Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

Long-term somatic embryogenesis and maturation of somatic embryos in Hevea brasiliensis

A high frequency of secondary embryogenesis was induced from isolated early cotyledonary-stage somatic embryos of Hevea brasiliensis. A long-term embryogenic line was established by the use of recurrent embryogenesis and maintained for 3 years on hormone-free medium by the transfer of selected proembryogenic masses every 10 days.

The addition of 234 mM sucrose as stress with sucrose and 10⁻⁵ M abscisic acid (ABA) to the culture medium enhanced the maturation of somatic embryos. Under these culture conditions, the embryo population was composed of 45% globular, 18% oblong and 37% torpedo-stage embryos. These somatic embryos had well-formed tissue structure, a well-defined epidermis, protein storage bodies, and a high accumulation of starch. The triglyceride content was five times as high in the torpedo-stage embryos that developed on medium supplemented with 234 mM sucrose and 10⁻⁵ M ABA as in embryos obtained on basal medium with 58 mM sucrose.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

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.     

Embryogenic cultures of the leguminous tree Albizia julibrissin and recovery of plants

A somatic embryogenic system was developed and plants regenerated in mimosa (Albizia julibrissin Durazz). Development of somatic embryos in the species has not previously been reported. Immature seeds, embryo cotyledons and embryo axes (cotyledons removed) at defined developmental stages were placed on induction media with different concentrations of 2,4-D. Two distinct embryogenic responses occurred: either proembryo masses or cotyledonary-stage embryos. Twenty five percent of all embryo axes cultured on basal medium produced cotyledonary somatic embryos. Six percent of in ovulo immature seed explants generated proembryo masses. These masses proliferated in liquid culture in the dark. Proembryos developed further when transferred to a growth-regulator-free semisolid medium in the light. Somatic embryos derived from either proembryo suspensions or cotyledonary embryo cultures on semisolid medium germinated to form plants that continued to grow vigorously following transfer to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Development and germination of American chestnut somatic embryos

American chestnut (Castanea dentata (Marsh.) Borkh.) plants were regenerated from developing ovules through somatic embryogenesis. On an initiation medium containing 18.18 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine (BA), 25 out of 1,576 ovules were induced to form proembryogenic masses (PEMs). These PEMs were cultivated on a development medium for 4 weeks. Individual somatic embryos were then grown on a maturation medium for at least one month, until shoot meristems and radicles were developed. Both development and maturation media consisted of Gamborg's B-5 basal medium, 0.5 μM BA, and 0.5 μM α-naphthaleneacetic acid, but the former contained 20 g l⁻¹ sucrose and the later contained 60 g l⁻¹ sucrose. A range of 86 to 586 embryos per gram PEMs was observed beyond the cotyledonary stage. These embryos then germinated, resulting in plantlets with a 3.3% conversion rate. An additional 6.3% of the mature embryos produced shoots, which could also result in plantlets by rooting of microcuttings. Proembryogenic masses that were established in continuous culture and maintained on initiation medium for 17 months retained regenerability, though the embryo yield decreased over time. Twenty plantlets were acclimatized and grown in potting mix in a greenhouse. The largest 6 were transplanted, along with seedling controls, into a nursery bed in 1997. As of July, 1999, 4 out of the 6 were surviving. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histological study of somatic embryogenesis induction on zygotic embryos of macaw palm (<Emphasis Type="Italic">Acrocomia aculeata</Emphasis> (Jacq.) Lodd. ex Martius)

The aim of this paper was to describe the histological events that led to somatic embryogenesis in macaw palm (Acrocomia aculeata (Jacq.) Lodd. ex Martius). Zygotic embryos were inoculated on Y₃ medium containing 9 μM 4-amino-3,5,6-trichloropicolonic acid (picloram). Somatic embryos regenerated from nodular callus on induction medium with activated charcoal under photoperiod or without activated charcoal under dark. Many proembryos originated from the fundamental meristem after 10–20 days of culture. When transferred to medium containing activated charcoal, under photoperiod, calli regenerated into somatic embryos of unicellular origin. These embryos had protoderm, plumule and procambial strands and some of them could germinate. After 30–40 days of culture, meristematic masses grew from procambial cells. The masses generated nodular callus, and after transfer to medium without activated charcoal, under dark, they generated somatic embryos of multicellular origin. Those embryos did not regenerate into plants.     

Maturation of avocado somatic embryos and plant recovery

Avocado proembryonic masses from suspension cultures were used to develop a protocol for somatic embryo development and maturation. Avocado somatic embryos could develop from proembryonic masses both in liquid and on semisolid medium but only the latter could develop to maturity. Size and number of opaque somatic embryos were affected by gellan gum concentration, with the optimum response obtained on medium supplemented with 6–7 g l⁻¹ gellan gum. The optimum sucrose concentration for recovery of opaque somatic embryos was 90 g l⁻¹; however, the development of embryos was suppressed at this concentration. Consequently, recovery of cotyledonary, opaque somatic embryos was achieved on medium with 30 g l⁻¹ sucrose. Somatic embryo development from dedifferentiating proembryonic masses required media with a high ratio of NO ₃ ⁻ :NH ₄ ⁺ (1:0 and 3:1) as opposed to the standard ratio (2:1) of MS medium. Germination of somatic embryos was sporadic. In order to increase the frequency of plant recovery, shoots that developed from somatic embryos were micropropagated using standard protocols. This revised version was published online in June 2006 with corrections to the Cover Date.     

Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens

Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.     

Somatic embryogenesis in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Embryonal-suspensor masses from immature embryos from cones of Sitka spruce (Picea sitchensis (Bong.) Carr.) proliferated on a modified Murashige & Skoog medium with N⁶-benzyl-aminopurine, kinetin, 2,4-dichlorophenoxyacetic acid and an organic nitrogen source. The slimy white embryonal-suspensor masses with proembryos were maintained on a solid proliferation medium with reduced amounts of growth regulators. Transfer of embryonal-suspensor masses to a non-woven polyester carrier with liquid maturation media containing ±2-cis-4-trans-abscisic acid and a reduced amount of inositol and organic nitrogen resulted in synchronized embryo formation. Further development was achieved on a medium without ±2-cis-4-trans-abscisic acid and organic nitrogen. Somatic embryos were successfully transferred ex vitrum.Abbreviations ABA ±2-cis-4-trans-abscisic acid - BAP N⁶-benzyl-aminopurine - ESM embryonal-suspensor masses - KIN kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid     

A simple method for the recovery of multiple fertile plants from individual somatic embryos of soybean [Glycine max (L.) merrill]

Summary We describe a method for obtaining and proliferating multiple, fertile plants from somatic embryos of several experimental and commercial soybean varieties. Shoot-bud cultures were initiated by placing cotyledonary and torpedo-stage somatic embryos derived from immature seedling cotyledons onto Cheng’s basal medium (CBO) containing 0.5 to 2.5 mg/liter 6-benzyladenine (6-BA). Prolific masses of adventitious shoots were produced within 6 to 18 wk. These cultures can be propagated indefinitely with regular subcultures to CBO containing 0.5 mg/liter 6-BA. Individual shoots were separated from the clusters and were rooted on CBO medium without exogenous growth regulators. By this method any number of plants can be produced from individual somatic embryos. The risk of losing valuable genotypes (e.g., derived from in vitro selection or transformation) due to inefficient embryo germination and embryo-to-plant conversion is thus greatly reduced. Plants were established in the greenhouse and progenies were field tested. Progenies from shoot-bud culture-derived plants showed no somaclonal variation for the seven recessive marker traits or quantitative agronomic characters evaluated under field conditions.     

Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.)

Summary Embryogenic masses were obtained from immature leaves of peanut (Arachis hypogaea L.) cultured on a medium containing 20 mg/l 2,4-D. Somatic embryos developed from these masses following transfer to a medium containing 3 mg/l 2,4-D. The embryo morphology was quite variable. Following transfer to hormone-free medium, these embryos germinated. Shoot elongation was obtained in 25% of the embryos following transfer to a medium supplemented with 0.5 mg/l each of BAP and Kn. The plants grown in vitro by this method survived in sand:soil mixture and were grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzyl amino purine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - Kn kinetin - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - Z zeatin     

Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis

Leaf explants of Phalaenopsis amabilis var. formosa formed clusters of somatic embryos directly from epidermal cells without an intervening callus within 20 – 30 d when cultured on 1/2-strength modified Murashige and Skoog medium supplemented with 0.1, 1 and 3 mg dm⁻³ TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium.     

湿地松体细胞胚胎发生和植株再生

以湿地松的未成熟合子胚为外植体,在附加８ｍｇ／Ｌ,２,４－Ｄ和４ｍｇ／Ｌ ＢＡ的ＬＰ培养基上诱导出胚性愈伤组织。在含１ｍｇ／Ｌ,２,４－Ｄ和０．５ｍｇ／Ｌ ＢＡ的培养基上保持并增殖。提高培养基的渗透压后,愈伤组织内大量的胚性胚柄细胞团和早期原胚。     

Isolation and culture of cereal protoplasts

Summary Protoplasts isolated from embryogenic suspension cultures derived from immature embryos of pearl millet (Pennisetum americanum) gave rise to cell masses. These cell masses upon transfer to a hormone-free medium formed embryoids, which further developed into plantlets with roots and shoots.     

Somatic Embryo Formation From Unemerged Inflorescences and Immature Embryos of a Graminaceous Crop Echinochloa

Formation of somatic embryos was dependent on concentrationof specific auxin and mineral nutrient formulation. On N₆ mediumwith low levels of 2,4-D somatic embryos were obtained fromunemerged inflorescences and immature embryos. Direct differentiationof somatic embryos, a rare feature of regeneration in graminaceousplants, was more apparent from immature embryos than from inflorescences.On the other hand, on MS medium with different levels of 2,4-Dcompact callus-like masses appeared which regenerated to formplantlets on auxin-free medium. At higher levels of 2,4-D andalso on N₆ medium compact tissues (morphogenic calli) appearedwhich were made up of thallus-like structures. Echinochloa, immature embryo, unemerged inflorescence, somatic embryo     

High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine

Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay' somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development did not advance beyond the heart stage in `Thompson Seedless' suspension cultures. Highly synchronized development of somatic embryos was obtained by inoculating <960-μm PEMs into liquid medium without 2,4-D. Somatic embryos were also produced in large numbers from suspension-derived PEMs of both cultivars on semisolid medium lacking 2,4-D. Somatic embryos matured and regenerated into plants in MS basal medium containing 3% sucrose. Using this method more than 60% of the somatic embryos regenerated plants. More than 90% of the regenerated plants were successfully transferred to the greenhouse. Received: 27 July 1998 / Revision received: 15 October 1998 / Accepted: 27 October 1998     

Somatic embryogenesis and plant regeneration in Acanthopanax senticosus

Mature embryos of Acanthopanax senticosus explanted on Murashige and Skoog (MS) medium with 0.5 mg/1 2,4-D developed somatic embryos directly from swollen cotyledon and embryo axes within one to two months. When the somatic embryos were transferred to medium supplemented with 2,4-D (0.5 mg/1) or IAA (1–3 mg/1) or Zeatin (0.5 mg/1) and NAA (0.2 mg/1), additional somatic embryos developed. Most (93%) embryos germinated on the above medium without 2,4-D. Sixty-two percent of the plantlets survived in soil. Histological observations revealed that the somatic embryos originated from cell masses of epidermal and sub-epidermal origin. There was no cytological separation zone between the somatic embryos and cultured expiants. Consequently, embryos were difficult to separate from their expiant tissue.