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

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.

Inheritance of plant regeneration from maize (Zea mays L.) shoot meristem cultures derived from germinated seeds and the identification of associated RAPD and SSR markers.

The inheritance of shoot regeneration through shoot-tip meristem culture derived from maize seedling was evaluated, and the markers (RAPD and SSR) associated with this regeneration character were identified both in a group of North American maize inbreds and a crossing population. A discrete distribution of percent regeneration and no. of shoots per explant was observed in the inbred group and the F(2) population. The results suggested that this regenerable trait was controlled by several major genes. Five RAPD markers were identified to be relevant to percent regeneration in maize shoot-tip culture system. One RAPD marker and three SSR markers were associated with no. of shoot per explant and its relevant traits. Of them marker BC603-1600 explained 18% of the variation for no. of shoot per explant and 16% of the variation for callus size. The BC603-1600 was sequenced and assigned in linkage group 7 based on a NCBI blast search. The information provided here should benefit to determine the genetic mechanisms involved in the maize regeneration response related to shoot meristem culture pathway and benefit to select high regenerable germplasm by using marker assisted selection.

Chromosomal location and expression of green fluorescent protein (gfp) gene in microspore derived transgenic barley (Hordeum vulgare L.).

Four doubled haploid barley lines (A, C, D, E) derived from gfp (green fluorescent protein) transformation and selection following particle bombardment of microspores were studied for gene expression pattern and the location of genome inserts. The integration sites were detected by fluorescence in situ hybridization (FISH) using the gfp plasmid DNA as a probe. Plants from events A, C, D and E all have a single insert site on chromosome 7L(5HL) at different locations while line E has a second insert site on chromosome 5S(7HS). All original transgenic plants were hemizygous for the transgenes and segregated in the T1 and T2 generations. Although line D had no GFP expression, FISH and PCR could detect gfp gene on its chromosome in transformed plants. Expression levels of GFP varied with lines and tissues examined. Plants from line C showed good expression in pollen and an intermediate level in root tips. Plants from A have intermediate expression of GFP in the pollen and light expression in the root tips. Line E showed strong expression in the root-tips and an intermediate level of GFP in the pollen. Lines A and C segregated as a single Mendelian locus while E segregated in a duplicate loci ratio (15:1) on seedling root tips but had low expression frequency in the pollen. PCR results were consistent with GFP expression on root tips in the three segregating lines. The expression of GFP for lines D and E was abnormal and may be related to the physical location of the transgene or the gene construct used.

The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley ( Hordeum vulgare L) uninucleate microspores.

The objective of this study was to correlate the time that DNA synthesis first occurs in haploid microspores of barley with cell cycle and plant morphological stages and to subsequently assess the influence of pretreatments on DNA synthesis at different stages of microspore development. Spikes with microspores in early, mid, and late uninucleate stages of the two-rowed barley cultivars Manley and Igri were subjected to two commonly used pretreatments. First, during cold pretreatment for 28 days there was a slow increase in relative DNA values as well as asymmetric nuclear divisions in some microspores. Second, during a 4-day cold plus 0.3 M mannitol pretreatment, there was very little change in the microspore stage or DNA values indicating that for the duration of this pretreatment the progression of the cell cycle was essentially suspended at all stages, both in Igri and Manley. The results are discussed relative to the potential for genetic transformation of microspores.

Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores.

A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 degrees C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 degrees C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55-60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 degrees C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 degrees C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85-97% of these ELS regenerated green shoots. The frequency of doubled haploids (74-83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 degrees C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).

Characterization of cDNAs expressed in the early stages of microspore embryogenesis in barley (Hordeum vulgare) L.

To gain insight into the molecular events occurring in the very early stages of barley microspore embryogenesis, cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture were isolated and characterized. A cDNA library established from barley microspores cultured for three days was differentially screened against probes generated from freshly isolated microspores. Three cDNAs representing genes not previously identified in barley were isolated. ECA1 (early culture abundant 1) lacked significant homology to known genes or proteins, and the transcript was only expressed during the early stages of culture. Expression was also reduced in low-density control cultures, therefore this gene may play a role in the early stages of barley microspore embryogenesis. ECGST (early culture glutathione S-transferase) had homology to parA-like genes, which are members of a newly discovered group of glutathione S-transferases (GSTs). The protein corresponding to ECGST may be important in protecting cells from oxidative stress during the culture process. ECLTP (early culture lipid transfer protein) had homology to lipid transfer proteins (LTPs), and had an expression pattern similar to that of an LTP known to be a marker of the early stages of embryogenesis in the carrot somatic embryogenesis system. The identification and characterization of the clones isolated in this study provides new information on the events involved in barley microspore embryogenesis.

Biotechnology and world food supply.

Biotechnology, the use of molecular and cellular tools to genetically modify and improve food supply, will play an increasing and important role in the continuing struggle to produce sufficient food for an ever increasing world population. Many of the approaches will supplement and enhance conventional breeding and also address environmental concerns and help to stabilize food production. This review provides a perspective dealing specifically with crops and three areas of biotechnology, namely, genome analysis with molecular markers, cell- and tissue-culture procedures, and the rapid and precise incorporation of genes via transformation. One must remember that biotechnology is still in its infancy, and that approaches will be greatly improved and more efficiently utilized in the future for the betterment of mankind.

Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.).

Transgenic barley plants were produced by the direct delivery of plasmid DNA into isolated microspores of barley cv. Igri using high velocity microprojectiles. The plasmid pAHC25 contained the uidA and bar genes, each under the control of a maize Ubi1 promoter. Bombarded microspores were cultured and selected on solid medium containing varying concentrations (2-5 mg/L) of the Basta herbicide active agent bialaphos. The effectiveness of selection with bialaphos depended on its interaction with the medium component glutamine. Six transgenic plants (R0) were obtained, and the presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants were confirmed by PCR and Southern blot analysis. Phosphinothricin acetyltransferase activity was observed in all six R0 transgenic plants, whereas none showed β-glucuronidase (GUS) activity in histochemical GUS assays. Two of the six R0 plants were haploid and sterile; one of them was trisomic and partially sterile; the remainder were diploid, but one of them was also sterile. Inheritance of the transgenes in progeny of three seed-producing transgenic plants was investigated. Southern blot analysis of genomic DNA from R1 plants showed that the introduced bar and uidA genes were hemizygous and stably cotransmitted to the R1 progeny derived from self-pollination. Analysis of Basta resistance and the integration of the bar gene by PCR analysis in R1 plants indicated that the bar gene was being inherited and expressed as a single dominant trait. Fluorescent in situ hybridization was performed on chromosomes of the trisomic plant to confirm the presence of transgenes in the genome.

Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture.

Embryogenesis from isolated microspore cultures of wheat was improved by ovary co-culture when compared to a completely defined medium. This indicates that essential factor(s) in addition to PAA or its analogs may be supplied by the ovaries. Isolated microspores cocultured with 20 ovaries of wheat on the top of semi-solid MMS3 induction medium for 21-30 days gave the best response. Both the number and quality of the embryos was significantly increased. The maximum frequencies of dividing microspores and of embryogenesis were 94% and 2.4%, respectively. Up to 2583 embryos were formed per 100 anthers of cv 'Chris' and between 18% and 43% of the larger embryos regenerated into green plants upon transfer. Genotype differences for both induction and embryogenesis phases were reduced using ovary co-culture. However, there was still a strong genotype influence on plant regeneration with cv 'Chris', with the F1 of 'Chris' × 'Sinton' displaying the highest frequencies. These results are important with respect to enhancing haploidy applications in wheat biotechnology and plant breeding.

Potential off biolistic transformation of barley microspores based on viability and transient β-glucuronidase activity.

Microspores could be an excellent target for plant transformation, owing to their haploid nature, the availability of a large population of fairly synchronous single cells, and their potential to regenerate into plants through embryogenesis. Therefore, the potential for microspore transformation by biolistic procedures was examined cytologically, based on the viability and β-glucuronidase (GUS) activity of bombarded microspores. The microspores were bombarded with gold particles coated with the plasmid pAHC25. On average, 10.7% of the total number of microspores bombarded contained particles. Of these, 4.7, 1.2, and 4.7% received one, two, and three or more particles, respectively. Of the microspores receiving particles, ca. 7% had one or more particles in the nucleus. Viability of bombarded microspores was followed for 7 days in culture. Over this period, the frequency of viable microspores with particles was significantly reduced from 1.56% at day 1, to 0.72% at day 3, and finally to 0.05% at day 7, with this last group having only a single particle. While microspores that received multiple particles did not survive after 1 week in culture, initially they could be scored as positive for transient GUS activity. Microspores with particles delivered directly into the nucleus (vs. other cell compartments) showed enhanced uidA transient expression and these microspores were most likely the source of integration of the introduced DNA into the recipient genome. The potential for the recovery of transgenic barley plants following biolistic bombardment is discussed.

Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories.

Random amplified polymorphic DNA (RAPD) analysis appears to offer a cost- and time-effective alternative to restriction fragment-length polymorphism (RFLP) analysis. However, concerns about the ability to compare RAPD results from one laboratory to another have not been addressed effectively. DNA fragments that were amplified by five primers and shown to be reproducibly polymorphic between two oat cultivars (within the Ottawa laboratory) were tested in six other laboratories in North America. Four of the six participants amplified very few or no fragments using the Ottawa protocol. These same participants were able to generate a considerable number of amplified fragments by using their own protocols. The reproducibility of results among laboratories was affected by two factors. First, different laboratories amplified different size ranges of DNA fragments, and, consequently, small and large polymorphic fragments were not always reproduced. Second, although reproducible results were obtained with four of the primers, reproducible results were not obtained with the fifth primer, using the same reaction conditions. It is suggested that if the overall temperature profiles (especially the annealing temperature) inside the tubes are identical among the laboratories, then RAPD fragments are likely to be reproducible.

Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare).

In an attempt to transfer traits of agronomic importance from H. bulbosum into H. vulgare we carried out crosses between four diploid barley cultivars and a tetraploid H. bulbosum. Eleven viable triploid F1 plants were produced by means of embryo rescue techniques. Meiotic pairing between H. vulgare and H. bulbosum chromosomes was evidenced by the formation of trivalents at a mean frequency of 1.3 with a maximum of five per cell. The resulting triploid hybrids were backcrossed to diploid barley, and nine DC1 plants were obtained. Three of the BC1 plants exhibited H. bulbosum DNA or disease resistance. A species specific 611-bp DNA probe, pSc119.2, located in telomeres of the H. bulbosum genome, clearly detected five H. bulbosum DNA fragments of about 2.1, 2.4, 3.4, 4.0 and 4.8 kb in size present in one of the BC1 plants (BC1-5) in BamHI-digésted genomic Southern blots. Plant BC1-5 also contained a heterozygous chromosomal interchange involving chromosomes 3 and 4 as identified by N-banding. One of the two translocated chromosomes had the H. bulbosum sequence in the telomeric region as detected using in situ hybridization with pSc119.2. Two other BC1 plants (BC1-1 and BC1-2) were resistant to the powdery mildew isolates to which the barley cultivars were susceptible. Seventy-nine BC2 plants from plant BC1-2 segregated 32 mildew resistant to 47 susceptible, which fits a ratio of 1∶1, indicating that the transferred resistance was conditioned by a single dominant gene. Reciprocal crosses showed a tendency towards gametoselection that was relative to the resistance. Mildew resistant plant BC1-2 also had a 1-kb H. bulbosum DNA fragment identified with a ten-base random primer using polymerase chain reaction (PCR). Forty-three BC1 plants, randomly sampled from the 79 BC1 plants, also segregated 23∶20 for the presence versus absence of this 1-kb H. bulbosum DNA fragment, thereby fitting a 1∶1 ratio and indicating that the PCR product originated from a single locus. The 1-kb DNA fragment and disease resistance were independently inherited as detected by PCR analysis of bulked DNA from 17 resistant and 17 susceptible plants as well as by trait segregation in the 43 individual plants. The progenies produced could serve as an important resistant source in plant breeding. This is the first conclusive report of the stable transfer of disease resistance and DNA from H. bulbosum to H. vulgare.

Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA).

The effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar Veery'S', while the average response from twelve F1 hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.

Improved plant regeneration from shed microspore culture in barley (Hordeum vulgare L.) cv. igri.

This report describes rapid regeneration of green plants from microspores of the barley cultivar Igri. Use of 0.3 M mannitol during maceration and isolation was essential for response from mechanically isolated microspores of barley cv. Igri grown under our conditions. A shed microspore culture system proved to be simple and gave a fast response; plants were obtained as early as 25 days after the material was taken from the donor plant. A 28-day cold-pretreatment of spikes can also be replaced with a 3-4 day pretreatment of anthers in mannitol. Shed microspores from 100 anthers produced an average of 292 plants with 91% of them green. Approximately 80% of the regenerated plants were spontaneously doubled-haploids.

Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgare).

Ethylene production was measured in cultured barley (Hordeum vulgare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels.

Microspore growth and anther staging in barley anther culture.

Nuclear growth, microspore cell growth and cell cycle stage were examined in microspores of anthers of Hordeum vulgare L. cv. Klages taken from florets of the middle of the spike as per anther staging methods. Although there was wide variation in nuclear size at all stages of the cell cycle, mean nuclear size appeared to be a good indicator of cell cycle stage for microspores within anthers. Microspore cell size increased considerably during Gl of the cell cycle. Anthers bearing microspores cytologically characterized as in the mid-uninucleate stage, which have proven to yield high levels of callus production, were determined to be in G1 of the cell cycle and were regularly found in spikes taken from tillers in which the base of the flag leaf had emerged 0 to 3 cm above the penultimate leaf.

Development of haploid cell lines from immature barley,Hordeum vulgare, embryos.

Callus and suspension cell lines were derived from haploid barley embryos produced by the Bulbosum method. Embryos 1 to 2 mm long callused on medium containing a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast-growing nodular, beige callus (Type 1), slow-growing, light brown, watery callus (Type 2) and a dense, light yellow, nodular callus (Type 3) were recovered. Type 3 callus was embryogenic and was produced on embryos 1 to 2 mm in length. Although callus cultures gradually became polyploid, a small proportion of haploid cells was retained and the majority of regenerated plantlets were haploid. The organogenic potential of long-term (Type 1) callus cultures was generally low and decreased with time. Attempts to inducede novo shoot formation in Type 1 cultures were not successful.

Genetic studies of the crossability of hexaploid wheat with rye and Hordeum bulbosum.

Crosses of wheat (Triticum aestivum L.) Group 5 chromosome substitution lines from three different cultivar backgrounds indicate that there may be multiple alleles for reduced crossability with rye (Secale cereale L.) and H. bulbosum on both 5A and 5B chromosomes. No reduction in seed set was observed from any of the 5D substitution lines. It was found that the Kr1 locus on chromosome 5B had a more pronounced affect on both rye and H. bulbosum crossability than the Kr2 locus on chromosome 5A and that the effects of both loci were cumulative. The Kr alleles affected both rye and H. bulbosum crossability in a linear, or additive, fashion. The Kr 2 allele was shown to have little effect on H. bulbosum crossability compared to a significant effect on rye crossability. Tests with tetrasomic and nulli-tetrasomic lines of 'Chinese Spring' indicated that the kr allele is 'null' or inactive in promoting crossability while the Kr allele is active in reducing crossability to both rye and H. bulbosum. Thus, extra doses of the kr allele do not increase rye or H. bulbosum crossability in the presence of the corresponding Kr allele.