Cytochemical studies of callus development from microspore in cultured anther of rice

Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day intervals from 0 to 40 days have been carried out. Glutaradehyde-OsO₄-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, lipids and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous lipid in the first few days of culture, are androgenic. Less than 30%, which have many lipid granules and some amorphous lipid, become nutritive microspores. Starch grains also accumulate in these nutritive microspores which degenerate at the stage when the androgenic multicellular microspores are in rapid development. The remaining microspores, which have no or little lipid, degenerate early. At about the 100-cell stage, each multicellular unit consists of two cell types, large and small. The large cells contain abundant amorphous lipid and starch grains which the small ones stain intensely with TBO.Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport lipid and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and leaf-like protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus.Abbreviations OsO₄ osmium tetroxide - TBO toluidine blue O - SBB sudan black B - PAS periodic acid-Schiff's reagent - NAA 1-naphthaleneacetic acid     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

Haploid plants regenerated via anther culture in wild barley (Hordeum spontaneum C. Kock)

Summary Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l^–1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.Abbreviations IAA indolacetic acid - BAP 6-benzylaminopurine     

Pollen dimorphism and anther culture in barley

Summary A dimorphism is observed in barley (Hordeum vulgare L., cv. Akka) pollen when stained with acetocarmine from the mid-binucleate stage onwards. The majority of grains have staining cytoplasms, while the remainder have cytoplasms which take up little or no stain (NS grains). The staining dimorphism cannot be detected at the late-uninucleate microspore stage when anthers are normally cultured, but the evidence suggests that the microspores have already diverged at this time and it is the cells destined to become NS grains in vivo that respond in culture to become pollen calluses. Evidence comes from a comparison of the frequencies of NS grains and pollen calluses and from their distribution between and within anthers.     

Genetic transformation of barley microspores using anther bombardment

Bombardment of intact anthers of commercial barley (Hordeum vulgare) varieties resulted in 0.5–1.0% of transformed microspores of which 20–40% continued in androgenic development (0.2% of all bombarded microspores). Using a system based on bombardment of anthers is therefore likely to be more technically efficient than the use of a microspore isolation, transformation and regeneration system. Bombardment of anthers has a number of technical and scientific advantages over existing systems for gene transfer and can be considered as a alternative method to existing methods for genetic transformation in barley.     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.     

Lycopersicon esculentum: Trifoliate plants recovered from anther cultures of heterozygous tftf plants

The effects of the genotype and growth medium composition on callus induction and shoot regeneration from tomato (Lycopersicon esculentum Mill) anthers were studied. Five male sterile varieties, homozygous for the recessive gene ms 10³⁵, their isogenic fertile counterparts, and nineteen sterile mutants from an F₂ population segregating for ms 10³⁵, were tested. Callus induction and shoot formation were found to be affected by the genotype. The presence of the mutant gene ms 10³⁵ greatly increased callus induction. A significant interaction concerning callus induction was found between the ms 10³⁵ gene and the general genetic background. In most of the plants shoot regeneration from the anthers was associated with various degrees of callus production. However, there was no correlation between callus production and the ability to regenerate plants from that callus. Anthers isolated from plants which were heterozygous for the recessive leaf marker trifoliate, regenerated diploid plants with trifoliate leaves. The plants retained the trifoliate phenotype for over six months in culture under non-aseptic condition. Since the trifoliate phenotype appears only in the homozygous recessive state, the evidence that these trifoliate plants are doubled haploids of sporogenic origin is discussed.     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre- treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3 ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

A cytological indicator allows rapid assessment of microspore maturity,leading to improved <Emphasis Type="Italic">in vitro</Emphasis> anther response in Indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The inadequate response of Indica rice (Oryza sativa L.) to anther culture hinders the utility of this technique for rice crop improvement. This study focused on intensifying the in vitro anther response of selected varieties from Sri Lankan germplasm by precisely targeting the most effective maturity stage of microspores for culture. Analysis of five varieties responsive to anther culture (“At 303”, “Bw 272-6B”, “Dahanala”, “Dikwee”, and “Herath Banda”), revealed that the microspore development stage at the time of culture was crucial for fine-tuning the callus induction response. The external guide used to determine microspore maturity which was the internode distance between the last two leaves of the panicle, could not be applied equally for all rice varieties. Anther response could be improved by, at least, four- to sixfold by precisely targeting anthers with late uninucleate microspores for culture. Close correlation was demonstrated between the stage of nuclear division in the microspores and the degree of starch accumulation. Therefore, the degree of starch deposition in the microspores can be used as an effective cytological marker, which can be applied more rapidly and reliably to assess microspore maturity, in place of the internode distance.     

Characterization of anther differentiation in cytoplasmic male sterile maize using a specific isozyme system (esterase)

Summary During anther development, characterized in maize plants with N cytoplasm, certain esterase isozymes in non-microspore cells decrease in amount with anther age and new isozymes appear in the developing microspores. In anthers from male sterile plants with cms T or cms C cytoplasm, neither of these changes in esterase patterns occurred. In anthers from plants with cms S cytoplasm, the decrease in the esterases of non-microsporogenous cells was observed but not the appearance of microspore esterases. In lines carrying cms S cytoplasm and nuclear restorer genes, esterase changes during anther development were as in normal fertile anthers. These results are discussed with respect to the phenomenon of cytoplasmic male sterility in the different maize genotypes.     

The development of haploid embryoids from anther cultures of Atropa belladonna L.

Summary Development of haploid embryoids from the microspores of Atropa belladonna occurs with relatively high frequency when anthers are excised from buds in which the petals are shorter than the sepals (at this stage microspores are predominantly uninucleate) and cultured on a medium containing iron as the ferric salt of ethylenediamine-di-O-hydroxyphenylacetic acid (FeEDDHA). Additions of combinations of kinetin, auxin and casamino-acids to the culture medium induce callusing in both haploid and diploid tissues, lead to the origin of embryoids from somatic tissues of the anther and should be avoided. Simple techniques for the maintenance of haploid clones are described.Stages in early embryogenesis in the pollen grains have been observed and these indicate that embryogenesis is most frequently initiated by an equal division in the uninucleate spore. The frequency of grains showing embryoid formation is very low and it is estimated that plantlets are formed from up to 50% of these grains.     

Microspore development in cultured maize anthers

The present study follows in vivo and in vitro microspore development utilizing an anther culture-responsive maize genotype (Pa91×FR16) and a DNA-specific fluorescent dye (mithramycin). Cultured anthers were sampled at various times and scored for abnormal microspore divisions, multicellular masses, and embryo-like structures. The frequency of abnormal microspore divisions reached a peak during the first 7 days in culture and then declined. The vegetative nucleus was mitotically active in culture with over 50% of the induced microspores exhibiting this type of division. Multicellular masses and embryo-like structures first appeared in the 14 and 25 day samples, respectively. Most of the microspores did not reach the multicellular stage and an even greater mortality occurred during the formation of embryo-like structures.     

Barley anther culture: The effect of position on pollen development in vivo and in vitro

Locule structure and organization were studied in vivo and in vitro to determine whether the disposition of pollen within barley anthers affected the response of pollen in culture. Following release from the meiotic tetrads, juvenile barley microspores become peripherally organized around the locule, with the single pollen pore oriented towards the tapetum. Scanning electron micrographs of transverse sections from freeze fractured anthers showed that some microspores failed to locate the tapetal surface and occupied a position in the centre of the locule where they continued to develop as small, abnormal pollen grains (dimorphic pollen). Previous evidence has suggested that in some species dimorphic pollen could be the source of embryonic pollen in vitro. Cultured anthers frequently dehisced to reveal a mass of dividing pollen grains, however those anthers that remained intact retained the original locule structure and could be freeze fractured permitting examination of the developing pollen in situ. This showed that pollen embryogenesis was not restricted to dimorphic pollen, and that any grain could become Embryogenic irrespective of position.     

Development of embryos and seedlings from pollen grains inLycium halimifolium Mill. in thein vitro culture

Anthers ofLycium halimifolium were grownin vitro at the following stages of development: tetrads, microspores and binucleate pollen grains. Pollen plantlets were obtained only from anthers inoculated at the stage of microspores. The growth of androgenic embryos was very slow. Mature embryos were formed in about 3% of the inoculated anthers. The largest number of embryos obtained from one anther was 4.