• Journal of Korean Society of Forest Science
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• v.86 no.3
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• pp.261-269
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• 1997

Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and ${\beta}$-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced about $25.75{\pm}2.77$(per ovule), $11.43{\pm}1.22$(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.

• Plant Resources
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• v.7 no.2
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• pp.87-92
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• 2004

Five different poplar hybrids were tested for rescuing embryo to elongate in vitro plantiets after hybridization. Ovaries and ovules were cultured on Woody Plant Medium (WPM) supplemented with cytokinins, 6-benzylamine (BA) and zeatin. Multiple shoots were initiated from half section of capsule with immature embryos after 21 days from pollination and tiny shoots were formed after the expansion of cotyledons in ovule cultures. Germinating response was better in intraspecific hybrids $(6.53\pm1.66)$ than interspecific crosses $(0.93\pm0.54)$ from half section of capsules on WPM medium. In general, zeatin was better than BA in inducing multiple shoots from isolated ovules. The highest average number $(19.40\pm4.53)$ of shoots was produced from immature ovules of 21 days post-pollination of WPM medium supplemented with 5.0 mg/L zeatin. The highest percentage of germination was 93% from the half section of in vitro ovary cultures. Soil acclimation was successfully conducted in cell tray containing artificially mixed soil with 96% survival rate.

• Korean Journal of Plant Tissue Culture
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• v.22 no.5
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• pp.261-266
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• 1995

This study was carried out to develop new varieties which are dwarf and tolerant to winter cold in the Aurantioideae by intergeneric crossing. to do that, the reciprocal crosses of Hwanggeumyooza and trifoliate orange, yooza and trifoliate orange were done and in vitro immature ovule culture of their hybrid was carried out .The callus formation from immature ovule was good in order of Hwanggeumyooza, Hwanggeumyooza $\times$ tifoliate orange, yooza, and trifoliate orange and best at 1 to 3 mg/L NAA+0.5mg/L zeatin on MT medium. In vitro germination percentage of 20week old hybrid of Hwanggeumyooza $\times$ tifoliate orange and trifoliate orange $\times$ Hwanggeumyooza were 41.3% and 37.7, respectively. The phenotype of hybrid (95%) of Hwanggeumyooza $\times$ trifoliate orange and that (100%) of trifoliate orange $\times$ Hwanggeumyooza were similar to that of trifoliate orange. After Hwanggeumyooza was pollinated by pollens of trifoliate orange, the pollen tubes grew on stigma after 3h of pollination and entered into micropyle after about 24~28 h. One gamete in pollen was fused with polar nuclei after 2 days and other one fused with egg nucleus at 3days after pollination. The fruit set percentage by intergeneric crossing was 14.0% in Hwanggeumyooza $\times$ trtfoliate orange and 17.5% in trifoliate orange $\times$ Hwanggeumyooza. The fruit set percentages of Hwanggeumyooza. and trifoliate orange were 34.2% and 39.5% by artificial self-fertilization, 34.2% and 39.5% by artificial cross fertilization, 3.1% and 1.4% by parthenocarpy and 13.0% and 3.0% by natural fertilization, respectively. The somatic and gametic chromosome numbers of Hwanggeumyooza, yooza, and trifoliate orange were 2n=18 and n=9.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.317-322
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• 1995

As a basic research for breeding new varieties, reciprocal -intergeneric crosses between Citrus junos and P.trifoliata were made. F$_1$ hybrid production using in vitro ovule culture, gametogenesis, and fertilization phenomena were investigated. Frequency of fruit set resulting from crossing of Citrus junos and Poncirus Trifoliata was 16.6% while that of Poncirus Trifoliata and Citrus junos was 11.7%. Callus formation occurred well when ovules at the 6th week after pollination were cultured on MT (Murashige and Tucker) medium supplemented with zeatin 0.5 mg/L and NAA 1.0 or 3.0 mg/L. Immature ovules developed into mature embryos of the MT medium supplemented with 2,4-D 0.1 or 3.0 mg/L. Immature ovules developed into mature embryos of the MT medium supplemented with 2,4 D 0.1 or 0.5 mg/L. The invitro germination rates of 20-week-old ovules set C. junos $\times$ P. Trifoliata and P. Trifoliata $\times$ C. junos were 54.5% and 48.6%, respectively. The emergence ratios of trifoliate hybrids obtained by C. junos $\times$ P. Trifoliata and P. Trifoliata $\times$ C. junos were 56.7% and 100%, respectively. The chromosome number of C. junos and P. Trifoliata was n = 9 or 2n = 18, and the sizes of their pollen grain were 33.75 $\mu$ and 25.0 $\mu$. The length and width of embryo sac in C. junos and P. Trifoliata were 69.38~79.23 $\mu$ and 27.50~38.56 $\mu$, and those of egg cells were 17.50~41.50 $\mu$ and 6.25~8.12$\mu$. Fertilization of C. junos and P. trifoliata terminated 72 h after pollination.

• Korean Journal of Medicinal Crop Science
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• v.2 no.3
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• pp.219-226
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• 1994

Immature ovule of intergeneric $F_1$ hybrid between Platycodon grandiflorum x Codonopsis lanceolata for producing embryogenic callus. somatic embryos and plant regeneration were cultured in vitro on various medium as well as MT(Murashige Tucker)medium treated with different concentration of plant growth regulators. Embryogenic callus induction was highest in the treatment of NAA 0.5 $mg/{\ell}$ and zeatin 0.01 $mg/{\ell}$ added on MT medium, whereas it was lower in treatments with auxins alone. MT medium were more effective in production of somatic embryos from incubated embryogenic callus. Most favorable plant growgh regulator for producing somatic embryos was 2. 4-D 0.5 $mg/{\ell}$ and zeation. BAP 0.01$mg/{\ell}$, but hormone-free and auxins alone were less effective. NAA 0.01$mg/{\ell}$ added with zeation 0.5 $mg/{\ell}$ was effective as high as NAA 0.01 $mg/{\ell}$ alone in normal plant regeneration from somatic embryo.

• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.335-339
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• 1998

The factors affecting the isolation and culture of the protoplast of embryogenic callus, derived from immature ovule in Citrus junos, were examined. An incubation time in enzyme solution of 16 hrs was preferable for protoplast isolation. Efficient protoplast yields were obtained from the treatment of equal concentration of 0.7 M $\textrm{BH}_{3}$ to the enzyme solution containing 1.0% cellulase, 1.0% macerozyme and 0.2% pectolyase. Protoplast cultured in MT medium with 0.1 mg/L 2,4-D showed vigorous division and some of them formed callus. Induced callus was subcultured on solid MT medium but the callus showed very slow growth. The above results show the possibility to culture from protoplast fusion in Citrus genera.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.81-86
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• 2008

This study describes conditions for plant regeneration from protoplasts-derived from embryogenic callus of satsuma mandarin. Plants were generated via somatic embryogenesis. Protoplasts isolated directly from nucellar callus induced from immature ovule of satsuma mandarin cv. Okitsu (Citrus unshiu Marc.) were cultured in 0.6M $BH_3$ medium. Cell division and plating efficiency were affected by protoplast culture method. The liquid over solid method was the most effective for formation of microcalli. Most of microcalli grew rapidly and transferred onto embryoid formation medium. Optimum embryoid formation medium was MT medium containing 1.5 g/L malt extract, 0.146 M sucrose and the medium for plantlet regeneration was MS medium containing 0.09M sucrose, 1.0 mg/L $GA_3$. No differences were noticed in growth habits and leaf characters such as shape, thickness, and colour between protoplast-derived plants and nucellar seedlings. This plant regeneration system from protoplasts-derived from embryogenic callus provides an alternative way for producing new scion and rootstock cultivar from citrus species which can not be crossed.

• FLOWER RESEARCH JOURNAL
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• v.17 no.4
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• pp.221-225
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• 2009

Interspecific crossing was conducted to obtain hybrid lilly with better quality of adapting to the unfavorable environment using oriental Lilium spp. as a female parent. Average interspecific crossing rate between oriental and asiatic lines by cut-style pollination was as low as 29%. Although the average germination rate of zygotic hybrid embryo between oriental 'Rodolfa' ${\times}$ asiatic 'Toronto' was increased from 76.6% to 78.3%. on 114 or 1/6 strength MS medium compared to the control, the rate of zygotic embryos formation between oriental 'Farolito' ${\times}$ asiatic 'Buff Pixie' was significantly enhanced up about 86~90% on 1/2 strength MS medium. Meanwhile, germination rate of interspecific hybrid embryo between oriental 'Snow Cristal' ${\times}$ asiatic 'Royal Trinity' showed up 100%. Germination rate of interspecific hybrids slightly increased by addition of $0.5-1.0mg{\cdot}L^{-1}$ GA. Germination rate of zygotic embryos between oriental 'Farolito' ${\times}$ asiatic 'Buff Pixie' and oriental 'Belcanto' ${\times}$ L. callosum was highest on the MS medium containing $0.01mg{\cdot}L^{-1}$ NAA and $0.1mg{\cdot}L^{-1}$ kinetin, as 66.6% and 45.2% respectively.

• Horticultural Science & Technology
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• v.35 no.1
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• pp.121-130
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• 2017

This study was carried out to investigate a method for producing cultured virus - free ovules for breeding high - quality Citrus cultivars. Ovules from the immature fruits of three citrus cultivars native to Jeju (Dongjeongkyool, Cheongkyool, and Jikak) and two cultivars of Citrus unshiu Marc. (Miyagawa wase and Haryejosaeng) that were thought to be infected with Citrus tristeza virus (CTV) were cultured on MS2 medium (Murashige - Skoog [MS] basal medium containing $500mg{\cdot}L^{-1}$ malt extract, $50g{\cdot}L^{-1}$ sucrose, $1.0 mg{\cdot}L^{-1}$ kinetin, and $8g{\cdot}L^{-1}$ agar). After four weeks of culture, 10, 21, 13, 5, and 7 somatic embryos and 2, 4, 2, 4, and 5 white callus cells (surrounding green somatic embryos) were obtained from Dongjeongkyool, Cheongkyool, Jikak, Miyagawa wase, and Haryejosaeng, respectively. After six weeks of culture, somatic embryos were obtained from cultured cells grown on MT basal medium supplemented with malt extract ($500mg{\cdot}L^{-1}$), lactose ($70g{\cdot}L^{-1}$), and agar ($16g{\cdot}L^{-1}$). Over 60% of the somatic embryos from citrus cultivars native to Jeju developed into normal plants on MS basal medium supplemented with malt extract ($500mg{\cdot}L^{-1}$), sucrose ($50g{\cdot}L^{-1}$), and agar ($8g{\cdot}L^{-1}$) after 10 weeks of culture. Normal plants were regenerated from two Citrus unshiu Marc. cultivars on MT basal medium supplemented with sorbitol (1.0 M), galactose (1.0 M), $GA_3$ ($1.0mg{\cdot}L^{-1}$), and Gelrite ($3g{\cdot}L^{-1}$). The absence of virus in plants generated from cultured ovules was confirmed by RT - PCR and antigen - antibody reactions. Therefore, virus - free Citrus cells can be obtained for breeding high - quality citrus cultivars using the biotechnological technique evaluated in this study.