Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l⁻¹ α-naphthalene acetic acid and later transferred to MS without growth regulators (MS_WH). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.     

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.     

The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

栓皮栎体细胞胚胎发生的细胞组织学观察

以栓皮栎未成熟合子胚为外植体,在添加0.25mg/L 2,4-D和0.5mg/L 6-BA的MS培养基上6周可诱导产生2种类型的胚性愈伤组织,一种表面具光泽、白色;另一种表面光滑湿润具光泽,色泽淡黄或无色透明。组织切片表明,胚性愈伤组织的细胞体积小,细胞核大,细胞质浓,细胞排列紧密;非胚性愈伤组织细胞的体积大,细胞核小,细胞质稀薄。胚性细胞团培养在不含激素的培养基上可诱导产生体细胞胚。体细胞胚直接起源于胚性细胞团表皮或近表皮的单细胞,经历与合子胚相似的球形胚、心形胚、鱼雷胚和子叶胚发育阶段。所有发育时期的体细胞胚的胚轴、子叶均产生次生体胚,它们起源于细胞质较浓的表皮单细胞。     

Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos

Immature zygotic embryos of ginseng produced somatic embryos on MS medium without growth regulators. However, in the culture of mature zygotic embryos, excision of the embryo was required for somatic embryo induction. Somatic embryos formed only on excised cotyledons without an embryo axis or on excised embryos without the plumule and radicle of the axis. This observation suggests that the axis tip of the embryo might suppress somatic embryo production although the cotyledon tissues have predetermined embryogenic competency. To clarify the role of the embryo axis on somatic embryo formation, excised plumules or radicles were placed in direct contact with the basal cut-ends of cotyledons. The adhesion of plumules or radicles highly suppressed somatic embryo formation from cotyledon explants. When an agar block containing exudate from excised plumules or radicles was placed in contact with the cut end of the cotyledon, a similar inhibition was observed. These results suggest that embryogenic competence is suppressed by endogenous inhibitors present in the axis tip of the zygotic embryo.     

Enhanced somatic single embryo formation by plasmolyzing pretreatment from cultured ginseng cotyledons

Cotyledon explants of Panax ginseng zygotic embryos directly produced somatic embryos on Murashige and Skoog medium without growth regulators. Somatic embryos were formed only near the proximal excised region of cotyledons. Multiple and/or single embryos were formed and the frequency of these formations differed according to the degree of maturity of the zygotic embryos used as the explant source. When cotyledon explants pre-plasmolysed (1.0 M sucrose for 24 h), the frequency of single embryo formation was enhanced regardless of cotyledon maturity. In addition, the distribution pattern of somatic embryos changed markedly because the embryos were formed over the whole surface of the cotyledons. Histological observation revealed that plasmolyzing pretreatment broke the plasmodesmatal connection between cells and when the embryogenic cell divisions commenced, plasmodesmatal strands were hardly observed except for newly formed cell walls. This indicates that the enhanced single embryo formation over the entire surfaces of cotyledon explants might be the result of an interruption of cell–cell interaction by plasmolyzing pretreatment.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Somatic embryogenesis in soybean via somatic embryo cycling

Summary The objectives of the present research were: a) to develop an efficient soybean embryogenic regeneration system characterized by a high frequency of explant response and a large number of somatic embryos per explant; b) to evaluate the factors affecting somatic embryogenesis via somatic embryo cycling; and c) to identify the origin of somatic embryos in the system. A highly improved and efficient system for soybean somatic embryogenesis was established using somatic embryo cotyledons and somatic embryo hypocotyl/radicle explants plated on α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented MS basal media. The system included somatic embryo cycling between liquid and solid medium and it consistently gave rise to a much higher frequency of explant response and a larger number of embryos per responding explant than those obtained from zygotic cotyledon explant tissues. Genotype, differences were observed for response in some of the treatments with cv “Fayette” being more responsive than “J103”. Histological studies revealed that somatic embryos induced in the somatic embryo cycling system originated almost exclusively from epidermal cells on both 2,4-D and NAA inductive media. The cells of the epidermis proliferated to produce somatic embryos directly without an intervening callus phase. A single-cell origin of somatic embryos was observed in cultures on a 40 mg/liter 2,4-D treatment. A large number of responding cells in the epidermis was also observed in the 10 mg/liter NAA treatment. The single-cell origin of somatic embryos from epidermal layers of the explant tissues should facilitate development of an efficient transformation system for soybean.     

Direct and indirect somatic embryogenesis in Freesia refracta]

Somatic embryogenesis can be induced in tissue cultures of Freesia refracta either directly from the epidermal cells of explant, or indirectly via intervening callus. In direct pathway, somatic embryos were in contact with maternal tissue in a suspensor-like structure. In indirect pathway, the explants first proliferacted to give rise to calluses before embryoids were induced. The two sorts of calluses were defined to embryogenic callus and non-embryogenic callus according to producing of somatic embryos. An indirect somatic embryo is developed from a pre-embryogenically determined cell. This kind of somatic embryo has no suspensor structure instead of a complex with maternal tissue. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.     

香雪兰的直接与间接体细胞胚胎发生

香雪兰的体细胞胚胎发生可通过两种途径进行,即直接发生与间接发生。在直接发生方式中,体细胞胚直接来源于尚未完全分化的外植体表皮细胞;体细胞胚与母体组织以一种类似胚柄的结构相联系。间接发生方式中,体细胞胚的形成要经过一个愈伤组织阶段。以是否能形成体细胞胚分类,可将愈伤组织分为胚性和非胚性愈伤组织。以间接方式形成的体细胞胚是由胚性愈伤组织中的一种决定细胞发育来的。这种体细胞胚不具有类似胚柄的结构,而与母体组织共同形成一个复合体。体细胞胚具有自己独立的维管束系统,在脱离母体组织后能够独立发育成株。     

虎眼万年青的直接体细胞胚胎发生和植株再生

以虎眼万年青(Ornithogalum caudatum Jacq.)子鳞茎的鳞叶为外植体,不经过愈伤组织阶段,直接诱导体细胞胚胎发生并再生植株。组织切片和扫描电镜的观察结果表明:体细胞胚来自鳞叶表皮最外层的单个细胞。这个细胞经第一次平周分裂,成为2-细胞原胚,然后依次经过4-细胞原胚、球形胚、香蕉形胚等阶段发育成小植株,其形态学发育过程与合子胚相似。小植株移入土中,1周后即可成活。此外,还观察到直接体细胞胚只产生于鳞叶的近轴面,而远轴面无体细胞胚产生,这两个部位在细胞结构和形态上有显著差异。     

雷竹体细胞胚诱导的初步研究

以雷竹（Phyllostachys violascens）种胚为外植体, 脱分化产生愈伤组织, 愈伤组织诱导产生体细胞胚, 并发育成苗。实验结果表明： 愈伤组织诱导体细胞胚基本培养基为MS无机盐＋20 g·L^-1葡萄糖（glucose）＋10 mg·L^-1腺嘌呤（adenine sulfate）＋ 0.5 g·L^-1麦芽抽提物（malt extract）＋0.1 mg·L^-1 6-BA＋0.01 mg·L^-1氨氯吡啶酸（picloram）＋10 g·L^-1type A agar; 将基本培养基中的氨氯吡啶酸浓度升高至0.1 mg·L^-1即为愈伤组织诱导的最佳培养基, 早期子叶胚是愈伤组织诱导的最佳胚体; 愈伤组织在添加0.001 mg·L^-1TDZ和0.3 mg·L^-1ABA的愈伤组织最佳培养基上光照培养4周后, 去除其中的ABA, 并添加0.1 mg·L^-1NAA继续培养1个月, 体细胞胚数量最多可达87.43%, 该培养基是体细胞胚发生的最佳培养基; 将上述体细胞胚发生培养基中的6-BA浓度升高至1 mg·L^-1, 继代培养2个月后有小苗出现。组织学观察显示, 体细胞胚细胞核大、质浓且多数呈球形原胚状。     

Direct somatic embryogenesis and plant regeneration from ray florets of chrysanthemum

Direct somatic embryogenesis from ray floret explants of five chrysanthemum cultivars has been obtained within 12 – 15 d on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Scanning electron microscopic observation also confirmed the direct origin of somatic embryos from explants. Somatic embryos developed asynchronously on the adaxial surface of explants. Among the five cultivars tested, Birbal Sahani was best responding (40 % explants responded on 4 mg dm^–3 2,4-D and 2 mg dm^–3 BA supplemented medium). Precocious germination of somatic embryos was noticed on the same medium. The best sucrose concentration in the medium was found to be 60 g dm^–3 where 70 % explants responded while 55 % embryogenic response was obtained on medium supplemented with 400 mg dm^–3 inositol. Plants developed from somatic embryos were transferred to soil and produced true-to-type flowers.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Morphohistological analysis of the origin and development of somatic embryos from leaves of mature Quercus robur

Summary In oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent.     

Relationship between auxin-induced cell proliferation and somatic embryogenesis in culture of carrot cotyledons

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon expiants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5 μ2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5 μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5 μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Enhancement of American chestnut somatic seedling production

Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree.     

欧石楠体细胞胚发生和植株再生

以欧石楠茎段为外植体,研究其体细胞胚胎发生和植株再生。对影响茎段不定芽分化及胚性愈伤组织诱导的主导因子进行比较分析,并研究其体胚萌发、生根及移栽;同时,采用树脂切片法对茎段脱分化产生胚性愈伤组织及体胚发育过程进行组织细胞学观察。结果表明,接种在1／2WPM基本培养基上的茎段,胚性愈伤组织诱导率为88．7％,显著高于其他处理,不定芽诱导率可达90．6％,平均分化倍数为3．6个,平均分化苗高3．82cm;体细胞经过成熟培养后。在添加1．0mg·L-1 ZT和0．3mg·L-1 IBA的1／2WPM培养基上萌发,萌发的体胚在I／2WPM附加0．2mg·L-1 NAA和0．3mg·L-1 IBA的培养基上形成完整的体胚苗植株,体胚苗生根率达到87．4％,经炼苗后移栽到蛭石：珍珠岩=3：1（V／V）的栽培基质中,成活率可达63．7％。在显微镜下可观察到球形胚、心形胚、鱼雷形胚和子叶形胚;体细胞胚以间接方式发生,表现为愈伤组织外层细胞直接发生和愈伤组织组织内部细胞发生。     

Somatic embryo cycling: Evaluation of a novel transformation and assay system for seed-specific gene expression in soybean

Somatic embryo cycling, a modification of soybean somatic embryogenic suspension culture, was developed as an efficient and rapid method of producing tissue suitable for stable transformation of soybean germplasm by biolistic particle bombardment. Instead of using immature seed explants, cotyledon-staged somatic embryo hypocotyls were placed on auxin-containing medium, where they initiated new somatic embryos primarily from single epidermal cells. By bombarding hypocotyls prior to initiation of subsequent embryo formation, we have effectively transformed soybean somatic embryos with the reporter genes neomycin phosphotransferase,gb-glucuronidase, and a mammalian stearyl CoA delta-9 desaturase, controlled by a seed-specific promoter. These embryos contain significantly reduced levels of saturated palmitic and stearic fatty acids, and significant amounts of monounsaturated palmitoleic acid, which is not normally abundant in soybean seeds. This study demonstrates the effectiveness of somatic embryo cycling for soybean transformation, and for testing expression of genes for seed-specific proteins. Abnormal flower development in recovered plants is a limitation for application of the technique to produce transgenic seed at present.