• Journal of Plant Biology
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• v.36 no.2
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• pp.183-192
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• 1993

Efficient plant regeneration system was established through the culture of rice (Oryza sativa L.) microspores. Microspores released by anther shedding culture developed into proembryos and calluses in B5 medium within two weeks of culture. The optimal hormone and carbon sources were dependent on the genotypes used. Microspore's viability decreased rapidly in culture time, therefore less than 3% of the total microspores were viable at the 9th day when the first microspore division was observed. Of two types of microspores (pollen dimorphism) observed in culture, only the P-grain, larger microspores than normal one was able to divide. Using 4',6-diamidino-2-phenylindole (DAPI) fluorescence staining, it was confirmed that the symmetrical division of uninucleate microspore was the first step leading to continuous microspore development. Microspore-derived proembryos and calluses were regenerated to plants in N6 medium containing 1 mg/L NAA and 5 mg/L kinetin, and 78.3% of the regenerated plants were haploids.

• Korean Journal of Agricultural Science
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• v.38 no.4
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• pp.613-618
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• 2011

To develop clubroot resistant Chinese cabbage inbreds, IT 033820, a clubroot resistant turnip, was cross pollinated with a Chinese cabbage inbred of BP 079. From 2005, conventional breeding and microspore culture method performed using these F1 plants as parental materials. In 2007, conventional breeding method resulted in 21 F3 inbreds. After inoculation of clubroot race 4, one inbred showing 83% resistant was selected and registered as 'onkyo 20036ho' in 2008. From 2005, we scanned hybrid cultivars using micro spore culture and developed many doubled haploid (DH) lines in Chinese cabbage. Using Chinese cultivar of 'oong-baek 2ho' we developed 26 DH inbreds in 2007. After inoculation of clubroot race 4, one DH inbred showing 77% resistant and yellow inner leaf color was selected and registered as 'onkyo 20034'in 2008. We found conventional breeding method was effective using introduced germplasm showing low germination. However, when using hybrid cultivar as starting material, microspore culture method was powerful for developing various inbred in short time.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.363-373
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• 2007

To establish an efficient and reliable microspore culture system for pepper (Capsicum annuum L.), the effect of light intensity used for donor plant's growth, microspore isolation methods, the composition of culture medium, and culture period in light on the production of embryos were investigated. The viability of microspores taken from the plants grown under the light intensity of 10,000 lux was almost same as that from the lower (5,500 lux) light intensity, and the embryo induction and development were a bit higher when donor plants were grown under the lower light intensity. This result implies that lower light intensity does not interfere with the embryo induction and development. However, it was very difficult to prepare microspores for culture since only a small number of flower buds could be harvested from plants grown under the light intensity of 5,500 lux. Microspore isolation methods greatly affected microspores viability; that is, when microspores were isolated by blending rather than maceration, the greater number of viable microspores were easily generated (about 13 times). Among media used for microspores culture in this study, MN medium was most efficient for embryo induction and development. Total number of embryos and the number of cotyledonary embryos were highest when microspores were cultured in dark for 4 weeks, and then in light for one week. These results will be provide valuable information to set up efficient microspore culture system of hot pepper with a high frequency of embryo production, which are applicable to gene transformation and mutagenesis.

• Journal of Plant Biotechnology
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• v.36 no.2
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• pp.184-192
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• 2009

The influences of pretreatment medium, the addition of fresh medium, and the size of culture plate on the production of embryos were investigated in isolated microspore culture of hot pepper (Capsicum annuum L.). Among the media used for heat shock pretreatment ($32{\pm}1{^{\circ}C}$), high frequency embryo production was obtained when the sucrosestarvation medium A was used. On the other hand, neither 0.37 M mannitol solution nor NLNS medium supplemented with sucrose was not efficient for embryo production. The addition of culture medium to pretreatment media considerably decreased the embryo production even though embryo development proceeded further. The embryo production was not improved by the addition of fresh medium after 2 or 3 weeks from starting culture. Increase in the size of the culture plate from $3.5{\times}1.0$ cm to $6.0{\times}1.5$ cm improved embryo quality. These results will provide valuable information for developing an efficient microspore culture system of hot pepper for high frequency embryo production.

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

• Horticultural Science & Technology
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• v.32 no.3
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• pp.382-389
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• 2014

Raphanus sativus L. cv. Taebaek, a efficiently microspore-derived embryo (MDE)-forming cultivar, and 'Chungwoon', a non-MDE-forming cultivar were selected as donor plants for isolated microspore culture. Radish flower bud of 2.0 (small, S), 4.0 (medium, M), and 6.0 (large, L) ${\pm}$ 0.5 mm in length were isolated to determine the temporal relationship between flower bud size and MED yield. Anatomical observations revealed no difference in the structure of the flower buds between the two cultivars. In both cultivars, the stigmas were much longer than the floral leaf in M-sized flower buds. The MDE yields for 'Taebaek' per petri dish were 6.6 and 1.3 for M- and L-sized of flower buds, respectively, but MDE formation was not induced in the S flower buds. On the other hand, 'Chungwoon' failed to form MDEs in all flower buds. The microspore density of 'Taebaek' was 1.3 times more than that of 'Chungwoon' for M sized flower buds. Of the M-sized buds from 'Taebaek' and 'Chungwoon', 92.1 and 81.6%, respectively, were in the late uninucleate microspore stage, which is characterized by the highest frequency of MDE formation. Anatomical observations of MDE formation revealed that the microspores were able to divide to form a primordium from which cell division took place continuously in the 'Teabeak' cultivar. However, the microspores of 'Chungwoon' failed to progress beyond the primodium stage, resulting in lack of MDE formation. By contrast, after the formation of the primordium, various developmental stages of embyos from microspore were observed in the 'Taebaek' cultivar. These results can be used to determine MDE forming potentials of radish cultivars.

• Korean Journal of Plant Tissue Culture
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• v.26 no.2
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• pp.71-76
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• 1999

Anthers of two hot pepper cultivars, Milyang-jare and Geryongsan-jare, were cultured on MS medium containing 0.1 mg/L 2,4-D and 0.1 mg/L kinetin. The influence of pretreatment at 4$^{\circ}C$ and 32$^{\circ}C$ on induction of microspore embryo was investigated. Milyang-jare was superior to the Geryongsan-jare in microspore embryo induction. The 32$^{\circ}C$ pretreatment increased embryo induction compared to the 4$^{\circ}C$ pretreatment while the 4$^{\circ}C$ pretreatment stimulated callus induction. Microspore embryos were regenerated to plantlets in the same medium or hormone free medium at 32$^{\circ}C$ treatment but most embryos failed to develop directly into plantlets at 4$^{\circ}C$ treatment. The optimal period of the 32$^{\circ}C$ pretreatment was 3 days in Milyang-jare and 6 days in Geryongsan-jare. The 32$^{\circ}C$ pretreatment was essential for induction and growth of microspore embryo in pepper.

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

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

• Proceedings of the Korean Society of Crop Science Conference
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• 1990.05a
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• pp.110-111
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• 1990