Somatic Embryo Induction Research Articles

Micropropagation via somatic embryogenesis of rare and endangered species, Acanthopanax seoulenses Nakai

A somatic embryogenesis and plant regeneration study was conducted with a rare and endangered species, Acanthopanax seoulenses, and various factors affecting somatic embryo induction were evaluated. The frequency of embryogenic callus induction was slightly better on wounded seeds compared with non-wounded ones. The optimum medium to induce somatic embryos (SEs) from embryogenic cells was MS medium supplemented with 3% sucrose and 0.1–0.2 mg/l abscisic acid (ABA), or MS medium with 3% sucrose and 0.1 mg/l ABA+0.02% activated charcoal. Gibberellic acid resulted in a positive effect on SE germination, but there were no differences in the range of 0.1 to 1.0 mg/l. SE germination and plant conversion rate were different in the two gelling agents: agar-gelled medium was slightly better than gelite-gelled medium, and the highest plant conversion, 78±18.2%, was achieved in agar-gelled medium. Regenerated plantlets were successfully acclimatized in artificial soil mixture, and more than 98% of the plants survived in vermiculite or an equal volume mixture of vermiculite and peat moss. Results suggest that the rare and endangered species is able to propagate effectively via the somatic embryogenesis system.

English

Batian is a true breeding commercial coffee variety that was released in Kenya in 2010. It is resistant to coffee berry disease and coffee leaf rust which are the main coffee diseases in Kenya. Coupled with early ripening, good beverage quality and high yields, demand for planting material has surpassed supply. Conventional propagation methods do not provide enough planting materials, hence the need to develop alternative methods. The objective of this study was to develop an effective in vitro protocol for propagating the coffee variety, Batian. Leaf explants were harvested and cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins benzyl amino purine (BAP) and thidiazuron (TDZ) separately, 100 mg/l myo-inositol 3% sucrose and gelled with 0.3% gelrite. The results show differences among cytokinins levels in induction of somatic embryos. BAP at 13.3 µM gave the highest mean of embryos per explants, 6.06 ± 1.18 and highest percentage of embryogenic cultures of 58.33%. Development of somatic embryos was achieved on hormone free MS media with highest mean length of 0.32 ± 0.03 mm. Indole butyric acid at 9.8 µM was best for induction of a well-developed root system with a mean length of 1.22 ± 0.09 mm. This protocol opens new prospects for massive propagation of Batian in nine months.   Key words: Batian, somatic embryo, Coffea arabica.

Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines induced by γ-irradiation (60Co) of adventitious roots

An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.

Microarray analysis of siberian ginseng cyclic somatic embryogenesis culture systems provides insight into molecular mechanisms of embryogenic cell cluster generation.

Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters.

The study on in vitro culture of embryogenic callus and somatic embryo tissue of Panax vietnamensis Ha et Grushv.

This paper presented the results on induction and multiplication of embryogenic calli in liquid medium; on induction of somatic embryo, shoot/root morphogenesis of somatic embryo through culture and on multiplication of somatic embryo tissue in liquid medium. The aim of this study is to lay out the basis for large scale multiplication of these two kinds of tissues with high capable of secondary metabolite production due to their more or less morphological differentiation status. Leaf disks (about 0.5 × 0.5 cm) were cultured on the MS medium with 2 mg/l 2,4-D for callus induction. Callus was subcultured on the MS medium with 1 mg/l 2,4-D + 1 mg/l NAA + 0.2 mg/l kinetin + 10% coconut water for induction of somatic embryo tissue. The embryogenic callus was proliferated in the MS½ liquid medium with 0.5 mg/l 2,4-D + 0.5 mg/l NAA. Depending on the initial medium and the subsequent media, the somatic embryo tissue was cultured for development, via two directions, into population of shoots or roots. The mentioned above tissues are being cultured on shaker in big flask/bioreactor for biomass propagation.

Control of Morphological Aberrations in Somatic Embryogenesis of Commiphora wightii (Arnott) Bhandari (Family: Bursaraceae) Through Secondary Somatic Embryogenesis

Somatic embryogenesis in Commiphora wightii was obtained using 5-8 mm long embryos from fruits (65-75 days old) collected during January to June. The embryos gave rise to embryogenic mass on Murashige and Skoog medium supplemented with 2,4-D (4.4 lM), sucrose (3.0 % w/v) and agar (0.6 % w/v), when incubated under dark conditions within 55-65 days. Further, these embryo- genic masses induced somatic embryos (1.00-9.00 mm long) within 40-45 days after transferring them to light on media devoid of 2,4-D. The somatic embryos were asyn- chronous with certain levels of abnormalities. The abnor- malities observed were different sizes of globular embryos, single cotyledonary and fasciated embryos. In order to cir- cumvent these abnormalities and to achieve synchronized induction, studies were carried out to induce secondary somatic embryos. The induction of Secondary Somatic Embryos was achieved on Murashige, Skoog and Schenk and Hildebrand media from primary cotyledonary somatic embryos. These were induced from the edge of cotyledon and the tip of hypocotyl, and were 1.00-3.00 mm in length. A total of 74 % of the two cotyledonary embryos were induced from secondary embryogenesis system against 24 % induced from primary somatic embryogenesis system.

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

English

The aim of this study was to establish an in vitro system for the induction, maturation and regeneration of somatic embryo in sugarcane from buds of cultivar RB 867515. Embryogenic calluses were obtained on semi-solid MS medium supplemented with 4.42 mg L-1 2,4-D. After four weeks of culture of explants on the callus induction medium, globular structures were obtained. At the end of 20 days in maturation medium, somatic embryos were observed. Histological analysis showed somatic embryos with caulinar and root apex, protodermal tissue, and the vascular system, which apparently has no connection with the vascular tissue explant that gave rise to it confirming the presence of the somatic embryo. The embryos were transferred to regeneration medium containing 1 mg L-1 GA3 and BAP, and after 1 to 2 weeks of culture, green points were observed, indicating the beginning of the formation of shoots.   Key words: Saccharum spp, bud culture, 2.4-D, morphogenetic pathway, embryogenesis, plant regeneration.

Improved Induction of Somatic Embryo in Watermelon [Citrullus lanatus (Thunb.) Matsum. et Nakai

In order to optimize somatic embryo induction of watermelon, three different explants from three different varieties were used to induce somatic embryo in medium contained BA in combination with either NAA or 2,4-D. ‘Kangbingjingxin’ cotyledon explant with all of PGR combination treatments has a high callus inductivity (75.6%-98.9%), but inductivity of somatic embryo was varied among different PGR combination, the highest inductivity (54.7%) was obtained in medium with BA 3.0 mg/L and NAA 0.05 mg/L. Somatic embryogenesis was different among ‘Kangbingjingxin’, ‘Zaojia 8424’ and ‘Heimeiren’, the inductivity was 46.2%, 34.1% and 15.7% respectively. Callus inductivity of three explants was 88.9%-98.9%, in which callus induced from cotyledon and cotyledon node was mostly embryonic callus while callus from hypocotyl was non-embryonic callus. Somatic embryo inductivity of cotyledon node (62.0%) was significantly higher than cotyledon (46.1%), there is no somatic embryo induced from hypocotyl. However, somatic embryos number per explant of cotyledon node (2.3) was significantly lower than cotyledon (6.0).

Induction of Somatic Embryos from Leaf and Stem Nodal Section Explants of Potato (Solanum tuberosum L.)

Somatic embryogenesis can be used as an alternative method to obtain high-quality potato seedlings. This research was conducted in order to evaluate: the potential of different types of explants to produce somatic mbryos;combinations of 2,4-D and BAP for somatic embryo induction; and the ability of BAP to act as growth regulator for the somatic embryo maturation process.Young leaf and stem nodal sections were cultured in MS media supplemented with 2,4-D and BAP. The developing calli were then transferred into media containing several concentrations of 2,4-D and BAP. The embryogenic calli were then transferred into media containing 1-5 μM BAP. The results show that MS media containing 2,5 μM 2,4-D + 5 μM BAP and μM 2,4-D + 0.5 μM BAP were the most suitable for inducing somatic embryos from leaf explants and stem nodal section explants, respectively. The somatic embryos were well-developed in the MS media supplemented with 5 μM BAP. The stem nodal section explants were able to produce a higher percentage of globular, heart and torpedo stage embryos compared with the leaf explants. Based on this research, stem nodal section explants have a high potential for producing somatic embryos induced in medium containing 1 μM 2,4-D + 0.5 μM BAP.

Somatic embryogenesis in Manchurian ash: performance of various explant stock trees, optimisation of the basal medium, and simplified plant growth regulator combinations

SummaryManchurian ash (Fraxinus mandshurica Rupr.) is an ecologically and economically important hardwood tree species distributed in Northeast China. Somatic embryogenesis has been achieved in this species. However, to improve the efficiency of somatic embryogenesis in this species, the effects of explant stock tree, basal medium type, and plant growth regulator combination were investigated. Somatic embryos (SEs) of Manchurian ash were induced on cotyledons from young zygotic embryos, and the highest rate of induction was obtained from zygotic cotyledons cultured on 0.5× Murashige and Skoog (MS) medium. When cultured on 0.5× MS medium containing 8.1 µM αnaphthaleneacetic acid (NAA) plus 2.2 µM 6-benzyladenine (BA), the cotyledon explants derived from different stock trees had varying potentials to produce somatic embryogenic tissue. Somatic embryos were formed on only 33 of the 46 stock trees (numbered 1 – 46) tested, and the mean rates of induction ranged from 3 – 67%. Significant differences were observed in the combination of plant growth regulators required for maximum induction of SEs on cotyledon explants obtained from four additional stock trees (numbered 59 – 62), for which the optimal combination was 5.4 – 10.7 µM NAA plus 2.2 µM BA. These tree-specific results imply that breeding source needs to be considered before the potential application of somatic embryogenesis for rapid and large-scale propagation of superior lines of Manchurian ash.

Embriogênese somática e morfoanatomia de embriões somáticos de <i>Ocotea porosa</i>

http://dx.doi.org/10.5902/1980509812343Ocotea porosa apresenta sementes com forte dormência tegumentar, comportamento recalcitrante, baixa germinação e irregularidade, o que dificulta a propagação natural. O objetivo deste trabalho foi estabelecer um protocolo de embriogênese somática para Ocotea porosa. Eixos embrionários zigóticos imaturos foram inoculados em meio de cultura WPM suplementado com 2,4-D (200 μM) combinado ou não com caseína hidrolisada ou glutamina (0,5 ou 1 g l-1) durante 90 dias. A embriogênese repetitiva foi induzida em meio contendo 2,4-D (22,62 μM) combinado com 2-iP (2,46 μM) seguido de transferência para meio de cultura com caseína hidrolisada ou glutamina (1 g l-1), durante 90 dias cada. A maturação dos embriões somáticos foi testada em meio de cultura contendo ANA (0,5 μM) e 2-iP (5, 10 ou 20 μM). A maior porcentagem de embriões somáticos induzidos (8,3%) foi observada em meio de cultura WPM contendo 200 μM de 2,4-D e 1 g l-1 de caseína hidrolisada e o desenvolvimento de embriões somáticos ocorreram de forma indireta. A embriogênese somática repetitiva foi promovida em meio de cultura WPM contendo caseína hidrolisada ou glutamina, no entanto, a caseína hidrolisada promoveu a manutenção da competência embriogênica por mais de dois anos. Na fase de maturação, houve baixa progressão dos embriões globulares para os estádios cordiforme e torpedo. Os diferentes estádios ontogenéticos de embriões somáticos de Ocotea porosa foram caracterizados pelo estudo histológico.

The Influence of the Developmental Stage and Culture Medium upon the Somatic Embryo Induction in <i>Quercus petraea</i>

The somatic embryogenesis is an advanced method for clonal propagation and a useful tool for ex situ conservation of genetic resources. In this paper an experimental device was designed, composed of: one oak species (Quercus petraea), one provenance, four types of explants (developmental stages of the zygotic embryo) and four variants of culture medium. It was released the two-way statistical analyses of the two analyzed factors affecting the embryo induction efficiency: the stage of explant and culture medium (4 stages x 4 culture media). The most important factor affecting the efficiency of somatic embryogenesis was found to be the developmental stage of the zygotic embryos used as juvenile explants. The high embryogenic ability of young zygotic embryos and low embryogenic ability of maturing zygotic ones showed the strong positive relationship between somatic embryo induction and accumulation of reserve nutrients in cotyledons. The best results were obtained with the youngest embryos (stage 1), with a linear decrement towards the stage 4. The embryo induction efficiency ranged between 44% and 10% in Q. petraea. Two nutritive culture media and two growth hormone combinations were tested. The effect of culture medium proved to be totally insignificant, besides the differences observed among the variants cultivated on different media. The situation was the same as well for the nutrients as for growth regulators.

DIRECT AND INDIRECT SOMATIC EMBRYOGENESIS ON ARABICA COFFEE (Coffea arabica)

Propagation of Coffea arabica L. through direct and indirect somatic embryogenesis technique is promising for producing large number of coffee seedlings. The objectives of the research were to evaluate methods for direct and indirect somatic embryo-genesis induction of C. arabica var. Kartika. The explants were the youngest fully expanded leaves of arabica coffee. The evalu-ated medium was modified Murashige and Skoog (MS) medium supplemented with a combination of 2.26 µM 2,4-D + 4.54 or 9.08 µM thidiazuron; 4.52 µM 2,4-D + 4.54 or 9.08 µM thidiazuron; or 9.04 µM 2,4-D + 9.08 µM thidiazuron. Both calli (100 mg) and pre-embryos developed from the edge of leaf explants were subcultured into regeneration medium (half strength MS with modified vitamin, supplemented with kinetine 9.30 µM and adenine sulfate 40 mg L-1). The results showed coffee leaf explant cultured on medium containing 2.26 µM 2,4-D + 4.54 or 9.08 µM thidiazuron to induce direct somatic embriogenesis from explant, while that of 4.52 or 9.04 µM 2,4-D + 9.08 µM thidiazuron to induced indirect somatic embrio-genesis. The medium for calli induction from coffee by explants was medium supplemented with 4.52 or 9.04 µM 2,4-D in combination with 9.08 µM thidiazuron. On the other hand, the best medium for activation of induction of somatic embryos was MS medium supplemented with 9.04 µM 2,4-D + 9.08 µM thidiazuron. Based on this results, the first step for developing micropropagation for coffee has been resolved. The subsequent studies will be directed to evaluate agronomic performance of the derived planting materials.

DIRECT AND INDIRECT SOMATIC EMBRYOGENESIS ON ARABICA COFFEE (Coffea arabica)

Propagation of Coffea arabica L. through direct and indirect somatic embryogenesis technique is promising for producing large number of coffee seedlings. The objectives of the research were to evaluate methods for direct and indirect somatic embryo-genesis induction of C. arabica var. Kartika. The explants were the youngest fully expanded leaves of arabica coffee. The evalu-ated medium was modified Murashige and Skoog (MS) medium supplemented with a combination of 2.26 µM 2,4-D + 4.54 or 9.08 µM thidiazuron; 4.52 µM 2,4-D + 4.54 or 9.08 µM thidiazuron; or 9.04 µM 2,4-D + 9.08 µM thidiazuron. Both calli (100 mg) and pre-embryos developed from the edge of leaf explants were subcultured into regeneration medium (half strength MS with modified vitamin, supplemented with kinetine 9.30 µM and adenine sulfate 40 mg L-1). The results showed coffee leaf explant cultured on medium containing 2.26 µM 2,4-D + 4.54 or 9.08 µM thidiazuron to induce direct somatic embriogenesis from explant, while that of 4.52 or 9.04 µM 2,4-D + 9.08 µM thidiazuron to induced indirect somatic embrio-genesis. The medium for calli induction from coffee by explants was medium supplemented with 4.52 or 9.04 µM 2,4-D in combination with 9.08 µM thidiazuron. On the other hand, the best medium for activation of induction of somatic embryos was MS medium supplemented with 9.04 µM 2,4-D + 9.08 µM thidiazuron. Based on this results, the first step for developing micropropagation for coffee has been resolved. The subsequent studies will be directed to evaluate agronomic performance of the derived planting materials.

낙엽송(Larix kaempferi) 현탁배양된 배발생세포로부터 체세포배 유도 및 발아를 위한 삼투압제 종류 및 농도 효과

본 연구는 낙엽송 배발생세포의 배양기간에 따른 현탁세포의 생장, 체세포배 유도 및 발아를 위한 다양한 삼투압제 종류 및 농도 효과를 조사하기 위해 수행되었다. 배발생 현탁세포의 생장은 배양일수가 증가함에 따라 settled cells volume (SCV)가 지속적으로 증가하는 경향을 보여 배양 18일 경에는 최대 10.1 ml의 SCV를 보였고, 그 이후로는 세포생장이 감소하였다. 현탁세포로부터 체세포배 발생을 위한 삼투압제 종류 및 농도에 따른 효과 비교에서 최대의 체세포배 유도 수는 0.2 M sucrose 처리구에서 352.3개(/g FW)로 나타났으며, 0.2 M maltose (203.7) 및 0.3 M maltose (193.7) 처리구에서 또한 효과가 있었다. 그러나 7.5% polyethylene glycol (PEG)이 첨가된 0.15 M sucrose 혹은 maltose 각각의 처리구에서는 체세포배 발생이 전혀 이루어지지 않았다. 다양한 삼투압제 종류 및 농도에서 발생된 체세포배의 발아율 비교에서는 최대 자엽 형성율(25.2%) 0.3 M maltose, 하배축 형성률(39%), 뿌리 발생율(30.3%) 및 식물체 재분화율(3.5%)은 0.2 M sucrose 처리구에서 각각 나타났다. This study was conducted to examine suspended embryogenic cells growth with days of culture, effects of various kinds/concentrations of osmoticum for induction of somatic embryos (SEs), following somatic embryos germination or plantlet regeneration. The proliferation pattern of embryogenic cells in suspension culture is characterized by settled cells volume (SCV) increased with the duration of culture with marked the maxium of SCV (10.1 ml) in 18 days of culture, however the SCV of cells gradually decreased after that. In comparison of kinds/concentrations of osmoticum on somatic embryo induction, the highest induction number (352.3/g FW) of the SE was showed in 0.2 M sucrose, in addition, we also observed some effects with treatments of 0.2 M maltose (203.7) and 0.3 M maltose (193.7), respectively. However, no somatic embryos produced in treatments of 7.5% PEG plus 0.15 M sucrose or maltose. In comparison of germination efficiency of SEs which occurred from the treatments of various kinds/ concentrations of osmoticum, the highest induction frequency of cotyledon (25.2%) was obtained from SEs that produced 0.3 M maltose, however, the best occurrence rates of hypocotyl (39%), radicle (30.3%) and plantlet regeneration (3.5%) were observed from the 0.2 M sucrose treatment, respectively.

Improvement of somatic embryogenesis and plantlet conversion in Oplopanax elatus, an endangered medicinal woody plant.

Oplopanax elatus is a medicinal plant on the verge of extinction because of overexploitation. In the present study, the effects of various factors on enhancing somatic embryogenesis and plantlet conversion were studied. Mature seeds were collected from a total of 13 plants from 4 mountains in South Korea, and the genetic distances were calculated to analyze the effect of genotype on somatic embryogenesis. Results of cluster analysis and the unweighted-pair-group method with arithmetic mean of 13 genotypes indicated the presence of 3 main groups. Both genotype and explant type affected the induction of somatic embryos (SEs). Sorak 2 and root were found to be the most suitable genotype and explant type, respectively, for SE induction in O. elatus. Among the different types of carbon sources tested, 5% sucrose induced the maximum number of SEs. The formation and development of SEs were significantly influenced by culture density; thus, 10 mg embryonic callus was found to be the most suitable for SE induction. The highest rates of germination and SE conversion were obtained in a germination medium containing 1.8 gelrite and 3.2 g·l-1 agar. In addition, 80% of the plantlets that were transplanted into artificial soil acclimatized successfully. Thus, our results showed that the percentage survival of O. elatus during in vitro proliferation could be increased by optimizing to the somatic embryogenesis system.