Androgenic response to preculture stress in microspore cultures of barley

Summary. Various stresses such as starvation and cold or heat shocks have been identified as triggers in the induction of the microspore embryogenesis. This study attempts to quantify the effects of different pretreatment conditions for successful microspore culture of malting barley (cv. Scarlett). While the sporophytic microspore development could be induced from treated and nontreated microspores, abiotic stress was essential for embryo formation and plant regeneration. The type of stress treatment applied affected the numbers and the ratios of albino and green plants regenerated, as well as their fertility. The highest number of green plants was obtained after the treatment of anthers in 0.3 M mannitol at 32 °C for 24 h before microspore culture. Correspondence and reprints: Department of Plant Biotechnology and Cytogenetics, Institute of Plant Breeding and Acclimatization, Radzików, 05-870 Blonie, Poland.     

Enhanced induction of microspore embryogenesis after <Emphasis Type="Italic">n</Emphasis>-butanol treatment in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) anther culture

The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

Ovary co-culture improves embryo and green plant production in anther culture of Australian spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A simple anther culture protocol for Australian spring wheat cultivars was developed using ovary co-culture. The inclusion of ovaries in the induction medium significantly increased the production of embryo-like structures (ELS), green and albino plants in two spring wheat cultivars tested. When five ovaries were added to the induction medium, the mean number of ELS per spike increased from 7.6 to 50.1 and green plants per spike increased from 0.6 to 8.9. The addition of 10 ovaries, however, did not further increase the production of ELS or green plants. The growth regulator combination of 2,4-D and KIN was compared with IAA and BA. There were no significant differences in the numbers of ELS or green plants although significantly fewer albino plants were produced with IAA and BA. Eight additional cultivars were screened using the protocol with either 5 or 10 ovaries in the induction medium. Green plants were obtained from nine varieties at frequencies ranging from 0.3 to 33.0 green plants per spike. Regenerant plants at maturity exhibited chromosome fertility rates in different cultivars ranging from 15% to 100%.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures

 The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10^–3–10^–5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M₂ doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M₂ doubled-haploid lines carrying mutations per line analysed was 3.8% when 10^–4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10^–5 and 10^–4 M sodium azide were applied to freshly isolated microspores from model cultivars. Received: 18 April 2000 / Revision received: 28 September 2000 / Accepted: 28 September 2000     

A comparison of barley isolated microspore and anther culture and the influence of cell culture density

Comparisons were made between the efficiency of barley plant regeneration from anther culture (AC) and isolated microspore culture (IMC) for the European winter cultivar `Igri' and the spring F<sub>1</sub> Australian breeder's hybrid Amagi Nijo×WI2585. In both cases, IMC produced a higher number of green regenerant plantlets per anther than AC. For `Igri' there was a 100- to 200-fold improvement and for Amagi Nijo×WI2585 there was a five- to ninefold improvement of IMC over AC. To improve the consistency and reliability of the IMC method, we investigated several parameters, including maltose concentration, subculture protocol, microspore plating density and colony plating density. Subculturing during the liquid culture phase produced no significant improvement in the number of microspores developing into colonies. The optimal concentration of maltose in the liquid induction medium was found to be 90 g l^–1. Both microspore plating density and colony plating density were found to influence plant regeneration. Microspores produced the highest numbers of colonies when plated at densities greater than 5×10⁴ ml^–1, and colonies produced optimal numbers of green plantlets when plated at 12.5–25 colonies/cm². Received: 23 March 1997 / Revision received: 29 May 1997 / Accepted: 25 June 1997     

Effect of growth regulators on microspore embryogenesis in coconut anthers

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y₃ medium containing 66 μM 2,4-D), followed by maturation medium (Y₃ medium without growth regulators) and germination medium (Y₃ medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA₃), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).     

Immunohistochemical Studies of the Cellular Changes in the Peripheral Olfactory System After Zinc Sulfate Nasal Irrigation

The peripheral olfactory system has a remarkable capacity for repair. We have performed an immunohistochemical study of the cellular changes that occur after zinc sulfate irrigation of the nasal cavity. The rapid loss of epithelial cells was followed by the proliferation of basal cells and the restoration of the epithelium with olfactory tissue. Horizontal basal cell markers, anti-cytokeratin 5/6 (CK5/6), and the Bandeiraea simplicifolia (BS-1) lectin initially co-localized on day 1 after treatment but rapidly displayed a disparity in their staining profile, with CK5/6 immunoreactive cells having a profile more akin to cells expressing the sustentacular marker cytokeratin 18 (CK18). This suggests CK5/6 and BS-1 label a different subset of horizontal basal cells. Axonal degeneration and regeneration was studied with a panel of markers to olfactory receptor neurons, their terminals, and olfactory bulb dendrites. The glial cells of the peripheral olfactory system, olfactory ensheathing cells, remained in position, with little change in immunoreactivity to laminin, although an increase in the expression of glial fibrillary acidic protein was observed. The events and the extent of reconstitution of the olfactory system after degeneration serves as a foundation for future studies designed to understand the unique regenerative capacity of the olfactory system.     

The relationship between induction of embryogenesis and chromosome doubling in microspore cultures

Summary. The objective of this paper is to review the relationship between induction of microspore embryogenesis and chromosome doubling. It has been augmented with relative data on chromosome doubling by nuclear fusion. Some of the treatments used for induction of embryogenesis may also lead to doubling of the chromosome number, either through nuclear fusion or endomitosis. High frequencies of spontaneous chromosome doubling in cereal species appear to be induced by treatments that block cell wall formation during the first cell divisions, resulting in coenocytic cells in which the nuclei are able to fuse. The use of mannitol as a pretreatment for induction of embryogenesis in barley, wheat, and maize microspore cultures provides examples of nuclear fusion. The use of antimicrotubule agents for embryo induction via treatments during the first few hours of microspore culture has also resulted in high frequencies of chromosome doubling. Factors such as the doubling agent concentration, temperature during treatment, and duration of treatment may be critical for individual species. Actin filament as well as microtubule assembly studies related to new cell wall formation provide further evidence at the molecular level for the relationship between microspore embryogenesis and chromosome doubling. Correspondence and reprints: Department of Plant Agriculture, University of Guelph, Crop Science Building, Guelph, ON N1G 2W1, Canada.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

A short severe heat shock is required to induce embryogenesis in late bicellular pollen of Brassica napus L.

 Until now it has been considered that in rape seed (Brassica napus) only late uninucleate microspores and early bicellular pollen are competent for induction of in vitro embryogenesis. Here we describe that pollen isolated at the late bicellular stage can also be induced to undergo embryogenesis. By the application of an additional short and more severe heat stress treatment, DNA synthesis was initiated in both generative and vegetative nuclei, but only vegetative cells were able to complete the cell cycle and to divide further. The ability of late bicellular pollen to respond to embryogenic induction treatment was accompanied by rearrangements of the microtubulular cytoskeleton and by the nuclear localization of 70 kDa heat shock proteins (HSP70). These findings confirm earlier observations that there is a strong correlation between the induction of embryogenesis and the synthesis and nuclear localization of HSP70. Received: 9 January 1997 / Revision accepted: 23 May 1997     

Polyploidization in embryogenic microspore cultures ofBrassica napus L. cv. Topas enables the generation of doubled haploid clones by somatic embryogenesis

Summary Embryogenic microspore and pollen culture followed by subculture of microspore-derived plantlets enabled the production of clones ofBrassica napus cv. Topas. Flow-cytometric analysis revealed that most microspore- and pollen-derived embryos (pEMs) were haploid initially. Spontaneous diploidization occurred at the globular stage of the pEMs, and was expressed as the relative increase of the 2C and 4C nuclear DNA content. Diploidization occurred throughout various organs of the pEMs and resulted in the formation of haploid and doubled haploid chimerics. In some embryos, nearly all cells were doubled haploid. From early cotyledon stage onward, pure haploid embryos were not observed anymore. At late cotyledon and germination stages, pure doubled haploid embryos and plantlets increased in number. Tetraploid pEMs were found occasionally. A culture regime was established to induce somatic embryos on the pEM-derived young plantlets. The ploidy of the somatic embryos varied highly and tended to be the same as that of the tissue at the initiation site on the pEM-plant. The results show that during the embryogenic development ofB. napus microspores, spontaneous diploidization occurs at globular stage, and increases progressively, resulting in the formation of chimerical haploid and doubled haploid plants as well as pure doubled haploid plants; ploidy neither affects pEM development at embryo developmental stages nor somatic embryogenesis, that starts on young pEM-derived plantlets; doubled haploid somatic embryos can be cloned from single pEM-derived plantlets; and doubled haploid embryos develop to fertile plants.     

L-羟脯氨酸-Zn(Ⅱ)的配合机制及其抗氧化性研究

以L-羟脯氨酸(Hyp)和ZnSO₄制备Hyp-Zn(Ⅱ)配合物,研究其配合机制及抗氧化能力。与L-羟脯氨酸相比,配合物在1100cm^－1处出现了一新的红外吸收峰,说明L-羟脯氨酸与Zn(Ⅱ)发生了配位作用;配合物的差热-热重图与L-羟脯氨酸相比,在290℃和375℃的吸热峰消失了,确证L-羟脯氨酸与Zn(Ⅱ)发生了配位作用;配合物核磁核磁共振图中3.5～3.9ppm的羧基氢和羟基氢的信号峰的消失,表明L-羟脯氨酸与Zn(Ⅱ)发生配合的位置是L-羟脯氨酸的α碳的羧基或γ碳的羟基氧;从配合物的原子力显微形貌相图可见数个L-羟脯氨酸围绕Zn(Ⅱ)形成的配合结构。透析实验结果证实L-羟脯氨酸与Zn(Ⅱ)配合的比例为4∶1（mol/mol）。上述测定和表征表明所形成配合物的分子式为Zn(Hyp)₄·H₂O。（Hyp）₄-Zn(Ⅱ)配合物抑制Zn(Ⅱ)产生的羟基自由基,抑制百分比为75.5%,其总抗氧化能力为80.167u/mL,抗超氧阴离子活力为53.19u/mL。     

Evidence for microspore embryogenesis in wheat anther culture

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

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.     

High frequency production of doubled haploid plants of Brassica napus cv. Topas derived from colchicine-induced microspore embryogenesis without heat shock

This report describes a very high genome doubling efficiency of Brassica napus cv. Topas plants, derived from microspores induced to undergo embryogenesis with a colchicine treatment, without the use of a heat treatment. The plants showed normal growth and development, and 90% were fertile. In contrast, only 6% of the plants derived from heat-induced embryos were fertile diploids. All cytological analysis of the progeny of fertile plants showed 2n=38 chromosomes. These results show that colchicine can simultaneously induce microspore embryogenesis and double the ploidy level to produce doubled haploid plants.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Comparison of frequency distributions of doubled haploid and single seed descent lines in barley

Summary Both doubled haploid (DH) and single seed descent (SSD) methods were used to derive homozygous lines from two crosses of barley. The frequency distributions of grain yield, heading date, and plant height of the DH and SSD lines were compared by the Mann-Whitney U test, Kolmogorov-Smirnov twosample test and Wald-Wolfowitz runs test. It was found that the DH lines distributed in the same manner as the SSD lines with respect to the three characters. The results indicated that although the SSD method had more opportunity for recombination than the DH method, it did not produce a sample of recombinants which differed significantly from the DH sample; thus both methods were equally efficient for use in deriving homozygous lines from F₁ hybrids in a relatively short time.Contribution no. 455 Charlottetown Research Station, Agriculture Canada, P.E.I. (Canada)