• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.51 no.spc1
• /
• pp.242-246
• /
• 2006

This study was showed into the pollen development with in vivo by bud size and genotype. Microspores of buds from 2.0 mm to 2.5 mm of all genotypes were composed of mainly tetrad cells and early uninucleate stage cells. Microspores derived from buds of 2.5-3.0 mm were exposed cells of early uninucleate, middle uninucleate, and late uninucleate. Microspores from buds of 3.0-3.5 mm contained mostly late uninucleate stage cells and showed some early binucleate stage cells. Microspores of buds with 3.5-4.0 mm in length were composed of mainly binucleate stage cells and decreased late uninucleate stage cells. Microspore with more than 4.0 mm were entered into binucleate stage cells of divided generative nucleus and vegetative nucleus. In 'Tamlayuchae', microspores derived from buds of 3.5-4.0 mm were observed cells of late uninucleate stage and early binucleate stage because of late microspore development. In MS-maintainer, the spring type, microspore derived from buds of 2.5-3.0 mm were observed tetrad stage cells.

• FLOWER RESEARCH JOURNAL
• /
• v.16 no.1
• /
• pp.12-16
• /
• 2008

This study was carried out to produce haploid plants to verify a systematic breeding program and genetic analysis. Effect of developmental stage of pollen grains and pre-treatment temperature on production of haploid plants was investigated. Microscopic investigation of the explants (Lilium Asiatic hybrid 'reamland' revealed that the length of flower bud at 23.0-24.9, 25.0-26.9, and 27.0-28.9 mm long coincided with tetrad, uninucleate, and binucleate, respectively. When the efficiency of the anther culture from microgametogenetic stages was tested, late uninucleate to early binucleate stage, having the length of 23.0 to 28.0 mm long flower bud, was the best. The frequencies of the callus induction and plant regeneration from the stage mentioned above were 17.8 and 6.7%, respectively. When calli were cultured on the MS medium containing picloram and zeatin at $25^{\circ}C$, shoots were obtained. Roots of regenerated plantlets were confirmed as haploid through an microscopic observation.

• Korean Journal of Plant Tissue Culture
• /
• v.21 no.5
• /
• pp.293-297
• /
• 1994

In order to induce immature pollen derived plants, anthers of Ranunculus japonicus Thunb. were cultured on Murashige and Skoog's medium supplemented with various combinations of auxins and cytokinins. The combinations of NAA and BA were more effective than those of 2,4-D and kinetin in the formation of calli and embryos. Up to 5t5% of the anthers cultured on medium containing 0.5 mg/L NAA and 1.0 mg/L BA gave rise to plantlets. The most suitable stage for anther culture in the induction of calli and/or embryos from immature pollens was at the uninucleate and early binucleate stage (3 days before anthesis). Immature pollens developed into embryos by repeated division of the vegetative nucleate after 60days of culture.

• Korean Journal of Plant Tissue Culture
• /
• v.26 no.3
• /
• pp.149-156
• /
• 1999

In order to investigate the effect of low temperature pretreatment on pollen dimorphism and embryo formation in anther culture of Platycodon grandiflorum, the anthers with microspore at the uninucleate stage were cultured on Murashige and Skoog medium supplemented with 0.5mg/L NAA and 1.0 mg/L BA. The low temperature pretreatment have clear effect on the frequencies of S pollen grains, symmetrical binucleate microspores (B type of S pollen), multinucleate and multicelled pollen grains. Especially, after low temperature pretreatment at 8$^{\circ}C$ for 5 days increased the frequency of S pollen grain (20.6%) in vivo. In addition, the highest frequency of callus induction (54.9%) and embryo formation (9.9%) were obtained from the anther pretreatment at 8$^{\circ}C$ or 5 days. Three distinct pathways could be recognized in the androgenesis, one involving mainly the vegetative cell, the second starting with the vegetative and the generative cell, respectively, and the third accompaning with two equal vegetative type cells in the pollen grains.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.47 no.1
• /
• pp.62-67
• /
• 2002

An efficient system of rice microspore culture could contribute to the production of genetically modified rice. The microspores were isolated by mechanical or shed methods. The number of microspores per 100 anthers isolated at uninucleate stage was higher than (or similar to) those at binucleate stage in isolation method with pestle or spatular, but microspore divisions were not easily observed on both stages. On the other hand, pollen division in shed pollen culture was observed more frequently at uninuclear than at binuclear stage. Cold pretreatment at 1$0^{\circ}C$ for 10 days resulted in the best multicellular division to produce microcalli at 12.5% efficiency in shed microspores. Heat shock at 33$^{\circ}C$ for one hour before or after pollen shedding enhanced cell division and callus formation. Out of twelve green regenerants, two were haploids and ten were diploids based on the chromosome analysis of root tips. The size of stoma was 12$^{m}$ m in haploids and 15 ${\mu}{\textrm}{m}$ in diploids determined by scanning electron microscope (SEM).

• Journal of Plant Biotechnology
• /
• v.2 no.3
• /
• pp.129-134
• /
• 2000

Clear visualization of pepper (Capsicum annuum L.) microspore nuclei with common stains such as acetocarmine or propionocarmine is difficult, hindering cytological analysis. The DAPI stain after the addition of ferric chloride solution to fixative resulted in clear visualization of nuclei. For clear visualization of nuclei and slight fluorescence of microspore wall, addition of 40-60 ${mu}ell$ of ferric chloride solution to the 1 $m\ell$ fixative was identified as most effective. At all stages of gametophytic development, the nuclei can be distinctly visualized. Starch granules does not intefere with the fluorochrome, and so the vegetative and generative nuclei were cleary visible in binucleate pollens. With its rapidity and reliability, this technique represents an efficient tool for routine staging or investigation of the nuclear status of the microspore during culture.

• Korean Journal of Plant Tissue Culture
• /
• v.26 no.1
• /
• pp.1-6
• /
• 1999

In order to obtain plantlet derived by anthers, the anthers of Lilium asiatic hybrid 'Gran Paradiso' were cultured on Murashige and Skoog's medium supplemented with various combinations of auxin and cytokinin. The most suitable pollen stage of anther culture for the callus induction was 3 days before anthesis at the early to late binucleate stage. Organogenic calli were induced on MS medium supplemented with 5.0 mg/L 2,4-D alone and the combination of 1.0 mg/L 2,4-D and 1.0 mg/L kinetin, however, the combination of NAA and BA was more effective than that of 2,4-D and kinetin on plant regeneration through organogenesis. Shoots were formed from the induced callus on the medium with 0.5 mg/L NAA and 1.0 mg/L BA after 180 days of culture. Multiple shoots with 3-4 leaves, roots, and bulblets were formed on the medium with the combination of 2.0 mg/L NAA and 2.0 mg/L BA after 250 days of culture. The chromosome from root tip of the regenerated plantlet showed the diploid (2n=2x=24). Diploid plants were transferred to the pots and all plants were flowered in two years.

• Proceedings of the Botanical Society of Korea Conference
• /
• 1996.07a
• /
• pp.48-60
• /
• 1996

We previously identified and characterized a predominantly pollen-expressed gene of Petunia inflata that encodes a receptor-like kinase named PRK1. The extracellular domain of PRK1 contains leucine-rich repeats which have been implicated in protein-protein interactions, and the cytoplasmic domain was found to autophosphorylate on serine and tyrosine. To investigate the function PRK1 in pollen development, we transformed P. inflata plants with a construct containing the promoter of a predominantly pollen-expressed gene of tomato, LAT52, fused to an antisense PRK1 cDNA corresponding to part of the extracellular domain of PRK1, There transgenic plants were found to each produce approximately equal amounts of normal and aborted pollen. Analysis of the inheritance of the transgene inserts in two of the transgenic plants, ASRK-13 and ASRK-20, to their progeny revealed that certain transgene inserts cosegregated with the pollen abortion phenotype. Microscopic examination of the aborted pollen grains showed that their outer wall, the exine, was essentially normal, but that their cytoplasm contained only starch-like granules. Staining of the nuclei of the microspores at different stages of uninucleate stage. However, at subsequent stages half of the microspores completed mitosis and developed into normal binucleate pollen, but the other half initially remained uninucleate, then lost their nucleio. Analysis of the amounts of PRK1 mRNA and the antisense PRK1 transcript suggested that the pollen abortion phenotype most likely resulted from down-regulation of the PRK1 gene by the antisense PRK1 transgene. These results suggest that PRK1 plays an essential role in a signal transduction pathway that mediates post-meiotic development of microspores.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.57 no.4
• /
• pp.317-323
• /
• 2012

For the establishment of an efficient embryogenesis from microspore culture in Brassica napus L. domestic cultivar 'Tammiyuchae', four different factors affecting microspore embryogenesis and plantlet regeneration were investigated. The highest embryogenesis rate was achieved when microspores at late uninucleate to early binucleate stage were isolated from flower buds with a length of 3.0~3.5 mm. On average, 388 embryos generated from 1 ml of microspores media. The highest number of embryos was obtained when microspores were subjected to $32.5^{\circ}C$ for 2 days. Embryogenesis of 'Tammiyuchae' was increased with increasing microspore culture density up to about $5{\times}10^4ea/mL$. Gradually higher culture density repressed embryogenesis of microspores. Regeneration rate of shoots from microspore-derived embryos was observed in MS solid medium supplemented with $0.5mg{\cdot}L^{-1}$ NAA and $1.0mg{\cdot}L^{-1}$ BA, and grew well in MS solid medium without plant growth regulators.

• Korean Journal of Plant Tissue Culture
• /
• v.28 no.5
• /
• pp.263-271
• /
• 2001

Inoculated anthers of Capsicum annuum L. were subjected to 4 and 32$^{\circ}C$ pretreatment and their influence on the microspore viability, early cytological changes and the induction frequency of microspore embryo was investigated. Viability of freshly isolated microspores was between 62 and 64%. During temperature pretreatment, microspore viability showed a rapid decrease and this tendency enhanced with the 32$^{\circ}C$ pretreatment. Irrespective of temperature pretreatment, microspore viability declined to nearly zero after nine days. Before temperature pretreatment, most of the microspores in anthers were at late uninucleate stage. Several types of multinuclear microspores appeared from the 2 day after culture onwards, together with many degenerated and non-induced microspores. The 32$^{\circ}C$ pretreatment gave higher proportions of embryogenic microspore than other treatment. However, the temperature pretreatment had no clear effect on the frequencies of symmetrical binucleate rnicrospore. The multinucleate grains might originate either by symmetrical or asymmetrical division. After 2 days of pretreatment at 25 and 32$^{\circ}C$ , degenerated microspore increased above 50%. In contrast, during 4$^{\circ}C$ treatment, nucleus of most microspores remained intact for 14 days. The 32$^{\circ}C$ pretreatment produced more embryos than 4$^{\circ}C$ treatment. The most effective period of 32$^{\circ}C$ pretreatment was 4 days. In contrast, effective period of 4$^{\circ}C$ pretreatment was 2 days and longer time had deleterious effect on induction of microspore embryo.