Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 – 40 M) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 – 40 M) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 M) plus indole-3-butyric acid (IBA 0.4 M). Plant regeneration occurred in the presence of IBA (0.8 M) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 M) and kinetin (k 2 M) were applied to the tissue culture shoots for 7 days in light.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Thidiazuron-induced in vitro shoot formation from roots of intact seedlings of Albizzia julibrissin

Seedlings of silktree (Albizzia julibrissin Durrazz.) were grown in vitro on MS-media containing B5 vitamins, 3% sucrose, 0.25% phytagel and various concentrations (0.1–10 M) of thidiazuron (TDZ). Addition of TDZ to the culture medium greatly reduced shoot and root elongation but did not influence shoot production from the cotyledonary node or apex. Within 8–10 days the seedling roots split open, formed large masses of callus, and developed green patches which eventually grew into normal shoots while still within the culture medium containing TDZ at 0.1–1.0 M. Such callus and shoot formation did not occur in control cultures lacking TDZ. At higher TDZ concentrations (2.5–10 M), the green patches formed in the callus did not further develop into shoots. Addition of other cytokinins (kinetin, benzylaminopurine, zeatin) to the culture medium also induced some shoot formation from the roots, but higher concentrations than TDZ were required to induce regeneration. Isopentenyladenine failed to induced shoot formation. Following excision and transfer to MS media with or without 4.9 M IBA, the shoots induced by kinetin or benzylaminopurine rooted 4–7 days earlier than those induced by TDZ, but all excised shoots developed into normal rooted plantlets within 3 weeks.Abbreviations TDZ thidiazuron     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Factors influencing secondary somatic embryogenesis inMalus x domestica Borkh. (cv ‘Gloster 69’)

A procedure for regeneration of apple plants through secondary somatic embryogenesis (SSE) was developed in apple Gloster 69. Primary somatic embryos were produced from cotyledon-derived cultures of immature zygotic embryos. These somatic embryos were multiplied by secondary somatic embryogenesis (SSE) on media with different Plant Growth Regulator (PGR) combinations. The highest SSE rate (55.5%) was obtained with a combination of NAA (5.3 M), BAP (0.9 M) and KIN (0.9 M) or with TDZ alone (10 M). In addition, effects of explant source, somatic embryo size, type and concentrations of carbohydrates and gelling agents on SSE were investigated. The optimum SSE (>73%) was obtained by the culture of large size somatic embryos or cotyledon-like structures on medium containing a combination of NAA/BAP/KIN or TDZ (10 M) alone, maltose (175 mM) and Phytagel (2.8 g/1).     

Role of endogenous purine metabolism in thidiazuron-induced somatic embryogenesis of peanut (Arachis hypogaea L.)

Somatic embryogenesis was induced at the hypocotyledonary notch region of intact peanut (Arachis hypogaea L.) seedings cultured on a medium containing 10 mol·L^–1 thidiazuron (TDZ). Inclusion of the purine analogs 2,6-diaminopurine (DAP), azaadenine or azaguanine to the thidiazuron amended medium inhibited the embryogenic response of the seedlings. DAP-mediated inhibition was not overcome by the addition of adenine sulphate. Inhibition of the embryogenic response by DAP provides evidence that the TDZ-induced accumulation of purine cytokinins is an essential component of TDZ-induced somatic embryogenesis process. Analyses of the endogenous level of purine metabolites indicated that supplementation of the media with TDZ resulted in an overall increase in the endogenous cytokinins while DAP inhibited the purine recycling resulting in decreased levels of endogenous adenine and zeatin.     

The initiation of callus culture ofMichelia champaca for essential oil production

Summary TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 M 2,4-D and 0.1–1 M BAP. The calli were grown well on media on media containing 0.1–1.0 M BAP and 2,4-D up to 50 M. As 1 M BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 M of 2,4-D and BAP were present in the medium.     

TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D

Caryopses cultures of rice on an auxin medium (2,4-D, 20 M) formed slow-growing tissues that failed to regenerate. The embryogenic tissue possibly lost its regeneration potential on the auxin medium. Reproducible regeneration, however, could be achieved by a short treatment with 20 M 2,4-D for 3 days. Transfer of these caryopses to the medium containing TDZ or BA at 10 M resulted in regeneration of somatic embryos and shoots in 30% of the cultures within 10–15 days. TDZ was better than BA for inducing shoot regeneration. Short treatment for 3 days at higher concentrations of auxin (40–80 M) and subsequent transfer to TDZ or BA medium resulted in an increased frequency (up to 50%) of regeneration.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Short-term changes in phosphorus storage in an oligotrophic Everglades wetland ecosystem receiving experimental nutrient enrichment

Natural, unenriched Evergladeswetlands are known to be limited by phosphorus(P) and responsive to P enrichment. However,whole-ecosystem evaluations of experimental Padditions are rare in Everglades or otherwetlands. We tested the response of theEverglades wetland ecosystem to continuous,low-level additions of P (0, 5, 15, and30 g L^–1 above ambient) in replicate,100 m flow-through flumes located in unenrichedEverglades National Park. After the first sixmonths of dosing, the concentration andstanding stock of phosphorus increased in thesurface water, periphyton, and flocculentdetrital layer, but not in the soil or macrophytes. Of the ecosystem components measured, total P concentration increased the most in the floating periphyton mat (30 g L^–1: mean = 1916 g P g^–1, control: mean =149 g P g^–1), while the flocculentdetrital layer stored most of the accumulated P(30 g L^–1: mean = 1.732 g P m^–2,control: mean = 0.769 g P m^–2). Significant short-term responsesof P concentration and standing stock wereobserved primarily in the high dose (30 gL^–1 above ambient) treatment. Inaddition, the biomass and estimated P standingstock of aquatic consumers increased in the 30and 5 g L^–1 treatments. Alterationsin P concentration and standing stock occurredonly at the upstream ends of the flumes nearestto the point source of added nutrient. Thetotal amount of P stored by the ecosystemwithin the flume increased with P dosing,although the ecosystem in the flumes retainedonly a small proportion of the P added over thefirst six months. These results indicate thatoligotrophic Everglades wetlands respondrapidly to short-term, low-level P enrichment,and the initial response is most noticeable inthe periphyton and flocculent detrital layer.     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron

The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10M) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate.     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica

Summary InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 M 4-PU and 1 M NAA at a cell density of 5 × 10⁴/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1–1 M 4-PU and 1 M NAA, and 10 M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 M 4-PU or 10 M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 M IBA and 0.1 M NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IBA indole-3-butyric acid - 2ip N ⁶-(2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - 4-PU N-(2-chloro-4-pyridyl)-N–phenylurea - TDZ thidiazuron     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

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

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

Shoot multiplication in <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5M TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5M indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.     

Plantlet regeneration from subculturable nodular callus of Pinus radiata

An efficient planlet regeneration system via nodular callus formation is described for Pinus radiata. Subculturable nodular callus was induced at its highest frequency (93%) on embryonic explants excised from seeds at an early stage of germination (radicle length 2–5 mm). The optimal medium for nodular callus tissue proliferation was LP basal medium that was modified by reducing the concentration of potassium nitrate to 500 mg l^–1 and supplemented with 22.2 M 6-benzyladenine (BAP) and 2.85 M indole-3-butyric acid (IBA). Bud differentiation from the nodules was achieved by reducing BAP and sucrose concentrations in the culture medium. The maximum frequency of adventitious bud formation occurred on LP basal medium containing 2% sucrose and 0.44 M BAP on which about 61% of the transferred nodules formed buds. During the next 6 weeks of culture on the same cytokinin-free medium multiple shoots elongated from the buds. These shoots were excised and transferred to root initiation medium (RIM2.1), consisting of full-stregth SH macro- and micro-salts, 1000 mg l^–1 myo-inositol, 0.4 mg l^–1 thiamine-HCl, 2% sucrose and a combination of naphthaleneacetic acid (NAA), IBA and BAP at concentrations of 2.69, 4.93 and 0.11 M, respectively. After 5–15 days, root meristems were initiated on the stem bases. The highest rooting frequency was achieved when shoots were treated for 10 days on RIM2.1 medium, before being transferred to half-strength Schenk and Hildebrandt medium with 1% sucrose and without growth regulators for root growth.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Regeneration in Phaseolus vulgaris L.: High-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and thidiazuron

Induction of prolific shoot formation in Phaseolus vulgaris L. cv. Kinghorn Wax was achieved by germinating mature seeds and growing seedlings on a medium supplemented with 10 M thidiazuron (TDZ), a substituted phenylurea, or 80 M N⁶-benzylaminopurine (BAP). Culture for 7 d in the presence of 10 M TDZ was sufficient to induce maximal shoot formation, whereas a continuous presence of BAP was required for the induction and development of shoots. The differentiation of adventitious shoots occurred within four weeks of seed culture, from tissues in the regions of axillary buds on the cotyledonary node and also areas surrounding the shoot apex of the intact seedling. The number of shoots regenerated from intact seedlings was significantly higher than that obtained with expiants. Regenerated shoots developed into flowering plants. Similar results were obtained in six other bean cultivars.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - TDZ N-phenyl-N 1-(1,2,3 thiadiazol-yl)urea (thidiazuron) To whom correspondence should be addressedThis research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and the University Research Board Grant Programs of the University of Guelph to P.K.S. We thank Drs. Jean Gerrath and R. Rastogi for helpful discussions. Technical assistance from Sangeeta Saxena is gratefully acknowledged.