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Farida Safadi and Harrison Hughes

Detached and intact leaves (first fully expanded leaf from the top) of tobacco (Nicotiana tabaccum L.) plantlets hardened in vitro with 2.0% polyethylene glycol (PEG) showed increased diffusive resistance (r) over those of nonhardened plantlets as measured by a steady state porometer. The leaves of the PEG hardened plants maintained a higher resistance throughout the one hour dessication period in approximately 30% relative humidity although both treatments showed an increase in diffusive resistance after 30 minutes. This indicates that the stomates are functioning in the in vitro tobacco plantlets. The higher (r) in the PEG treated plants may be due to more complete closure of stomates, higher cuticle wax content or a combination of both.

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Kee-Yoeup Paek and Eun-Joo Hahn

Adventitious buds regenerated from homogenized leaf tissue of Saintpaulia ionantha Wendl. `Crimson Frost' were micropropagated to determine types and frequencies of the variants obtained. Plants grown for one year in a greenhouse showed 67% variation and 33% normality. A higher rate of variation was observed in leaf color rather than in leaf shape. The variations in leaf color and leaf shape were 67% and 19%, respectively. In regard to flower type, greater numbers of semidouble and double types were obtained as compared to single types. Both flower types showed a much higher rate of normal (mixed) color (81%) as compared to pink, red or white (19%). An sodium dodecylsulfate polyacrylamide gel electrophoresis profile of protein extracted from leaves of the stock plants and the variants indicated no difference between them and did not reflect the variation in phenotype.

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S. Mohan Jain, Carola Lasus, and Karita Alen

The aim of this study was to isolate Phytopthora cactorum-resistant strawberry plants, regenerated from gamma-irradiated explants on a shoot regeneration medium. Three gamma doses (0, 5, 10, 15 krad) were used to irradiate strawberry axillary buds taken from in vitro-grown plants. After irradiation, axillary buds were cultured on a shoot regeneration medium containing 0.75 mg BA/liter and 0.4 mg IBA/liter. Shoot regeneration occurred mainly from axillary buds irradiated with 5 and 10 krad. The highest dose (15 krad) produced few shoots. The shoot regeneration rate was highest at the 50-krad dose. All the regenerated plants were transferred in the greenhouse. The crude extract of P. cactorum, isolated from the strawberry field, was prepared in sterile water; 1 ml of it was put directly in the center of the crown of each of 400 regenerated plants. After 2 weeks, leaves of most of the plants wilted. Only 20 plants survived the first round of selection; they grew slowly when compared with the control and also showed some tolerance to drought. Further investigations are in progress to reconfirm the resistance of selected putative disease-resistant strawberry plants.

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P.W. Simon, C.E. Peterson, and W.H. Gabelman

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Fabiola Domínguez, Xavier Lozoya, and James Simon

An efficient whole plant regeneration method from callus cultures of Piper auritum was achieved through organogenesis derived from leaf tissue. Proliferating callus and shoot cultures derived from leaf tissue explants placed on Murashige and Skoog (MS) medium supplemented with 2.0 mg·L–1 2, 4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 mg·L–1 kinetin. Optimum combination of hormones (mg·L–1) for shoot induction was 0.5 2,4-D: 1.5 mg·L–1 kinetin (by volume), that resulted in a high rooting rate (49.6 shoots per explant). All of the plants elongated when using a medium consisting of 0.1 mg·L–1 2,4-D plus 1 mg·L–1 kinetin. Elongated shoots were successfully rooted (100%) on half-strength MS medium supplemented with 2.0 mg·L–1 indole-3-acetic acid. All plantlets survived to the growing conditions of a greenhouse. This study demonstrates that leaf tissue of P. auritum is competent for adventitious shoot regeneration and establishes an efficient and useful protocol for the multiplication and conservation of P. autirum for further investigation of its medicinally active constituents.

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J.L. Robbins, X.Y. Li, and A.K. Yadav

Shoot tip explants from field-grown guava (Psidium guajava L.) trees, which frequently show a high rate of contamination, were cultured on the MS medium with neem leaf (Azadirechta indica L.) extract in H2O. Ten grams of neem powder prepared from crushed dried leaves was made to 200 mL aqueous solution and was left on a laboratory bench for 24 h. The amber-color neem extract was filtered to separate and discard solid. The liquid was refrigerated until needed. To 950 mL basic medium in deionized H2O containing 25 mL macronutrients, 5 mL micronutrients, 5 mL vitamins, 5 mL EDTA + Fe, 2 mL BA, 30 g sucrose, and 7 g agar, 50 mL neem extract was added either before or after autoclaving. No neem extract was added to the control. Medium was adjusted to 5.7 pH. Unused medium was refrigerated. Shoot tips from actively growing young twigs of field-grown L-49 guava trees that showed variable contamination in previous studies were harvested during midmorning hours. Samples were washed in running water and disinfected with 15% Clorox solution with few drops of Tween-20 for 15 min. There were 20 explants in each treatment. Explants were subcultured a week later. None of the four test studies showed contamination-free cultures from field trees. There was no consistency due to autoclaving neem extract. The neem leaf extract did not show complete elimination of contamination, although it delayed it for few days. It was concluded that neem extract was not effective at concentrations used in getting rid of contamination. Therefore, further investigate effect of neem for this purpose higher concentrations need to be examined.

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Proceedings of the Workshop Novel Laboratory Exercises in Plant Tissue Culture

held at the 90th Annual Meeting Nashville, Tennessee 26 July 1993

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A.R. Kuehnle and N. Sugii

Leaf explants of seven cultivars of Hawaiian anthuriums (Anthurium andraeanum Linden ex André cv. Kaumana, Kozohara, Marian Seefurth, Mauna Kea, Nitta, Ozaki, and Paradise Pink) produced callus most successfully after 2 to 3 months on a modified Pierik medium containing 0.36 μm 2,4-D and 4.4 μm BA. Petiole explants callused best on Pierik modified Pierik, and Finnie and van Staden media. Long-term cultures of callus from Univ. of Hawaii anthurium selections UH965, UH1060, and UH1003 were maintained for 12 to 13 months and were still capable of plantlet regeneration. Adventitious plantlets were recovered from callus plated on a Kunisaki medium containing 2.2 or 22 μm BA. Regeneration appeared to be organogenic rather than embryogenic and varied among the genotypes tested. Chemical names used: N- (phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D).

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MC.G. Chávez and R. Ferrera-Cerrato

The responses of four micropropagated strawberry (Fragaria × ananassa Duch.) cultivars (`Douglas', `Tioga', `Aiko', and `Pajaro') to colonization by three vesicular-arbuscular mycorrhizal (VAM) fungi were determined under nursery conditions. Species of VAM endophytes were Glomus sp. CPH-23, Glomus macrocarpum Tul. & Tul., and Glomus versiforme Berth & Trappe. Yield in VAM plants tended to exceed that of nonVAM plants during the latter part of the harvest, but VAM effects differed widely with host-endophyte combinations. Cultivar-endophyte combinations producing the best yield were `Douglas'-Glomus sp. CPH-23, `Tioga'- G. macrocarpum, and `Aiko'- G. versiforme. The number of strawberries per plant differed significantly (P < 0.01) for `Tioga', depending on the cndophytes used. Root colonization by the endophytes varied from 25% to 75%. Yield was not related to colonization.

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Jeffrey W. Adelberg, Bill B. Rhodes, Halina T. Skorupska, and William C. Bridges

Adventitious and axillary shoots of melon (Cucumis melo L.) were cultured from explants on a modified Murashige and Skoog medium containing 10 μm BA. Explants were diversified with regard to genetic source (breeding lines Miniloup, L-14, and B-line), seed parts (apical and cotyledon tissue), seed maturity (10-40 days after pollination; DAP), and cotyledon sections with respect to apical-radicle axis (distal and proximal). Plants were screened for ploidy level by pollen morphometry. Immature cotyledons produced more tetraploid regenerants than mature cotyledons from seed of breeding line Miniloup; the highest frequency of tetraploid regenerant plants was from cotyledons of embryos harvested 18 and 22 DAP. Explants from the apical meristem of the same seeds produced fewer or no tetraploid plants. Proximal sections from immature cotyledons of three genotypes (Miniloup, L-14, B-line) produced higher frequencies of tetraploids than whole mature cotyledons or whole immature cotyledons.