• Journal of Plant Biotechnology
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• v.44 no.1
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• pp.27-34
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• 2017

Our aim was to find the effective duration and culture conditions for microspore culture in a genetically variant cabbage (Brassica oleraceae L. var. capitata). We discovered that the '$2{\times}NLN$ medium containing $AgNO_3$ 1 mg/l, sucrose 13%' was most efficient in producing the cabbage embryos. The number of induced embryos was higher in $F_2$ or $F_3$ progeny generation than the $F_1$ hybrid cultivar used as plant materials. Thus, we recommend microspore culturing using progeny plants of failed $F_1$ cultivar. The acquisition ratio of embryos and plants derived from microspore was higher in the early flowering period as compared to the late flowering period. When transplanted in the soil, 71.2% plants developed from the early flowering period, compared to 27.0% from the middle period and 1.8% plants from the late period. Thus, we recommend microspore culture using buds collected during the early flowering period.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.30-41
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• 2011

The influences of the agitation as well as the addition of medium during culture on the production of embryos were invested in isolated microspore culture of hot pepper (Capsicum annuum L.). When the culture medium was added during initial liquid culture step of liquid-double layer culture, the embryo yield and quality greatly increased. The most effective time point for medium addition was 5 days after the culture commenced. On the other hand, the effect of medium addition at later double layer culture step in liquid-double layer culture on the embryo production was less compared to that of medium addition during the initial liquid culture step. Agitating the culture for 1 week during later double layer culture step in liquid-double layer culture effectively increased the production of normal cotyledonary embryos. In the case of liquid culture, agitating the culture for 1 week from 7 days after the culture commenced was also effective for embryo development. However, when the total agitation time was longer (2 to 3 weeks) during liquid-double layer culture or liquid culture, the embryos developed abnormally in both cases. The normal cotyledonary embryos obtained in this study successfully developed to plants when transferred to regeneration media. These regenerated plants were either diploid or haploid, and there was a difference in the number of chloroplasts between guard cells of diploid and haploid. These results can be used as an important data for developing an efficient microspore culture system with high quality embryo production in hot pepper.

• Journal of Plant Biotechnology
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• v.35 no.2
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• pp.109-114
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• 2008

The aim of this research is to establish the conditions for Agrobacterium-mediated genetic transformation using microspore. The embryo induction from the microspore was examined under several Kanamycin concentration in media, and the induction rate decreased about 4, 8, 10 times when the Kanamycin concentration increased 10, 50, 100 mg/L, respectively. This indicates that the transformation rate would be much lower if the Kanamycin was used for selection marker. In order to apply the GFP gene as a reporter gene for Agrobacterium-mediated genetic transformation, GFP expression from the microspore-mediated embryos was observed using GFP filter under microscope. The GFP expression occurred when the microspore cultured toward the embryo development for 12, 24 and 48 days. The microspore formed a cluster by microspore division from 12 days culture and continuously became a bigger mass. We obtained a total of 8 GFP-expressing embryos suggesting that the transformation of microspore occurred. However, those young embryos were not fully developed. Further study pertinent to culture conditions is required to fulfill the Agrobacterium-mediated genetic transformation using microspore.

• Journal of Plant Biotechnology
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• v.2 no.3
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• pp.129-134
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• 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 CROP SCIENCE
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• v.51 no.spc1
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• pp.237-241
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• 2006

This experiment was carried out comparison with haploid plants productivity by microspore culture among domestic cultivars of Brassica napus L. Isolated microspore from flower buds were cultured on NLN medium supplemented with 13% sucrose, $0.05mg/{\ell}$ BA and $0.5mg/{\ell}$ NAA. Genotype was important factor in haploid embryo productivity 'Tamlayuchae' showed the highest haploid embryo production frequency (176 embryos formed from 1 flower bud). But, 'Hallayuchae' and 'Youngsanyuchae' were not generated embryo even cell division. When suspension culture on NLN liquid medium at 100 rpm, embryos were developed multilobe abnormal embryo cluster. Multilobe abnormal embryos on MS medium basal solid medium were regenerated multiple shoots. Regenerated haploid plant with well developed shoots and roots on MS basal medium were successfully transferred to pots.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.494-504
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• 2010

The effect of the addition of the fresh medium, volume of under solid medium in double layer culture as well as the medium change after pretreating microspores on the production of embryos in microspore culture of hot pepper (Capsicum annuum L.) has been studied. When cultured after heat pre-treatment, changing pretreatment media with fresh culture media proved to be more effective for embryo production rather than supplementing additional culture media. Heat-pretreating for 3 days turned out more effective for embryo production than pretreating for 1 or 2 days. In the case of anther pretreatment, the addition of fresh medium after culture was not effective for embryo production. In pretreating microspores, however, supplementing additional fresh culture media greatly improved embryo yield and quality. The best time point of media addition was 4 days after culture commenced, and the most effective number of times of media addition was one time addition. Moreover, the effective volume of added medium in double layer culture for embryo production was 1.5 ml. The addition of media more than 1.5 ml reduced both embryo yield and quality. Double layer medium was more effective for embryo development than liquid medium. When the volume of under solid medium increased ranging from 3 ml to 7 ml, more cotyledonary embryos were produced in either 5 ml or 7 ml compared to 3 ml, even though the total number of embryos were highest in 3 ml. These results can be used as an important data for establishing an efficient microspore culture system for producing high frequency of normal embryos in hot pepper.

• Journal of Plant Biotechnology
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• v.29 no.3
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• pp.193-198
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• 2002

The objective of this study was to determine the effects of phenylacetic acid (PAA) on embryo production in anther and microspore culture of herbaceous peony (Paeonia lactiflora Pall.). The anthers of herbaceous peony were cultured on MS medium with 0 to 100 mg/L PAA according to two-step culture method. The ruptured anthers were transferred onto embryo formation medium without growth regulators. The MS medium with 2 mg/L PAA was effective in enhancing of direct embryogenesis and producing of normal embryo with two cotyledons from the cultured anthers. However, the increase of PAA concentration more than 5 mg/L PAA inhibited the embryo formation and promoted to callus formation from the anthers. The PAA affects significantly on the division of microspore and embryo formation in shed pollen culture and the best result was obtained from a medium supplement with 2 mg/L PAA. The preculture of anther for 10 days on solid medium with 2 mg/L PAA was effective for embryo formation from shed microspore of herbaceous peony.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.347-353
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• 2009

Procedures that induce microspore embryogenesis have been described for a range of Brassica species, but embryo yield remains low for a number of genotypes. We have carried out experiments with the microspores from a weakly responsive line of B. napus to determine the culture conditions that optimize their in vitro embryogenesis by treating them with effectors of ethylene synthesis and action. The results revealed that isolated microspores subjected to an initial heat stress in a medium supplemented with inhibitors of ethylene synthesis such as AVG and $CoCl_2$ exhibited significantly increased embryo yields. This suggested that regulatory effects are exerted by the ethylene produced by the isolated microspores on the early processes of gametogenesis. As a consequence, treatment of microspores with SAM, an ethylene synthesis precursor, or with the ethylene-releasing agent ethephon, led to decreases in embryo yield. A special response to ethylene during the early stages of microspore development was finally shown to occur through experiments where isolated microspores were treated for increasing periods of time with $CoCl_2$. Collectively, our data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis.

• Horticultural Science & Technology
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• v.30 no.5
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• pp.573-578
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• 2012

A total of eleven Chinese cabbage accessions were used for microspore culture and were grown to take basal data. Based on the collected data, breeding materials were chosen to develop new improved Chinese cabbage cultivars. The range of microspore-derived embryoid taken from flower buds was 1.6 to 35.4 embryoids. The embryoids from IT26110 and IT26153 among the Chinese cabbages were more than 34 per flower bud. The viability rate after cold treatment was low from 0.2 to 11.7%. The range of fertility rate was 7.7 to 58.8% in general but the IT26118, IT26122, IT26128, IT26130, and IT26164 were more than 50%. The result of their seed production ability by selfing was 11.9 seeds per siliqua in IT26128 while the others were less than 10 seeds. In the microspore culture using parents of different hereditary, the number of embryoids, the number of plants, the rate of fertility and their pure seed production ability appeared to be very different in doubled haploid lines obtained from fertile plants of Chinese cabbage.

• Proceedings of the Korean Society of Crop Science Conference
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• 2020.06a
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• pp.134-134
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• 2020