Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

Somatic embryogenesis and plant regeneration from leaf-derived cell suspension of a mature tree — Thevetia peruviana L.

Cell suspension cultures, which retained embryogenic potential for almost 2 years, were established from young, expanding, juvenile leaves of a mature Thevetia peruviana L. tree. Calli were obtained by culturing young leaf discs on MS medium supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1 mg/L kinetin. Suspension cultures were initiated by transfer of calli to liquid medium containing 1 mg/L 2,4-D + 0.1 mg/L kinetin, and the cultures were maintained by subculturing to fresh medium at 2 week intervals. Embryogenic frequency of cell aggregates was more than 80% when plated on semi-solid medium containing 0.1 mg/L 2, 4-D and 2 mg/L 2-isopentenyladenine (2-iP). Cell aggregates with developing embryos were transferred to fresh medium lacking growth regulators for embryo maturation. Early embryo development was synchronous and a large number of somatic embryos were produced. These somatic embryos developed into plantlets upon subsequent transfer to modified half-strength MS medium. More than 200 green and rooted plants, at an average of 80 plants per 100 mg of embryogenic callus, were obtained with 60% survival under glass house conditions.Abbreviations 2, 4-D 2 4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine - IAA Indole — 3 — acetic acid - KN Kinetin - MS Murashige and Skoog (1962) basal medium - NAA 1 -Napthalene acetic acid     

Plant regeneration from cell suspension-derived protoplasts of Populus × beijingensis

A protocol for plant regeneration from cell suspension-derived protoplasts of Populus × beijingensis is described. Protoplasts were isolated from cell suspension cultures 6 d after subculture and further cultured in liquid NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 0.6 M glucose, 9.05 μM 2,4-dichlorophenoxyacetic acid, and 0.89 μM 6-benzyladenine at a density of 2?×?10⁵ protoplasts per milliliter. The initial plating efficiency and final plating efficiency recorded after 10 and 30 d reached 33.7 and 1.07%, respectively. The proliferated calli transferred to regeneration medium supplemented with 2.22 μM 6-benzyladenine and 0.54 μM α-naphthaleneacetic acid gave the highest rate of shoot formation (44.4%). All protoplast-derived shoots were able to form roots on half-strength MS medium supplemented with 2.46 μM indole-3-butyric acid.     

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L^?1, and for the ovary, it is MS + picloram 1.5 mg L^?1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L^?1 + TDZ 0.1 mg L^?1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.     

Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla

Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 M TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA₃ for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 M) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.     

Characterization of somatic embryogenesis in Pelargonium××hortorum mediated by a bacterium

Several cultivars of hybrid seed geranium (Pelargonium×hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos at high frequency when explants were co-cultivated with a morphogenesis promoting bacterium. This bacterium was isolated as an in vitro contaminant from cultures of geranium seedling explants and identified as belonging to the genus Bacillus and species circulans. Co-cultivation of hypocotyl explants with the bacterium promoted somatic embryo formation and improved both the frequency and quality of somatic embryos. In the cultivar Ringo Rose, the least responsive among the cultivars screened, the embryogenic response was more than four times that of axenic cultures. Nearly 70% of these embryos converted into plantlets, while the somatic embryos induced under axenic conditions developed poorly and plantlet formation was inconsistent. Among the different treatments of bacterial culture tested (autoclaved culture, culture filtrate, sonicated bacterial culture, sonication of bacterial culture followed by filtration, HPLC fractionation of crude bacterial lysate), only two HPLC fractions promoted embryogenesis to a marginal degree. Co-cultivation of the explants with bacterium during the first week of induction was crucial for obtaining high-frequency embryogenesis, indicating the role of bacterial stimuli during the induction process. Received: 23 June 1998 / Revision received: 20 August 1998 / Accepted: 27 October 1998     

In vitro organogenesis and somatic embryogenesis from punctured leaf of Populus nigra × P. maximowiczii

Various explant sources of Populus nigra × P. maximowiczii were used to examine the effects of growth hormones on morphogenesis in vitro. Initial experiments indicated that punctured leaves were superior to non-punctured ones for both callus growth and formation of shoots and roots on MS medium containing various types and concentrations of growth hormones. After 6 weeks in culture, an average of 178 shoots, 129 roots and 3.1 g fresh weight of callus were directly produced from the abaxial side of each punctured leaf. The best combinations of growth hormones for shoot, root and callus proliferation were 0.88 M BA plus 0.05 M 2,4-D, 0.44 M BA plus 2.69 M NAA and 0.44 M BA plus 2.26 M 2,4-D, respectively. Embryoids were also formed on callus derived from punctured leaves. The number of embryoids varied from 0 to 30 per punctured leaf. Adventitious shoots also developed simultaneously with the embryos. Embryoids were removed with a scalpel at the early developmental stages and placed on MS medium lacking growth regulators. Regenerated plantlets were transferred to pots containing vermiculite for normal growth in the greenhouse.     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

Does ethylene play a role in thidiazuron-regulated somatic embryogenesis of geranium (Pelargonium × hortorum bailey) hypocotyl cultures?

Summary The accumulation of ethylene in headspace of hypocotyl cultures of geranium (Pelargonium × hortorum Bailey) and its possible role in thidiazuron-mediated somatic embryogenesis was investigated. The action of ethylene as determined by various ethylene synthesis and action inhibitors was varied. Silver nitrate (AgNo₃), aminoethoxyvinylglycine (AVG), and silver thiosulphate (STS) had no significant influence on the embryogenic response, while 1-methylcyclopropene (1-MCP) applied during the initial 3 d of induction or the expression phase, significantly increased the number of somatic embryos formed. Thidiazuron-treated tissues accumulated large quantities of ethylene within 6 h of culture, but the levels decreased after 12 h and reached very low levels after 3 d in culture. In the presence of acetylsalicylic acid (ASA), the levels of ethylene decreased by 20 to 50% during the first 48 h of culture. Analysis of endogenous auxin, cytokinins, and abscisic acid (ABA) indicated possible interactions of ethylene with other phytohormones during the induction of somatic embryos on geranium hypocotyl explants. Thidiazuron (10 μM) increased, while ASA decreased the levels of endogenous auxin, cytokinins, and abscisic acid during this period of induction.     

Plant regeneration from embryogenic cell suspensions and protoplasts of dessert banana cv. ‘Da Jiao’ (<Emphasis Type=Italic>Musa paradisiacal</Emphasis> ABB Linn.) via somatic embryogenesis

The ‘Da Jiao’ cultivar of banana (Musa paradisiacal ABB Linn.) is an ideal germplasm to produce new banana varieties resistant to Fusarium oxysporum f. sp. cubense (FOC) race 4, for this cultivar is not only a popular dessert banana in south China, but also bears high resistance to FOC race 4. In this study, we established a homogeneous embryogenic cell suspension (ECS) of ‘Da Jiao’ and obtained regenerated plants from ECS-derived protoplasts via somatic embryogenesis. The ECS was initiated from yellow friable callus induced from immature male inflorescence on M1 medium. A pre-culture was used to select ECS in M2 medium without 2,4-dichlorophenoxyacetic acid for 10 d. Addition of 1.0 mg L⁻¹ abscisic acid to M3 medium could enhance the frequency of somatic embryogenesis by about 2.6-fold. Protoplasts, with a yield range of 5–6 × 10⁶ per milliliter, were isolated from the ECS. About 0.35% of the protoplasts formed microcallus, which contained about 100 cells, after 1 mo of feeder layer culture with ECS of Musa acuminate cv. Mas (AA) as nurse cells. Healthy plantlets (0.14%) were regenerated from the microcallus through somatic embryogenesis.     

Quantitative treatment of a bioreactor employing a recombinant cell system — A theoretical analysis

Summary A theoretical analysis on the product distribution in a bioreactor employing a recombinant cell system has been made. Simulated results of a batch or plug flow reactor based on Monod's kinetics show that a higher initial concentration of plasmidless cell should be avoided to prevent rapid consumption of substrate. This enables one to get maximum concentration of metabolite, the desired product.     

RLCC-isolation of Raucaffricine from its most efficient source — cell suspension cultures of Rauwolfia serpentina Benth

Raucaffricine (vomilenine-galactoside) was shown to be the major indole alkaloid of Rauwolfia serpentina Benth. cell suspension cultures grown in AP-medium (alkaloid production medium). Several grams of this glycoalkaloid can conveniently be isolated by RLCC (Rotation Locular Countercurrent Chromatography). A newly discovered enzyme efficiently converts the glycoalkaloid to its aglycon, vomilenine, which occupies a key function in the biosynthesis of ajmaline. This is the first demonstration of the occurrence of raucaffricine in Rauwolfia serpentina.     

Cell suspension culture of Rhizoma zedoariae in a two-stage perfusion bioreactor system for β-elemene production

We presented a two-stage combined bioreactor system consisting of a stir-tank and an airlift column, and challenged with Rhizoma zedoariae cell suspensions for β-elemene production. Two-stage culture was initiated when the cell concentration in both vessels was maintained at an appropriate density. The cells were proliferated in stirred-tank with the maximal growth rate of 0.17 d⁻¹ to present enough cells for β-elemene synthesis. In the airlift column, continuous cell separation from culture medium was achieved by using a cell retention device based on centrifugal and gravity settling when the system was performed in perfusion mode. The results indicated that additives can efficiently promote the accumulation of β-elemene in R. zedoariae cells. In addition, the β-elemene content showed higher levels in cell lines of overexpressing 3-hydroxy-3-methylglutaryl coenzyme-A reductase, Farnesyldi phosphate synthase, and ST02C genes.

     

Plant regeneration from interspecific hybrid and backcross progeny of Helianthus eggertii × Helianthus annuus

An efficient protocol for plant regeneration from leaves of the interspecific hybrid Helianthus eggertii Small. × Helianthus annuus L. was developed. The regeneration capacity of the first backcross progeny is reported. Leaves from the F1 interspecific hybrid were cultured on Murashige and Skoog basal media (MS) supplemented with -naphthalenacetic acid (NAA), N ⁶-benzyladenine (BA), AgNO₃, KNO₃, casein hydrolysate and adenine sulfate. Embryo-like structures and/or shoots regeneration were observed on most of the tested media. The best results were obtained on media with a higher concentration of cytokinin (8.8 M BA) and lower concentration of auxin (1.08 M NAA). The addition of casein hydrolysate in the media increased the regeneration efficiency. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. The regeneration potential of leaf, stem and root explants of eighteen first backcross lines was studied. Most of the tested lines were highly regenerable and some of them had DNA content closely related to that of Helianthus annuus L.     

Geranylhydroquinone 3′′-hydroxylase, a cytochrome P-450 monooxygenase from Lithospermum erythrorhizon cell suspension cultures

Geranylhydroquinone 3′′-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae). The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor of shikonin. Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed that hydroxylation takes place specifically at position 3′′, i.e. at the methyl group involved in the cyclization reaction. The enzyme is membrane-bound and was found in the microsomal fraction. It requires NADPH and molecular oxygen as cofactors, and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO. The inhibitory effect of CO is reversed by illumination. These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase. The optimum pH of GHQ 3′′-hydroxylase is 7.4, and the apparent K _m value for GHQ is 1.5 μM. The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower than that obtained with geranylhydroquinone. Received: 20 March 1999 / Accepted: 20 July 1999     

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L⁻¹ picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L⁻¹ 2,4-D, 0.16 g L⁻¹ putrescine, 0.5 g L⁻¹ casein amino acids and 2.0 g L⁻¹ activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L⁻¹) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.     

High-efficiency direct somatic embryogenesis and plant regeneration from leaf base explants of “peperina” (Minthostachys verticillata)

In Vitro Cellular & Developmental Biology - Plant - In vitro culture has been recognized as a potential plant clonal propagation tool for a broad variety of commercial, ornamental, and...     

The accumulation of podophyllotoxin-β-d-glucoside by cell suspension cultures derived from the conifer Callitris drummondii

Summary Podophyllotoxin was produced by cell cultures derived from needles of Callitris drummondii. The needles of this conifer contained 1.56% podophyllotoxin on a dry weight basis, 32% being present in the -glucosidic form. Trace amounts of desoxypodophyllotoxin and matairesinol were also detected. In dark-grown cell cultures, ca. 0.02 % podophyllotoxin was accumulated, 85–90 % in the -D-glucosidic form. Moreover, traces of the lignans matairesinol, hinokinin and asarinin were detected. Illumination stimulated the endogenous production of podophyllotoxin--D-glucoside; contents of up to 0.11 % could be measured.