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     

Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) tissue cultures

Summary Thidiazuron (TDZ) effectively induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey) during only a 3-day period of induction. The presence of acetylsalicylic acid (ASA) during this period caused a two-fold increase in the number of somatic embryos and enhanced synchronization of embryo development compared to the TDZ treatment alone. Salicylic acid was ineffective in modulating similar embryogenic responses as ASA. The ASA-induced enhancement and synchronization of somatic embryogenesis could possibly be used as an experimental system to study the interplay of growth regulators in somatic embryogenesis.Abbreviations ASA acetylsalicylic acid - SA salicylic acid     

Inhibitory effect of GA3 on the development of thidiazuron-induced somatic embryogenesis in geranium (Pelargonium xhortorum Bailey) hypocotyl cultures

Somatic embryogenesis in geranium (Pelargonium xhortorum Bailey cv Scarlet Orbit Improved) can be achieved by incubating hypocotyl explants on MS medium supplemented with thidiazuron (TDZ; 10 M for 3 days followed by subculture on medium devoid of any plant growth regulators. The presence of gibberellins (GAs) during both the induction and expression phases of embryogenesis was significantly detrimental to somatic embryo formation on the hypocotyl explants. The addition of the GA-synthesis inhibitors paclobutrazol, uniconazole or ancymidol during the period of growth and differentiation of somatic embryos increased the number of somatic embryos formed on each explant. However, paclobutrazol added during the period of induction had no significant influence on somatic embryo formation. Results suggest that both exogenously supplied as well as endogenous GAs play a role, albeit a negative one, on somatic embryogenesis of geranium.Abbreviations MS Murashige and Skoog (1962) medium - MSO basal medium devoid of any plant growth regulator - TDZ N-phenyl-N1,2,3-thidiazol-5-ylurea (thidiazuron)     

Modulation of somatic embryogenesis in hypocotyl-derived cultures of geranium (Pelargonium X hortorum bailey) CV ringo rose by a bacterium

Summary The Ringo Rose cultivar of zonal geranium (Pelargonium x hortorum Bailey) has been shown to be morphogenetically unresponsive. Attempts to improve somatic embryogenesis using various seed stress treatments before germination proved ineffective. However, bacterial contamination of one of the seed-stress treatments led to infected explants that had a significant increase in frequency of high-quality somatic embryos. The co-cultivation of explants with the isolated bacterium (tentatively identified asBacillus sp.) was found to be repeatable, and potentially represents a novel way to improve morphogenesis in geranium and possibly other species.     

Shoot regeneration of hybrid seed geranium (Pelargonium x hortorum) and regal geranium (Pelargonium x domesticum) from primary callus cultures

Procedures have been developed that increase the rate of shoot regeneration of hybrid seed geranium from month-old primary callus cultures. Hybrid geranium callus tissue covered with green nodular structures was initiated by placing shoot tip explants on solidified Murashige & Skoog medium (MS) supplemented with 2.0 mgl^-1 zeatin and 1.9 mgl^-1 indoleacetic acid. Hybrids Red Orbit, White Orbit and Scarlet Orbit were shown to produce 5–50 shoot primordia per explant when callus was initiated on this medium. Regal geranium callus was initiated by placing leaf explants on MS medium supplemented with 2.0 mgl^-1 6-benzylaminopurine and 2.0 mgl^-1 naphthaleneacetic acid. Regal geranium cultivars Tiny Tot and Lavender Grand Slam were shown to produce between 2–50 shoot primordia per explant when initiated on the same medium.     

Triazole treatment of explant source provides stress tolerance in progeny of Geranium (Pelargonium hortorum Bailey) plants regenerated by somatic embryogenesis

Thehypothesis that chemically induced stress tolerance in plants can betransferredto a larger clonal population regenerated by somatic embryogenesis wasevaluatedusing the triazole compound paclobutrazol as a chemical inducer of stresstolerance in Geranium (Pelargonium horturum Bailey). Seedswere imbibed in 3.4, 10.2 or 17.0 M (1, 3, 5 mgL^–1) paclobutrazol for 24 h and germinatedfor 7 days. Hypocotyl explants were cultured in vitro toinduce somatic embryogenesis. Plants regenerated from somatic embryos wereexposed to heat stress at 56°C. Explants treated with3.4 M paclobutrazol yielded a substantially higher number ofsomatic embryos compared with untreated explants. In contrast, 17.0M paclobutrazol treatment inhibited embryogenesis producing asignificantly lower number of somatic embryos. There was no difference in theembryo number between control and 10.2 M treatment. Somaticembryos derived from 3.4 and 10.2 M paclobutrazol treatedexplants developed into plants at a faster rate than the control and 17.0M treatments. Plants derived from paclobutrazol-treatedexplants displayed a greater tolerance to heat stress compared with thecontrols. Observations in this study provide a technique for regeneratingplantsin tissue/cell culture with additional desirable traits such as stresstolerancewith minimal chemical contamination of the environment.     

Role of purine metabolism in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium × hortorum) hypocotyl cultures

Somatic embryogenesis in geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μ M thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6-diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μ M adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP-induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy-tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.     

Induction of high-frequency somatic embryogenesis in geranium (Pelargonium x hortorum Bailey cv Ringo Rose) cotyledonary cultures

Summary The cv Ringo Rose of hybrid seed geranium (Pelargonium x hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos when cotyledonary explants were cultured on regeneration medium containing thidiazuron (TDZ), forchlorfenuron (CPPU), or a combination of indole-3-acetic acid and N⁶ benzylaminopurine (IAA+BAP). Amendment of the basal medium with TDZ (0.5 M) was the most effective treatment. Addition of amino acids to the medium promoted the growth of somatic embryos. Retention of the proximal region of the cotyledon was crucial for regeneration, but the removal of the distal 1/3 to 1/2 cotyledon had no significant effect on somatic embryogenesis. Cotyledonary explants formed somatic embryos in higher frequency and much earlier than hypocotyl explants cultured on the same medium. The somatic embryos induced on cotyledonary explants were germinated on basal medium. More than 70% of the somatic embryos were converted into plants and transferred to soilAbbreviations BAP N⁶-benzylaminopurine - CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea (forchlorfenuron) - IAA indole-3-acetic acid - TDZ N-phenyl-N'-1,2,3,-thiadiazol-5ylurea (thidiazuron)     

Role of purine metabolism in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium×hortorum) hypocotyl cultures

Somatic embryogenesis in geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μ M thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6-diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μ M adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP-induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy-tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.     

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.     

Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene

Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h^–1g^–1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh^–1 g^–1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO₃and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.     

Regeneration of plantlets from leaf and petiole explants of Pelargonium x hortorum

Summary The in vitro plant regeneration potential of vegetatively propagated geraniums (Pelargonium x hortorum) has been investigated. Using various combinations of growth regulators and a choice of different explants, a regeneration protocol has been developed to raise in vitro plantlets from young petiole and leaf explants from three different cultivars of geraniums. In all three cultivars, very young petiole explants exhibited a higher regeneration potential as compared with leaf explants. Regeneration efficiencies were found to be highly dependent on the cultivar, with cv. Samba showing the highest regeneration potential, followed by cvs. Yours Truly and then Sincerity. Samba also showed the highest number of shoots from both the petiole [57 shoot buds per petiole explant in the presence of 3 μM zeatin and 1 μM indole-3-acetic acid (IAA) and leaf explants (43 shoots per leaf explant with 10 μM zeatin and 2 μM IAA). Shoot buds transferred to Murashige and Skoog (MS) medium supplemented with 0.44 μM N⁶-benzyladenine and 0.11 μM IAA grew vigorously and attained 1–2 cm in length in 3–4 wk. These shoots rooted with 100% efficiency on MS basal medium, and plants developed that showed normal growth and flowering under greenhouse conditions.     

Histological analysis of somatic embryogenesis induced in leaf explants ofHelianthus smithii Heiser

Summary According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants ofHelianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed.Abbrevations BAP 6-benzylaminopurine - MES 2-(N-morpholino)ethane-sulfonic acid - MS medium of Murashige and Skoog - NAA 1 -naphthaleneacetic acid - TDZ thidiazuron     

Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium x hortorum Bailey)

Summary Root outgrowths formed on the root tissue of geranium (Pelargonium x hortorum Bailey cv. Kim and cv. Shone Helena) plants in response to treatment with the phenylurea derivative, thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea; TDZ). Treatment with the cytokinin N⁶-benzylaminopurine (BAP) or the auxin -naphthaleneacetic acid (NAA) did not result in stimulation of similar abnormal structures on the root tissue. Significantly more outgrowths developed on roots of plants treated with 10 M and 20 M TDZ than on control plants or those treated with 1 M TDZ for the eight-week treatment period. Some outgrowths produced shoots and plantlets while still attached to roots, and regenerants were easily separated from the root tissue and transferred to soil in the greenhouse where they grew to maturity. Histological observations suggested these outgrowths originated from the vascular cambium region of the root.     

Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants

Gentiana kurroo (Royle), Gentiana cruciata (L.), Gentiana tibetica (King. ex Hook. f.), Gentiana lutea (L.), and Gentiana pannonica (Scop.) leaves derived from axenic shoot culture were used as explants. For culture initiation, leaves from the first and second whorls from the apical dome were dissected and cultured on Murashige and Skoog (MS) basal medium supplemented with three different auxins: 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid (NAA), or 3,6-dichloro-o-anisic acid (dicamba) in concentrations of 0.5, 1.0, or 2.0 mg/l; and five different cytokinins: zeatin, 6-furfurylamonopurine (kinetin), N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ), N-(2-chloro-4-pyridyl)N′-phenylurea, or 6-benzylaminopurine (BAP). The cytokinin concentrations used were dependent on the type of cytokinin and varied between 0.25 and 3.0 mg/l. After 2 mo. of culture, the morphogenic response of explants was assessed. Frequency of embryogenesis was the highest for G. kurroo (54.7%) and dependent on plant growth hormones (PGRs). This gentian was the only species showing morphogenic capabilities on media supplemented with all applied combinations of PGRs, while none of the 189 induction media permutations stimulated somatic embryogenesis from G. lutea explants. G. tibetica and G. cruciata both produced an average of 6.6 somatic embryos per explant, while G. pannonica and G. kurroo regenerated at 15.7 and 14.2 somatic embryos per explant, respectively. Optimum regeneration was achieved in the presence of NAA combined with BAP or TDZ. This auxin also stimulated abundant rhizogenesis. Somatic embryos were also regenerated from adventitious roots of G. kurroo, G. cruciata, and G. pannonica. Somatic embryos converted into plantlets on half strength MS medium.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

High frequency somatic embryogenesis and plant regeneration from papaya hypocotyl callus

High frequency somatic embryogenesis in papaya (Carica papaya L.) tissue cultures was achieved by culturing hypocotyl sections from ten-day-old seedlings on half-strength Murashige and Skoog salts (MS) medium containing modified MS vitamins, 2.3 to 112.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), 400 mg l^-1 glutamine, and 6% sucrose. Four hermaphroditic Hawaiian cultivars produced embryogenic calluses after ten to 14 weeks of culture at 27°C in the dark. Efficiency in embryogenic response of genotypes differed, Kapoho > Sunset > Sunrise > Waimanalo. The frequency of embryogenesis in induction medium containing 4.5 M 2,4-d was lowest with 3% sucrose and highest with 7% sucrose. Somatic embryos developed directly from embryogenic calluses on induction medium, or, more often, they differentiated from calluses subcultured on a medium devoid of growth regulators. Between 50 and 500 embryos were produced from each 2-mm hypocotyl section after at least two months on induction medium and two months on maturation medium. Embryos subsequently developed into normal-looking plants on MS medium. Shoot cuttings from germinated embryos and micropropagated plants were rooted with 5.0 M indole-3-butyric acid (IBA), grown in the greenhouse, and transferred to the field.Journal Series no. 3732 of the Hawaii Institute of Tropical Agriculture and Human Resourees     

High Frequency Plant Regeneration from Astragalus melilotoides hypocotyl and stem explants via somatic embryogenesis and organogenesis

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem segments on MS medium containing 9.05 µM 2,4-D and 2.22–4.44 µM BA. Both somatic embryos and adventitious buds were initiated from hypocotyl-derived calli while only adventitious buds were formed from stem-derived calli in MS medium supplemented with 2.69 µM NAA and 4.44–8.89 µM BA. Somatic embryos or adventitious buds developed into plantlets following being cultured for 3 weeks on MS medium without any growth regulators or with 14.78 µM IBA, respectively. All the regenerated plants were normal with respect to morphology and growth characters, and produced fertile seeds after planting in soil.