In vitro culture of adult and juvenile bud explants of Passiflora species

Cultivar E23, an F1 hybrid of P. edulis and P. edulis f. flavicarpa is usually propagated by shoot-tip grafting. Various media were tested to evaluate the potential of E23 for in vitro propagation. Adult tissue was difficult to culture and did not respond to media containing low (<10 µM) concentrations of growth regulators. Growth of adult buds on intact stem sections was promoted by 1 week of dark incubation on MS basal medium plus 150 µM 2iP, 200 µM adenine sulphate and 17.1 µM IAA (3 mg l^–1), and further developed into shoots on MS medium plus 4.9 µM 2iP (1 mg l^–1) and 5.7 µM IAA (1 mg l^–1). By contrast, juvenile shoots of E23, and Passiflora species: edulis f. flavicarpa, edulis, alata, caerulea, mollissima, coccinea, herbertiana and suberosa grew rapidly on MS medium plus 10 µM kinetin and 5 µM IAA. Rapid multiplication was achieved on MS plus 20 µM BA, 10 µM kinetin, 5 µM IAA, and roots initiated on MS plus 5 µM IAA.Abbreviations IAA indole-3-acetic acid - 2iP N6-iso pentenyl adenine - BA N6-benzyl adenine     

Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid

Multiple shoots were produced from nodal explants of cassava (Manihot esculenta Crantz) by a two-step procedure: a 6- to 8-day exposure to 0.11–0.22 µM thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by culture on agar-solidified MS medium supplemented with 2.2 µM 6-benzyladenine (BA) and 1.6 M gibberellic acid (GA₃). TDZ caused the nodal explants to expand and this expansion (growth) continued during culture with BA and GA₃. From this expanded explant, clusters of buds and fasciated stems developed continuously and these gave rise to shoots. The shoot proliferation process was open-ended, yielding an average of 31.5 shoots per nodal explant after 10 weeks of culture with genotype CG 1–56. A positive response was also obtained from seven other genotypes evaluated with this protocol.Abbreviations BA 6-benzyladenine - BM basal medium - DPU 1,3-diphenylurea - GA₃ gibberellie acid - 2iP isopentenyladenine - MSM multiple shoot medium - NAA 1-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - Z zeatin     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tsatsai</Emphasis>)

Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.     

Factors Affecting In Vitro Multiplication of Date Palm

Rapid method of in vitro multiplication of date palm was developed. Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm^–3 dimethylaminopurine (2iP) + 1 mg dm^–3 naphthalene acetic acid (NAA). Shoot buds were proliferated from white nodular cultures on hormone free medium. Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm^–3 2iP + 0.5 mg dm^–3 NAA. Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium. Among four concentrations of sucrose used, 30 g dm^–3 speeded up the bud proliferation more than 10, 20 and 40 g dm^–3. However, the largest shoot buds were observed with 40 g dm^–3 sucrose. Solidification of culture media by 1.75 g dm^–3 Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm^–3 Phytagel.     

Vegetative propagation of Begonia venosa Skan in vitro from inflorescence explants

Methods are described for the vegetative propagation of Begonia venosa Skan. Young flower buds are capable of producing callus which, contrasting to callus from leaves of adult plants, is very organogenic. For callus induction are required: BA and NAA at a conc. of 0.5 mgl^–1, 21 °C and low irradiance. Subculture of organogenic callus is optimal on a medium with 0.1 mgl^–1 BA and 2% glucose, whereas NAA is ommitted. Shoot development from preformed adventitious buds is enhanced by lowering the BA and glucose conc. Optimal rooting of excised shoots is obtained on a medium without regulators and a low glucose level. No visible mutations can be detected in the plant material, even not at the flowering stage.Abbreviations (BA) 6-Benzylaminopurine - (2iP) N⁶-(2-isopentenyl)adenine - (MS)(1) Murashige and Skoog - (NAA) 1-Naphthaleneacetic acid Publ. 535     

Thidiazuron promotes adventitious shoot regeneration from pothos (Epipremnum aureum) leaf and petiole explants

Summary Regeneration of adventitious shoots of pothos (Epipremnum aureum Linden and Andre) ‘Jade’ was obtained using leaf and petiole explants preprated from shoot tips of 3-yr-old greenhouse-grown plants. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), 6-(4-hydroxy-3-methy-trans-2-butenyl-amino)purine (zeatin) or N-isopentenylaminopurine (2iP) individually with α-naphthaleneacetic acid (NAA) in 18 combinations. Callus was initiated from cut surface and along the midrib or major vein of leaf sections. Shoot regeneration from leaf and petoole explants occurred in 30d on medium containing 1, 5 or 10μM TDZ with 0.5 or 1.0μM NAA except petioles on medium with 10 μM TDZ and 1.0 μM NAA where regeneration failed. More time (50d) was needed for shoot regeneration when explants were cultured on medium containing either 2iP or zeatin with NAA. Regeneration frequencies were up to 20% and 50% for leaf and petiole explants, respectively. Shoot numbers per responding explant attained 30 for leaf and petiole explants on medium containing TDZ but only one to four on medium containing either 2iP or zeatin. These results indicate that TDZ is a more effective cytokinin for in vitro regeneration of pothos than either zeatin or 2iP.. Shoots elongated readily and rooted well on MS basal medium, without plant growth regulators. Plantlets acclimatized rapidly and grew vigorously in the greenhouse after transfer to pots containing a commerecial potting medium.     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

In vitro Shoot Regeneration from Leaves of Sweet Cherry (Prunus avium) 'Lapins' and 'Sweetheart'

Shoot regeneration was achieved from leaves of in vitro cultures of Prunus avium L. cv. 'Lapins' and 'Sweetheart' using woody plant medium (WPM) supplemented with 1-naphthalene-acetic acid (NAA) and thidiazuron (TDZ) or benzyladenine (BA). Percent regeneration was influenced by plant growth regulators and by explant type, orientation and wounding. Optimal regeneration was observed with whole-leaf explants wounded by transverse cuts along the midrib and incubated abaxial surfaces uppermost, on media supplemented with 2.27 or 4.54 µM TDZ plus 0.27 µM NAA. The percent regeneration of the two cultivars was not significantly different. Optimum conditions for regeneration resulted in 71.4% of 'Lapins' and 54% of 'Sweetheart' explants producing one or more shoots per explant.     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Germplasm Preservation of Wild Arachis Species through Culture of Shoot Apices and Axillary Buds from In Vitro Plants

A study was conducted to evaluate in vitro techniques for germplasm preservation of wild species of Arachis. Nodal segments excised from in vitro-grown plants of A. retusa, A. macedoi and A. burchellii were used to examine the effects of explant position and age of the donor plant. Explants were excised from plants maintained in culture for 30, 60, 90 or 180 d, numbered I – V from top to bottom and cultured on MS medium supplemented with 2.7 µM NAA or different BAP concentrations (0, 4.4, 13.2 and 22 µM). The age of the donor plant has not influenced the responses of the four genotypes studied. In contrast, shoot regeneration ability was significantly affected by the original explant position, decreasing from top to bottom. In media supplemented with different BAP concentrations, multishoot formation was induced from apical segments at low frequencies (10 – 20%) and segments of all positions originated calluses at the explant basis after 30 d of culture. The culture of nodal segments in the presence of 2.7 µM NAA as the sole growth regulator is recommended for the multiplication of in vitro collections of wild groundnut species in order to avoid callusing and adventitious shoot formation.     

In vitro organogenesis and plant regeneration from leaves of Solanum candidum Lindl,S. quitoense Lam. (naranjilla) and S. sessiliflorum Dunal

Adventitious shoots and roots were regenerated from leaf segments of 3 Solanum species: S. candidum Lindl., S. quitoense Lam. and S. sessiliflorum Dunal. Leaf explants differentiated shoots on modified MS medium supplemented with 23–163 M kinetin and 0–5.7 µM indoleacetic acid (IAA). Excised shoots were induced to form roots by transfer to media with benzyladenine (BA) and naphthaleneacetic acid (NAA) at 0.09 and 0.11 µM respectively for S. quitoense and 0.01 µM NAA for S. candidum and S. sessiliflorum. Adventitious roots were produced directly from leaf explants with 0–140 µM kinetin and 0–5.7 µM IAA in combination. Rooted plants were successfully established in the greenhouse.     

Micropropagation of Two Australian Native Fruit Species,Davidsonia pruriens and Davidsonia jerseyana G. Harden & J.B. Williams

Nodal explants from in vitro grown seedlings of Davidsonia pruriens and D. jerseyana, established on MS media were treated with various concentrations of three cytokinins. D. pruriens developed optimum shoot growth in terms of shoot height and number of leaves per shoot when 1.0 µM BA was added to basal MS medium while optimum shoot growth for D. jerseyana was obtained when 0.01 µM 2iP was added to the medium. Optimum root initiation and development was obtained when actively growing axillary shoots were cultured on 1/2MS medium plus 32.2 µM IBA for 3–5 days for D. pruriens and 2–3 days for D. jerseyana before transfer to PGR-free medium containing 10 µM riboflavin. Root initiation of more than 80% was achieved with multiple genotypes of D. pruriens and three genotypes of D. jerseyana using juvenile material. The plantlets were transferred to pots and grown in the greenhouse with a success rate of 60% for D. pruriens and 75% for D. jerseyana. Adult D. jerseyana stem explants produced 2–5 shoots per nodal explant upon treatment with 0.1 µM BA. Side shoots from adult D. jerseyana produced similar results for shoot multiplication as for juvenile material. Protocol for multiplication of adult D. pruriens was achieved with much greater difficulty by using material from the green house. Axillary shoots were initiated when 100 µM TDZ was applied to the stem of an adult pot plant and the resultant side shoots were cultured on MS medium containing 1.0 µM BA and 1.0 µM GA₃.     

Plant regeneration from callus and protoplasts in Medicago polymorpha

Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l^-1 2iP and 0.1 mg l^-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l^-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.Abbreviations NAA -naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - 2iP N⁶-²-isopentenyl-adenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - GFMS growth regulator free MS medium - Prol proline - Malt maltose     

In vitro multiplication ofVella lucentina M. B. crespo (Brassicaceae), a threatened Spanish endemic species

Summary Vella lucentina M. B. Crespo is a threatened Spanish species that is endemic to a small area in eastern Alicante Province (SE Spain). Micropropagation techniques were applied forex situ conservation of this plant. Aseptic epicotyls bearing the apical bud were grown in Murashige and Skoog medium supplemented with 6-furfurylaminopurine (Kin), N⁶-benzyladenine (BA) or 6-(γ,γ,-dimethilalylamino) purine (2iP). High multiplication rates were obtained with 0.5, 1, or 2 mg·liter⁻¹ BA, or 1 or 2 mg·liter⁻¹ 2iP. Indole-3-acetic acid and indole-3-butyric acid were utilized for rooting in half-strength Murashige and Skoog medium. Regenerated plants were transferred to a potting mix and gradually acclimated to field conditions. No morphological differences were observed amongin vitro andin vivo plants.     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.