Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

The cryopreservation of shoot tips of Rosa multiflora

Shoot tips of in vitro grown plantlets of Rosa multiflora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.Abbreviations RSC1 modified Murashige and Skoog medium for Rosa multiflora shoot culture     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids

Summary A method for the production of somatic embryos and subsequent plant regeneration for Anthurium andraeanum Linden ex André (Monocotyledonae) hybrids is described. Whole leaf blade explants, derived from plantlets grown in vitro, formed translucent embryogénic calli at their basal ends within one month of culture in the dark. Secondary somatic embryos formed frequently and without an intervening callus on surfaces of primary embryos. Embryogenesis was induced with three genotypes using a modified half-strength Murashige and Skoog (MS) medium supplemented with 1.0 to 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.33 to 1.0 mg l^–1 kinetin. A combination of 2% sucrose with 1% glucose in the medium favored embryogenesis over 3% sucrose alone. Whole leaf blades on medium solidified with 0.18% Gelrite produced more somatic embryos than leaves on medium with 0.7% Bacto-agar. Within two to three months after culture initiation, embryos were transferred to modified MS medium containing 0.2 mg l^–1 6-benzyladenine (BA) and 2% sucrose and placed in the light for conversion into plantlets. Rooted plantlets were recovered and transferred into pots with tree fern fiber medium and grown in the greenhouse.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine     

ZEA MAYS EMBRYO SACS IN CULTURE. I. PLANT REGENERATION FROM 1 DAY AFTER POLLINATION EMBRYOS

One day after pollination, embryo sacs of Zea mays, containing zygotes and a few endosperm nuclei, were partially isolated and grown in culture. The zygotes underwent normal embryo development, and 40% germinated on a modified Murashige and Skoog medium with 0.1 mg/liter 6-benzylaminopurine. After the appearance of a root and a shoot, individual plantlets were transferred to soil and grown to sexual maturity. The techniques for partial mechanical isolation and successful growth to maturity of such young embryos provide the tools necessary for the recovery of plants from in vitro-fertilìzed embryo sacs.     

In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr.

A method is presented for the in vitro propagation of Datura insignis Barb. Rodr. Nodal explants were cultured on Murashige and Skoog medium supplemented either with BA alone or in combination with 2,4-D or IAA. Shoot multiplication and elongation were obtained in various growth regulator concentrations. Best results were obtained in a medium with 1.0 mgl^-1 of BA. Individual shoots were excised and transfered to growth regulator-free medium for rooting. Additional multiplication was obtained by single-node culture using explants from these in vitro rooted shoots. Rooted plantlets were successfully grown in soil.     

Micropropagation of seed-derived plants ofCynara scolymus L., cv. ‘Green Globe’

Growth of Cymbidium kanran rhizome was enhanced by higher NAA:BAP ratios in modified Murashige & Skoog (MS) media. Only vegetative shoots resulted from rhizomes cultured in vitro when lower NAA:BAP ratios were used. The rhizomes were induced from the axils of leaves when shoots were explanted to medium containing higher concentrations of NAA. Root formation of C. kanran was inhibited by the addition of either auxin or cytokinin to the culture media. Differentiation of the rhizomes into plantlets occurred when the concentrations of ammonium nitrate and potassium nitrate in MS medium wewe reduced. The modified MS medium containing lesser amounts of potassium nitrate and ammonium nitrate than those of the original MS media, and was optimal for the production of plantlets from rhizomes of C. kanran without addition of auxin and cytokinin.Abbreviations NAA -naphthaleneacetic acid - BAP N⁶-benzylaminopurine - MS medium Murashige & S Skoog medium     

Improvement of somatic embryogenesis and plant recovery in cassava

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA Benzyl aminopurine - GA Giberellic acid - MS Murashige and Skoog - NAA Naphthalene acetic acid     

Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos

Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation. The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog     

In vitro control of adventitious bud differentiation by inorganic medium components and silver thiosulfate in explants of Passiflora edulis F. flavicarpa

Summary Hypocotyl and leaf explants from Passiflora edulis F. flavicarpa were evaluated for morphogenesis when cultured on several nutrient media supplemented with benzyladenine and indoleacetic acid. The effect of silver thiosulfate on growth-regulator-induced morphogenesis was also investigated. Murashige and Skoog medium was more effective than woody plant medium in promoting adventitious bud differentiation. The omission of ammonium or nitrate from the Murashige and Skoog medium and a disequilibrium from the Murashige and Skoog nitrate: ammonium ratio drastically reduced the bud-forming capacity of the explants. The inclusion of silver thiosulfate in the culture medium significantly increased the differentiation and development of adventitious shoots. Regenerated shoots were excised and induced to root on basal Murashige and Skoog medium. Plants were transplanted to pots and grown ex vitro.     

Meristem culture and virus eradication in Alstroemeria

Stem apical meristems, rhizome apical meristems and rhizome axillary meristems excised from Alstroemeria plants were grown in vitro on modified Murashige and Skoog (MS) media containing different concentrations of gibberellic acid and 6-benzylaminopurine (BA). Plantlets developed from stem apical meristems never regenerated a rhizome and eventually died. The highest regeneration rate (74.1%) of plantlets with a rhizome was observed when rhizome axillary meristems were grown on modified MS medium containing M 8.9 of BA. Alstroemeria mosaic potyvirus (AlMV) could be eradicated from infected Alstroemeria plants through meristem culture. The rate of virus eradication was 73.7 and 14.7% for plantlets developing from explants measuring 0.7 mm and 2.0 mm, respectively. Greenhouse evaluation of virus-negative and AlMV-infected Alstroemeria plants showed that healthy plants produced more floral stems, more vegetative stems, longer floral stems and gave a higher fresh weight than infected plants.     

In vitro production of solasodine from Solanum trilobatum

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g⁻¹ (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g⁻¹ (d.m.) of solasodine.     

Micropropagation of Thymus piperella

Explants from aseptically germinated seeds of Thymus piperella L. were induced to form shoots on modified Murashige and Skoog medium, the best yield being 5.1 shoots per explant when the medium contained 6.6 M BA plus 2.8 M IAA. Shoots could be rooted on the same basal medium supplemented with 2.8 M IAA, and 71% of the plantlets were successfully acclimatized.Abbreviations BA benzyladenine - CMS modified MS culture medium - IAA indoleacetic acid - MS Murashige & Skoog (1962) culture medium - NAA -naphthaleneacetic acid     

Anther culture of some perennial triticeae

Three field grown Agropyron spp. (crested wheatgrasses) and two Thinopyrum spp. (intermediate and tall wheatgrasses) were evaluated for anther culture response. Hormonally modified potato extract and 85D12 media induced pollen embryogenesis. Modified Murashige and Skoog media were tested for their effects on callus proliferation and plantlet regeneration. Callus induction frequency and plantlet production were highest (25.0% and 45.8%, respectively) for Thinopyrum ponticum (2N=70) (tall wheatgrass). One-hundred and nine albino plantlets were produced from T. ponticum Jose both by direct regeneration on 85D12 medium and through a callus phase from potato extract media. This is the first report of plantlet production from anther culture of a Triticeae perennial forage grass. Further experimentation with environmental and cultural conditions may result in the production of green plantlets.Abbreviations MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-ip 2-isopentenyladenosine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid Cooperative investigations of the USDA-Agricultural Experiment Station and the Utah Agricultural Experiment Station, Logan, UT 84322. Approved as Journal Paper No. 3596     

Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L.

Protoplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark at 24°C with or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasts. Microcolonies developed after 10 d of culture, which in turn formed callus upon transfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in thin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlets were grown to flowering plants upon transfer to soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA Fluoresceine diacetate - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige & Skoog medium (1962)     

Similarity of growth patterns between plantlets and seedlings of Brassica campestris L. under different in vitro environmental conditions

Explants and seeds of Brassica campestris L. were cultured on Murashige & Skoog (1962) medium with and without sucrose in a vessel with different numbers of air changes per hour under different PPF (photosynthetic photon flux) conditions. The growth and development of plantlets in the vessel were similar to those of seedlings when cultured under the same in vitro environmental conditions. The growth and development of seedlings when cultured under the same in vitro environmental conditions. The growth and development of plantlets/seedlings were greater for treatments with a higher number of air changes per hour and a higher PPF regardless of the sucrose concentration in the culture medium.The CO₂ concentration in the vessel with a lower number of air changes per hour decreased to approximately 100 ppm during the photoperiod on day 21 due to the photosynthetic activities of the plantlets/seedlings. The low CO₂ concentration, in turn, reduced the net photosynthetic rate of plantlets/seedlings in the vessel, and thus affected their growth and development.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration in the culture room - MS mineral composition of Murashige & Skoog (1962) medium - PPF photosynthetic photon flux     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

Embryogenesis and plantlet development in the bamboo Phyllostachys viridis (Young) McClure

Leaf explants of Phyllostachys viridis (Young) McClure were cultured on Murashige and Skoog medium supplemented with 9×10^-6 M 2,4-dichlorophenoxyacetic acid. Numerous embryoids were observed. On transfer to Murashige and Skoog medium lacking hormones, plantlets developed within two weeks and were later successfully transferred to the field.