Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.)

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, -naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 M indole-3-butyric acid (IBA) and 3–12 M N₆-benzylaminopurine, thidiazuron (TDZ), or 6-(,-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4°C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 M TDZ and 2 M IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.     

Thidiazuron Induced Multiple Shoot Formation in Chickpea (Cicer arietinum L)

Thidiazuron (TDZ), a substituted urea with cytokinin-like activity stimulated shoot proliferation in chickpea (Cicer arietinum L). Embryonic axis with half portion of both cotyledons was more responsive explant, producing ～22 shootslexplant at 1.0 µM TDZ concentration but higher concentration of TDZ (5.0 µM) reduced both the shoot proliferation and growth. An incubation time of 12 h at 1.0 µM TDZ was sufficient for induction of multiple shoot formation.TD2 induced high frequency of shoot formation as compared to BAP and also minor salts of MS medium played an important role in increasing the number of shoots. Roots could be induced in these shoots in MS medium supplemented with 0.5 µM IBA.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants

The goal of this study was to develop an efficient regeneration protocol to be used for genetic transformation of sesame. Published regeneration methods using benzyladenine (BA) and 1-naphthalene acetic acid (NAA) were unsuccessful for the cultivars used herein. Experiments were carried out using cotyledon and hypocotyl explants from the cultivar Mtwara-2. Later the optimised culture conditions were used to investigate the regeneration response of different genotypes. There was significant interaction between hormone treatments and macronutrients for shoot and root regeneration. Results also showed that shoot regeneration was significantly influenced by explant type. Shoots were only obtained from cotyledons whereas both cotyledons and hypocotyls could produce roots. Modified Murashige and Skoog (MS) medium with N6 macronutrients resulted in twice the shoot regeneration frequency obtained with ½MS macronutrients in the presence of thidiazuron (TDZ). The shoot regeneration frequency was significantly reduced when BA was used in place of TDZ. On shoot regeneration medium containing BA and NAA, only roots were formed. Replacing NAA with indole-3-acetic acid (IAA) greatly improved the regeneration of shoots. The optimum growth regulator combination for shoot regeneration was 20 μM TDZ together with 2.5 μM IAA, which gave a frequency of 63% and 4.4 shoots per regenerating explant for the best cultivar Ex-El. Genotypic differences were significant both for the number of explants regenerating shoots and the number of shoots produced per regenerating explant.     

Screening for in vitro shoot-forming capacity of seedling explants in bell pepper (Capsicum annuum L.) genotypes and efficient plant regeneration using thidiazuron

Summary In vitro shoot regeneration ability of 17 (7 Italian and 10 Hungarian) bell pepper genotypes was investigated using excised cotyledons and rooted hypocotyls as explants. Most of the Italian genotypes and two of the Hungarian genotypes responded well, producing shoots from rooted hypocotyls. Only two genotypes (one Italian and one Hungarian) gave a weak response using cotyledons. For direct shoot induction in these explants, in addition to the methods cited in the relevant papers, a new method was applied using thidiazuron as a cytokinin. Shoots were successfully regenerated from cotyledons of two Italian and two Hungarian genotypes using thidiazuron which were considered to be non responsive to the usual methods.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS medium (Murashige and Skoog 1962) - TDZ N-phenyl-N-thiadiazol-l,2,3-5-ylurea (thidiazuron) - PVC polyvinyl chloride     

In vitro induction of multiple shoots and plant regeneration inVigna

Summary Cotyledonary nodes, excised cotyledons, and hypocotyl segments of six varieties ofVigna mungo andV. radiata have been tested for their morphogenic potential on media containing a range of hormonal combinations including benzyladenine, kinetin, thidiazuron (TDZ), and zeatin. Multiple shoots developed on cotyledonary node explants in all varieties tested on basal medium containing cytokinin. Presence of both the cotyledons, either full or half, resulted in a maximum number of shoots produced. Shoot bud regeneration was achieved via meristem formation on excised cotyledons on Murashige-skoog basal medium with B₅ vitamins supplemented with TDZ. Mature plants had normal phenotypes.V. mungo var. PS1 andV. radiata var. Pusa 105 were found to be the most responsive varieties for shoot regneration. The histology ofin vitro organogenesis was studied.     

Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron

Morphogenesis from several explant types excised from in-vitro-grown plantlets of a Brazilian eggplant (Solanum melongena L.) variety (F-100) was evaluated in response to thidiazuron (TDZ). Leaves and cotyledons were found to be the most responsive explants. Optimal shoot bud induction rates (75–100 buds/explant) were achieved in the presence of 0.2 μm TDZ. Organogenic calli were transferred to growth regulator free MS medium before shoot excision. Rooting was induced on half-strength MS medium supplemented with 0.6 μm IAA. Received: 1 March 1997 / Revision received: 29 October 1997 / Accepted: 15 November 1997     

Multiple shoot formation from normal and malformed somatic embryo explants of Eastern redbud (Cercis canadensis L.)

A procedure for multiple shoot formation from somatic embryo explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. TDZ in combination with BA produced more shoots than either treatment alone. The highest number of shoots (3.3 to 3.4 shoots per explant) was obtained from partially desiccated and wounded explants treated with a combination of 5 or 10 M BA and 0.5 or 1.0 M TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 1.5 to 3.2 shoots per explant by cutting through the cotyledonary node prior to culture. In addition, the frequency of explants forming shoots was increased by desiccation of somatic embryo explants to approximately 50% moisture and by using somatic embryos with two well formed cotyledons as explants.Abbreviations ABA abscisic acid - BA benzyladenine - CRD Completely randomized design - DKW Driver and Kuniyuki medium - LSD Least significant differences - TDZ thidiazuron     

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     

Thidiazuron enhances axillary and adventitious shoot proliferation in juvenile explants of Mediterranean provenances of maritime pine <Emphasis Type="Italic">Pinus pinaster</Emphasis>

The effectiveness of thidiazuron (TDZ) in promoting in vitro organogenesis in cultured embryo axes and cotyledons from two Spanish Mediterranean provenances of Pinus pinaster Aiton is reported. Application of 44.4 μM TDZ as a 6-d pulse on DCR medium and subsequent transference of maritime pine explants to the same medium without growth regulators, but supplemented with activated charcoal, produced between 17 and 19 axillary shoots per embryo axis with no differences between the two provenances. The same sequential treatment allowed adventitious shoot differentiation on 75% of the cotyledons, with a mean of 12.2 shoots regenerated per explant. Axillary and adventitious shoots were rooted ex vitro after an auxin pulse of indole-3-butyric acid and 1-naphthaleneacetic acid. The micropropagation protocols presented here should enable vegetative multiplication of selected families, and elite trees from these Spanish Mediterranean provenances of maritime pine.     

The influence of thidiazuron on shoot regeneration from leaf explants of fifteen cultivars of Rhododendron

The influence of cytokinin thidiazuron (TDZ) and auxin indole-3-acetic acid (IAA) on in vitro shoot organogenesis of fifteen Rhododendron genotypes was investigated and a protocol for high frequency adventitious shoot regeneration from leaf explants was developed. High genotypic variation was observed and regeneration frequencies ranged from 0 to 100 %. Genotype Ovation had the highest number of shoots (26.4 per explant) after 12 weeks on medium with 0.57 μM IAA and 1.20 μM TDZ, but only 65 % of explants regenerated. Catawbiense Grandiflorum had 17.7 shoots per explant and 75 % regeneration on medium with 5.70 μM IAA and 0.45 μM TDZ and Van Werden Poelman had 14.3 shoots per explant and 100 % regeneration on medium with 0 57 μM IAA and 0.45 μM TDZ.     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Direct differentiation of somatic embryos on different regions of intact seedlings of Azadirachta in response to thidiazuron

Direct differentiation of somatic embryos occurs in high-frequency and at high density in response to 1.0 microM TDZ, on different regions-hypocotyl, epicotyl, cotyledonary-node, cotyledons and leaves-of intact seedlings of Azadirachta. One-week-old seedlings on this medium exhibited stress symptoms as visible by the loss of root formation and reduction in the elongation of hypocotyl and epicotyl. Globular somatic embryos were more abundant on hypocotyl, epicotyl, stem tip and leaves. The arrest of embryos at this stage was possibly due to their presence in high density. Well-developed somatic embryos were present on the cotyledons and the cotyledonary-node. These embryos on isolation and transfer to hormone-free medium regenerated readily to form plantlets. The possible role of stress in thidiazuron-induced somatic embryo formation is discussed.     

Adventitious shoot induction and somatic embryogenesis with intact seedlings of several hybrid seed geranium (Pelargonium x hortorum Bailey) varieties

Summary Intact seedlings of hybrid seed geranium (Pelargonium x hortorum Bailey) were tested for their ability to produce adventitious shoots and somatic embryos by direct culture of mature seeds on Murashige and Skoog (1962) medium (MS) supplemented with growth regulators BAP, BAP + IAA, or thidiazuron (TDZ). Ten varieties were tested in the presence of different BAP concentrations, four with BAP + IAA, and two with TDZ. Varieties used in this study differed in their response to BAP in the medium. Multiple adventitious shoots were produced by seven of the ten varieties tested. Multiple adventitious shoots were induced at all levels of TDZ in the medium. TDZ also induced callusing from roots and direct embryogenesis from intact hypocotyls. Adventitious shoots were separated, rooted and transferred to soil where they grew as normal healthy plants and flowered.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - TDZ thidiazuron     

Jasmonic acid inhibition of in vitro shoot organogenesis in Pinus radiata cotyledons

The effect of jasmonic acid (JA) onde novo shoot formation in excised cotyledons of radiata pine (Pinus radiata D. Don), was examined. JA had no effect on shoot-forming (SF) tissues at concentrations up to 10⁻⁶ mol · L⁻¹. At concentrations greater than that, JA caused a reduction in the number of shoots formed, as well as the lengths of the shoots and fresh and dry masses of the tissues. Reciprocal transfer of excised radiata pine cotyledons from a SF medium with 10⁻⁵ mol · L⁻¹ JA to a SF medium without JA and vice versa showed that any exposure of the cotyledons to JA either during the shoot induction phase (days 0-21) or the shoot development phase (beyond day 21) led to a reduction in shoot formation. However, the JA effect was significantly less if the cotyledons were not exposed to JA during the initial 10 days in culture; indicating that the JA effect was mainly during shoot primordia formation and the subsequent development into shoots.     

Regulation of organogenesis and somatic embryogenesis by auxin in melon,Cucumis melo L.

Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to the 2,4-D concentration in the media, that is, adventitious shoots were formed at low concentration, callus proliferated without differentiation at intermediate concentration and somatic embryos were induced at high concentration. Cotyledons of mature seeds formed both adventitious shoots and somatic embryos more efficiently than any other tissues cultured.Effects of three auxins, 2,4-D, NAA and IAA, on organogenesis and embryogenesis were compared using cotyledons of mature seeds. Adventitious shoots were formed at low level of auxins (0 to 0.01 mg/l 2,4-D; 0 to 0.1 mg/l NAA; 0 to 1.0 mg/l IAA), and embryos were formed at high level of auxins (1.0 to 2.0 mg/l 2,4-D; 3.0 to 10.0 mg/l NAA; 20.0 to 100.0 mg/l IAA). IAA gave more efficient shoot formation and embryogenesis than the other auxins.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA 3indoleacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog     

TDZ-induced direct shoot organogenesis and somatic embryogenesis on cotyledonary node explants of lentil (Lens culinaris Medik.)

An efficient and simple procedure for inducing high frequency direct shoot organogenesis and somatic embryogenesis in lentil from cotyledonary node explants (without both the cotyledons) in response to TDZ alone is reported. TDZ at concentration lower than 2.0 μM induced shoot organogenesis whereas at higher concentration (2.5–15 μM) it caused a shift in regeneration from shoot organogenesis to somatic embryogenesis. The cotyledonary node and seedling cultures developed only shoots even at high concentrations of BAP and TDZ, respectively. TDZ at 0.5 and 5.0 μM was found to be optimal for inducing an average of 4–5 shoots per cotyledonary node in 93 % of the cultures and 55 somatic embryos in 68 % of the cultures, respectively. The somatic embryos were germinated when transferred to lower TDZ concentration (0.5–1.0 μM). The shoots were rooted on MS basal medium containing 2.5 μM IBA. The plantlets were obtained within 8 weeks from initiation of culture and were morphologically similar to seed-raised plants. The possible role of stress in thidiazuron induced somatic embryogenesis is discussed.Key words: Thidiazuron, Lens culinaris, Somatic embryogenesis, Organogenesis     

Induction of high-frequency somatic embryogenesis in geranium (Pelargonium x hortorum Bailey cv Ringo Rose) cotyledonary cultures

Summary The cv Ringo Rose of hybrid seed geranium (Pelargonium x hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos when cotyledonary explants were cultured on regeneration medium containing thidiazuron (TDZ), forchlorfenuron (CPPU), or a combination of indole-3-acetic acid and N⁶ benzylaminopurine (IAA+BAP). Amendment of the basal medium with TDZ (0.5 M) was the most effective treatment. Addition of amino acids to the medium promoted the growth of somatic embryos. Retention of the proximal region of the cotyledon was crucial for regeneration, but the removal of the distal 1/3 to 1/2 cotyledon had no significant effect on somatic embryogenesis. Cotyledonary explants formed somatic embryos in higher frequency and much earlier than hypocotyl explants cultured on the same medium. The somatic embryos induced on cotyledonary explants were germinated on basal medium. More than 70% of the somatic embryos were converted into plants and transferred to soilAbbreviations BAP N⁶-benzylaminopurine - CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea (forchlorfenuron) - IAA indole-3-acetic acid - TDZ N-phenyl-N'-1,2,3,-thiadiazol-5ylurea (thidiazuron)     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.