Zinc sulphate improved microspore embryogenesis in barley

The effect of ZnSO₄ concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control) to 600 μM in the stress pre-treatment medium alone or in combination with 30 (control) to 600 μM in the embryo induction medium were assayed in anther culture. Incorporation of Zn²⁺ in the pre-treatment medium itself did not affect microspore embryogenesis. The optimum concentration in the stress pre-treatment and induction media was 180 μM for cultivars (cvs.) Igri and Reinette, and 90 μM for cv. Hop. A significant increase of 30 and 300% in cv. Igri and Reinette, respectively, were produced with 180 μM ZnSO₄ in both the number of embryos and green plants. In order to confirm the effect of Zn²⁺ on microspore embryogenesis this micronutrient was incorporated in the induction medium of isolated microspore cultures of cv. Igri. Concentrations of 90–300 μM ZnSO₄ resulted in an increase of 40–53% in the number of embryos and green plants. All these results indicate that the beneficial effect of Zn²⁺ is exerted mainly during the culture phase, increasing the number of embryos, leading to an increased number of green plants, but it had no effect on percentage of regeneration or green plants.     

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     

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     

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%.     

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.     

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.     

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.     

Improved microspore culture and doubled-haploid plant regeneration in the brown condiment mustard (Brassica juncea)

 The availability of doubled haploids could greatly contribute to improving seed quality in condiment mustard (Brassica juncea). We have developed an efficient and reliable protocol of microspore culture, modified from that of Baillie et al. (1992), based on a modification of the sucrose concentration of culture media. A comparison of microspore culture media differing in their sucrose content showed that a decrease from 17% (w/v) sucrose during the first 48 h to 10% (w/v) thereafter favoured an increase in the production of embryos whatever the responding genotype tested. Thus, out of the 13 B. juncea genotypes studied, 12 gave rise to embryos, and seven of these embryos could be converted into plants. Doubled-haploid plants were produced after treatment with colchicine. Received: 16 January 2000 / Revision received: 8 August 2000 / Accepted: 20 September 2000     

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     

Doubled haploid production in Flax (Linum usitatissimum L.)

There is a requirement of haploid and double haploid material and homozygous lines for cell culture studies and breeding in flax. Anther culture is currently the most successful method producing doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this review we focus on tissue and plants regeneration using anther culture, and cultivation of ovaries containing unfertilized ovules. The effect of genotype, physiological status of donor plants, donor material pre-treatment and cultivation conditions for flax anthers and ovaries is discussed here. The process of plant regeneration from anther and ovary derived calli is also in the focus of this review. Attention is paid to the ploidy level of regenerated tissue and to the use of molecular markers for determining of gametic origin of flax plants derived from anther and ovary cultures. Finally, some future prospects on the use of doubled haploids in flax biotechnology are outlined here.     

Haploid formation in maize, barley, flax, and potato

Summary. The article is reviewing some significant features and issues in the process of haploid formation in two important monocotyledonous crop plants – maize and barley – and in two dicotyledonous plants – flax and potato. Exotic maize lines with higher androgenic response turned up as a good source for this heritable trait and this valuable trait can be incorporated into elite maize lines via crossing. Lots of attempts were devoted to identifying some cytological and/or morphological markers for androgenic response in maize microspore cultures. The “starlike” organization of the cytoplasm inside the induced maize microspores together with the enlarged size of induced microspores can be considered as morphological markers for androgenic response. In barley, microspores with rich cytoplasm that was of granular appearance with the nucleus located near the cell wall and with no visible vacuole had the largest survival rate and many of these cells continued in development and produced embryos. In flax, a dramatic increase of induction rate in anther cultures (up to 25%) was achieved when flax anthers were pretreated for 3 days at 4 °C and afterwards kept for 1 day at 35 °C. Also gynogenesis in flax has been reported already and complete plants were obtained. In potato microspore cultures, formation of two dissimilar cells indicated a strong polarization in the system and as a result of this polarization a prominent suspensor developed that persisted until the torpedo stage of the androgenic embryo. This was the first time the formation of a well developed suspensor was described in connection with androgenesis. Correspondence and reprints: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07 Nitra, Slovak Republic.     

Direct gene delivery into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants

A procedure for direct gene transfer into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants is presented. By modifying the microspore culture method and adopting the firefly luciferase (Luc) gene as a non-destructive marker, we could obtain stably transformed androgenetic embryos from bombarded microspores. Luc-positive embryos were easily isolated from the large non-transformed population using a high-sensitivity bioluminescent image analyzer. PCR and Southern blot analyses confirmed that the introduced transgene was integrated stably into the genome of the selected embryos. Diploidized plants obtained from the haploid embryos were self-pollinated, and all of the offspring tested were Luc-positive, indicating rapid fixation of the transgene which is characteristic of doubled haploids. Received: 14 May 1997 / Revision received: 15 July 1997 / Accepted: 28 July 1997     

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.     

Influence of Fe concentration in the medium on multicellular pollen grains and haploid plants induced by mannitol pretreatment in barley (Hordeum vulgare L.)

Summary. This study aims to clarify the short- and long-term effects of the iron concentration in the medium on androgenesis induced in barley by isolated microspore culture. The ultrastructural features and pectin composition of the intine wall were studied in the initial stages of androgenesis. The evolution of electron-dense iron deposits on the intine was analysed in multicellular pollen grains obtained by isolated microspore culture performed for 3, 6, and 9 days using various concentrations of FeNa₂ EDTA. Finally, the number of embryo-like structures and green plants obtained by microspore culture using different Fe concentrations was evaluated in order to estimate the optimum concentration for isolated microspore culture. Correspondence and reprints: Departamento de Bioquimica, Biologia Celular y Molecular de Plantas, Estación Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, Profesor Albareda 1, 18008 Granada, Spain.     

The inheritance of genetic markers in microspore-derived plants of barley Hordeum vulgare L.

Summary Biochemical, molecular and morphological markers have been used to monitor the segregation of alleles at major gene loci in microspore-derived lines of four spring barley crosses and their parents. Significant deviations from the expected Mendelian ratios were observed for four of the ten markers studied in the cross. Distorted ratios were associated with loci located on chromosomes 4H and 6H. The differential transmission of alleles was in favour of the responsive parent (Blenheim) used in the anther culture studies. For the -Amy-1 locus on chromosome 6H, the preferential transmission of Blenheim alleles was most pronounced in the haploid regenerants that were colchicine treated. These results are discussed in relation to the genetic control of androgenetic response in barley and with respect to the exploitation of another culture in barley improvement.     

Androgenic response of barley accessions and F1s with<Emphasis Type="Italic"> Fusarium</Emphasis> head blight resistance

Most Fusarium head blight (FHB) resistant barley (Hordeum vulgare L.) accessions perform relatively poorly from an agronomic point of view. Due to the polygenic inheritance of FHB resistance, introgression of this complex trait into well-adapted elite germplasm will likely require multiple cycles of hybridization and selection to combine resistance and agronomic performance. The use of anther culture to produce doubled haploids would seem well justified to reduce the time required to achieve this goal. Unfortunately, little is known concerning the androgenic response of the small number of genotypes with known partial FHB resistance. To make the best use of such FHB resistance donors in a barley improvement program, we first characterized the FHB resistance of eight reported FHB resistance sources (Chevron, Gobernadora, Seijo II, Shyri, Svanhals, Zhedar I, F104-250-9 and C97-21-38-3) in our own FHB nursery in Quebec City (QC, Canada). In parallel, we assessed the androgenic response of these same eight lines with that of three cultivars (ACCA, Léger and Cadette) of known androgenic response. Finally, the androgenic response of F₁ hybrids involving some of these genotypes used as parents was measured and compared to that of the parental genotypes. Very large and significant differences were observed in the number of green plants produced by the different accessions and F₁s. Although anther culture seemed very promising for some accessions, for others, the androgenic response was so low that a conventional approach would seem more appropriate.     

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)     

Desiccated doubled-haploid embryos obtained from microspore culture of barley cv. Igri

 Barley microspore-derived doubled-haploid embryos have been produced in vitro. The development of embryo desiccation technology will allow long-term storage, germplasm preservation and low delivery cost. Treatment of the microspore-derived embryos was essential to induce desiccation tolerance and to arrest further development and plant regeneration. At the concentrations used, a treatment with trehalose was more efficient than with sucrose, and mannitol was harmful to the embryos. Up to 80% of the desiccated embryos produced complete green plants when transferred to regeneration medium, by the application of a 0.6 m trehalose or a 10^–5 m abscisic acid treatment to the embryos in the culture induction medium. The morphology of these plants was similar to plants produced directly from non-desiccated embryos. Received: 28 September 1998 / Revision received: 27 November 1998 / Accepted: 5 January 1999