Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Changes in the fatty acid unsaturation after hardening in wheat chromosome substitution lines with different cold tolerance

Two wheat (Triticum aestivum L.) varieties, Cheyenne (Ch, winter wheat with excellent frost tolerance) and Chinese Spring (CS, spring wheat with weak frost tolerance), and chromosome substitution lines (CS/Ch 5A, CS/Ch 5D, CS/Ch 7A) created from Cheyenne and Chinese Spring were used to study the effect of chromosome substitutions on the membrane lipid composition in the leaves and crowns before and after cold hardening. The percentage of fatty acid unsaturation in phosphatidylethanolamine was greater in the crown of hardened Cheyenne than in Chinese Spring. The value of CS/Ch 5A was similar to Cheyenne and that of CS/Ch 5D to Chinese Spring, while the value of CS/Ch 7A was in between those of Cheyenne and Chinese Spring. A smaller difference was found between the unsaturation level in the phosphatidylcholine from Cheyenne and Chinese Spring after hardening, while the value obtained for the substitution line CS/Ch 7A was similar to Cheyenne. The percentage decrease in thetrans-Δ³-hexadecenoic acid content was found to be correlated with the frost tolerance of the wheat genotypes.     

Pollination in vitro: effects on the growth of pollen tubes,seed set and gametophytic self-incompatibility in Trifolium pratense L. and T. repens L.

Summary Growth of pollen tubes and seed set were compared after hand pollination in situ and in vitro in two self-incompatible species, Trifolium pratense and Trifolium repens. Adhesion of pollen grains to the stigma was greater in vitro for both species. After cross-pollination, in vitro culture gave a significant increase in the cumulative growth of pollen tubes in pistils of T. pratense compared to in situ conditions. After selfing in T. repens, pollen tube growth was significantly increased by in vitro culture of florets. Seed set after crossing in situ and in vitro was similar for both species. Seed set after selfing in vitro was not increased in T. pratense. Several genotypes of T. repens were classified as very good, good and poor selfers based on their capacity for seed set following selfing in situ. In vitro pollination increased self seed formation by 1.7-, 18.0- and 31.0-fold for each class, respectively. Ovules located nearest to the style were fertilized more often after selfing than after crossing.     

Developmental expression of ASG- 1 during gametogenesis in apomictic guinea grass (Panicum maximum)

We have used Western blue-visualized in situ-hybridization (ISH) to monitor the expression of apomixis-specific gene-1 (ASG-1, GenBank accession number AB000809) during gametogenesis in obligate-sexual and facultative-apomictic (aposporic) genotypes of guinea grass (Panicum maximum). The in situ-analysis revealed that ASG-1 is not expressed in the ovule during early floral development in both, the facultative apomicts (A1 stage) and the obligate sexuals (S1 stage). With the appearance of the aposporous initial cell(s) in the ovule of the apomictic type (A2-1 stage), ASG-1 expression is strong and specific to this apomixis-specific cell. ASG-1 expression continued through different stages of aposporous embryo sac development (A2-2 stage), indicating that the gene may play a role in this developmental process. Regular embryo sacs in sexual types did not show hybridization signals (S2 stage). However, strong ASG-1 expression was detected in immature pollen grains and young embryos in both reproductive types, suggesting that ASG-1 may be an allele derived from the obligate-sexual wild type. Expression in pollen grains faded with maturation. In a heterologous system, using Paspalum notatum, a facultative-aposporic tropical grass (bahia grass), identical results were obtained. The results are discussed in view of the fact, that ASG-1 shows some homology to genes known to be seed- or embryo-specific or involved in processes related to cell growth.     

Pollen morphology and in vitro germination characteristics of nodulating and nonnodulating soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) genotypes

Artificial hybridization in highly self-pollinated crop species such as soybean (Glycine max L.) is important for both generating genetic variability and segregation for selection. In higher plants, pollen is an agent for transmission of genetic information over generations. The objective of this study was to measure and compare both morphological (length, width) and in vitro germination (germination percentage tube length) characteristics of pollen from the nodulating soybean cultivar, Bragg, and a nonnodulating Bragg mutant line, Nod 139, obtained following ethyl methanesulphonate treatment. Highly significant (P = 0.007) differences in pollen length were observed between these two genotypes. Similarly, in vitro germination percent (G%) indicated highly significant (P = 0.0001) differences between these genotypes, suggesting that the nodulation trait produces variation in in vitro germination capacity of the pollen. It appears the nonnodulation trait in soybean alters pollen grain length and G%.     

Mechanism of durable resistance: a new approach

Summary Wheat genotypes, including backcross derivatives of Thatcher carrying Lr10 and Lr23, substitution lines for Lr10 and Lr23 in Chinese Spring background and Chinese Spring and Thatcher were analysed against 21 pathotypes of leaf rust in seedling tests. Adult plant responses in all these stocks were observed in the field nurseries under exposure to the inoculum of the Indian virulent races of leaf rust. The seedling data demonstrated that both the substitution lines and the backcross derivatives for each gene carry identical pattern of infection for resistance. The high level of adult plant resistance in the substitution lines, in contrast to the backcross derivatives in Thatcher, has been postulated to be due to the combination of resistance contributed by Lr10 and adult plant Chinese Spring resistance or to Lr23 and Chinese Spring adult plant resistance. It has been suggested that genes Lr10 and Lr23 added to the Chinese Spring background provide sources for durable resistance, since Chinese Spring has continued to provide a moderate level of adult plant resistance to leaf rust for a very long time.     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Pollen performance as affected by the pistilar genotype in sweet cherry (Prunus avium L.)

Summary Differences in pollen performance in higher plants can result in significant selective advantages for some particular genotypes leading to both gametophytic and sexual selection. However, the possibility of selection among male gametophytes has been questioned since natural selection could lead to the fixation of alleles for the best competing male genotypes. These two apparently conflicting hypotheses could be reconciled if pollen performance, rather than operating in absolute terms, could be modulated by the pistilar genotype. Thus, pollen performance in vivo and in vitro has been compared in four sweet cherry (Primus avium L.) cultivars. Differences among the cultivars studied have been recorded in the speed and final pollen germination percentages both in vivo and in vitro. The results obtained show that the female genotype also modulates the final result of pollen performance. These two factors are not merely additive but, on the contrary, the interaction between them affects pollen behavior in vivo. This fact has clear implications for gametophytic and sexual selection since the best male-female combinations can be favored and this could explain the variability observed for pollen performance in nature.     

The effects of pollen diversity on plant reproduction: insights from apple

For many plants, the number of pollen genotypes deposited on a flower’s stigma is positively related to fruit maturation and seed number; however, the mechanisms underlying this effect are less understood. Here we examined whether diversity of pollen (1, 3, or 5 donors) affects reproductive success in self-incompatible apple (Malus × domestica). Using paternity analysis, we then assessed the siring rate of individual donors to test whether diversity effects are related to the presence of a superior pollen donor or to donor × donor interactions. Increasing the diversity of compatible pollen enhanced seed number and reduced seed abortion in some but not all recipient genotypes. This effect was associated with an increased number of sires per fruit and non-random siring success among pollen donors. Two donors had consistently high siring rates, regardless of recipient genotype; however, the siring success of pollen donors was not correlated with their siring success in single-donor pollinations. Rather, siring success was affected by the identity of other pollen genotypes present on the stigma. Our results therefore suggest that the effects of pollen diversity are variable but may enhance fecundity by fostering interactions between pollen donors.     

In vitro maturation and germination of <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> microspores

Microspores cultured in vitro can be regarded as a system to study gene regulation, cell fate determination and cell differentiation during pollen development as well as an alternative method of genetic transformation in plants. In our study, pollen development and viability in Orychophragmus violaceus in vivo were determined and then pollen from the late unicellular stage was cultured in vitro. MS liquid medium + White vitamins + 2% (V/V) coconut milk + 0.5 M maltose, pH = 7.0 was the most appropriate for in vitro culture of Orychophragmus violaceus microspores. With this medium, the rates of in maturation and germination were 19.3% and 4.7%, respectively. Liquid medium with 0.6 M maltose + 1.6 mM boric acid + 2.9 mM Ca(NO₃)₂ + 29.6 μM vitamin B1, pH = 7.0 was optimal for germination of pollen matured in vivo. The rate of germination was 70.7%. Pollen matured in vitro cultured in similar medium exhibited a rate of germination of 62.7%. Hence, the experimental study showed that in vitro maturation of microspores is feasible and this experimental system can be applied to further theoretical and practical research.     

In-vitro culture of tobacco pollen: gene expression and protein synthesis

Summary Immature pollen grains of Nicotiana tabacum L., cv Petit Havana were isolated at the mid-binucleate stage and cultured in vitro. During the first 66 h of in-vitro culture the pollen developed the same ability to set seed and germinate as pollen matured in vivo. No fertile pollen was produced when protein synthesis was inhibited temporarily at an early stage of development. In the case of inhibition at day 2 of development a delay in the total time necessary for maturation was observed that was equal to the length of time the inhibitor was applied. Hybridization experiments with a pollen-specific cDNA probe showed that the pattern of gene expression in vitro was similar to that in vivo. However, the model system differs from natural pollen development with respect to the dehydration period, which is absent in the model system, and the synthesis of proteins. Protein synthesis of in-vitro cultured pollen differed significantly from that of pollen developing in vivo, even though pollen maturation in vitro proceeded in the same time as in vivo, and led to fully matured fertile pollen. Pollen development in vitro is thus an ideal model system for studying gene expression in relation to fertility and for experimental manipulation of microsporogenesis.     

Ultrastructural changes in maturing pollen grains ofApium nodiflorum L. (Apiaceae), with special reference to the endoplasmic reticulum

Summary The ultrastructural events in 3-cellular pollen grains ofApium nodiflorum L. are investigated during pollen maturation. Three distinct developmental stages are distinguished from the formation of sperm cells up to anthesis, whereby the rough endoplasmic reticulum (RER) is mainly involved. The most conspicious form is the highly dilated RER in the vegetative cytoplasm of the youngest pollen grains, which changes to vesicular RER in the following stage. In mature pollen grains the RER has a narrow cisternal configuration and often forms stacks. Pollen activation is preceded by the accumulation of polysaccharide particles.     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

Effect of in vitro and in vivo colchicine treatments on pollen production and fruit set of melon plants obtained by pollination with irradiated pollen

Haploid/doubled haploid (DH) technology can aid plant breeding programs by accelerating production of homozygous lines, provided enough viable DH progeny can be obtained from diverse haploid genotypes. In cases where there is a low frequency of spontaneous doubling, chromosome doubling procedures are required to achieve fecundity. We produced 63 parthenogenetic melon plantlets via pollination with γ-irradiated pollen, cloned them by nodal cuttings, and tested the effects of in vitro and in vivo colchicine treatment on survival, ploidy, pollen production, and fruit recovery. The most effective procedure was in vitro exposure of 3 cm shoot tip explants to 500 mg/l colchicine for 3 h. This treatment gave 83% survival of explants and 26% conversion to diploidy. Fruit recovery rate was 60% among plants with good pollen production. In vivo exposure of the tops of young plants to 5000 mg/l for 2 and 4 h yielded some fruits but also resulted in less survival and more morphological abnormalities. Strategies for recovery of progeny from parthenogenetic melon plants are recommended. To our knowledge, this study represents the first comprehensive study of recovery of fruits and viable seeds from parthenogenetic melon plants.     

In vitro pollen germination and transient transformation of<Emphasis Type="Italic">Zea mays</Emphasis> and other plant species

To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such asPennisetum americanum andTradescantia species, and dicot species, such asArabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, andVicia faba. On average, reproducible germination rates ranging from 50–100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducibleLuc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin-and pollen-specificZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed. An erratum to this article is available at .     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.