Somatic embryogenesis from immature embryos of redbud (Cercis canadensis)

Somatic embryos developed directly from 96 and 110 day post-anthesis Cercis canadensis L. (redbud) zygotic embryos from one of two trees sampled that were explanted onto modified Schenk and Hildebrandt medium amended with either 1, 2, 3 or 5 mg/1 2,4-D in combination with either 7.6 or 12. 6 mM ammonium ion. Although somatic embryogenesis was expressed on most media, the number of explants that produced somatic embryos and the mean number of embryos formed per explant were greatest on media that contained either 2 or 3 mg/1 2,4-D; 12.6 mM ammonium ion inhibited embryogenesis from 96 day post-anthesis explants. Zygotic embryos explanted 117 days after anthesis produced only callus and roots. Somatic embryos that were bottle-shaped or had distinct cotyledons organized roots on germination media, but only one embryo formed a shoot. No additional development occurred. Histological examination of somatic embryos showed that shoot apical meristems were poorly developed.Abbreviations 2,4-D 2, 4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - FPA Formalin-propionic acid-ethanol (50%)     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Relation of the developmental stage of zygotic embryos of yellow-poplar to their somatic embryogenic potential

The goal of the study was to characterize the optimal developmental stage of zygotic embryo expiants of the hardwood forest tree species yellow-poplar (Liriodendron tulipifera L.) for the initiation of embryogenic cultures, using morphological measurements and polypeptide profiles of the embryos. Developing zygotic embryos from seeds of six full-sib families, collected every two weeks from 4 weeks postpollination until seed maturity (18 weeks postpollination) were divided into 2 subsamples for each collection date. One group was used to initiate tissue cultures. Embryos in the other group were measured (total length, cotyledon length and hypocotyl thickness) and soluble polypeptide profiles of the embryos were analyzed by SDS-polyacrylamide gel electrophoresis. Potential of an expiant to produce an embryogenic culture peaked during the eighth week following pollination, with an average of 28% of the expiants producing proembryogenic masses, and declined to near zero for mature zygotic embryos. The maximum embryogenic potential corresponded to the globular stage of embryo developmet. Soluble protein profiles of zygotic embryos from 5 sampling dates indicated that decline in embryogenic potential appeared to parallel an increase in the level of a polypeptide of approximately 55 kDa, possibly a storage protein.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - CH Casein hydrolysate - SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis - PEM Proembryogenic mass     

Single-cell origin and development of somatic embryos in Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce)

The origin and development of somatic embryos in calli initiated from immature zygotic embryos of Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce) was studied. Immature zygotic embryos cultured on callus induction medium produced two types of white calli that were phenotypically different from one another. The callus that proliferated from the hypocotyl region was white to translucent, glossy, mucilaginous and embryogenic. The callus mass which originated from the radicle end was reddish-white, nonmucilaginous and nonembryogenic. Whole mount preparations of the entire explant with two different types of calli showed the presence of embryogenic cells in the mucilaginous callus mass derived from the hypocotyl region of the zygotic embryo. The origin of somatic embryos in both Norway and white spruce could be traced to single cells of the hypocotyl callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Nodule culture and regeneration of Eucalyptus grandis hybrids

Callus of three superior Eucalyptus grandis hybrids was induced from immature inflorescences, floral parts, shoot tips, zygotic embryos, and hypocotyl explants on various auxin (2,4-D or NAA) and cytokinin (kinetin) supplemented media. Hypocotyl callus initiated on 4 mg/l NAA and 1 mg/l kinetin formed massive nodular structures, and shoots and roots after four weeks on hormone free-medium. Callus from all other expiants turned brown and died upon transfer to hormone free or reduced hormone media. The nodular structures originating from hypocotyl-callus were maintained by subculture for over three years and retained the ability to form thousands of shoots. Shoots were successfully rooted (98% rooting) and plantlets developed were transferred to mist-greenhouse and then to greenhouse conditions with 95% survival. Plantlets were grown for six months in the greenhouse without sign of abnormal growth.Abbreviations NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - MS Murashige and Skoog Medium (1962) - IBA indolebutyric acid     

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.     

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。     

In vitro somatic embryogenesis and plant regeneration of cassava

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4–16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA₃, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Plant regeneration via somatic embryogenesis in the seeded diploid banana Musa ornata Roxb.

Somatic embryos of a seeded diploid ornamental banana (Musa ornata Roxb.) were obtained from zygotic embryos cultured on semi-solid Murashige and Skoog (MS) (1962) medium with the auxin 2,4-D (0.5, 1, 2 mg/l) and 5% CW. Removal of 2,4-D and transferral to Schenk and Hildebrandt (SH) (1972) salts with CW followed by basal MS led to embryo germination and growth. Plantlet production was obtained using filter paper bridges in liquid half-strength SH medium with 1% sucrose. The remarkable phenotypic fidelity of somatic embryos to that of zygotic embryos and the presence of a haustorium-like outgrowth on the somatic embryos is described.     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).     

马尾松成熟合子胚的体细胞胚胎发生和植株再生

采用马尾松（Ｐｉｎｕｓｍ ａｓｓｏｎｉａｎａ Ｌａｍ ｂ．）成熟合子胚为起始外植体,在含２,４－Ｄ １０ ｍ ｇ／Ｌ,ＫＴ和ＢＡ 各４ ｍ ｇ／Ｌ的ＤＣＲ培养基上得到胚发生培养物。将白色半透明的愈伤组织（含早期原胚）在含２,４－Ｄ１．０ ｍ ｇ／Ｌ,ＫＴ和ＢＡ 各０．４ ｍ ｇ／Ｌ的ＤＣＲ培养基上保持并增殖。在附加９０００ ｍ ｇ／Ｌ肌醇的ＤＣＲ高渗培养基上得到粗壮的后期原胚。ＡＢＡ 和活性炭同时使用能促进子叶胚的形成,最高频率为３５．１％ 。在无激素培养基上,成熟体细胞胚萌发并进一步形成完整小植株     

Plant regeneration from hordeum spontaneum and hordeum bulbosum immature embryo derived calli

Callus cultures were initiated from isolated immature embryos of Hordeum spontaneum and Hordeum bulbosum on MS or B5 basal medium supplemented with 2 mg/1 2,4-D. Shoot regeneration occurred on transfer of tissue to media containing 1 mg/1 IAA and 1 mg/1 zeatin. The regenerated shoot buds were rooted on basal medium without hormones. The in vitro regenerated plants were transferred to soil and were grown to fertile mature plants. A low percentage of albino plants was observed among the regenerated plants. No major differences were detected between the two species in respect to their potency to form callus or to the regeneration capacity. The regeneration capacity of calli decreased gradually and ended after 6 months in culture.Abbreviations IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - MS Murashige and Skoog medium     

Somatic Embryogenesis and Plantlet Regeneration from Callus of Mature Zygotic Embryos of Masson Pine

Embryogenic cultures were initiated from mature zygotic embryos of masson pine (Pinus massoniana Lamb. ) on DCR medium supplemented with 2, 4-D 10 mg/L, KT and BA each at 4 mg/L. Pale and translucent calli with early stage proembryos were maintained and multiplicated on DCR medium supplemented with 2, 4-D 1.0 mg/L KT and BA each at 0.4 mg/L. Robust late-stage proembryos were obtained when the calli were cultured on DCR medium containing 9000 mg/L myo-inositol. Abscisic acid and activated charcoal promoted the formation of cotyledonary embryos at the highest frequency of 35.1 %. Mature somatic embryos could germinate and develop further into plantlets when they were isolated and cultured on a hormone-free DCR medium.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

Apical proliferation of embryogenic tissue of soybean [Glycine max (L.) Merrill]

Somatic embryos and embryogenic tissues were initiated from immature zygotic embryos of soybean [Glycine max (L.) Merrill cv. Fayette]. Zygotic embryos were placed on a medium containing 40 mg/l of 2,4-dichlorophenoxyacetic acid and 6% sucrose. Somatic embryos were first seen 4 weeks after cultures were initiated. Following transfer, secondary somatic embryos proliferated directly from the apical or terminal portions of the older primary somatic embryos. Single somatic embryos or clusters of embryos were seen growing directly from the top of older somatic embryos. Light microscopy revealed that these embryos were of surface or subsurface origin. The apical soybean somatic embryo tissue may represent cotyledonary tissue (which has been shown to be most responsive) at a very young and manipulatable state.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid Salaries and research support were provided by state and federal funds appropriated to OARDC-OSU. Journal Article No. 131-87     

Somatic embryogenesis and plant regeneration from immature embryo explant of papaya (Carica papaya L. cv. washington and honey dew)

Immature zygotic embryo explants of Carica papaya were cultured on MS medium supplemented with 2,4-D (2.0 mg/l) and formed globular embryos on explants without callus formation in 4-6 weeks. Maturation and conversion of somatic embryos was also achieved on the same medium. Cotyledonary stage embryos germinated to 63.66 and 68.33% in cv. honey dew and washington respectively in MS basal medium supplemented ABA (0.5 microm/l). Robust development and proliferation of plantlet roots in vitro was obtained on MS basal medium. Hardened plantlets have 60% survival rate.     

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     

Plant regeneration via somatic embryogenesis in pea (Pisum sativum L.)

Whole plant regeneration via somatic embryogenesis was obtained in pea (Pisum sativum L.) using explants from immature embryos or shoot apex segments. The induction of somatic embryos required picloram or 2,4-D. Germination of fully-developed embryos was accomplished by subculture on medium with only cytokinin and then on medium supplemented with cytokinins in combination with a reduced auxin concentration. Plantlets obtained from both zygotic embryos and shoot apices were transferred to soil and were grown to maturity. Nine plants were examined cytologically, revealing three tetraploids (2n=4x=28) and six diploids (2n=2x=14).Abbreviations Picloram 4-amino-3,5,6-trichloropicolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - IBA indole-3-butyric acid KAES Journal Article No. 87-3-4     

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