Clonal propagation of Cephaelis ipecacuanha

Shoot cultures of Cephaelis ipecacuanha A. Richard were established by using shoot tips as initial explants. Multiple shoots were obtained from node segments upon culture on B5 medium supplemented with NAA-BA (0.01–3, 5 mg/l). These shoots were rooted on B5 and 1/2 MS media containing IAA or NAA, and the regenerated plants were transferred to soil and grown in a greenhouse. The emetic alkaloids of the regenerated plants, mother plants and leaves of shoot cultures were analyzed by TLC and HPLC. Seven months of growth under greenhouse condition, the contents of the emetic alkaloids in the regenerated plants were comparable to those of the mother plants.Abbreviations B5 Gamborg B5 (1968) medium - MS Murashige-Skoog (1962) medium - 1/2 MS a half strength MS medium - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - Kin kinetin - BA 6-benzylaminopurine - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

Efficient and stable regeneration from protoplasts of <Emphasis Type="Italic">Cyclamen coum</Emphasis> Miller via somatic embryogenesis

Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic embryos per gram fresh mass. Up to 4.24 × 10⁵ protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis. Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated from the embryogenic cultures.     

Fertile plant regeneration from cell suspension and protoplast cultures of barley (t Hordeum vulgare cv. Schooner)

Fertile plants were regenerated from both cell suspension and protoplast-derived cultures of the two-row barley, Hordeum vulgare L. cv. Schooner. Embryogenic calluses, derived from immature embryos, were used to establish suspension cultures. More than 100 plants, with variable seed set, have been regenerated from six embryogenic cell suspension cultures. Protoplasts isolated from three suspension cultures divided and when the resultant embryogenic proto-calluses were transferred to regeneration medium both green and albino shoots were produced. The green shoots were transferred to growth regulator-free medium and plantlets that developed strong root systems were potted in soil and grown to maturity in the glasshouse. Root tip analysis of plants regenerated from cell suspension cultures revealed the expected 2N = 14 complement of chromosomes. However, chromosomal analysis of protoplast-derived plants showed numerical variation among a proportion of the regenerants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration in zygotic embryo cultures of balloon flower

Mature zygotic embryos of balloon flower (Platycodon grandiflorum) formed embryogenic calluses at a frequency of 43% when cultured on Murashige and Skoog medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Cell suspension cultures were established from embryogenic calluses using MS liquid medium with 4.52 μM 2,4-D. Following transfer to solid MS basal medium, cell suspension cultures gave rise to somatic embryos, which then developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment and characterization of long-term embryogenic maize callus and cell suspension cultures

Friable callus (type 2) was selected from three genotypes (A188, hybrid A188 × B73, and hybrid B73 × A188) of Zea mays L. The three genotypes of type 2 callus doubled in fresh weight after 1 week, and growth was better on N6 than on Murashige-Skoog (MS) medium. Type 2 callus of hybrid B73 × A188 was maintained in culture longer than A188 type 2 callus, and it regenerated higher numbers of plants than the other two genotypes. Type 2 callus of the hybrid B73 × A188 was used to establish cell suspensions. Suspension cells initially grew better on N6 than on MS medium, but after several months of subculture, cells in either N6 or MS medium grew at similar rates. Suspension cells were in mid-log phase by 5–7 days and in stationary phase by about 10 days depending on inoculum density. Growth rate was optimal when cells were transferred at mid-low phase and dry weight of the suspension cells increased at least 10-fold during a 10-day period. Suspension cells from 9-month-old cultures plated on solid medium regenerated plants at an efficiency similar to that of the friable type 2 callus but with more phenotypic abnormalities. Thus, cell suspensions derived from type 2 B73 × A188 callus, in culture for over 1 year, were capable of regenerating plants when 9-months old.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

农杆菌转化的小冠花发状根的诱导及其植株再生

利用野生型发根农杆菌15834菌株感染小冠花15日龄无菌苗子叶和下胚轴切段,建立了高效的发状根培养及其体细胞胚胎发生再生体系。发状根可直接从受伤的外植体表面产生,也能在外植体诱导的愈伤组织上发生,在无外源激素的MS固体和液体培养基上,转化根能自主生长,表现出典型的发根特征。用适宜浓度的乙酰丁香酮处理对数生长期的农杆菌菌液2h,感染预培养2d的子叶获得了最高的转化频率(87.4%)。在附加0.2mgL2,4_D,0.5mgLNAA和0.5mgLKT的MS培养基上,发状根能100%形成胚性愈伤组织,并于含0.5mgLKT,0.2mgLIBA和300mgL脯氨酸的MS培养基上顺序经过体细胞胚胎发育的各个典型时期,转换成完整植株。再生植株除具有发达的侧根外,其它形态特征与未转化植株未见明显的差异,但在获得的5个转化克隆中,其中1个的发状根及其再生植株叶片中有毒物质3_硝基丙酸的含量显著下降,分别为未转化对照的57.68%和58.17%。冠瘿碱纸电泳检测和rolB基因PCR扩增检测均证明农杆菌Ri质粒上的T_DNA已经整合到小冠花转化细胞的基因组中。     

Neo-formation of flower buds and other morphogenetic responses in tissue cultures of Melia azedarach

Melia azedarach has great interest because of its insecticidal properties. Recently, the occurrence of precocious flowering in tissue cultures of this species was reported. This paper describes some in vitro morphogenetic responses using hypocotyl segments as explants and MS basal medium. Amongst the results we report are: (a) in basal medium, 5% of the explants neo-formed floral buds and flowers, and 80% formed vegetative shoots; (b) flower neo-formation could not be controlled or increased by addition of benzyladenine, or lowering the nitrogen level; (c) benzyladenine increased the regeneration of vegetative shoots; (d) compact green calluses were eventually formed in basal medium, and vigorous friable calluses can be easily induced with 0.5 mM 2,4-D; (e) green calluses could be subcultured and regenerated into plants, and, from friable calluses, cell suspensions were started; (f) histological studies showed that neo-formations originate in the wound tissue or from the inner tissue of the hypocotyl.     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

Plant regeneration from protoplasts of durum wheat (Triticum durum Desf. cv. D6962)

Suspension cultures of durum wheat were established from embryogenic callus maintained in liquid medium for 30 months. Protoplasts were readily isolated from the suspension cultures with yields as high as 3 X 10⁷ protoplasts per g fresh weight suspension cells. When incubated in a modified MS medium containing half strength of the macroelements, 5 M 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.6 M glucose, protoplast-derived cells divided at frequencies ranging from 1.4 to 10.0 %. After transfer to a solid subculture medium, the protoplast-derived colonies formed embryogenic protuberances, from which green plants have been regenerated.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Plant regeneration from hairy roots and calluses of periwinkle Vinca minor L. and foxglove purple Digitalis purpurea L.

Plants regenerated from hairy root culture and calluses of foxglove purple and periwinkle have been obtained. It was found that organogenesis in hairy root culture occurs spontaneously on hormone-free medium but with varying efficiency. The frequency of direct shoot formation from root cultures was up to 60% in Digitalis and 3.7% in Vinca. The addition of 1 mg/L BA, 0.1 mg/L NAA, and 5% sucrose to B5 medium increases regenerative capacity of periwinkle up to 19.1%. Regenerated plants showed morphological features typically seen in Ri-transgenic plants. They include growth and plagiotropism of the root system, increased shoot formation, changed leaf morphology, and short internodes.     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Rapid micropropagation system in vitro and antioxidant activity of Scabiosa tschiliensis Grunning

A rapid micropropagation system for Scabiosa tschiliensis Grunning, an ethnic medicinal plant, has been developed. Calluses were induced from leaf and petiole explants on Murashige and Skoog (MS) medium supplemented with 2.0 mg l^?1 thidiazuron and 0.5 mg l^?1 2,4-dicholorophenoxyacetic acid. In this medium, callus induction rate was about 94.05 %. Adventitious shoots developed from leaf (86.30 %) and petiole (83.33 %) calluses when cultured on MS medium containing 4.0 or 2.0 mg l^?1 N⁶-benzyladenine (BA), respectively. Up to 73.85 % of the regenerated shoots formed complete plantlets on MS medium supplemented with 2.0 mg l^?1 indole-3-butyric acid, with an average of 3.25 roots per shoot. Quantitative analysis of flavonoids showed that the phytochemical profiles of calluses and regenerated plants were similar to that of wild-type plants. The 2, 2-diphenyl-1-picrylhydrazyl assay revealed that the flavonoid extracts of calluses, adventitious shoots and wild-type plants had stronger antioxidant activities, the inhibitory concentrations being 23.944, 31.329 and 26.502 μg ml^?1, respectively, where 50 % of DPPH was scavenged (IC₅₀). Results showed that this perennial herb could be used as a potential source of new natural antioxidants.     

High frequency plant regeneration from immature ovule-derived embryogenic cell suspension cultures of Chelidonium majus var. asiaticum

Culture conditions for high frequency plant regeneration via somatic embryogenesis in cell suspension cultures of Chelidonium majus var. asiaticum are described. Immature ovules formed embryogenic calluses at a frequency of 40% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum ovule size for embryogenic callus formation ranged from 1 to 1.5 mm in length. Cell suspension cultures were established from embryogenic calluses using MS liquid medium containing 4.52 μM 2,4-D. Upon plating onto MS basal medium, cell aggregates from cell suspension cultures produced somatic embryos which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a growth chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant Regeneration from Immature Zygotic Embryo-Derived Embryogenic Calluses and Cell Suspension Cultures of Catharanthus roseus

Culture conditions for plant regeneration in immature zygotic embryo-derived embryogenic cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) Little Bright Eye are described. Immature zygotic embryos formed off-white, friable calluses at a frequency of 20% on Murashige and Skoog (MS) medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) after 8 weeks of culture. After a second subculture using MS basal medium at 4-week intervals, off-white friable calluses formed a small quantity of yellowish, compact embryogenic calluses. Upon transfer to MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Cell suspension cultures were established with embryogenic calluses using liquid MS medium supplemented with 4.52 µM 2,4-D. Embryogenic cell clumps from cell suspension cultures developed into plantlets at a frequency of 56.7% when plated onto MS basal medium. Plantlets were transplanted to potting soil and grown to maturity in a growth chamber.     

Plant regeneration on cultured root segments of Cephaelis ipecacuanha A. Richard

Summary Simple one step micropropagation system for Cephaelis ipecacuanha A. Richard was developed using root cultures grown in vitro. Adventitious shoots were directly formed on the cut end of root segments without callus formation, on phytohormone-free B5 solid medium in the dark. When the shoots attached with root segments were further cultured under 16 h light / 8 h dark, they developed into plantlets, which could be transplanted to soil. The regenerated plants grew well in a greenhouse with showing normal appearance and accumulated alkaloids. The influence of auxin on adventitious shoot formation was also investigated.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - B5 Gamborg B5(Gamborg et al. 1968) - WP woody plant (Lloyd and McCowm 1980) - RC root culture (Thomas and Davey 1982) - HF phytohormone free - IAA indole-3-acetic acid - NAA 1-naphtaleneacetic acid - TIBA 2,3,5-triiodobenzoic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - SEM scanning electron microscopy - C.V. coefficient of variation