Effect of polyamines on adventitious root formation from Tobacco (Nicotiana tabaccum) leaf segments

To elucidate the effect of polyamines on adventitious root formation, we investigated the relationship between the frequency of adventitious root formation and the endogenous content of free polyamines in tobacco leaf segments which had been treated with polyamine biosynthesis inhibitors and polyamines. Adventitious root formation was inhibited in rooting medium (10 μM IAA) with methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine (CHA), and promoted with spermidine and putrescine. Treatment with high IAA (100 μM) medium plus CHA or MGBG promoted rooting up reversion of the rooting inhibition than the one treated with high IAA concentration alone. Spermidine promoted adventitious root numbers on low IAA (1 μM) medium when applied during culture period. The rooting inductive phase (in the presence of IAA) was determined by periodical transfer of leaf segments from IAA-containing medium to IAA free medium, and by changing polyamine contents, to be inductive phase. Putrescine and spermidine were accumulated to a maximum during the inductive phase. Therefore, the results point out the involvement of polyamines in inductive phase of adventitious root formation in tobacco leaf segments.     

Micropropagation of garlic through in vitro bulblet formation

We developed a novel micropropagation method for garlic (Allium sativum L.) by the combination of initial shoot-tip culture, shoot multiplication and in vitro bulblet formation. Garlic shoot-tips were cultured on LS medium containing 1 M indole-3-acetic acid (IAA) and 1 M 6-benzyladenine (BA) to regenerate proliferative shoots. These shoot-tips produced multiple shoots when transferred to modified LS medium containing 5 M 1-naphthaleneacetic acid (NAA) and 10 M BA, and cultured at 20°C under 12-h light conditions. Higher ratios of KNO₃/NH₄Cl in the media promoted multiple shoot formation, together with suppressing vitrification of these shoots. The proliferated shoots of early maturing cultivars produced bulblets by culture on LS growth regulator-free medium at 25°C under 16-h light. On the other hand, the late maturing cultivar, Howaito-roppen, formed bulblets after a low temperature treatment of the proliferated shoots for 6 months followed by culture on LS medium containing 6 to 12% sucrose for two months. The dormancy of the bulblets of cv. Howaito-roppen was broken by successive treatments at a high (35°C), a middle (20°C), and then a low (5°C) temperature.Abbreviations IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - LS Linsmaier and Skoog macro- and microelements     

Production of triploid plants of papaya by endosperm culture

Triploid papaya (Carica papaya) plants were obtained by immature endosperm culture. Visible callusing of the endosperm occurred 21 days after initiation of cultures. A continuously growing callus was observed and a maximum of 68.7% of callus induction frequency was obtained when immature endosperm with embryo was cultured on Murashige and Skoog (MS) medium containing 6.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.5 μM α-naphthaleneacetic acid (NAA) and 4.0 μM 6-furfurylamino purine (Kn). Shoot buds were produced when the callus was subcultured on a medium supplemented with 6-benzyladenine (BA) or thidiazuron (TDZ) along with NAA. Shoots were detached from the callus and transferred to the elongation medium supplemented with indole-3-acetic acid (IAA) and BA. The combination of 3.0 μM IAA and 1.5 μM BA was the best in terms of the number of cultures (93.8%) showing axillary shoot proliferation. The addition of 2.0 μM indole-3-butyric acid (IBA) to the 1/2 MS medium was most effective at inducing root formation with 90% of the shoots developing four to five roots. Healthy rooted plantlets were transferred to pots containing sterilized bed soil and perlite (3:1) mixture in the greenhouse and 78% of the micropropagated plants survived transplantation. The leaves from endosperm-derived plants showed larger stomata and more chloroplasts in guard cells than that from the parent plants. Over 75% of the endosperm-derived plants were triploid with chromosome number 2n = 3x = 27.     

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μ M α-naphthaleneacetic acid and 1 μ M kinetin, and (2) 1 μ M α-naphthaleneacetic acid and 1 μ M kinetin. The contents of alkaloids, K⁺ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N-demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K⁺ were observed in calli grown on the high-auxin medium, both cultures were K⁺ deficient. The fact that the low-auxin medium limited K⁺ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K⁺ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low-auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot-buds is discussed.     

Plant regeneration by organogenesis in Glycine max

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5M BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.Abbreviations BA benzyladenine - IAA indoleacetic acid - SAS secondary axillary shoots - MS Murashige and Skoog (1962) medium     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea ) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).     

High frequency plantlet regeneration from cotyledonary node cultures of <Emphasis Type="Italic">Aegle marmelos</Emphasis> (L.) Corr.

High frequency plantlet regeneration was achieved in cotyledonary nodes of Aegle marmelos. Cotyledonary nodes from 1 mo. old in vitro grown seedlings of A. marmelos were cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) (0–8.8 μM), kinetin (KIN) (0–9.4 μM), and indole-3-acetic acid (IAA) (0–1.14 μM) either alone or in combinations. The highest regenerative response was observed on medium containing 6.6 μM BA + 1.14 μM IAA where approximately 86.6% of the cultures responded with an average shoot numbers of 487.5 per explant in 7-wk time. Cultures maintained on KIN-supplemented medium showed very poor response. In vitro responded shoots were transferred to root induction medium consisting of half-strength MS supplemented with auxins IAA, indole-3-butyric acid (IBA), or α-naphthalene acetic acid (NAA). Rooting was best in medium supplemented with 14.7 μM IBA. Rooted plantlets were acclimatized and transferred to the field with 80% survival rate.     

In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir.

An efficient micropropagation system for Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant, has been developed. Shoot organogenesis occurred from S. involucrata leaf explants inoculated on medium with appropriate supplements of plant growth regulators. 66.0% of shoot regeneration frequency and 5.2 shoots per leaf explant were achieved when cultured on a medium containing 10 μM 6-benzylaminopurine (BAP) and 2.5 μM 1-naphthaleneacetic acid (NAA). Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature, and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4°C by 5-day pretreatment period. Up to 87.0% of the regenerated shoots formed complete plantlets on a medium containing 2.5 μM indole-3-acetic acid (IAA) within 28 days, and 85.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition. The phytochemical profile of the micropropagated plants was similar to that of wild plants. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.     

Influence of boron on the growth and biochemical changes in plant growth promoting rhizobacteria (PGPR) inoculated banana plantlets

The effects of rhizobacteria inoculation in modified MS medium containing boron (1 and 10 μM) on the biochemical components, physiological characteristics and mineral content of the in vitro banana plantlets were carried out. The presence of rhizobacteria in the medium supplemented with boron at two concentrations: 1 and 10 μM resulted in an improvement in growth and root biomass compared to the control (uninoculated). Rhizobacteria inoculation also produced an increase in protein, nitrate, soluble nitrogen and chlorophyll contents of the plantlets cultured in MS modified medium containing boron. An increase in percentage of growth (>295%) was shown when boron was applied into medium inoculated with Bacillus sphaericus UPMB10. The effectiveness of inoculation is increased when associated with boron, nitrogen or carbon into the medium. Thus, these bacterial strains could be used as a bioenhancer for growth of in vitro banana plantlets.     

Somatic embryogenesis in Clematis integrifolia × C. viticella

Nodal sections of Clematis integrifolia × C. viticellawere cultured at 24 °C in darkness on a medium containing the salts and vitamins of Murashige and Skoog (1962) supplemented with sucrose (30 g l⁻¹), 2 μM 6-benzylaminopurine (BAP) and 0.5 μM 4-indole-3-yl-butyric acid (IBA). These explants produced a white friable callus on their surfaces. Somatic embryos were first observed on the surface of the callus after eighteen months in culture. Thereafter, pieces (125 mm³) of the embryogenic mass were subcultured every 4 weeks and continued to produce somatic embryos over the two-year period of observation. A mean (± SE) of 64±4 cotyledonary stage embryos were observed per 125 mm³ callus four weeks after transfer to fresh medium. Comparison of the effect of growth regulators on conversion showed that the highest frequency of unipolar and bipolar conversions occurred on medium containing 10 μM kinetin and 1 μM BAP. Shoots were excised from embryos and inserted in Sorbarods saturated with liquid medium containing 0.05 μM IBA and 0.05 μM 1-naphthalenacetic acid. After four weeks 88% had formed root and survived transfer to compost in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

Efficient regeneration of Eucalyptus urophylla from seedling-derived hypocotyls

Seedling hypocotyls were used as explants to establish a regeneration protocol for Eucalyptus urophylla and N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU), one kind of di-substituted urea, was found useful growth regulator. The hypocotyls incubated on a modified Murashige and Skoog medium (SPCa), supplemented with 6.6 μM PBU and 0.57 μM indole-3-acetic acid (IAA) dedifferentiated and form calli (100 % after 7 d). Compared with other growth regulator combinations, PBU stimulated more vigorous calli and restrained their darkening. In addition, the calli induced by PBU showed high frequency of adventitious buds formation (57%). Shoot proliferation and elongation was then stimulated on SPCa medium containing 0.44 μM 6-benzyladenine (BA), 0.54 μM naphthalene acetic acid (NAA) and 0.3 μM gibberellic acid (GA3). For rooting, shoots were cultivated on root induction medium containing 2.5 μM indole-3-butyric acid (IBA). Plantlets were then successfully transplanted to greenhouse.     

Morphogenic Potentialities of Flower Buds of Kalanchoe pinnata Pers. grown in vitro

Excised flower buds of Kalanchoe pinnata Pers. representingtwo early stages of development (designated sets I and II),were cultured on modified White's medium (WB). They failed toattain full development on WB or on WB containing any of thefollowing supplements: indole-3yl-acetic acid (IAA), 2,4-dichlorophenoxyaceticacid (2,4-D), naphthaleneacetic acid (NAA), kinetin, or coconutmilk (CM). A slight stimulation of the growth of corolla wascaused by kinetin (10 ppm). IAA (I ppm) and NAA (I ppm) inducedrooting from the cut end of the pedicel and from the proliferatedtorus tissue situated between the sepals and petals. 2,4-D (Ippm) either singly or in concert with CM (10 per cent) stimulatedthe formation of shoot buds and root growth. Addition of kinetin(I and 10 ppm) to WB favoured shoot formation, but suppressedrooting. Flower buds of set II developed shoot buds more readilythan those of set I. Thus, the primordia floral organs presentin the immature buds lose their ability for normal morphogenesisunder culture conditions. Buds destined to form flowers canbe made to revert to vegetative growth.     

Plant regeneration of common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis> L.) by somatic embryogenesis

This is the first report on somatic embryogenesis in common ash (Fraxinus excelsior L.). Experiments on somatic embryogenesis induction were carried out on zygotic embryos at different phases of development and maturation. The embryo axes were isolated and cultured on media containing different plant growth regulators (PGRs). Embryogenic tissues were obtained from embryos collected at an incomplete maturation phase and cultured on a modified Murashige and Skoog medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine (BA). Embryos isolated from seeds at an advanced stage of maturation showed only organogenetic phenomena. Embryogenic tissues were successfully subcultured and multiplied on medium containing a reduced concentration of PGRs. After their isolation, somatic embryos were induced to develop and mature by transfer to PGR-free medium and subsequent culture on medium containing 0.1 μM BA. Somatic embryos developed completely and also germinated spontaneously. Embryo germination and conversion were significantly improved when subjected to a period of storage at 4°C and transplant onto woody plant medium. Plantlets were successfully transferred to soil and acclimatized in a “misted” greenhouse.     

Benzyladenine and low temperature promote phase transition from juvenile to vegetative adult in bulblets of Lilium?×?formolongi ‘White Aga’ cultured in vitro

Scales excised from lily bulblets were cultured on MS medium supplemented with 0.044 or 4.4 μM BA in the dark for 180 days. The culture period was divided into stage 1 (day 0–30), stage 2 (day 31–90) and stage 3 (day 91–180). The scales were cultured at 25°C in stage 1, 25°C or 8°C in stage 2, and 25°C in stage 3. When the scales were cultured on medium with 4.4 μM BA at 25°C for 180 days, bulblets with and without an elongated stem were produced. The percentage of bulblets with elongated stems greatly increased when the scales had been cultured at 8°C in stage 2. On medium with 0.044 μM BA, only bulblets without elongated stems were produced. The diameter of shoot primodia significantly enlarged in bulblets produced on medium with 4.4 μM BA at 8°C in stage 2 and no such enlargement occurred under the other conditions. Nearly square parenchyma cells were observed in the non-elongated shoot primodia in the former bulblets but not in the latter. These cells changed into longitudinally rectangular ones in the internode of elongated stems. Procambium was arranged almost parallel to the shoot axis in the stem of bulblets in the medium with 4.4 μM BA, but not in the medium with 0.044 μM BA.     

Néoformation caulinaire et radiculaire chez les fragments de feuille deBegonia rex Putz.: Action de divers facteurs régulateurs trophiques et d'environnement

The effects of several growth and trophic substances on bud and root neoformation on leaf fragments ofBegonia rex were studied in precisely defined environmental conditions. IAA, depending on the type of treatment, had different effects. In aseptic cultures, a notable stimulation of bud formation was observed at certain concentrations. However, non aseptic treatments of IAA had no visible effects except at very high concentrations.(10^?3 M) where bud formation was totally inhibited and root formation was favored. NAA, at 10^?6 M and 10^?5 M strongly stimulated root formation and inhibited shoot formation. All the cytokinins used stimulated bud formation and inhibited partially or totally root formation. Gibberellic acid inhibited bud and root formation. Glucose and sucrose clearly stimulated bud and root formation and sucrose, when applied simultaneously with other growth substances, modified the effects of these substances alone. The most favorable environmental conditions were at 24°C in a 24 h photoperiod but other temperatures (17 to 27°C) and photoperiods (9 or 16 h) did not prevent neoformation.     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog