Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

In vitro culture of Dianthus zeyheri subsp. natalensis,a South African carnation

A South African carnation species Dianthus zeyheri subsp. natalensis was cultured in vitro using techniques similar to those developed for the cut carnation (Dianthus caryophyllus L.). The ease of callus and suspension culture establishment makes this species a useful tool for fundamental biochemical studies.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - KIN kinetin - pCPA p-chlorophenoxyacetic acid     

Somaclonal variation and resistance to Verticillium wilt in lucerne,Medicago sativa L., plants regenerated from callus

Somaclonal variation in disease reaction type to infection by the vascular wilt pathogen Verticillium albo-atrum Reinke & Berth was assessed in a population of lucerne plants regenerated from callus lines obtained from a susceptible cultivar. Disease severity in the regenerant population was reduced by comparison with parental controls. Seed progeny and plants recovered via a second tissue culture cycle reverted to mainly susceptible reaction types. In a further experiment a low molecular weight toxic fraction from culture filtrates of the fungus was incorporated into the callus medium prior to regeneration. Toxin treatment reduced the regenerative capacity of callus, and there was little evidence for a higher frequency of wilt resistant plants in populations selected at low toxin concentrations. The results suggest that somaclonal variation as an alternative breeding strategy for disease resistance in lucerne offers no advantages over conventional recurrent selection.     

Influence of pH on Suppression of Fusarium Wilt of Carnation by Pseudomonas fluorescens WCS417r

The influence of the nutrient solution pH on suppression of fusarium wilt by Pseudomonas flurescens WCS417r in carnation grown in rockwool was investigated. Experiments were conducted with carnation cultivars Lena and Pallas, susceptible and moderately resistant to fusarium wilt, respectively. WCS417r significantly reduced fusarium wilt in the susceptible cv. Lena, that was root-inoculated with Fusarium oxysporum f.sp. dianthi (Fod), at pH 7.5, but not at pH 6.5 and 5.5 This corresponded with a higher in vitro siderophore production and antagonism of Fod by WCS417r at pH 7.5 than at pH 6.5 and 5.5. Fusarium wilt in the moderately resistant cv. Pallas, however, was also significantly reduced by treatment with WCS417r at pH 5.5 This corresponded with the low influence of pH on induced resistance by WCS417r in plants of cv. Pallas that were stem-inoculated with Fod. The results indicate that the influence of pH on control of fusarium wilt of carnation by Pseudomonas fluorescens WCS417r differs between carnation cultivars that differ in their level of resistance against fusarium wilt. In susceptible cv. Lena, fusarium wilt is suppressed by antagonism by WCS417r, that is most effective at pH 7.5. In the moderately resistant cv. Pallas, fusarium wilt is suppressed by both antagonism and induced resistance by WCS417r. The latter is also effective at lower pH.     

Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.)

To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole-acetic acid - BA 6-benzyladeninepurine - S.E.M. standard error of mean     

In vitro detection of Cornus florida callus insensitive to toxic metabolites of Discula destructiva

Summary Dogwood anthracnose, caused by the fungus Discula destructiva Redlin, is a severe disease of flowering dogwood (Cornus florida L.) and Pacific dogwood (C. nuttallii Aud.). Disease control is inadequate in nurseries and landscapes and absent in the forest, and resistant cultivars are not commercially available. The ability to select tissues insensitive to culture filtrates from D. destructiva in vitro offers a novel and important approach for the selection of dogwood genotypes that are resistant to or tolerant of this devastating fungus. Embryo-derived dogwood callus cultures were established on Murashige and Skoog medium amended with benzyladenine (BA) and either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA). Selection for insensitivity to D. destructiva metabolites was done by placement of individual cultures on media amended with progressively higher concentrations of a partially purified culture filtrate (PPCF) containing lowmolecular-weight compounds. Following this selection process, cultures were challenged in a dose-response format with PPCF to determine whether the sensitivity of the callus to the culture filtrate had changed. During the selection period, the fresh weight of callus grown on medium containing 2,4-D and amended with PPCF was always less than that of callus grown on medium amended with the same concentration of potato-dextrose broth (PDB, negative control). Fresh weight of callus was greater on medium containing NAA amended with PPCF than on medium with the same concentration of PDB. Callus selected in the presence of NAA showed decreased sensitivity to toxic metabolites at higher concentrations of culture filtrate. The in vitro system described may assist in the identification of disease-resistant germplasm important to the long-term survival of flowering dogwood.     

Further Investigation of the Specificity of Interactions Between Wild Lactuca spp. and Bremia lactucae Isolates from Lactuca serriola

Callus cultures derived from isogenic lines of the tomato cultivars Moneymaker and Craigella, resistant or susceptible to F. oxysporum f. sp. lycopersici, were inoculated with Fusarium oxysporum f. sp. lycopersici race 1. Fungal growth was restricted on callus derived from resistant plants, after inoculation with a conidial suspension, whereas callus derived from susceptible plants was totally overgrown by the fungus within 7 days. The concentration of the phytoalexin rishitin was significantly higher in the callus culture derived from a resistant tomato line compared with the callus culture from a susceptible line, 2 and 3 days after inoculation with mycelium. The results of the experiments were compared with experiments with whole plants. Rishitin production as well as growth of the fungus was comparable with responses in plant-fungus interaction. Therefore callus culture may be useful in studying the interaction between tomato plants and race 1 of F. oxysporum f. sp. lycopersici.     

Callus initiation and plant regeneration in ragi (Eleusine coracana Gaertn.)

Callus was successfully initiated on root, mesocotyl and leaf base segments of 3- to 4-day-old seedlings of ragi (Eleusine coracana Gaertn.). 2,4-D along with casein hydrolysate for Murashige and Skoog's basal medium was found to be most effective for callus initiation and maintenance. Mesocotyl and leaf base tissue derived calli gave shoot buds in medium in which the 2,4-D concentration was lowered.     

Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets

Protoplasts were isolated from field and in vitro-grown leaves, cotyledons and cell suspension cultures (of ovule callus origin) of the scion apple cultivars Starkrimson, Rainier, Qiujin and Liaofu. Fast-growing calluses were obtained from leaf, cotyledon and cell suspension derived protoplasts of the four genotypes. The best proliferation responses were obtained from cell suspension protoplasts. For all genotypes tested, nodular calluses were obtained from protoplasts that had originally been cultured on K8P medium, but only those of cultivar Starkrimson underwent organogenesis. In this cultivar shoot buds were produced on callus derived from both cotyledon and cell suspension protoplasts and complete plants. This is the first example of whole plant regeneration from protoplasts isolated from an undifferentiated tissue in apple.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole acetic acid - IBA 3-indole butyric acid - LH lactalbumin hydrolysate - MS Murashige & Skoog (1962) - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - VC L(+) ascorbic acid     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Plant regeneration via somatic embryogenesis in ginger

Embryogenic callus cultures of ginger were induced from young leaf segments taken from in vitro shoot cultures. Among the four auxins tested in Murashige & Skoog medium, dicamba at 2.7 M was most effective in inducing and maintaining embryogenic cultures. Efficient plant regeneration was achieved when embryogenic cultures were transferred to Murashige & Skoog medium containing 8.9 M benzyladenine. Histological studies revealed various stages of somatic embryogenesis characteristic of the monocot system. The in vitro-raised plants have been established in soil.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

Use of culture filtrates of Ceratocystis ulmi as a bioassay to screen for disease tolerant Ulmus americana

Cullus cultures of elm (Ulmus americana L.) derived from Dutch elm disease susceptible, intermediate-resistant, and resistant genotypes were exposed to the culture filtrates of three patogenic isolates of Ceratocystis ulmi, the causal agent of Dutch elm disease. Callus fresh weights, cell viability, and reactions of stem cuttings were determined after exposure to various concentrations of the filtrates. Calli from the susceptible elm failed to increase in fresh weight and lost viability after exposure to media containing culture filtrate. Calli from the resistant and the intermediate-resistant elms exhibited growth rates and maintained viability similar to controls not exposed to culture filtrate. Stem cuttings of the susceptible elm wilted after exposure to the culture filtrate. The symptoms were similar to wilt seen with the disease. Cuttings from the resistant elm had no disease symptoms whereas, the intermediate elm had some leaf chlorosis. Callus screening may thus be useful for selection of Ulmus germplasm for Dutch elm disease resistance.     

Effect of growth regulators concentrations on morphological development of meristem-tips in Dioscorea cayenensis-D. rotundata complex and D. praehensilis

The use of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-d) has played an important role in the production and maintenance of totipotent cereal callus. However, 2,4-d has been implicated in the loss of totipotency from barley callus. To examine the effect of 2,4-d on barley callus, regenerability and karyotype were examined over time as influenced by cultivar differences and 2,4-d levels, during a period in which initially vigorous plant regeneration typically declines dramatically. Higher (20.4–27.1 M) versus lower (6.8–13.6 M) concentrations of 2,4-d were positively associated with the number of green plantlets recovered from calli maintained for 10 and 16 weeks before transfer to regeneration media, and with the longevity of regenerability. There was a positive relationship between 2,4-d concentration and normal karyotype. We also investigated the use of phenylacetic acid for the initiation of regenerable barley callus. Very poor callus growth and plant regeneration was supported by phenylacetic acid.Abbreviations PAA phenylacetic acid - SPDL(s) single plant-derived lines(s) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - MSO Murashige and Skoog medium lacking growth regulators     

Production of solamargine by in vitro cultures of Solanum paludosum

Callus cultures of Solanum paludosum were established from roots, hypocotyles, cotyledons and leaf limbs of plantlets cultivated in sterile conditions on a Murashige and Skoog's modified medium. Non organogenous calluses were obtained with addition of BA or kinetin (10^-5M to 10^-6M) as the cytokinin and 2,4-d or NAA (10^-5M to 10^-6M) as the auxin. These calluses permitted the establishment of a cell suspension culture with BA (10-6M) and 2,4-d (10^-6M). Zeatin (10^-6M) with IAA (10^-6M) gave rise to organogenous calluses. These organogenous callus cultures developed multiple shoots which either proliferated if they were cultivated on a medium containing zeatin with IAA or IBA or were able to regenerate into whole plants when zeatin was used as the only hormone. The different plant material produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits. The best production was obtained with the fruits of regenerated plants from organogenous callus cultures after reintroduction of these plants in their brasilian biotope. The solamargine content of the two types of plant materials was about 0.06% and 2.5% (dry weight) respectively for the callus cultures and the fruits from in vitro plants. The fruits were harvested a year after the beginning of the plantlet regeneration step.Abbreviations HPTLC high performance thin layer chromatography - HPLC high performance liquid chromatography - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - IAA 3-indolebutyric acid - NAA -naphthaleneacetic acid - IBA 3-indolebutyric acid - IPA isopentenyladenine     

Improved Technique for Rapid and Non-Destructive In Vitro Differentiation between Resistant and Susceptible Banana Clones of Fusarium Oxysporum f. sp. Cubense

Culture filtrates of Fusarium oxysporum f. sp. cubense were applied to field-grown banana leaves of susceptible and resistant clones. The difference in leaf lesions, measured after 48 h, varied from 13 to 51 mm2 depending on the composition of the growth medium.     

Colonization of Fusarium Wilt‐Resistant and Susceptible Watermelon Roots by a Green‐Fluorescent‐Protein‐tagged Isolate of Fusarium oxysporum f.sp. niveum

Interactions between watermelon and a green fluorescent protein (GFP)‐tagged isolate of Fusarium oxysporum f.sp. niveum race 1 (Fon‐1) were studied to determine the differences in infection and colonization of watermelon roots in cultivars resistant to and susceptible to Fusarium wilt. The roots of watermelon seedlings were inoculated with a conidial suspension of the GFP‐tagged isolate, and confocal laser scanning microscopy was used to visualize colonization, infection and disease development. The initial infection stages were similar in both the resistant and susceptible cultivars, but the resistant cultivar responded differentially after the pathogen had penetrated the root. The pathogen penetrated and colonized resistant watermelon roots, but further fungal advance appeared to be halted, and the fungus did not enter the taproot, suggesting that resistance is initiated postpenetration. However, the tertiary and secondary lateral roots of resistant watermelon also were colonized, although not as extensively as susceptible roots, and the hyphae had penetrated into the central cylinder of lateral roots forming a dense hyphal mat, which was followed by a subsequent collapse of the lateral roots. The initial infection zone for both the wilt‐susceptible and wilt‐resistant watermelon roots appeared to be the epidermal cells within the root hair zone, which the fungus penetrated directly after forming appressoria. Areas where secondary roots emerged and wounded root tissue also were penetrated preferentially.     

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.     

Relative Wilt-Inducing Capacity of the Culture Filtrates of Isolates of Fusarium oxysporum f.sp. radicis-lycopersici, the Tomato Crown and Root Rot Pathogen

Cell-free 30-day-old culture filtrates of 24 isolates of Fusarium oxysporum f.sp. radicis-lycopersici (FORL) differed considerably in their capacity to induce wilting in 28-day-old tomato seedings and to inhibiting the germination of tomato seeds. The wilt effects ranged from mild on leaves and lateral stems, to total collapse of the seedlings in 24 h. Wilt, leaf curl and leaf chlorosis, appearing in this sequence, were the three symptoms clicited by the culture filtrates. Boiled and non-boiled filtrates elicited similar sympotoms. The high wilt capacity filtrates were pH 7.2; the others were generally below pH 6. The high wilt capacity filtrates showed polyphenoloxidase activity but the overall pattern of this activity did not correlate consistently with wilt capacity. The majority of the lower wilt capacity filtrates showed a net inhibition of dihydroxy-phenylalanine (DOPA) oxidation. The study suggests that the symptoms in the tomato seedlings were elicited by toxins in the culture filtrates. Further, it appears that the differences obtained in the wilt capacity of filtrates from the isolates were due, at least in part, to inherent differences in the concentration of the toxic factors. The rapidity of the onset of wilt, the total, collaps of filtrate-treated seedlings and the absence of fungi in wilted seedlings suggest further that the operative mechanisms are physiological and biochemical and not impairment of the seedlings’ translocation system by physical blockage with mycelia.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.