Somatic embryogenesis and plant regeneration from immature embryos of western larch

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21–93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - IBA indole-3-butyric acid - ABA abscisic acid     

Callus induction and plant regeneration from immature embryos of rye and triticale

Callus cultures were established from the scutellum, scutellar node and radicle region of immature embryos of rye and octoploid triticale on modified Murashige-Skoog basal medium supplemented with various growth regulators. 2, 4-D, 2, 4, 5-T and 2, 4, 5-Cl, POP were found suitable for initiation and maintenance of callus cultures. Cytokinins had no or inhibitory effect on callus induction and growth. On basal medium containing 5 mg/l of 2,4,5-Cl₃ POP, 16% of triticale and 17% of rye primary cultures exhibited shoot bud regeneration after 3–4 weeks. Transfer of such cultures to basal medium supplemented with zeatin or zeatin in combination with IAA further promoted shoot elongation and plantlet formation. Plantlets were rooted on basal medium containing 1 mg/l NAA and were eventually transferred to soil. Chlorophyll variants were observed in about 6% of triticale cultures.     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

Organogenesis and plant regeneration from immature embryos of Rosa hybrida L.

Intact, flowering, rose plants have been regenerated in vitro from excised embryos of crosses between Bridal Pink (the maternal parent) and several pollen parents. Explanted embryonic tissues developed into an organogenic callus which formed adventitious shoots after several months only on a modified half-strength Murashige & Skoog medium containing 1.0 M BA and 0.05 M NAA. These shoots could be separated, grown individually, rooted in a medium with no BA or NAA, with 1.0 M IBA, and transplanted to greenhouse media. Embryos ranging in age from 21 to 35 days post-pollination formed organogenic callus that eventually regenerated adventitious shoots. Histological examination of normally-developing embryos showed that well-defined embryonic axes were beginning to develop at approximately 20–25 days postpollination. Analysis of populations of regenerated plants from different crosses showed differences in flower color, growth habit, peduncle length, and petal number. This system may be useful for irradiation-mutation breeding and/or for the development of transgenic rose plants using Agrobacterium tumefaciens.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutanoic acid - MS Murashige & Skoog - NAA -naphthaleneacetic acid - 2iP N⁶-(2-isopentenyl)adenosine     

Plant regeneration from immature embryos of peanut

Plant regeneration from immature embryos of peanut (Arachis hypogaea L.) can be accomplished through somatic embryogenesis. The highest frequency of somatic embryo formation occurred on B5 medium plus 0.5–1.0 mg/l picloram. Shoots and plants developed from the somatic embryos only after extended culture on basal medium. Shoots were excised from thick embryonic roots and rerooted on Murashige and Skoog medium containing half the normal concentration of inorganic salts. This technique should be useful for the production of interspecific hybrid plants from immatureArachis embryos.     

Adventitious shoot regeneration from immature embryos of sorghum

Eleven genotypes of sorghum were examined for their response in tissue culture, and the tissue culture system was optimized. The cultures were initiated from immature embryos taken approximately two weeks after flowering. The response of immature embryos varied with the genotype. `C. Kafir' and `PE932 025' showed the highest frequency of callus induction and regenerable callus formation under appropriate culture conditions. Regeneration occurred at high frequencies when cytokinins (kinetin or 6-benzyladenine) had been added in the callus induction medium, followed by regeneration medium devoid of growth regulators. The addition of proline and polyvinylpyrrolidone also enhanced shoot formation, but the addition of cytokinins to regeneration media did not improve shoot formation. On the revised culture medium, plants were regenerated from up to 100% of sorghum immature embryos.     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

Direct somatic embryogenesis and plant regeneration from immature embryos of hybrid sunflower (Helianthus annuus L.) on a high sucrose-containing medium

Somatic embryos of sunflower (Helianthus annuus) were obtained by placing immature zygotic embryos on a high sucrose (12%) containing medium. The somatic embryos were first observed 6 days after culture and a callus intermediate was not formed. Histological examination revealed the classical stages of embryo development. The somatic embryos proliferated directly from the surface of the zygotic embryos and germinated after placement on a low sucrose medium. Seeds have been obtained from regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - BAP 6-benzylamino purine.Salaries and research support were provided by State and Federal funds appropriated to OSU-OARDC. Journal Article No. 67–87     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Japanese larch (Larix leptolepis)

Embryogenic tissue was initiated using LM, LP and MS media from open-pollinated immature embryos of Larix leptolepis. The initiation frequency varied with collection dates. The highest frequencies of embryogenic tissue initiation (60, 67 and 59% on LM, LP and MS media, respectively) were observed from cones collected on July 30. At this time, all the excised embryos were at the cotyledonary stage. ABA over a wide concentration and length of exposure range did not promote maturation, but was beneficial in reducing precocious germination. Of over 400 plants regenerated, 72 were transplanted into soil mixtures and to date, 69 of these (95%) have survived. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses

Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.     

Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.)

Summary An efficient protocol was developed to regenerate entire plants from immature embryos of elite genotypes of rye as a prerequisite to plant transformation. Three winter genotypes and one spring genotype were tested using both immature and mature embryos as explants. Four types of callus initiation media and five kinds of regeneration media were tested in all possible combinations. Immature embryos gave much higher levels of plant regeneration than mature embryos, but mature embryos could be induced to regenerate plants for all genotypes and media tested, although at low levels. A minimum stage of embryo development must be reached before embryos can be cultured successfully. Genotypic effects were less pronounced than those reported for inbred cereal species such as wheat and barley, but there was an effect of genotype on percentage of callus formation. There was a significant interaction between genotype and initiation media. Composition of the initiation media affected both the percentage of callus formation from embryos and subsequent frequencies of plant regeneration. Composition of the regeneration media had no effect on level of plant regeneration. Immature embryos of all genotypes tested could be induced to produce 90–100% callus on appropriate initiation media and all regenerated shoots from approximately one-half to three-quarters of the calluses produced.     

Callus induction and plant regeneration from immature embryos of Brachypodium distachyon with different chromosome numbers

The paper reports the in vitro cultivation of two commercial lines and 23 wild populations (with 10, 20 and 30 chromosomes) of Brachypodium distachyon. Callus induction was assayed on Murashige and Skoog medium containing 1 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) with 30 g dm⁻³ of sucrose (MSs) or maltose (MSm). No significant differences were seen between the two media with respect to callus induction. Calli were transferred to MSm medium without 2,4-D but containing 0.1 mg dm⁻³ of 6-benzylaminopurine for plant regeneration. The plant regeneration response was very variable depending on the original induction medium, although no overall preference for one or the other medium was seen. The three main culture stages (callus induction, plant regeneration, and green plantlets formation) are probably differently controlled in the plants with different chromosome numbers. This supports the idea that the three cytotypes of Brachypodium cultured actually belong to different species.     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Callus induction and plant regeneration from maize mature embryos

Calli were induced from mature embryos of maize (Zea mays L.) inbred lines A632, B73 and Mol7 on MS medium supplemented with 1–2 mg/1 2,4-dichlorophenoxyacetic acid. Callus induction frequency ranged from 23–100%, with Mol7 having the highest frequency. Plants were regenerated from 4–5% of the B73 and Mol7 explants. Embryogenic and organogenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R₀ plant with abnormal pollen was detected, and no morphological variants were observed in the R₁ progeny.Abbreviations Dicamba 3,6-Dichloro-o-anisic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Ze zeatin     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

High frequency plant regeneration from immature cotyledons of mungbean

An efficient and simple method for high frequency plant regeneration from immature cotyledons of mungbean is described. Immature cotyledons isolated from embryos, one week prior to harvest were cultured on MS medium with combinations of growth regulators such as benzyladenine (1 or 2 mg l⁻¹), thidiazuron (0.1 or 0.5 mg l⁻¹), gibberellic acid (0.1 mg l⁻¹) and indole-3-acetic acid (0.1 or 0.5 mg l⁻¹). A large number of greenish shoot primordia were initiated from the entire surface of the cotyledons in some of the growth regulators. Medium supplemented with benzyladenine (2 mg l⁻¹) in combination with indole-3-acetic acid (0.5 mg l⁻¹) produced the best response. On subculture to the same medium, well developed shoots were obtained. Addition of 0.5% activated charcoal to the shoot initiation medium completely inhibited initiation of shoot primordia. The shoot buds could be rooted on medium supplemented with 0.1 mg l⁻¹ indole butyric acid and plants transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos

The developmental potential of the inner cell mass (ICM) of the cultured mouse embryo was determined by testing the ability of the ICM to regenerate trophoblast in vitro. ICM's isolated by immunosurgery from either single or chimeric embryos were able to regenerate trophoblast when they were isolated at 69 hours of culture from the 2-cell stage, but they had lost this capacity by 93 hours of culture. Trophoblast regeneration by isolated ICM's did not appear to require either a critical cell mass at the time of isolation or cell proliferation during regeneration.     

Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.