Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

Factors important for somatic embryogenesis in zygotic embryo explants ofCapsicum annuum L.

We used four cultivars ofCapsicum annuum L.—Sweet Banana, California Wonder, Yolo Wonder, and Ace—to reexamine the critical factors influencing somatic embryogenesis from zygotic embryo explants, as reported in the literature. When we followed the protocol of Buyukalaca and Mavituna (1996), which had induced somatic embryogenesis from mature zygotic embryos of cv. Ace, only callus was formed without embryogenesis from our mature zygotic embryo expiants. Using the procedures of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), with some modifications, we were able to induce somatic embryogenesis in all four cultivars. Rates of conversion were significantly reduced, from 75% and 65% to 40% and 28% in ’Sweet Banana’ and ’California Wonder’, respectively, when the immature zygotic embryo expiants were held on the induction medium for longer than two weeks. Likewise, somatic embryogenesis of ’Yolo Wonder’ was not observed if the induction medium was supplemented with 10% glucose or fructose, or without 10% sucrose. For somatic embryo induction and eventual plantlet conversion in Yolo Wonder’, maltose could adequately replace sucrose. In all four cultivars, somatic embryos were initiated from immature zygotic explants on media with or without coconut water, under both light and dark conditions.     

Somatic embryogenesis and plantlet formation from a rare and endangered tree species,Oplopanax elatus

We tested the possibility of plantlet formation via somatic embryogenesis with leaf segments and mature zygotic embryos from a rare and endangered tree species,Oplopanax elatus. To induce calli, expiants were cultured under darkness in a solid MS medium containing 3% sucrose, 1g L^-1 glutamine, and 0.3% gelrite. Treatment supplements included 2,4-D alone or in combination with thidiazuron. Generally, callus induction and growth were good from leaf expiants, whereas embryogenic calli could be induced only from zygotic embryos. These embryogenic calli were white or pale yellow and very friable. ABA and activated charcoal appeared to be important factors when inducing somatic embryos, with optimum levels being 0.1 mg L^-1 and 0.02%, respectively. Many somatic embryos showed abnormalities during their development on the germination medium, but 35% could be converted if placed on a medium containing gibberellic acid (GA₃). The germinating embryos sometimes formed secondary embryos at the lower portion of the hypocotyls. Normal or converted plantlets were acclimatized in an artificial soil mixture; their survival was about 60% after two months. This culturing system provides a feasible approach for regenerating plants, via somatic embryogenesis, from mature zygotic embryos.     

华山松胚性愈伤组织诱导与幼胚离体培养

探索华山松(Pinus armandii)体细胞胚胎发生技术对其实施规模化无性繁殖和开展遗传转化具有重要意义。本文以1/2LM为基本培养基, 通过激素调节等措施对华山松的胚性愈伤组织诱导和幼胚的离体培养技术进行了初步研究。研究结果: 胚性愈伤组织诱导率最高可达52.71%, 但愈伤组织继代培养后没有体细胞胚胎的分化; 首次从其子叶期的幼胚中直接诱导出具有根和茎的完整植株, 诱导率达92%以上。文章确认了采集的幼胚发育状态对胚性愈伤组织的诱导有重要影响, 并对诱导的培养条件等进行了探讨。     

Plastid ultrastructure and DNA related to albinism in androgenetic embryos of various barley (Hordeum vulgare L.) cultivars

In order to understand the occurrence of albinism during androgenesis in barley, a number of plastid parameters were analyzed in microspore-derived embryos and androgenetic plantlets, and the results were compared in albino and non-albino producing cultivars. In the winter cv. Igri, plastids in microspore-derived embryos are characterized by numerous divisions, differentiated thylakoids, low amount of starch and a high DNA content examined by immunoelectron microscopy. After regeneration, the androgenetic plantlets were mostly chlorophyllous. In contrast, in the spring lines tested, the plastids of microspore-derived embryos were rarely dividing amyloplasts in which thylakoids and DNA were scarce and albino plantlets were mainly regenerated. After 2 weeks on the regeneration medium, plastids of Igri chlorophyllous androgenetic plantlets were typical chloroplasts, whereas in spring lines plastids of albino androgenetic plantlets were proplastids with the same characteristics as those in the corresponding microspore-derived embryos. These results strongly suggest that the origin of androgenetic albinism differs in winter and spring cvs.: in the winter cv. Igri plastid alteration may take place during the regeneration step of androgenesis whereas in the tested spring lines plastids are already affected in the microspore-derived embryos meaning that albinism is not initiated during regeneration but originates earlier during the androgenetic process likely as early as the sampling stage.     

Efficient plant regeneration of Malaysian <Emphasis Type="Italic">indica</Emphasis> rice MR 219 and 232 via somatic embryogenesis system

Improvement on rice plant regeneration system from an embryogenic callus using two Malaysian indica rice MR 219 and MR 232 was developed in this study. Different stages of rice explants (zygotic embryos) were tested for callus induction and regeneration using various carbon sources and amino acids. The present study shows that dough stage of zygotic embryos was the best stage of explants for the embryogenic callus induction and regeneration of both rice cultivars. The embryogenic callus nature with the compact structure was confirmed by scanning electron microscopy (SEM) analysis. Inclusion of maltose at the concentration between 10 and 20 mg/L shown higher responsive for the development of green somatic embryos and initiation of rice regeneration with an average of 29–37 (87–91%) regenerated plantlets, respectively. All in vitro regenerated rice plantlets were green, morphological and physiologically healthy condition. Rice plantlets were hardened in acclimatization room for 3 weeks and later transferred into soil with 95% survival in both varieties were recorded. Hence, regeneration system from zygotic rice embryos via somatic embryogenesis system was carried out successfully by using 10 g/L of maltose and combinations of glutamine, asparagine and arginine amino acids.     

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g?L^?1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g?L^?1 sucrose, 2.5 g?L^?1 activated charcoal, and 2.5 g?L^?1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.     

Micropropagation of Elaeis guineensis Jacq. 'Dura': Comparison of three basal media for efficient regeneration

Three basal plant tissue culture media, namely, N6, MS, and modified Y3, were compared to optimize micropropagation protocol for E. guineensis. Full strength media were used separately to regenerate plantlets directly using immature zygotic embryos (IZEs), and through somatic embryogenesis of calli obtained from IZEs. The plantlets regenerated by direct regeneration on three media were examined for shoot length and rooting percentage. For the induction of callus, somatic embryogenesis, and rooting modified Y3 medium was the most effective. In conclusion, the results indicate that modified Y3 medium is the most suitable for direct regeneration, callus induction and somatic embryogenesis in E. guineensis.     

Somatic embryogenesis and plant regeneration from immature seeds of Magnolia obovata Thunberg

We have tested plantlet formation by somatic embryogenesis using immature seeds of Magnolia obovata. Seed collection date appeared to be critical for embryogenic cell induction. The optimal collection date was 3–4 weeks postanthesis. The embryogenic cells proliferated, formed somatic embryos, and were subsequently converted into normal plantlets under optimized culture conditions. Somatic embryo formation from the embryogenic calli was better on sucrose medium than on glucose medium. The optimum level of sucrose appeared to be 3% with an average of 28 somatic embryos per plate. About 25% of somatic embryos were converted into normal plantlets in 1/2 MS medium containing gibberellic acid (GA₃). During somatic embryo germination, secondary embryogenesis was frequently observed in the lower part of the hypocotyl or radicle ends of germinating somatic embryos. Finally, about 85% of converted plantlets survived in an artificial soil mixture, were transferred to a nursery, and have grown normally.     

水稻花粉白苗的若干特性及其成因的探讨

水稻花粉白苗的再分化所生成的再生植株全为白苗,这说明花粉白苗的形成是不可逆的,可能是基因突变的结果。不同品种或杂交组合的水稻花粉白苗叶片丙酮提取液的吸收光谱相互间显示出了相似性,这种相似性就进而提示突变可能发生在基因组的特定位点上。有可能花粉白苗是质体DNA在花粉的脱分化过程中发生缺失形成的。这种缺失主要影响质体rRNA的合成水平,并不导致蛋白质和叶绿素合成能力的全面丧失。花粉植株细胞中的质体可能是不均一的。     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

辣椒花药培养胚状体发生的组织学和细胞学研究

采用荧光显微镜、扫描电镜和透射电镜技术．系统研究了辣椒花药培养胚状体发生的组织学和细胞学变化特征。辣椒单个花药中花粉发育具有强烈的不同步性。随着培养时期的变化．不同时期花粉的百分率也发生变化。处于单核靠边期的小孢子培养以后按两种发育途径之一进行发育。在多数情况下,孢子体不对称分裂,产生典型双核花粉。胚性花粉粒是由营养核的重复分裂形成的。当小孢子从四分体中释放出来．特殊类型的外壁已经形成。在随后的花粉发育过程中．小孢子体积增大,外壁继续加厚。培养24h后,小孢子体积增大。胚性发生的小孢子表现出两种不同的形态变化。当胚状体发育到心形胚时．胚状体的表皮细胞排列规则。用光学和电子显微镜分析了小孢子胚状体形态形成过程．及胚状体诱导后细胞组织发生的一系列结构变化的时序性特征,这些变化主要影响质体、液泡室、细胞壁和细胞核,进一步分化的程序模拟合子胚的发育。     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

Deletion of core components of the plastid protein import machinery causes differential arrest of embryo development in Arabidopsis thaliana

Among the genes that have recently been pinpointed to be essential for plant embryo development a large number encodes plastid proteins suggesting that embryogenesis is linked to plastid localized processes. However, nuclear encoded plastid proteins are synthesized as precursors in the cytosol and subsequently have to be transported across the plastid envelopes by a complex import machinery. We supposed that deletion of components of this machinery should allow a more general assessment of the role of plastids in embryogenesis since it will not only affect single proteins but instead inhibit the accumulation of most plastid proteins. Here we have characterized three Arabidopsis thaliana mutants lacking core components of the Toc complex, the protein translocase in the outer plastid envelope membrane, which indeed show embryo lethal phenotypes. Remarkably, embryo development in the atToc75-III mutant, lacking the pore forming component of the translocase, was arrested extremely early at the two-cell stage. In contrast, despite the complete or almost complete lack of the import receptors Toc34 and Toc159, embryo development in the a tToc33/34 and atToc132/159 mutants proceeded slowly and was arrested later at the transition to the globular and the heart stage, respectively. These data demonstrate a strict dependence of cell division and embryo development on functional plastids as well as specific functions of plastids at different stages of embryogenesis. In addition, our analysis suggest that not all components of the translocase are equally essential for plastid protein import in vivo.     

Secondary somatic embryogenesis of carnation (<Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L.)

This is the first report describing culture conditions necessary to induce secondary embryogenesis in two carnation cultivars, Nelson and Spirit. In the first step, embryogenic calli were induced on petal explants followed by development of primary somatic embryos from the calli. In the second stage, secondary somatic embryos were obtained when precotyledonary and cotyledonary primary embryos were isolated and transferred onto a series of culture media all containing MS basal salt mixture, and supplemented with different concentrations of 2,4-D, BA, sucrose and mannitol. The highest rate of secondary embryogenesis occurred on mannitol containing media. Secondary somatic embryos were converted into plantlets when they were transferred onto growth regulator-free half-strength MS medium and successfully acclimated in the greenhouse.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.     

Quantitative analysis of ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.)

Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.     

低温预处理对水曲柳体细胞胚胎发生的影响

用4℃低温预处理未成熟的水曲柳种子0-30d,取出种子内的合子胚为外植体诱导体胚发生,研究低温预处理影响体胚发生的结果表明：低温预处理过的外植体其体胚发生总数和子叶胚发生数均低于未作低温预处理的;随着预处理时间的延长,畸形胚发生数和发生比率与总的体胚发生数和发生率的变化趋势基本相同;处理20d的正常胚发生数和发生比率的绝对数虽然很低,但远高于不作低温预处理的。说明4℃低温预处理对水曲柳体胚发生没有促进作用,对畸形胚的发生也不能控制,总的来讲,适当的低温处理有一定的改善正常体胚发生的潜力。