimental design with 42 replications was used with genotypes as whole plots and sugar combinations as subplots. Galactose and mannose did not support embryoid initiation and were dropped from the analysis. Averaged over the three genotypes, maltose was the best sugar (105 embryoids/100 anthers), followed by glucose (47 embryoids/100 anthers) and maltose + glucose (37 embryoids/100 anthers). These three sugar combinations were superior to the standard medium sugar, sucrose (24 embryoids/100 anthers), and to fructose (12 embryoids/100 anthers). The embryoids were divided into two groups for plant regeneration. The first group was transferred to regeneration medium (Liang 85D12 salts, sugars at 0.06 M, and wheat starch at 7 % w/v as gelling agent) with the same sugar (R-sugar) used as in initiation. The second group was transferred to regeneration media with sucrose. I+R-maltose (0.55)     

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.     

Dihaploid Plant Production from 4 × -Genotypes of Potato by the Use of Efficient Anther Plants Producing Tetraploid Strains (4 × EAPP-Clones) — Proposal of a Breeding Methodology

Tetraploid clones of potato with a superior efficiency in producing androgenetic plants (4 × EAPP-clones) have been obtained by culturing in vitro anthers of 2 × EAPP-clones. The latter were isolated by three cycles of recurrent selection from diploid breeding material (UHRIG 1985 a). In this paper we report on the capacity of 4 × EAPP-clones, when crossed to in vitro unresponsive 4 × genotypes, to transmit to their F₁ their androgenetic potential, Five 4 ×× 4 × F₁ crosses were considered, which produced on average 35 embryoids per flower – a value higher than that of nonresponsive 4 × genotypes (no embryoids obtained), but-also significantly better than the value found for 4× EAPP-clones (9.8 embryoids per flower). The hybrid families behaved differently from each other, with one producing up to 63 embryoids per flower. A range of per plant values was, moreover, found, revealing the existence of a large variability among sister plants belonging to the same F₁ cross. The presented data indicate a rather simple inheritance of dominant genetic factors acting in favor ot androgenesis. They also suggest that the utilized 4× EAPP-clones were possibly heterozygous for such genes. Data are also reported on the ploidy level of anther plants obtained from 4× and 2× EAPP-clones. In this respect 2× EAPP-clones show the interesting capacity of generating, via anther culture, a consistent fraction of tetraploid plants (13,7 %). Based on the findings reported in this paper we propose, for tetraploid S. tuherosum L., a cyclic breeding procedure making use of anther culture and where ploidy level alternates, within a cycle, between 2× and 4×.     

Effects of Growth Regulators and Genotypes on Callus and Embryoid Induction from Maize Anther Culture *

Fifty genotypes and ten growth-regulator combinations were used in two experiments (I and II) to investigate genotype and hormonal effects on production of callus and embryoids via anther culture in maize (Zea mays L.). Hormonal effects across all genotypes were not significant in either experiment. However, highest callus-induction frequencies in both experiments occurred on YP basal medium plus 2,4-D at 2.0 mg 1^?1, and kinetin at 1.5 mg 1^?1 indicating that this combination was more effective than others. Genotypic differences in callus or embryoid induction across all media were significant. The most responsive genotypes in experiment I were single-cross hybrids Yuanwu × 592 and K727 × K305, which produced 18.3 and 6.7 % calli, respectively, with their appropriate media. The most responsive entry in experiment II was CIMMYT Pool 29, which produced 15.0 % calli on appropriate medium and an average of 10.0 % calli across 10 media. Twenty-three plantlets was regenerated from this study. Most of them developed embryogenically.     

Impact of Phosphorus from Dairy Manure and Commercial Fertilizer on Perennial Grass Forage Production

Increased recovery and recycling of manure phosphorus (P) by crops on dairy farms is needed to minimize environmental problems. The main objective of this study was to compare P utilization by orchardgrass (Dactylis glomerata L.) and tall fescue (Festuca arundinaceae Schreb.) from dairy manure or inorganic fertilizer. The study was conducted from 1994 to 2000 at the Cornell University Baker Farm, Willsboro, NY, on a somewhat poorly drained Kingsbury clay (very–fine, illitic, mesic Aeric Epiaqualfs). The design was a split‐plot in a randomized complete block with two manure rates (16 800 and 33 600 kg ha^?1) and one nitrogen (N) fertilizer rate (84 kg N ha^?1 at spring greenup and 56 kg N ha^?1 prior to each regrowth harvest) as the main plots and grass species as subplots replicated six times. Fertilizer P [Ca(H₂PO₄)₂] was applied to the fertilizer treatment in 1995 and 1996 at 11 kg P ha^?1 year^?1. Orchardgrass P removal averaged 21 % higher than tall fescue P removal for the spring harvest, but orchardgrass averaged 24 % lower P removal than tall fescue removal for all regrowth harvests from 1995–99. Phosphorus herbage concentration in the fertilizer treatment was in the range of 1.9–2.7 g P kg^?1 compared with 2.2–5.3 g P kg^?1 in the manure treatments. Seasonal P removal ranged from as low as 9.2 kg P ha^?1 to as high as 48.5 kg P ha^?1. Morgan extractable soil P in the top 0–0.20 m remained high through 1999, with 29.1 kg P ha^?1 at the highest manure rate in tall fescue compared with 8.4 kg P ha^?1 measured in 1993 prior to the experiment. In 2000, soil P at the highest manure rate in tall fescue dropped to 10.1 kg P ha^?1, following cessation of manure application in 1998. Intensively managed harvested orchardgrass and tall fescue have the potential to remove large quantities of manure P.     

In vitro Selection for Tolerance to Fusarium in F1 Microspore Populations of Wheat

Microspore populations of eight Fhybrids of winter wheat (Triticum aestivum L.) whose parents had different levels of resistance to Fusarium were screened in vitro, using phytotoxins of Fusarium as biochemical probe. Two selection methods were compared for the in vitro selection: either embryoids and calli were first initiated from anthers in toxin-free medium and then grown on medium with 0.3—0.9 %Fusarium toxin; or anthers were immediately cultured in modified liquid potato-2 medium in the presence of 1.5, 2.0 and 2.5ml Fusarium toxin per liter culture medium, a concentration which reduced the number of calli and embryoids to about 10 % compared to the toxin-free controls. Microspores from donor hybrids which were produced from very susceptible cultivars were killed by lower toxin concentrations than micro-spores from hybrids of less susceptible parents. From surviving calli and embryoids, originally initiated from 242,000 anthers in both procedures a total of 375 green lines could be regenerated. The results indicate that it is possible to enrich the fraction of regenerating microspores by those which contain the gene complex responsible for reduced susceptibility to Fusarium by the use of a pathotoxin.     

Selection in vitro for erucic-acid content in segregating populations of microspore-derived embryoids of Brassica napus

Microspore culture of Brassica napus under optimized conditions leads to the regeneration of microspore-derived embryoids that, at the late cotyledonary stage, contain large amounts of storage lipids, equal or similar in composition to those found in seeds of the homozygous donor plants. At that stage, the microspore-derived embryoids are large enough to allow the dissection of one cotyledon under aseptic conditions and the determination of its fatty-acid composition. The remaining part of the embryoid can be cultured further and regenerated to give a plant. This offers the possibility of early selection for fatty-acid composition in segregating populations of microspore-derived embryoids. In order to verify this hypothesis, embryoids were generated from microspores of F| plants derived from a cross between doubled haploid lines of the low-erucicacid cv. ‘Duplo’ and the high-erucic-acid cv. ‘Janetzki’. The contents of eicosenoic acid (C20: 1) and erucic acid (C22: 1) in the cotyledons and in the seeds derived from plants regenerated from the remaining parts of the embryoids were highly correlated (rs = 0.85**, P = 0.01). This indicates that, in breeding programmes for high erucic acid, the majority of the microspore—derived embryoids can be discarded at an early stage in vitro. Only microspore-derived embryoids with a high content of C20: 1+C22:1 in the cotyledons need to be transferred to the greenhouse. This report also deals with the addition of abscisic acid (ABA) to the embryoid culture medium to increase the correlation, and discusses the possible application of this system for the selection of high-oleic or low-linolenic types in corresponding microspore-derived embryoid populations.     

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

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

Free Air CO2 Enrichment (FACE) of grapevine (Vitis vinifera L.): I. Development and testing of the system for CO2 enrichment

The control of CO₂ levels is reported for a Free-Air Carbon dioxide Enrichment (FACE) facility employed in a vineyard at Rapolano, Italy in 1996 and 1997. This control is required for evaluating the validity of a biological experiment conducted on grapevine in CO₂ enriched and control experimental plots. Six rectangular emission arrays enclosing seven plants each were installed in the vineyard imposing three CO₂ exposure levels (ambient, 550 and 700 μmol mol⁻¹) and monitoring CO₂ levels at the centre of each experimental plot. In the 2 years, average seasonal (May–September) CO₂ levels during treatment hours (05:00–19:00 h) varied from 697 to 698 μmol mol⁻¹ for 700 μmol mol⁻¹ target treatment and from 549 to 550 for 550 μmol mol⁻¹ target treatment. The averaged 1 min measurements of CO₂ concentration were within 20% of the target concentrations for more the 80% of the time. The FACE facility also provided a good spatial control of CO₂ concentration for an experimental sampling volume of 15.7 m³ (8 m×1.4 m×1.4 m), including five plants per plot.     

Alfalfa Phytoestrogen Content: Impact of Plant Maturity and Herbage Components

Legumes contain a range of non‐nutritional phytochemicals that may have health‐promoting effects in humans. In this study, we determined the concentrations of four phytoestrogens (coumestrol, apigenin, luteolin and quercetin) in field‐grown alfalfa (Medicago sativa L.). Differences between plants of different stages of maturity, between plant parts, and different canopy segments were assessed. The concentration of individual phytoestrogen in whole herbage varied between 15 and 225 μg g^?1 dry matter (DM) and was strongly affected by stage of maturity. Coumestrol and apigenin concentrations were highest at early vegetative stages, luteolin and quercetin at early vegetative and late flowering stages. All phytoestrogens were found in lowest concentrations at the early flowering stage (average 68 μg g^?1 DM); stage at which alfalfa is usually harvested when used as a forage source for animals. At vegetative stages, apigenin was the predominant phytoestrogen in herbage followed by coumestrol, the reverse being observed upon initiation of flowering; luteolin and quercetin were found at all stages in similarly lower concentrations. Concentrations of luteolin, quercetin and apigenin were 225, 410 and 690 % greater, respectively, in flowers than in leaves or stems; coumestrol concentration was similar between plant parts. In flowers and stems the predominant phytoestrogens were apigenin and quercetin, followed by coumestrol and luteolin. Similar concentrations (average 26 μg g^?1 DM) of each of the four phytoestrogens were found in leaves. Concentrations through the herbage canopy varied and were greatest at >60 cm from the soil surface for apigenin and coumestrol, but greatest at >60 and 0–20 cm for quercetin and at 0–20 cm for luteolin. The results suggest that if alfalfa is to be used as a source of phytoestrogens and is harvested for the production of herbal supplements or nutraceuticals, management will need to be adapted.     

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.     

Genetic analysis for haploid-regenerations responses of the hexaploid-wheat anther cultures

Genetic variability in response to anther culture was investigated in 49 winter hexaploid wheats, comprising 33 pure lines (F₁₀) derived from a composite cross programme and their 16 parental genotypes. All genotypes were grown in a randomized block design with three replications in a controlled greenhouse. The number of embryoids and total plant regeneration per 100 anthers, as well as the numbers of green and albino plants regenerated per 100 embryoids, were measured. Significant genetic variability was observed among the 49 genotypes for all the traits studied. All traits showed high heritability. Among the genotypes compared, DC²30N and 1BPT-40 gave the best results for the production of embryoids and IBPT-78 had the highest value for the production of green plants. The genotype IBPT-34 developed a large number of albino plants, and it should be useful as a parent in studies to determine the genetic control of albino plants in wheat.     

Effects of Salinity and Soil–Drying on Radiation Use Efficiency,Water Productivity and Yield of Quinoa (Chenopodium quinoa Willd.)

Drought and salinity reduce crop productivity especially in arid and semi‐arid regions, and finding a crop which produces yield under these adverse conditions is therefore very important. Quinoa (Chenopodium quinoa Willd.) is such a crop. Hence, a study was conducted in field lysimeters to investigate the effect of salinity and soil–drying on radiation use efficiency, yield and water productivity of quinoa. Quinoa was exposed to five salinity levels (0, 10, 20, 30 and 40 dS m^?1) of irrigation water from flower initiation onwards. During the seed‐filling phase the five salinity levels were divided between two levels of irrigation, either full irrigation (FI; 95 % of field capacity) or non‐irrigated progressive drought (PD). The intercepted photosynthetically active radiation was hardly affected by salinity (8 % decrease at 40 dS m^?1) and did not differ significantly between FI and PD. Radiation use efficiency of dry matter was similar between salinity levels and between FI and PD. In line with this, no negative effect of severe salinity and soil–drying on total dry matter could be detected. Salinity levels between 20 and 40 dS m^?1 significantly reduced the seed yield by ca. 33 % compared with 0 dS m^?1 treatment owing to a 15–30 % reduction in seed number per m², whereas the seed yield of PD was 8 % less than FI. Consequently, nitrogen harvested in seed was decreased by salinity although the total N‐uptake was increased. Both salinity and drought increased the water productivity of dry matter. Increasing salinity from 20 to 40 dS m^?1 did not further decrease the seed number per m² and seed yield, which shows that quinoa (cv. Titicaca) acclimated to saline conditions when exposed to salinity levels between 20 and 40 dS m^?1.     

Productivity and Sustainability of Cotton (Gossypium hirsutum L.)–Wheat (Triticum aestivum L.) Cropping System as Influenced by Prilled Urea, Farmyard Manure and Azotobacter

Field experiments were conducted at Indian Agricultural Research Institute, New Delhi, during 2001–2002 and 2002–2003, to study the effect of inorganic, organic and Azotobacter combined sources of N on cotton (Gossypium hirsutum L.) and their residual effect on succeeding wheat (Triticum aestivum L.) crop. The results indicated considerable increase in yield attributes and mean seed cotton yield (2.33 Mg ha^?1) with the combined application of 30 kg N and farmyard manure (FYM) at 12 Mg ha^?1 along with Azotobacter (M₄). The treatment in cotton that included FYM, especially when fertilizer N was also applied could either improve or maintain the soil fertility status in terms of available N, P and K. Distinct increase in yield attributes and grain yield of wheat was observed with the residual effect of integrated application of 30 kg N ha^?1 + FYM at 12 Mg ha^?1 + Azotobacter. Direct application of 120 kg N ha^?1 resulted 67.4 and 17.7 % increase in mean grain yield of wheat over no N and 60 kg N ha^?1, respectively. Integrated application of organic and inorganic fertilizer is therefore, recommended for higher productivity and sustainability of the cotton–wheat system.     

Identification of QTLs associated with spring vegetative budbreak time after dormancy release in pear (Pyrus communis L.)

Dormancy release is greatly affected by chilling unit (CU) accumulation. Lack of CU has a major impact on spring vegetative budbreak (VB). To understand the genetic mechanism governing the chilling requirement (CR), we conducted a QTL analysis of VB date in F₁ population, derived from a cross between ‘Spadona’ (low CR) and ‘Harrow Sweet’ (high CR). Using a unique methodology of tree mobility, replicates of the same genotypes were exposed during the winter, over two consecutive years, to climates that differ greatly in their CU and to the same heat conditions to induce VB, in order to evaluate CR genetic impact and to distinguish it from the heat factor. Broad‐sense heritability within locations ranged from 0.62 to 0.66. Due to a strong impact of GxE interaction, it was reduced to 0.46 for the overall mean. We examined the previously discovered apple QTLs detected in linkage groups (LG) 9 and 8, based on the synteny between the species. Our analysis confirms significant QTLs in LG8 (R² = 12%–24%) and LG9 (R² = 20%–38%) for all locations and years     

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.     

Plant Regeneration Through Anther Culture of Three Wild Species of Hordeum (H. murinum, H. marinum and H. bulbosum)

Plant regeneration was achieved through anther culture of three wild species of Hordeum (H. murinum, H. marinum and H, bulbosum). Calli or embryoids were formed from microspores in anthers cultured on a medium containing 6-benzylammopurine (BAP) and ficoll. These calli or embryoids regenerated green or albino shoots and roots after transfer to regeneration media. Green plantlets which developed on regeneration media were transferred to soil where they showed further growth.     

Energy Input and Output Analysis of four Field Crops in California1

Four crops, corn (Zea mays L.), sweet sorghum (Sorghum bicolor L.), fodder beet (Beta vulgaris L.) and sugarbeet (Beta vulgaris L.) were grown in irrigated plots at the experimental farm of the University of California, Davis, in 1980 and 1981. Six fertilizer N levels ranging from 0 to 280 kg ha^?1 were used to estimate the most efficient N input for each of the tested crop in terms of energy input and output analysis. Calculations of cultural energy input costs in relation to potential ethanol yield showed production requirements of: corn 30.9 GJ ha^?1, sweet sorghum 30.4 GJ ha^?1, fodder beet 49.4 GJ ha^?1 and sugarbeet 41.0 GJ ha^?1. Highest average energy inputs were for liquid fuels for operations 35%, irrigation 23% and fertilizer nitrogen 19%. Fodder beet had the highest fermentable carbohydrate yield at 13.05 Mg ha^?1 followed by sugarbeet at 11.5 Mg ha^?1. Sweet sorghum and corn yields were lower at 9.71 and 8.09 Mg ha^?1, respectively. Crop production inputs of energy per liter of potential ethanol were: corn 6.42 MJL^?1 sweet sorghum 5.25 MJL^?1, fodder beet 6.35 MJL^?1 and sugarbeet 5.95 MJL^?1.