In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

In vitro clonal propagation of annatto (Bixa oreliana L.)

Summary A protocol for in vitro propagation of Bixa orellana is described. Plants were regenerated from shoot apex and nodal explants on B5 medium supplemented with 4.9 μM 2-isopentenyl adenine. The multiplication factor of shoot apex explants was higher (nine shoots per explant) than that of the nodal explants (five shoots per explant). Regardless of the position of the nodes, all the nodal explants gave similar responses. However, the size of the nodal explant was an important factor in producing multiple shoots: 0.5 cm nodal explants produced the maximum multiple shoots. Regenerated shoots from shoot apex explants rooted best on MS medium supplemented with 0.05 μM α-naphthalene acetic acid (NAA). whereas shoots regenerated from nodal explants needed 2.7 μM NAA for rooting. Eighty per cent survival of in vivo transferred plants occurred on the best potting substrate, coco peat. Since the multiplication factor was nine per explant, this protocol can be use for commercial microprogation. However, the regeneration capacity declined after 10 subcultures. Approximately, 3350 rooted plants could be generated in 10 mo. after eight subcultures, from one shoot with a shoot apex and four nodes.     

Putrescine-enhanced somatic embryos and plant numbers from elite oat (Avena spp. L.) and reciprocal crosses

Summary Mature embryos from hulled, regenerable GP-1 (A. sativa L.), hull-less, recalcitrant Tibor (A. nuda L.) and reciprocal crosses were cultured in vitro on a putrescine- (Put) containing medium. Hormone-free Murashige and Skoog medium (MS-0) or shoot proliferation medium (SPM) [2.0 mgl⁻¹ (9.0 μM) 2,4-dichlorophenoxyacetic acid (2,4-D)], with and without 0.5 mM Put or 1 mM Put, were tested for effects on somatic embryogenesis and plant regeneration. Put/SPM (0.5mM) was the best medium for both somatic embryos (SEs) and plant numbers per gram of callus, regardless of genotype. This effect was most evident in Tibor, which produced no somatic embryos or plants on SPM, a previously published regeneration medium, and in Tibor ×GP-1, which produced reduced numbers of SE and plants on the remaining media. The number of SEs per gram of callus for GP-1 and GP-1× Tibor showed little significant differences between the different media. Put treatments produced plants from the four genotypes but the regeneration efficiency on Put-containing medium was similar or even better than on SPM for explants containing maternal GP-1 germplasm. This suggests that Put-containing MS-0 medium can be used for testing regeneration of other oat lines. In addition, SPM containing 0.5 mM Put can be used to induce significant regeneration of plants from normally recalcitrant genotypes. This improvement greatly increases the number of potential germplasms for further transformation efforts.     

Regeneration of plantlets from leaf and petiole explants of Pelargonium x hortorum

Summary The in vitro plant regeneration potential of vegetatively propagated geraniums (Pelargonium x hortorum) has been investigated. Using various combinations of growth regulators and a choice of different explants, a regeneration protocol has been developed to raise in vitro plantlets from young petiole and leaf explants from three different cultivars of geraniums. In all three cultivars, very young petiole explants exhibited a higher regeneration potential as compared with leaf explants. Regeneration efficiencies were found to be highly dependent on the cultivar, with cv. Samba showing the highest regeneration potential, followed by cvs. Yours Truly and then Sincerity. Samba also showed the highest number of shoots from both the petiole [57 shoot buds per petiole explant in the presence of 3 μM zeatin and 1 μM indole-3-acetic acid (IAA) and leaf explants (43 shoots per leaf explant with 10 μM zeatin and 2 μM IAA). Shoot buds transferred to Murashige and Skoog (MS) medium supplemented with 0.44 μM N⁶-benzyladenine and 0.11 μM IAA grew vigorously and attained 1–2 cm in length in 3–4 wk. These shoots rooted with 100% efficiency on MS basal medium, and plants developed that showed normal growth and flowering under greenhouse conditions.     

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.     

Direct shoot regeneration from leaf and internode explants of Aloysia polystachya [GRIS.] mold. (Verbenaceae)

Summary Adventitious bud regeneration from leaf and internode explants of Aloysia polystachya was achieved. Shoots from nodal segments grown in vitro were cut into pieces and used as sources of explants. Organogenesis was induced from both explants cultured on quarter-strength Murashige and Skoog (MS) semisolid medium (plus sucrose 5 g l⁻¹) containing different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 116 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD), 14-h photoperiod, and at a temperature of 27±2°C. The type of explant markedly influenced organogenesis and growth of the regenerated shoots. The regeneration frequencies were higher with leaf explants, while the number of shoots formed per responsive explant was greater with internode explants. However, the growth of regenerated shoots from internodes was seriously affected by vitrification. The number of shoots produced per responsive leaf explant increased from one to seven as the percentage of leaf explants producing shoots increased from 20 to more than 80%. NAA at 0.05 μM in combination with BA at 0.5μM induced the highest regeneration rate (87±8.8%) after 20 d of culture, yielding 5.9±0.8 shoots per responsive leaf explant. Histological examination confirmed the occurrence of direct organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium without plant growth regulators for 30 d. Finally, the elongated shoots were rooted by pre-treatment in an aqueous solution of NAA at 500 μM for 2 h and transferred to 1/4 MS. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 2-yr-old in vitro-regenerated plants was established.     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

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     

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.     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

Regeneration of <Emphasis Type="Italic">Aloe arborescens</Emphasis> via somatic organogenesis from young inflorescences

A system for in vitro regeneration of Aloe arborescens was developed using young inflorescences as explants. Different phytohormone combinations of N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ), benzyladenine (BA), 6-(γ,γ-dimethylallyl-amino)purine riboside (2iPR), zeatin ribozide (ZR), N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and kinetin (K), with or without ancymidol, were examined in order to induce plant regeneration. Efficient shoot regeneration was initiated on Murashige and Skoog (MS) medium supplemented with BA or TDZ. MS medium enriched with 19.6, 22.2 μM BA and 3.92 μM ancymidol (MSBA5/1 medium), promoted organogenesis enabling 87.3% of the explants to regenerate 6.04 ± 1.79 shoots/explant. Subsequent shoot elongation and plant regeneration were strongly affected by the medium composition used for shoot induction. Optimal elongation (three to four shoots per explant) was obtained when shoots, initiated on MSBA5/1 medium, were subsequently transferred onto MS containing only 4.4 μM BA. Rooting was performed on MS media lacking growth regulators. Histological analysis revealed that the initiated shoots originated from the receptacle tissue surrounding the residual vascular tissue of the flower buds.     

Effect of genotype,explant, subculture interval and environmental conditions on regeneration of shoots from in vitro monoploids of a diploid potato species,Solanum phureja Juz. & Buk.

In vitro anther-derived monoploids (2n=x=12) of Solanum phureja were compared for shoot regeneration from leaf and stem explants under various environmental conditions. Monoploids from the same or different diploid clones varied for frequency and earliness of shoot regeneration and number of shoots formed per explant. Leaf explants regenerated at higher frequencies than stem explants. Explants from stock plantlets subcultured at a 2- or 4-week interval regenerated earlier and at a higher frequency than those from plantlets subcultured at longer intervals. Regeneration frequency and number of shoots per explant were greater when explants were incubated at 20°C compared to 25°C. Explants from stock plantlets maintained under a 16 h as opposed to an 11 h photoperiod exhibited increased shoot regeneration; however, neither photoperiod nor the maintenance temperature of the stock plantlets influenced regeneration frequency. Genotypic differences were observed for the frequency of chromosome doubling among regenerated shoots whereas temperature treatments had no influence on chromosome doubling.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthale-neacetic acid     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

The Effect of a Submersion Pretreatment on In Vitro Explant Growth and Shoot Regeneration from Hypocotyls of Flax (Linum Usitatissimum)

This study was carried out to determine the effect of temporary submersion of hypocotyl segments in water on in vitro explant growth and shoot regeneration on MS (Murashige and Skoog, 1962) medium supplemented with 1 mg l⁻¹ BAP (6-benzylaminopurine) and 0.02 mg l⁻¹ NAA (naphthaleneacetic acid) in three flax cultivars. It was observed that water-treated hypocotyl explants gave rise to the highest values with respect to shoot regeneration percentage, shoot number per hypocotyl, shoot length and total shoot number per Petri dish, successful rooting and plantlet establishment. This procedure may be applicable for other species cultured in vitro.     

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.     

Biochemical and histological changes associated with <Emphasis Type="Italic">in vitro</Emphasis> responses in sunflower cotyledonary explants

In vitro shoot regeneration from sunflower cotyledonary explants can be obtained in the presence of kinetin and indole-3-acetic acid. In contrast, callus proliferation is obtained in the presence of 2,4-dichlorophenoxyacetic acid on culture medium. The purpose of this study was to investigate changes in protein profiles during callus and shoot development from cotyledonary explants and to correlate them with ontogenic stages during in vitro culture. Cotyledons cultured in the presence of 2,4-dichlorophenoxyacetic acid produced friable callus as a result of early division of parenchymatic cells associated with the vascular bundles of the explant. The callogenic ability was independent of the cotyledonary region used as starting explant. Direct shoot organogenesis was observed from the same type of cells growing in culture media supplemented with kinetin and indole-3-acetic acid. In this case, the regeneration potential varied among regions from which the explants were obtained. Protein profiles revealed differences associated with shoots or callus developmental programs. A 27-kDa polypeptide was uniquely detected in the explants undergoing shoot organogenesis. The amount of this polypeptide during the first 4 d of culture increased and was followed by the appearance of meristematic centers in histologically analyzed samples. This polypeptide could be used as a specific marker for in vitro shoot development in this species.     

Shoot production per responsive leaf explant increases exponentially with explant organogenic potential in Nicotiana species

As part of the effort to develop optimal plant varieties for the production and molecular farming of plant-made pharmaceuticals, this study evaluated shoot organogenic potential of a total of 115 Nicotiana accessions, representing 53 species. To induce shoots, leaves from seedling grown in vitro were cut into pieces, cultured on shoot-induction medium under low light for 3 weeks, and then subcultured onto the same medium for another 4 weeks under normal light. Statistical analysis detected significant differences among the 115 accessions for the percentage of leaf explants producing shoots and the number of shoots produced per responsive leaf explant. Importantly, regression analysis also found an exponential relationship between the number of shoots produced per responsive leaf explant and the percentage of leaf explants producing shoots. The number of shoots produced per responsive leaf explant increased rather slowly, ranging from zero to around five, as the percentage of leaf explants producing shoots increased from 0 to 80%, but the increase became dramatic as the percentage increased from 80% to 100%, reaching as high as 35 shoots per responsive leaf explant. This exponential relationship is the first of its kind to be established in plant regeneration studies using either organogenesis or somatic embryogenesis systems. A possible mechanism that governs the establishment of the exponential relationship is discussed.Abbreviations 2ip 6-(,-Dimethylallylamino)-purine - BA Benzylaminopurine - IAA Indole-3-acetic acid - LS Linsmaier and Skoog - MS Murashige and Skoog - PI Plant introduction number - PMP Plant-made pharmaceuticals - SIM Shoot induction medium - USDA US Department of Agriculture     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

In vitro regeneration and cytological characterization of shoots from leaf explants of three accessions of Solanum commersonii

In vitro culture of explants were used to apply genetic or cell engineering techniques to the sexually incompatible potato relative Solanum commersonii (2n=2x=24) Three accessions of S. commersonii were tested for regeneration from leaf explants using six different protocols. A two step-regeneration procedure gave the best results. Genetic variability for regeneration ability was found between accessions, and between clones within accessions. The accession PI 472834 regenerated at highest frequency. Clones with high regeneration ability were selected. Approximately 60% of regenerated plants were diploids and 40% were tetraploids. A very low frequency of chimeras was found. Leaf shape and chloroplast counts in guard cells were shown to be quick and reliable methods for estimating ploidy levels. Use of the diploid and tetraploid regenerants obtained for potato breeding is discussed.Abbreviations BAP 6-benzylaminopurine - EBN Endosperm Balance Number - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene-acetic acid - ZEA zeatin     

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.