Plant regeneration through somatic embryogenesis in the forage grass Caucasian bluestem (Bothriochloa caucasica)

Plantlets were regenerated from cultured seed explants of the forage grass Caucasian bluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] via somatic embryogenesis. Embryogenic callus was produced in four weeks when surface sterilized seeds were cultured on a medium containing MS-salts, B-5 vitamins, 12 mM L-proline, 2% sucrose, 0.8% agar and 5M 2,4-D. Plantlets were regenerated in 6–8 weeks after culture initiation. Healthy root and shoot systems were produced within three weeks after the plantlets were transferred to a medium lacking 2,4-D. Approximately 95% of the plantlets survived greenhouse acclimation and produced healthy plants and viable seeds. Caucasian bluestem callus cultures exhibit natural resistance to kanamycin. High levels of kanamycin (up to 800 mg/l) did not completely inhibit callus growth. However, the regeneration of healthy-plantlets was completely inhibited by kanamycin even at low levels (50 mg/l).     

Embryogenesis and Regeneration of Green Plantlets from Wheat (Triticum aestivum) Leaf Base

A short-term regeneration system from leaf-base-derived callus of wheat (Triticum aestivum L.) was developed. Embryogenic callus formation and shoot regeneration were achieved from the first basal segments of 3–4-day-old seedlings. Callus formation frequency as well as plantlet regeneration frequency was dependent on the composition of basal medium and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). MS medium with 2,4-D 4.5–9.0 mol l^–1 was optimal for the culture of wheat leaf base. Effects of different combinations of plant growth regulators, which were added in either callus induction medium or shoot regeneration medium, were tested. Adding of BAP in callus induction medium shortened the time of shoot emergence but could not improve the producing of embryogenic calli and green plantlets. Optimal ratio of 2,4-D, BAP and NAA gave similar regeneration frequency to control. Existence of cytokinins in regeneration medium had no effect on increasing the regeneration frequency. The regenerants could grow to normal, fertile plants after they were transferred into soil.     

Selective enhancement of Ipomoea batatas Poir. embryogenic and non-embryogenic callus growth and production of embryos in liquid culture

Embryogenic callus cultures of Ipomoea batatas Poir. produce fast growing non-embryogenic material which soon dominates the cultures. Our objective was to selectively enhance the proliferation of the embryogenic fraction. For this, the effect of BAP and 2,4-D concentrations on growth of embryogenic and non-embryogenic callus were studied and consequently, nutrient media for the production and indefinite maintenance of embryogenic callus without embryo formation were defined. Selective proliferation of embryogenic callus was obtained on solid media with 10 M 2,4-D and 1 M BAP and in liquid media with 5 M 2,4-D. Selective proliferation of non-embryogenic callus occurred in liquid medium with 1 M 2,4-D. In embryogenic liquid culture, embryos were produced with 0–2 M 2,4-D. Increasing 2,4-D concentration from 0 to 2 M in these cultures restricted embryo development.Abbreviations 2,4-D = 2,4-dichlorophenoxyacetic acid - BAP = 6-benzylaminopurine     

Factors affecting the establishment and maintenance of embryogenic callus and suspension cultures of wheat (Triticum aestivum L.)

Improved suspension cell culture systems are needed to facilitate the application of recombinant DNA technology for wheat germplasm enhancement. This study evaluated three wheat (Triticum aestivum L.) cultivars, and the effects of medium basal salts, 2,4-D, sucrose, and L-proline concentrations on the establishment of rapidly growing and highly embryogenic callus and suspension cultures. Percent embryogenic calli was visually estimated and verified with light and scanning electron microscopy. The most highly embryogenic callus was produced by cultivar Bobwhite on medium with MS basal salts, 5.6 M 2,4-D, 58 mM sucrose, and zero proline. The suspension cultures that produced the greatest number of regenerated plants utilized callus tissue produced on solid medium with MS basal salts, 87 mM sucrose, 9 M 2,4-D, and no proline.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid; RG, relative growth - %EC percent embryogenic calli - RV Redway and Vasil medium (1990a) - DPA days postanthesis     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses

Objectives of this research were to test the effects of plant genotypes and auxin 2,4-D (2,4-dichlorophenoxyacetic acid) medium concentrations on embryogenic (E) callus production of two grass species. Two Old World bluestem,Bothriochloa ischaemum, accessions (A-8793 and A-8911c) and three bermudagrass,Cynodon dactylon (L.) Pers., accessions (A-10978b, A12164, and Brazos) supplied the explant material. Immature inflorescences 9 mm in length were placed on modified Murashige-Skoog (MS) agar medium containing 0, 1, 3, or 5 mg L^-1 of 2,4-D. Explants of all genotypes produced callus by the end of a 4-week dark incubation period at 25°C. When subcultured onto fresh media and maintained at 25°C with a 16 hr photoperiod, calli became embryogenic within 8 weeks of inoculation. Three mg L^-1 of 2,4-D in the media maximized E callus production in both bluestem genotypes and in A-10978b and A-12164 bermudagrass genotypes. Maximum E callus production from Brazos bermudagrass resulted from the 1 mg L^-1 treatment. Somatic embryos developed after subculture under light. Embryos showed scutellum-like structures and coleoptile-coleorhiza bipolar organization. Plantlets were regenerated from all genotypes except Brazos, whose embryoids failed to germinate. All callus from Brazos eventually senesced. Light and scanning electron microscopy confirmed regeneration through somatic embryogenesis.     

Anthocyanin production in callus cultures of roselle (Hibiscus sabdariffa L.)

Callus tissues derived from seedlings of roselle (Hibiscus sabdariffa L.) were shown to produce two cyanidin glycosides as major anthocyanin pigments. Both callus growth and anthocyanin synthesis were remarkably stimulated by 2,4-dichlorophenoxyacetic acid. The highest anthocyanin yield was observed when 1 M 2,4-D in combination with 0.1–1 M kinetin was supplemented to the culture medium. In contrast, gibberellic acid showed inhibitory effect on anthocyanin production.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - GA gibberellic acid     

Proliferative activity of callus cultures of Taxus baccata L. in relation to anticancer diterpenoid taxol biosynthesis

Summary Stable growing callus cultures were recovered from stem segments of Taxus baccata. Callus developed from the peridermal cells. The content of taxol in callus tissues varied in the range of [0.0003–0.001% on dry weight basis] and was higher in fast growing calli compared to slow growing calli.Abbreviations BAP 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA -naphthyl-1-acetic acid - PVP polyvinylpyrrolidone - SEM scanning electron microscopy     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

Cumin regeneration from seedling derived embryogenic callus in response to amended kinetin

Callus was induced from hypocotyl and primary leaf explants of cumin (Cuminum cyminum L.) seedlings on a medium with 4 M 2,4-D alone or plus 2 or 4 M kinetin. An embryogenic callus developed within 2 weeks after transferring the callus to medium lacking plant growth regulators (PGR). The presence of kinetin in the callus induction medium with 2,4-D enhanced both the callus proliferation and the subsequent differentiation of the embryoids on the PGR-free medium. Plumules with or without simultaneously developed roots were observed 3–4 weeks after subculturing the embryogenic callus on medium containing 0.5 or 1.0 M kinetin. Subsequently, they were transferred onto half-strength medium supplemented with 1 M indole-3-butyric acid (IBA) and 2% polyethylene glycol (PEG, 6000) for root induction and/or proliferation, and in vitro hardening of the regenerated plants. The survival rate ex vitro was 70%. No plants developed from the embryogenic callus continuously incubated on medium lacking kinetin. We concluded that kinetin is crucial for plant regeneration from the induced embryoids of cumin.     

Physiological and molecular effects of brassinosteroids on Arabidopsis thaliana

We examined the effects of brassinosteroids on Arabidopsis thaliana (L.) Henyh. ecotype Columbia in order to develop a model system for studying gene regulation by plant steroids. Submicromolar concentrations of two brassinosteroids, brassinolide and 24-epibrassinolide, stimulated elongation of Arabidopsis peduncles and inhibited root elongation, respectively. Furthermore, brassinolide altered the abundance of specific in vitro translatable mRNAs from peduncles and whole plants of Arabidopsis. Root elongation in the auxin-insensitive Arabidopsis mutant axr1 was inhibited by 24-epibrassinolide but not by 2,4-D, indicating an independent mode of action for these growth regulators in this physiological response.Abbreviations BR brassinolide - EBR 24-epibrassinolide; 2.4-D,2,4-dichlorophenoxyacetic acid - KPSC 10 mM potassium phosphate, pH 6.0, 2% sucrose, 50 g/ml chloramphenicol - PAGE polyacrylamide gel electrophoresis     

Stably transformed herbicide resistant callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts

Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.Abbreviations GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - PMSF phenylmethylsulfonyl fluoride - MES 2[N-Morpholino]ethanesulfonic acid - HEPES [N-2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] - PAT Phosphinothricin acetyltransferase - CTAB cetyltrimethylammonium bromide     

Callus induction and plantlet regeneration in damiana (Turnera diffusa,Willd.)

Experiments were performed to determine optimal conditions for callus induction and plantlet regeneration from damiana (Turnera diffusa, Willd.) leaf explants. MS media [7] and B5 media [4] were used; both of them were supplemented with different combinations of 2,4-dichlorophenoxyacetic, acid (2,4-D) and 6-benzyl-adenine (BA). In all cases the samples were incubated under continous fluorescent light, 100 mol.m^-2.sec^-1, at 25±2°C for 60 days. Highest callus percentage was obtained in MS media supplemented with 2,4-D 10^–5 M plus BA 10^-8M. Maximum percentage and multiple shoots and leaves were produced in MS media with BA 10^-5M and BA 10^-5M plus 2,4-D 10^-7M. These explants were transferred for rooting to MS medium without plant growth regulators. Later, rooted plantlets were successfully established in soil.     

Plant regeneration through somatic embryogenesis from callus induced on immature embryos of Alstroemeria spp. L.

Summary The plant regeneration ability of callus obtained from zygotic embryos of the monocot Alstroemeria spp. was studied. The best explants for somatic embryogenesis were immature zygotic embryos in half-ovules when the endosperm was still soft and white. For 2 genotypes embryogenic callus was induced on callus induction medium with a success rate of 54%. The best callus induction period was 10 weeks. The morphology of embryogenic callus was nodular. Somatic embryos were formed after transfer of the callus to regeneration medium. These somatic embryos revealed later on the typical features of zygotic Alstroemeria embryos. The total duration of the plant regeneration protocol, from inoculation till rooted plantlets ready for transfer to the greenhouse, was 28 weeks.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Effect of phytohormones on plant regeneration in callus culture of <Emphasis Type="Italic">Iris ensata</Emphasis> Thunb

The effect of phytohormones on plant regeneration in callus culture of Iris ensata Thunb. was studied. 2,4-Dichlorophenoxyacetic acid (2,4-D) proved to be the most suitable auxin for the induction and subculturing of morphogenic callus. Organogenic calluses were induced from isolated embryos at the waxphase in MS medium supplemented with 2,4-D (1.0–2.0 mg/l) and kinetin (0.2–0.5 mg/l). Changes in the medium hormonal composition favor the development of adventitious structures. The setting of adventitious shoot buds took place in the presence of 6-benzylaminopurine, while the development of shoots and root primordia was observed after 2,4-D replacement with indoleacetic acid (2.0 mg/l). The root initiation in regenerating plants required a hormone-free medium. The development of intact seedling and regenerated plants of I. ensata were studied. Analysis of the shoot structure demonstrated that in vitro cultured plants are at the juvenile stage.     

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 M dicamba, addition of 2.25 to 9 M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 M dicamba and 2.25 M BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.Abbreviations MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - dicamba 3, 6-dichloro-o-anisic acid     

Effect of silver nitrate on long-term culture and regeneration of callus from Brassica oleracea var. gemmifera

Incorporation of AgNO₃ (1–10 mg1^-1) into the culture medium of Brassica oleracea var. gemmifera callus significantly improved growth and allowed long-term callus culture. In the absence of AgNO₃, callus died shortly after removal from the hypocotyl explants. Regeneration of shoots from callus on low-hormone medium was also enhanced by AgNO₃. Significant differences in shoot production were found between the three genotypes examined. Cv. Aries produced large numbers of shoots even in the absence of AgNO₃. Investigation of callus production from the inbred parent lines of cv. Aries indicated that tissue culturability may be determined genetically.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid