Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Micropropagation for fraser photinia (Photinia × fraseri)

Embryo suspensor masses were induced by culture of isolated mature zygotic embryos of Fraser fir (Abies fraseri [Pursh] Poir.). Maximum induction frequencies were observed after 10 weeks culture on one-half strength Murashige and Skoog medium containing 10 μM thidiazuron and on one-half strength Verhagen and Wann medium containing 10 μM cytokinin [6-(dimethylallylamino)purine, 6-benzyladenine, or thidiazuron). Proliferation of embryo suspensor masses occurred on one-half strength Verhagen and Wann medium supplemented with 10 μM cytokinin. When embryo suspensor masses were transferred to media containing 5-80 μM abscisic acid, cotyledonary-stage embryos were formed. Somatic embryos germinated on medium lacking plant growth regulators, but abnormal cotyledonary-stage somatic embryos with stunted cotyledons and reduced embryo axes were also observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvement of somatic embryogenesis inFeijoa sellowiana berg (Myrtaceae) by manipulation of culture media composition

Summary Somatic embryos could be induced from the cotyledons of zygotic embryos from immature fruits ofFeijoa sellowiana Berg (Feijoa) in the presence of a wide range of concentrations of fructose, glucose, maltose, and sucrose. Mannitol or sorbitol alone were ineffective. The highest frequencies of induction (99%) and the greatest number of somatic embryos per explant (134) were obtained with 0.4M fructose and 0.3M sucrose, respectively. This sucrose concentration also showed greater induction capacity than equimolar combinations of its monosaccharide constituents combined. Somatic embryo development was arrested at the globular stage at concentrations higher than 0.5M of all the sugars tested. When transferred to solid germination medium containing 2.0 mg/liter (5.77μM) gibberellic acid, 0.5 mg/liter (2.32μM) kinetin, and 0.029M sucrose, somatic embryos formed under 0.3 or 0.4M sucrose had better germination capacity than those induced under lower (0.1 and 0.2M) concentrations, as assessed by the frequency of explants presenting germinated embryos and by the number of plants obtained from those explants. On liquid media of similar composition somatic embryos did not germinate. Our data suggest that high (0.3 to 0.4M) carbohydrate levels improve somatic embryogenesis by acting both as carbon source and as osmotic regulator.     

Evidence of somatic embryogenesis from root tip explants of the rattan Calamus manan

Summary Somatic embryogenesis of Calamus manan, a single-stemmed rattan species, in tissue culture was scientifically demonstrated for the first time. Root tips of in vitro plantlets produced friable callus when the explants were cultivated for several mo. on a Murashige and Skoog induction medium containing 7.5 mg Picloram per l (31.1 μM). Histological analyses established the presence of proembryos within the callus which differentiated subsequently into somatic embryos using the same culture medium. Histological examination revealed that these somatic embryos completely lacked starch and protein reserves, which did not prevent them, however, from germinating, and showing bipolar development. These somatic embryos further developed into young plants, similarly to zygotic embryos.     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.     

Effect of explant orientation, pH, solidifying agent and wounding on initiation of soybean somatic embryos

Summary Several methods have been developed to obtain somatic embryos of soybean. We report here a new procedure that results in high frequency somatic embryo initiation in a short period of time. Somatic embryos were induced from immature cotyledons of the cultivars “Jack,” “Thorne,” “Resnik,” and “Chapman.” Immature cotyledons were cultured on a medium containing MS salts, B₅ vitamins, 6% sucrose, and 40 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Culture modifications included: orientation of the explants (adaxial or abaxial side of the cotyledon in contact with the medium), adjustment of medium pH (5.7 or 7.0), wounding of cotyledons with scalpel blades, inclusion of ethylene modulators, and use of Noble agar or Gelrite™ as the solidifying agent. The treatment that resulted in the highest embryo induction across the cultivars consisted of abaxial side of the explant facing the medium, pH 7.0 and 0.2% Gelrite™. “Jack” was the most responsive cultivar showing the first embryos as early as 14 d after culture. After 21 d, an average of 44 embryos per cotyledon was obtained with this cultivar. The inclusion of silver nitrate (AgNO₃) in the culture medium did not enhance the number of primary somatic embryos induced per cotyledon, but the addition of 15 μM AgNO₃ did result in a faster production of secondary embryos using the cultivar “Jack.” Wounding of the explants with a scalpel resulted in an earlier induction of somatic embryos. Embryo initials were first observed after only 7 d. Histological examination of cultured cotyledons indicated that the somatic embryos originated from the subepidermal tissues and were of multicellular origin. This somatic embryo induction procedure could be useful for direct transformation work and permits the production of embryogenic tissue within 2 wk.     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Effect and analysis of phenolic compounds during somatic embryogenesis induction in Feijoa sellowiana Berg

Summary. The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140–560 μM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 μM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 μM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 μM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed. Correspondence: J. M. Canhoto, Departamento de Botanica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Cal?ada Martim de Freitas, 3001-455 Coimbra, Portugal.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Embryogenesis induction in petals of Araujia sericifera

The embryogenic capacity of Araujia sericifera petals and some of the factors involved in the induction of embryos was investigated. The influence of 6-benzyladenine and α-naphthalene acetic acid, light intensity (90 or 5 μmol m^-2 s^-1) and silver thiosulphate (inhibitor of ethylene action) were studied. It was found that petals are an easy system in which to induce somatic embryogenesis. Plants were recovered from somatic embryos. Although 6-benzyladenine is essential for inducing an efficient response, a high dosage increased callogenesis and reduced embryogenesis. The highest rate of embryogenesis is induced with high light intensity (90–100 μmol m^-2 s^-1), even though the presence of silver thiosulphate in the medium markedly reduced embryo induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polyethylene glycol-promoted development of somatic embryos in loblolly pine (Pinus taeda L.)

Summary The effect of polyethylene glycol (PEG) combined with abscisic acid (ABA) and KCl on somatic embryo development in loblolly pine was investigated. Two embryogenic cell lines, which had not produced cotyledonary stage embryos using previously published methods, were employed in this study. As a maturation medium, basal medium was supplemented with 0 to 10% PEG (MW 3350), 10 to 40 mg/l (37.8 to 151.3 μM) ABA, 0 or 10 mM KCl, 1.5 g/l activated charcoal, 30 g/l sucrose, and 6 g/l agar. Without PEG in the maturation medium, most somatic embryos at stage 1 could not mature further. Embryogenic tissues on the maturation medium with 5 to 7.5% PEG consistently produced stage 2 and stage 3 somatic embryos. PEG at 10% significantly decreased the number of stage 2 and 3 embryos compared to PEG at 5 to 7.5% ABA at 40 mg/l (151.3 μM), combined with 7.5% PEG and 10 mM KCl, gave the maximum number of stage 3 embryos in both cell lines. ABA at 10 mg/l (37.8 μM) induced an over-proliferation of embryogenic tissues and generally failed to produce mature embryos. KCl also significantly enhanced initial stage embryo formation and subsequent embryo maturation.     

Direct somatic embryogenesis from <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr. under the influence of ethylene action inhibitor-silver nitrate

For the first time direct somatic embryogenesis from hypocotyl explants of in vitro regenerated plantlets of C. arabica and C. canephora was achieved on modified MS medium containing 10 – 70 μM silver nitrate supplemented with 1.1 μM N⁶ benzyladenine and 2.85 μM indole-3-acetic acid. A maximum of 144.1±7.3 and 68.7±3.3 embryos per explant were produced at 40 μM silver nitrate in C. canephora and C. arabica respectively. Only yellow friable embryogenic callus obtained from the cut edges of most of leaf explants of both C. arabica and C. canephora at all concentrations of silver nitrate were tried in this experiment. Formation of secondary embryos from stage I primary embryos (small yellow, round, globular embryos) was more (28.23±1.3) at 60 μM silver nitrate in C. canephora, while 40 μM silver nitrate supported more of secondary embryo formation in C. arabica (40.5±1.2). When stage II (green globular round matured embryos) and stage III primary embryos (tubular stage embryos) were used, secondary embryo formation was very small and many of these embryos developed into plantlets and some of them even rooted. By using these protocols within 45 – 60 days it is possible to get secondary embryos from primary embryos and direct somatic embryos from hypocotyls of in vitro plantlets in both these Coffea species.     

Somatic embryogenesis from chickpea (Cicer arietinum L.) inmature cotyledons: The effect of zeatin, gibberellic acid and indole-3-butyric acid

Studies were conduced to test the effects of various cytokinins on somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons. Zeatin (13.7 μmol) added, to B5 basal medium, supplemented with 1.5 % sucrose and 0.2 μmol indole-3-acetic acid, was the most effective cytokinin. Lobular structures obtained from cotyledons cultures were transferred to B5 basal medium supplemented with gibberellic acid and indole-3-butyric acid at different concentrations. The most effective treatment was B5 medium containing 14.4 μmol gibberellic acid plus 1.0 μmol indole-3-butyric acid in which 42.8 % of lobular structures cultured formed normal somatic embryos. High conversion of embryos into plantlets (61.0–65.2 % embryos regenerated plants) was observed when germinated embryos were placed on plant development medium.     

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.     

Effects of light regimes on anther culture response in bread wheat

This experiment was initiated to further test the effects of light regimes during callus induction and plant regeneration on anther culture response of spring wheat (Triticum aestivum L.). Spring wheat cultivars 'Edwall' and 'WA 7176' with high callus induction from anther culture but low green plant production were used. Different gro-lux light and dark regimes during callus induction, and gro-lux light and fluorescent light regimes during plant regeneration were used. Callus induction decreased significantly at relatively high light intensity (315 μmol m⁻² s⁻¹) applied at any period of culture when compared to continuous dark. Light regimes used continuously and from the 15^th to the last day of callus induction also had a significant negative effect on plant regeneration compared to continuous dark and light application in the first half of callus induction. During plant regeneration, '15 day dark + 7 day gro-lux light' significantly increased plant regeneration compared to both 'gro-lux' and 'fluorescent light' regimes. Light regimes during both callus induction and plant regeneration and their interaction effects were found to be highly significant on green plant proportion and green plant yield. 'Continuous light' application during callus induction increased green plant proportion more than other applications in contrast to its negative effect on plant regeneration. During plant regeneration, '15 day dark + 7 day gro-lux light' had the higher green plant proportion compared to only 'fluorescent light' and only 'gro-lux light'. The highest green plant yields were obtained from '15 day dark + 7 day gro-lux light' during plant regeneration in combination with either 'continuous dark' or 'continuous light' regimes during callus induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Summary Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Murashige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g l⁻¹, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20–30 g l⁻¹. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneactic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.     

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm⁻³) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 μM gibberellic acid (GA₃), plumular poles of few embryos elongated resulting in the development of shoots.     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro response of encapsulated somatic embryos of camellia

Plant regeneration via somatic embryogenesis was achieved in leafbase and leaf tip explants derived from 10-day-oldin vitro-grown seedlings ofEchinochloa colona. Somatic embryogenesis was induced in the callus on Murashige and Skoog (1962) medium supplemented with 4.44 μM 6-benzyladenine, 4.64 μM kinetin and 8.05 μM 1-naphthaleneacetic acid after 4 weeks of culture incubated for 14 days in continuous dark and subsequently under a 14-h photoperiod. The incidence of somatic embryogenesis was greater in leafbase- than in leaf tip-derived calluses. Histological observations revealed various stages of development of somatic embryogenesis. The embryos matured and germinated on fresh medium lacking growth regulators. The somatic embryo-derived plantlets were established in soil.