Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Micropropagation of candelilla,Euphorbia antisyphilitica Zucc

Axillary shoot proliferation was induced in vitro from shoot explants of greenhouse grown candellila (Euphorbia antisyphilitica Zucc). Optimum shoot proliferation was obtained by supplementing a modified Murashige and Skoog [7] medium with 0.13 M naphthalene-acetic acid and 4.44 M 6-benzylaminopurine. Rooting occurred on 100% of shoots transferred to a medium containing half strength salts supplemented with 0.49 M indole-3-butyric acid. Fully rooted plants were transferred to potting soil and established under greenhouse conditions without special acclimatization techniques.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.     

Micropropagation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees

In vitro propagation protocols were established for two leguminous trees, Bauhinia variegata and Parkinsonia aculeata. In each case axillary shoot proliferation was achieved from nodal explants from mature (6-2-8 years) trees using Murashige & Skoog's medium supplemented with 2.22–31.1 M of 6-benzyladenine. Subsequent rooting of the regenerated shoots was achieved on medium containing 2.46–14.8 M of indole-3-butyric acid. Successful transfer of the regenerants to soil has been accomplished.     

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 M isatin plus 3 M zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA isopentyladenosine - NAA -napthaleneacetic acid     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Callus induction and plantlet regeneration in ornamental Alocasia micholitziana

Callus induction from petiole explants has been achieved in Alocasia micholitziana `Green Velvet'. The highest percentage (71%) of explants inducing callus was obtained on MS medium supplemented with 0.5 M 2,4-D and 0.5 M kinetin in the dark after 4 months of culture. Shoots were regenerated at the highest frequency of 33.3% under light condition when 0.5 M BA was added to MS medium with the average of 7.8±2.3 shoots per callus explant. The callus-derived shoots rooted on hormone free MS medium and within 4 weeks the plantlets were ready for acclimatization. The regenerated plants appeared morphologically similar to mother plants.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

Micropropagation ofGmelina arborea

Multiple shoots were obtained from single node explants of matureGmelina arborea Roxb. on MS medium supplemented with 6-benzyladenine (BA). Seven to nine shoots were formed whenin vitro-derived single node explants were subcultured on MS medium supplemented with 1.1 M BA. For root initiation the cut ends of microshoots were pulsed for 5 min with 246 M indole-3-butyric acid and transferred to a plastic cup containing sterile vermiculite. The shoots were covered with polyethylene bag and maintained in a culture room. After hardening, plantlets were transferred to earthen pots containing a mixture of garden soil: compost and have been established in the field.     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Different regeneration potential of mesophyll protoplasts from cultivated and a wild species of tomato

Mesophyll protoplasts were isolated from leaves of three cultivars of Lycopersicon esculentum (L.) Mill., namely Hilda 72, Rutgers and Rentita, and from the wild tomato species Lycopersicon peruvianum (L.) Mill. Protoplasts from L. peruvianum divided and grew actively in a liquid medium according to Zapata et al. (1977), whereas protoplasts from the tomato cultivars Hilda 72 and Rutgers showed comparable rates for cell division only, when the content of major elements in this medium was reduced to one half of the original concentration and when mannitol as osmoticum was replaced by glucose. In Rentita protoplasts no cell division could be observed in about 15 different modifications of the five basic culture media tested. The morphogenetic potential of these tomato cells was assessed by comparing the root and shoot formation of protoplasts and of leaf explants. L. peruvianum exhibited the highest potential. Calli derived from protoplasts regenerated roots on Murastrige-Skoog agar containing 1 M benzylaminopurine (BAP) plus 10 M indole-3-acetic acid (IAA) and 0.1 M BAP plus 1 M IAA. Shoot formation occurred in the combinations of 10 M BAP with 0.1, 1.0, and 10 M IAA. Plantlets regenerated from the L. peruvianum calli could be grown in soil. No shoots or roots were regenerated from calli of Hilda 72 and Rutgers protoplasts in all combinations of BAP and IAA tested in the range from 0.1 M to 100 M, thus indicating the rather low morphogenetic potential of these protoplasts as compared to protoplasts from L. peruvianum leaves.Abbreviations BAP benzylaminopurine - IAA indole-acetic acid - TMV tobacco mosaic virus