In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

In vitro plantlet regeneration from embryonic explants ofPinus pinea L

Summary Clonal propagation ofPinus pinea L. was achieved by organogenesis on cotyledon explants and the influence of several factors on adventitious bud production and development was investigated. Gupta and Durzan (DCR) medium with benzyladenine (5 μM) induced higher bud production. Bud development and shoot elongation required subcultures on medium with activated charcoal. Rooting was obtained after 10 d culture on medium containing IBA (10 μM).     

Crown architecture of grafted Stone pine (Pinus pinea L.): shoot growth and bud differentiation

The singular umbrella-like crown shape of Stone pine can be interpreted as a consequence of primary shoot-growth patterns and posterior axis differentiation due to differential secondary growth and down-bending of branches. This paper centres on the first aspect, analysing the growth, branching and flowering behaviour of about 5,000 individual shoots on 27 grafted Stone pines. The data measurement on standing trees allowed to study correlations of topologic and geometric variables in the shoot and their ancestors. The only significant correlations were found with parameters of the mother shoot formed the previous year and with the number of cones born 3 years before by the respective ancestor. The fitted relationships between geometric and topologic shoot and branch variables are the first step of a structural model construction that can be completed with functional components like a radiation and a carbon allocation submodel, stressing the importance of the heavy Stone pine cones as carbon sinks, with a total annual allocation similar to stem wood. In conclusion, the Stone pine crown shape emerges as consequence of the lack of initial vigour differentiation between stem and main-branch apical meristems that favour the generalized sylleptic reiteration in the open-grown trees.     

Bud break and multiple shoot formation from tissues of mature trees ofPinus caribaea andPinus kesiya

Summary Proliferation of terminal and axillary buds of 20 yr-oldPinus caribaea andP. kesiya trees was obtained on half strength DCR medium supplemented with 0.5 mg·liter^−1 6-benzylaminopurine (BA). These sprouts further elongated with the formation of multiple shoots with the ratio of 1:3, on transfer to medium in which the 0.25 mg·liter^−1 BA of the initiation medium was replaced by 0.25 mg·liter^−1 kinetin. Rooting was obtained on the same medium. Plantlets thus formed were transferred to perlite:peat:vermiculite mixture (1:1:1) in polybags (10×5 cm) under 80±5% humidity in a polyhouse. Plantlets ofP. caribaea andP. kesiya were established with 72.5 and 83.3% survival, respectively.     

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Antibody against the light-dependent NADPH-protochlorophyllide oxidoreductase of oat was used to detect a protein of the same molecular weight in cotyledons of 40-day-old dark-grown seedlings of Pinus pinea L. Exposure of the seedlings to light resulted in a rapid decrease in protochlorophyllide content without the concomitant decrease in 38 kDa protein which is observed on transfer of dark-grown angiosperm seedlings to light. The stability of the light-dependent NADPH-protochlorophyllide oxidoreductase in pine in the absence of accumulated substrate is consistent with either (1) a different mechanism of regulation of chlorophyll synthesis in gymnosperms or (2) a higher proportion of stable extra-plastidic protein reacting with the antibody to the light-dependent NADPH-protochlorophyllide oxidoreductase than is the case in angiosperms.Abbreviations Chl chlorophyll - Chlide chlorophyllide - NADPH-Pchlide oxidoreductase NADPH protochlorophyllide oxidoreductase - NC nitrocellulose - PBS phosphate buffered saline - Pchlide protochlorophyllide - SDS sodum dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

The Pinus pinea L. woodlands along the coast of South-western Spain: data for a new geobotanical interpretation

The origin and natural range of the Stone pine (Pinus pinea L.) has been questioned for more than a century. In this work, we focus the investigation on one of the most important and controversial regions, viz., the Iberian Peninsula and, specifically, the Huelva and Cadiz populations in Andalusia, one of the most representative population cores. Although some authors maintain that it is an autochthonous Iberian species, most of them consider it to be exotic. From this idea, many works have been done and a sintaxonomic scheme has been created, which is accepted by the majority of the scientific community, not including Pinus pinea, nor its formations, since they are considered as man-induced forest crops. However, Stone has been present for several thousand years in the Iberian Peninsula and in the territory studied, as several paleobotanic and historical data show, proving that Pinus pinea is an autochthonous species of this region. This is a clear consequence to the field of geobotany, since – at least – the Stone pine woodlands from the Iberian Southeast must be considered as communities predominated by an autochthonous species that must be included in the sintaxonomichal schemes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Flora Palinologica Italiana,Sezione Aeropalinologica: S 205 -Pinus pinea L. (Pinaceae)

Summary According to the program Palynological Italian Flora, Aeropalynological section, the pollen morphological card ofPinus pinea L. is presented. The study is carried out on pollen coming from three Italian localities and regards fresh and acetolyzed pollen. For each sample, measurements are carried out on 30 fresh pollen grains in glicerol jelly with fuchsin and on 30 acetolyzed pollen grains in water/glicerol (1/1); general observations regard 1000 fresh and 1000 acetolyzed pollen grains/sample. Some observations on the main differences between fresh and acetolyzed pollen are mentioned.

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Riassunto Nell'ambito della Flora Palinologica Italiana, Sezione Aeropalinologica, è presentata la scheda morfopalinologica diPinus pinea L. nella versione su polline fresco e polline acetolizzato, su tre campioni di diversa provenienza. Vengono notate le principali differenze tra polline fresco e polline acetolizzato.

     

Indirect shoot organogenesis from leaves of Dieffenbachia cv. Camouflage

A novel protocol for indirect shoot organogenesis of Dieffenbachia cv. Camouflage was established using leaf explants excised from in vitro shoot cultures. The frequency of callus formation reached 96% for explants cultured on Murashige and Skoog (1962) basal medium supplemented with 5 μM thidiazuron and 1 μM 2,4-dichlorophenozyacetic acid. The number of shoots regenerated was high, with up to 7.9 shoots produced per callus cultured on basal medium supplemented with 40 μM N ⁶-(Δ²-isopentenyl)adenine and 2 μM indole-3-acetic acid. Regenerated shoots rooted well in a soilless substrate, acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions. Somaclonal variations in leaf variegation, color, and morphology have been observed in regenerated plants.     

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

Transformation of maize using microprojectile bombardment: An update and perspective

Summary Using microprojectile bombardment of maize suspension cultures and bialaphos selection, transformed embryogenic calli have been recovered in numerous independent experiments. Fertile transgenic plants have been regenerated from several transformed callus lines. Stable inheritance and expression ofbar and functional activity of the enzyme phosphinothricin acetyl transferase were observed in three subsequent generations of transformed plants. Evidence to date indicates that the transformation process and the presence of the foreign gene per se do not detrimentally influence either plant vigor or fertility. This represents a practical method for introducing foreign genes into maize, which may be applicable to other monocot species. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

A simple technique to overcome vitrification inGypsophila paniculata L.

Node cultures ofGypsophila paniculata showed vitrified growth when cultured under standard conditions, resulting in poor survival of tissue culture derived plants. The deviant growth pattern appeared to be determined in the lag phase of the cultures. A limited evaporation period in this stage induced normal growth. Re-establishment of high relative humidity and poorer gas exchange thereafter did not lead to vitrified growth. Vitrification was also depressed when evaporation was continued during the whole culture period, but shorter plantlets resulted. A simple adaptation of the plastic lid that is commonly used in micropropagation, allowed controlled and contamination-free evaporation.     

Uptake and metabolism of benzyladenine during shoot organogenesis in Petunia leaf explants

Benzyladenine (BAP) uptake and metabolism were characterized during the key stages of shoot organogenesis in leaf explants of Petunia MD1. Using leaf explant transfer experiments, it was shown that exposure to 2.2 M BAP for 6, 8 or 10 days induced shoot formation on 27, 80 and 100% of the explants respectively, with a concomitant increase in the number of shoots per explant. BAP uptake and metabolism were characterized in leaf explants after 1, 3, 6 or 10 days exposure to [³H]BAP or 10 days exposure plus an additional 2 days on basal medium (10+2). BAP and 9--D-ribofuranosyl-BAP ([9R]BAP) were detected at days 1 and 3 only. Therefore, the BAP free base was not detectable during the shoot induction period between days 6 and 10, as defined by leaf transfer experiments. The BAP ribotide pool was largest on day 1 and decreased to day 10+2. It is possible that the BAP ribotide pool provided either the active cytokinin itself or acted as a short-term storage form for the active cytokinin in petunia shoot organogenesis. Other metabolites detected in petunia leaf tissue included 7--D-glucopyranosyl-BAP ([7G]BAP), 9--D-glucopyranosyl-BAP ([9G]BAP) and an unidentified metabolite C.Abbreviations BAP benzyladenine - [7G]BAP 7--D-glucopyranosyl-BAP - [9G]BAP 9--D-glucopyranosyl-BAP - [9R]BAP 9--D-ribofuranosyl-BAP - [9R-5P]BAP 5-monophosphate of [9R]BAP - [9R-5PP]BAP 5-diphosphate of [9R]BAP - [9R-5PPP]BAP 5-triphosphate of [9R]BAP - TEA Triethylamine This research was supported in part by NSF Grant DCB-8917378 to J.D.C. and USDA-CRGO Grant 89-37261-4791 to T.J.C.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

Thidiazuron-induced de novo shoot organogenesis on seedlings,etiolated hypocotyls and stem segments of Huang-qin

Development of an efficient in vitro propagation system for Huang-qin (Scutellaria baicalensis), a traditional Chinese medicinal plant used in the treatment of a wide range of human ailments, is described. Thidiazuron [TDZ: N-phenyl-N′- (1,2,3-thidiazol-5-ylurea)] effectively induced regeneration on cultured intact seedlings, etiolated hypocotyl explants and sterile stem segments of Huang-qin. Histological examinations of excised hypocotyl or nodal explants revealed that adventitious shoots formed through an intermediate callus. Comparison of TDZ-induced regeneration in the three tissue types indicated that isolation of explants was not essential for optimal regenerative efficiency. Significantly more regenerants formed along hypocotyls of intact seedlings (20 shoots/explant) than were observed on excised hypocotyls (9.7 shoots/explant) indicating that endogenous metabolites produced in adjacent tissues provided resources for the shoot initiation. More than 95% of de novo regenerants formed roots and then intact plantlets under either sterile culture or greenhouse conditions. Regeneration protocols developed in this study may provide the basis for improvement of this crop through the identification of medicinally active constituents and eventual development optimized pharmaceutical products. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of exogenous proline and proline analogues on in vitro shoot organogenesis in Arabidopsis

In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mMproline, and to a lesser extent by 5 mM proline, butinhibited by inclusion of 10 mM proline in thehormonally-supplemented regeneration medium. Theability of low concentrations of the prolineanalogues, azetidine-2-carboxylate and thioproline toovercome the stimulatory effect of 1 mM proline, anda slight increase in the stimulative effects of 1 mMproline by D-proline, are consistent with an importantrole for the interconversions of proline and itsprecursors in regulating cell division anddifferentiation.     

Transport and metabolism of [3H]iso-pentenyladenine following application to the tips of intact and decapitated tap roots of Pinus pinea

[³H]iso-Pentenyladenine ([³H]iP) was fed for 24 h to the tips of intact and root tip-decapitated Pinus pinea seedlings. Twelve and 24 h after application to the roots of intact plants most of the applied radioactivity (±60%) was transported to the shoot. Root tip removal increased transport of the applied radioactivity to the shoot, but the overall pattern of distribution of radioactivity in the seedling did not change. Large amounts of radioactivity were recovered from the elongation zone of the root. Some radioactivity also accumulated in the older part of the root with well-developed lateral roots. When [³H]iP was applied one day after decapitation, no significant changes in the pattern of radioactivity distribution were found between the intact and decapitated root systems. However, when applied 7 days after decapitation there was a significant increase of radioactivity in the region of the root where lateral roots were emerging. HPLC separation of extracts from the different root sections showed that [³H]iP was extensively metabolized in the root. Six peaks of radioactivity, which co-chromatographed with authentic cytokinin standards, were detected.Abbreviations ABA abscisic acid - ADE adenine - IAA indole-acetic acid - iP iso-pentenyladenine - HPLC high performance liquid chromatography - [OG]DHZ O-glycosyldihydrozeatin - [9R-MP]DHZ ribosyldihydrozeatin monophosphate - [9G]iP iso-pentenyladenine-9-glucoside - [9R]Z ribosylzeatin - [9R]iP iso-pentenyladenosine - TLC thin layer chromatography     

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.