Recurrent somatic embryogenesis and development of somatic embryos in Akebia trifoliata (Thunb.) Koidz (Lardizabalaceae)

Plant Cell, Tissue and Organ Culture (PCTOC) - A simple and effective protocol was established for recurrent somatic embryogenesis and plant regeneration in Akebia trifoliata (Thunb.) Koidz....     

Ovule morphology, embryogenesis and seed development in three Hylocereus species (Cactaceae)

Pre-embryonic and embryonic stages and seed developments were studied in the diploids Hylocereus monacanthus and Hylocereus undatus and the tetraploid Hylocereus megalanthus. Ovule morphology was similar among species except for micropyle entrance. H. monacanthus had the thickest and most robust suspensor. Embryo developmental time, measured from fertilization to maturity, was significantly more prolonged in H. megalanthus. Typical to Cactaceae, the seed coat was formed by one layer of sclerenchymatous cells, but was more lignified in H. megalanthus. Morphological features common to all species included (1) cellular type endosperm with independent patterns of development in the chalazal and micropylar zones, forming a haustorium layer from the chalazal zone to the embryo; (2) an endothelial layer surrounding the embryo sac almost complete; (3) a nucellar summit growing into the micropyle; and (4) a placental obturator and a funicle connecting the ovarian tissue to the ovule. Seed development was typically endospermic (exendospermic orthodox seeds). Anomalies included two egg cells in the same embryo sac, two embryos developing in the same ovule, and embryos developing from the chalazal pole region. Total seed number and seed viability were significantly lower in H. megalanthus than in the other two taxa. Embryos at different developmental stages were observed in aborted H. megalanthus seeds.     

The relationship between seed dormancy,seed size and weediness,in Crepis tectorum (Asteraceae)

Summary I examined the germination characteristics of weed and outcrop populations of Crepis tectorum to test the hypothesis that the presumably more ephemeral weed habitat favors the highest levels of seed dormancy. The winter annual habit characterizing most plants of this species was reflected in a rapid germination of seeds sown in late summer. A slightly higher fraction of surface-sown seeds of weed plants delayed germination. Buried seeds of weed plants also survived better than seeds produced by plants in most outcrop populations, supporting the idea that weediness favors seed dormancy and a persistent seed bank. However, the differences in seed dormancy between the two ecotypes were small and not entirely consistent. Furthermore, high levels of seed dormancy were induced during burial in the outcrop group, suggesting that there is a potential for a dormant seed population in this habitat as well. Demographic data from one of the outcrop populations verified the presence of a large between-year seed bank. Possible environmental factors favoring seed dormancy in outcrop populations are discussed. The unusually large seeds of weedy Crepis contrasts with the relatively small difference in seed dormancy between the two ecotypes.     

High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature,dry seed

Peanut (Arachis hypogaea L.) somatic embryos were produced from the embryo axes of mature, dry seeds of cultivar GK-7. Percent embryogenic explants ranged from 88–100% using 10–40 mg/1 of 2,4-D in the induction medium. Neither 2,4-D concentration nor photoperiod during the induction period had a large effect on percent embryogenesis, mean number of embryos per explant, or embryo morphology. However, embryos obtained from cultures grown in the dark were easier to remove from the explant than those under a 16-h photoperiod. Somatic embryos developed on the epicotyl portion of the embryo axis, primarily on the young, expanding leaves. A survey of 14 genotypes indicated that genotype had a large influence on embryogenic capacity, with all genotypes being embryogenic to some extent. The ability to recover somatic embryos from axes of harvested, stored seeds represents significant advantages for the establishment of peanut embryogenic cultures, including the use of simple sterilization procedures and a constant source of explant tissue.Abbreviations B5 medium of Gamborget al. (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts medium     

A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants

Summary In order to improve the late phases of Theobroma cacao L. embryogenesis from tissues of maternal origin, zygotic embryogenesis and somatic embryogenesis were compared, with respect to morphological, histological, and physiological parameters. Zygotic embryogenesis could be divided into three steps: (a) embryogenesis sensu stricto, (b) a growth period in which cotyledonary embryos reached their final dimensions, and (c) a maturation period in which embryos accumulated protein and starch reserves, dehydrated to a water content equal to 30%, and underwent a modification in soluble sugar composition. Monosaccharides and sucrose contents decreased to the benefit of the oligosaccharides raffinose and stachyose. The formation of somatic embryos by use of basic protocols was studied to define the limiting factors that could lie behind their poor development. Morphological abnormalities of somatic embryos, which represented 80% of the total population, were described. A histological study showed that somatic embryos lacked starch and protein reserves; moreover, their water content was much higher than that of their zygotic counterparts. Introducing a growth period into the culture protocol made for better embryo development. Adding sucrose and abscisic acid to the maturation medium was effective in increasing reserve synthesis and resulted in higher germination, conversion, and acclimatization rates.     

Totipotency, somatic embryogenesis, and Harry Waris (1893–1973)

F. C. Steward and Jakob Reinert in the late 1950s, independently and with different degrees of scientific exactness, demonstrated that somatic cells of cultivated carrot can produce embryo-like structures in aseptic culture. Growth substances in the nutrient medium were viewed as central to the process. The now classic papers of Steward and Reinert have found a special and enduring place in the literature of plant development. But Harry Waris also deserves credit for his observation that vegetative cells sloughed off from aseptically germinated seedlings reared in liquid nutrient medium can produce 'embryos'. In his studies, seedlings of Oenanthe aquatica (Umbelliferae) were maintained in culture for protracted periods under nutrient conditions designed to foster imbalance in protein metabolism, but without exogenous growth hormones. Seedlings placed in media with high concentrations of glycine grew normally for 3–4 months; after this a "period of morbidity" occurred, followed by production of new plants from the root tips. These new plants, later called "neomorphs", in turn reproduced by colorless outgrowths of leaf epidermis. Such outgrowths, and "nodules" formed in a callus produced by the original seedlings, passed through stages described as "nodule", "fusiform", and a stage with two or more "lobes". Transfer of the neomorphs to a medium lacking glycine resulted in the development of normal plants. We show that Waris was among the first, if not the first, to observe and recognize somatic embryo production in aseptic culture, and indeed to call them "embryos". We also discuss his investigations in the context of understanding development at the cellular level, then and now.     

Embryology in Norway spruce (Picea abies). An analysis of the composition of seed storage proteins and deposition of storage reserves during seed development and somatic embryogenesis

Seed cones were collected from open-pollinated trees of Norway spruce ( Picea abies ) in a seed orchard from pollination until maturation of the seeds. Immature embryos were isolated for embryogenic tissue cultures that were maintained either on solidified medium or as liquid cultures. By transferring young somatic embryos to medium containing 90 m M sucrose and 7. 6 μ M ABA growth continued to mature embryos that accumulated storage reserves in both the hypocotyl-shoot axis and the cotyledons. Both zygotic and somatic embryos at different developmental stages were processed for microscopy as were the megagametophytes. Total protein was extracted from the seed material at intervals during development and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These analyses revealed that storage protein started to accumulate in the megagametophytes at the time when embryos were growing into the gametophytic tissue, while it occurred a few weeks later in the embryos at rapid embryo growth and organ differentiation. Lipid bodies also became abundant in the mature plant material. Although plastids with prominent starch grains were very frequent in both megagametophytes and embryos during development they were not observed in the desiccated tissues. Zygotic and somatic embryos displayed a similar developmental pattern.
By sequential salt-extraction and dilution two fractions highly enriched in storage protein were obtained. One fraction (G-1), requiring higher salt concentration for protein solubilization, was dominated by a protein migrating to around M, 55000–60000 when separated under non-reduced condition. After exposure to reducing agent this protein was replaced by two new ones with M, 33000 and 22000 giving evidence of disulfide bonded polypeptides. The other fraction (G-2), was dominated by polypeptides around M, 42000 and low molecular mass polypeptides (<14000).     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Asexual reproduction,regeneration, and somatic embryogenesis in the planarian Dugesia tigrina (Turbellaria)

In reviewing recent research published in Russian on regeneration and asexual reproduction, the following morphogenetic processes in the planarian Dugesia tigrina are considered: 1) regeneration of lost parts of the body; 2) regeneration of the whole worm from fragments of the body, either by normal regeneration when the inital polarity of the fragment is retained or by somatic embryogenesis when one or more new axes of polarity arise; 3) somatic embryogenesis, or development of individuals from somatic cells; 4) hypermorphosis, or the presence of more than the usual number of organs or body parts, a process that can be interpreted in terms of somatic embryogenesis; and 5) asexual reproduction. Some morphological, biochemical, and physiological studies of the division zone in D. tigrina demonstrate peculiarities of a local breakdown of integrative functions, a breakdown which in turn causes division of the individual to take place at this zone; timing of division is controlled by the organism as an integrated whole.     

High frequency somatic embryogenesis and synthetic seed production of the endangered species Swertia chirayita

An efficient protocol for plant regeneration through somatic embryogenesis was established from in vivo leaf explants of Swertia chirayita, a critically endangered medicinal herb. The highest frequency (76%) of embryogenic callus was induced on Murashige & Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (Kn) from in vivo leaf explants. Globular somatic embryos were induced and further matured from such embryogenic calli by subsequent culture on the same medium. The highest number of somatic embryos (48.83 ± 4.6) was recovered from embryogenic calli derived from leaf explants after 6 weeks of culture. Synthetic seeds were produced by encapsulating of torpedo stage embryos in sodium alginate (4% W/V) gel, dropped into 100 mM calcium chloride (CaCl₂ · 2H₂O) solution. The synthetic seeds were germinated on MS medium. The highest frequency of synthetic seed germination (84%) was observed on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA. Regenerants were successfully acclimatized under ex vitro condition. This is the first report on synthetic seed production of S. chirayita. Application of these protocols would be helpful in reducing stress in natural habitat, and in long-term storage of elite genotypes through synthetic seed production.     

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis

Summary Immature embryos from 5 Cladrastis lutea (Michx.) K. Koch (yellowwood) trees were initially cultured on modified Schenk and Hildebrandt medium (SH) containing either 4.5, 9.0, 13.5 or 23 M 2,4-D. One-third of the explants were transferred to SH medium supplemented with 25.0 M NAA after 2 and 3 weeks respectively. The remaining explants were incubated on the initial 2,4-D containing media for 6 weeks. Groups of somatic embryos formed directly only at the proximal end of cotyledons; only a few formed as single embryos. The greatest numbers were formed from zygotic embryos explanted from 6–8 weeks post-anthesis and initially cultured on medium containing 9 or 13 M 2,4-D. However, all treatments supported somatic embryogenesis. In the second year, explants were initially cultured on SH medium containing either 9.0, 13.5, or 23 M 2,4-D and then transferred to SH medium containing 4.0 M ABA after 2 or 3 weeks. ABA did not affect the development of somatic embryos. Six of 21 somatic embryos germinated on half-strength SH medium without growth regulators. Three entire plantlets were formed, but only one was established in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - CRAF III chromium trioxide-acetic acidformalin     

Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.)

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5–7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 μm 2,4-dichlorophenoxyacetic acid and 0.93 μm kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology. Received: 29 December 1997 / Revision received: 9 April 1998 / Accepted: 24 April 1998     

Adventitious shoot induction and somatic embryogenesis with intact seedlings of several hybrid seed geranium (Pelargonium x hortorum Bailey) varieties

Summary Intact seedlings of hybrid seed geranium (Pelargonium x hortorum Bailey) were tested for their ability to produce adventitious shoots and somatic embryos by direct culture of mature seeds on Murashige and Skoog (1962) medium (MS) supplemented with growth regulators BAP, BAP + IAA, or thidiazuron (TDZ). Ten varieties were tested in the presence of different BAP concentrations, four with BAP + IAA, and two with TDZ. Varieties used in this study differed in their response to BAP in the medium. Multiple adventitious shoots were produced by seven of the ten varieties tested. Multiple adventitious shoots were induced at all levels of TDZ in the medium. TDZ also induced callusing from roots and direct embryogenesis from intact hypocotyls. Adventitious shoots were separated, rooted and transferred to soil where they grew as normal healthy plants and flowered.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - TDZ thidiazuron     

柑橘愈伤组织生长速度与体细胞胚胎发生的关系

为了探讨柑橘愈伤组织不能再生的原因,试图寻找柑橘愈伤组织生长速度与其体细胞胚胎发生之间的关系,对7种柑橘类型的29种基因型的愈伤组织的生长速度进行了测定,并对愈伤组织生长速度与体细胞胚胎发生之间的相关性进行了统计分析。结果表明,柑橘愈伤组织生长速度与体细胞胚胎发生之间的相关系数为r=-0.3683。由此推断在这两者之间还存在其它影响因素。     

Regeneration via somatic embryogenesis of the endangered wild arum (Arum palaestinum)

Somatic embryogenesis was obtained from callus of wild arum (Arum palaestinum). Callus was induced from sterilized corm bud sprouts cultured on basal medium containing 4.4???M 6-benzyladenine and 5.4???M 1-naphthaleneacetic acid. Callus was maintained under dark conditions using basal medium with 4.4 or 8.8???M 6-benzyladenine and 5.4 or 10.8???M 1-naphthaleneacetic acid. The highest callus weight and most desirable callus phenotype were achieved using basal medium containing 8.8???M 6-benzyladenine and 5.4???M 1-naphthaleneacetic acid. Friable calli were cultured in the dark on basal medium containing 4.5???M 2, 4-dichlorophenoxyacetic acid, 0.46???M 6-furfurylaminopurine, 5.4???M 1-naphthaleneacetic acid, and 1.7?mM proline to induce embryogenesis before transfer to regeneration medium. Embryos that developed on regeneration medium were transferred to medium minus plant growth regulators for germination. Ninety percent of the germinating embryos developed into rooted plantlets. Rooted plants were grown in the greenhouse and acclimatized successfully with a 95?% survival rate. This is the first report of successful somatic embryogenesis and plant regeneration in A. palaestinum.     

防风体细胞胚发生和发育的组织学研究

采用石蜡切片和半薄切片法研究组织培养中防风体细胞胚发生和发育的形态和结构特征,以了解体细胞再生形成胚胎结构的细胞分化和形态发生特征。结果表明:(1)胚性细胞内含有丰富的淀粉体,随细胞分裂和分化逐渐减少;(2)体细胞胚的发生发育经历了类似合子胚的各阶段;(3)多细胞原胚形成的分裂方式以平周分裂为主,并由此表现出有序生长;(4)在体细胞胚迅速发育过程中,不同阶段的体细胞胚始终与周围细胞存在明显界限;(5)胚柄发育不明显;(6)畸形胚常见,其中连体胚是胚性细胞发生密度过高造成的,而次生胚则在原来胚的基础上又产生新的胚的发生中心。     

Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz)

Summary In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium.Abbreviations BM Basal Medium - MIE medium volume per initial embryo - E/IE number of Embryos per Initial Embryo     

Induction of somatic embryogenesis and embryo development in Rumex acetosella L.

Axillary buds of the dioecious plant Rumex acetosella L. were isolated and cultured in vitro. The callus tissue which developed at the basal parts of the explants displayed a high capacity for shoot formation. This morphogenetic pattern was predominant on Murashige and Skoog (MS) medium supplemented with 2% sucrose, 2.2 mgl^-1 benzylaminopurine and 0.17 mgl^-1 indole-3-acetic acid. Somatic embryogenesis was induced when the osmolality of the medium was increased by adding 6% sucrose instead of 2%, or hexitols in addition to 2% sucrose. Most of the embryogenic calli were formed on the basal parts of leaf laminae and bracts. Development and maturation was strongly promoted by transferring the tissue to a solid or liquid medium lacking benzylaminopurine and indole-3-acetic acid and supplemented with 10 mgl^-1 gibberellic acid. The embryos germinated and developed into normal rosette plants when transferred to vermiculite moistened with hormone-free, half-strength MS salt solution. The histology of successive embryogenic stages is presented.