Axillary bud proliferation and organogenesis of <Emphasis Type="Italic">Euphorbia pulchurrima</Emphasis> winter rose

Summary Protocols for both axillary bud proliferation and shoot organogenesis of Euphorbia pulchurrima Winter Rose^TM were developed using terminal buds and leaf tissues. Greenhouse-grown terminal buds were placed on Murashige-Skoog (MS) basal medium supplemented with various concentrations of either benzlyaminopurine (BA) or thidiazuron (TDZ). Explants produced the greatest number of axillary buds on media containing between 2.2 and 8.8 μM BA. The number of explants that produced axillary buds increased with increasing BA concentration. TDZ at concentrations between 2.3 and 23.0 μM caused hyperhydricity of shoots and were not effective in promoting shoot proliferation. The most calluses and shoots were produced from leaf midvein sections from in vitro grown plants placed on the medium containing 8.8–13.3 μM BA and 17.1 μM indole-3-acetic acid (IAA) for 1 mo. before transferring to the medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Five-mo.-old shoots derived from shoot culture or organogenesis rooted readily in artificial soil with or without treatment with indolebutyric acid, and were acclimatized in the greenhouse.     

Morphogenetic effect of glycerol on tissue cultures of the red seaweed Grateloupia doryphora

Explants of Grateloupia doryphora were cultivated in Provasoli Enriched Seawater culture medium (PES) supplemented with glycerol (0.1, 0.3, 0.5 or 0.8 mol 1^–1) or carbohydrates (0.1 or 0.3 mol 1^–1 mannose, glucose and galactose) and agar (3, 8, 15 g 1^–1 ). The osmolality of the medium was adjusted by dilution of the seawater (70 or 100%, v/v). The increase in fresh weight of explants cultivated in liquid medium with glycerol (0.3 mol 1^–1) and without glycerol was compared. All experiments were carried out in the light, except for one assay in which the explants were cultivated in the dark. Glycerol was an effective carbon source for the vegetative propagation of G. doryphora in solid and liquid media. Mannose, glucose and galactose all had no effect on growth or morphogenesis of the explants. In solid media the main effect of glycerol was as a morphogenetic inductor, with PES70 (70% seawater) + 0.1 or 0.3 mol 1^–1 glycerol + 3 or 8 g 1^–1 agar the best formulation. An increase in the concentration of agar in glycerol-containing medium reduced the morphogenetic capacity of the explants, which developed into compact cell masses. The effects of glycerol were observed only in explants cultivated under light.     

Influence of agar and activated charcoal on uptake of gibberellin and plant morphogenesis In vitro

Summary Agar and activated charcoal (AC) are commonly used in tissue culture. However, their deeper actions and functions are largely unknown. This experiment investigated the effect of agar and AC, singly and jointly, on gibberellin (GA) uptake by corn shoots. Corn seeds were germinated on Murashige and Skoog medium (MS). Shoot excised from 1-wk-old seedlings were cultured on liquid (0.0 g l⁻¹ agar) or solid (8 g l⁻¹ agar) MS containing 3 μM indole-3-acetic acid, 13.3 μM N⁶-benzyladenine, and 6000 CPM ml⁻¹ [³H]GA₄ as tracer. Both liquid and solid media had two treatments, one without AC and one supplemented with 5 g l⁻¹AC. Uptake of [³H]GA₄ and morphogenesis of corn shoots were recorded after 2 wk of culture. Corn explants cultured in AC-free media acquired high levels of [³H]GA₄, while explants from AC-containing media showed only traces of [³H]GA₄. Explants cultured in AC-free liquid medium contained about twice the amount of [³H]GA₄ as those from AC-free solid medium. Addition of agar reduced shoot length, while addition of AC increased both shool and root length. It is concluded that: (1) agar reduced the uptake of GA₄; and (2) GA₄ was irreversibly adsorbed by AC, and thus became unavailable to corn explants.     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

Shoot regeneration from leaf explants of <Emphasis Type="Italic">Brunfelsia calycina</Emphasis>

A protocol for plantlet regeneration through shoot formation was developed for the neotropical shrub Brunfelsia calycina. This shrub is unique in its change in flower color from dark purple to white. Explants from young and mature leaves were incubated on MS medium (pH 5.7, 30 g/l sucrose, 7.5 g/l agar) with various combinations of Indole-3-acetic acid (IAA) and 6-Benzyladenine (BA) under a 16 h photoperiod at a constant temperature of 25°C. Shoot emergence was best at 4.44 μM BA and 2.85 μM IAA for young leaf explants, and at 8.88 μM BA, 2.85 μM IAA for mature leaf explants. When shoots were transferred to MS medium supplemented with 1.23–2.46 μM indole butrytic acid (IBA), they developed roots.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

Enhancement of direct somatic embryogenesis and plantlet growth from leaf explants of <Emphasis Type="Italic">Phalaenopsis</Emphasis> by adjusting culture period and explant length

Two Phalaenopsis orchids, Phalaenopsis amabilis and Phalaenopsis ‘Nebula’, were used to test the effects of induction period (30, 45 and 60 days), subculture period (30, 45 and 60 days), and explant length (1, 1.5 and 2 cm) on direct somatic embryogenesis from different regions (leaf tip, adaxial side, abaxial side and cut end) of leaf explants from in vitro grown seedlings. The results showed that the cut end had a highest competence to form embryos than the other regions of the leaf explants from both orchids. In addition, the suitable culture conditions were 60 days for induction period in darkness, 45 days for subculture period in light and 1 cm for explant length. Besides, the combinations of N⁶-benzyladenine (BA) and naphthaleneacetic acid were tested on their effects on plantlet conversion and further development of leaf-derived embryo. It was found that 0.5 mg/l of BA showed the highest response on plantlet conversion rate and the lowest browning rate of explants. In this communication, the embryo structures and development were proved by scanning electron microscopy.     

Plant tissue cultures from four Tuscan globe artichoke cultivars

The aim of the study was to examine the possibility of propagating in vitro four of the most common cultivars in Tuscany (central Italy): Terom, Violetto di Toscana, Chiusure and Empolese. The first three belong to the “Violetti” group, while cv Empolese belongs to the “Romaneschi” group. Explants were cultured on an induction medium (IM), which is a modified MS medium consisting of nitrate concentrations reduced by one quarter, 0.8 mg L⁻¹ 6-benzylaminopurine (BA) and 0.2 mg L⁻¹ 3-indole butyric acid (IBA). Explants were then transferred to a proliferation medium (PM) consisting of the same basal medium together with 0.03 mg L⁻¹ BA and 0.05 mg L⁻¹ gibberellic acid (GA₃). A rooting double-phase was then established. The pre-rooting medium (PRM), consisting of a basal MS medium with half strength nitrate concentrations, 0.5 mg L⁻¹ indole-3-acetic acid (IAA) and 1 mg L⁻¹ paclobutrazol (PBZ) was used for two weeks. Over the next four weeks, a rooting medium (MR) was used, consisting of a basal MS medium with 2 mg L⁻¹ β-cyclodextrin and 2 mg L⁻¹ α-naphthaleneacetic acid sodium salt (NAA). The cv Empolese provided the highest number of proliferated explants and rooted plantlets using the method described.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

Summary An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm² in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, β-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlied-atmosphere chamber which was gassed with 95% O₂ and 5% CO₂. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30°C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin B₁, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.     

Effects of auxin and light treatments of donor plants on shoot production fromindica-type rice (Oryza sativa L.)

An efficient clonal propagation procedure for a Brazilianindica rice subspecies was developed with shoot apex explants. Shoot apices were excised from 4-d-old seedlings and cultured on MS medium supplemented with 8.9 μM 6-benzyladenine. The efficiency of shoot production was influenced by growth regulators and light treatments to the donor plant. Explants derived from seedlings growth in the presence of 10.7 μM naphthaleneacetic acid and in the absence of light showed significantly increased regeneration capacity as compared to control explants. Anatomical analysis of the new shoot meristems revealed that they originated from preexisting apical and axillary meristem as well as from the mesocotyl parenchyma.     

Efficient plant regeneration of garlic (Allium Sativum L.) by root-tip culture

Summary Root apices from in vitro cultured garlic (Allium sativum) cloves of cvs. ABEN and GT96-1 were used as axenic explants for organogenic callus production and plant regeneration experiments. Explants cultured in media based on those of Chu and co-workers (N6) or Murashige and Skoog (MS) could induce organogenic callus after 8 wk culture in darkness. Both media were supplemented with 2,4-dichlorophenoxyacetic acid (2.2–4.5 μM), alone or combined with 6-furfurylaminopurine (kinetin, 2.3–4.6 μM). Shoots started to grow 3 wk after culturing in the presence of light and the addition to culture media of 4.4 μM N⁶-benzyladenine. Plants capable of producing microbulbs regenerated 6 wk later. Up to 170 plants g⁻¹ FW callus were obtained when culturing was initiated in MS medium supplemented with 4.6 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid.     

Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ (<Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">P. davidiana</Emphasis>)

A complete protocol for adventitious shoot regeneration was developed from the leaves of peach rootstock ‘Nemaguard’(Prunus persica × P. davidiana) grown in vitro. Shoot explants were cultured in vitro in Murashige and Skoog medium supplemented with 3.55 μM 6-benzyladenine and 7.38 μM indole-3-butyric acid (IBA). Non-expanded leaves along with their petioles from 3-week-old in vitro-grown shoots were used as explants. Regeneration percentage was influenced by plant growth regulators, basal medium, explant type, dark period, and gelling agents. Optimal regeneration was observed with leaf explants wounded by transverse cuts twice along the midrib and first incubated with abaxial surfaces facing upward in the dark for 3 weeks, and then transferred to the light and cultured with the adaxial side in contact with regeneration medium, as seen on 1/2 MS, woody plant medium or Schenk and Hildebrandt medium supplemented with 9.08 μM thidiazuron, 0.54 μM IBA and 0.25% agar. This produced the highest regeneration percentage at 71.7% and a mean of 5.74 ± 3.24 shoots on 1/2 MS medium. Adventitious shoots were rooted (98.3–100%) and rooted plantlets survived after acclimatization to the greenhouse.     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

葛(豆科)叶的解剖学研究

利用光学显微镜和扫描电镜观察了葛(Pueraria lobata)叶的解剖学特征。结果表明,葛叶片的上、下表皮都只有一层表皮细胞,上表皮比下表皮厚。上、下表皮都有腺毛和非腺毛。气孔主要分布在下表皮,下表皮的气孔密度为(261±17)mm-2,上表皮只有(6±3)mm-2。叶肉由两层栅栏组织细胞和一层海绵组织细胞构成。叶肉细胞中有丰富的叶绿体。在栅栏组织和海绵组织之间有一层平行于叶脉的薄壁细胞。叶脉中含有大量的草酸钙晶体。葛叶的这些形态特征与其喜阳、耐旱的特点相适应。     

Establishment of hairy root lines and analysis of gentiopicroside in the medicinal plant <Emphasis Type="Italic">Gentiana macrophylla</Emphasis>

The leaf explants from the eight-week-old Gentiana macrophylla Pall sterile seedlings were precultured for three days on Murashige and Skoog (MS) agar medium with 1 mg/l of 6-benzyladenine, and then incubated in MS liquid medium containing Agrobacterium rhizogenes R1000 for 2 h with shaking. After 10 days of culture on hormone-free MS agar medium with 500 mg/l cefotaxime under illumination of 300 μmol/(m² s) with a 16-h photoperiod at 24°C, up to 18.3% of the infected explants produced typical hairy roots (HRs). Finally, three stable HR lines with no morphologically visible differences were obtained. The HR lines could vigorously grow and propagate on/in hormone-free half-strength MS solid/liquid medium. Moreover, they changed from white to green when moved from darkness to the light and spontaneously produced calli, which were able later to produce adventitious buds. PCR analysis revealed that three HR lines, which were undergone to continuous subculturing over two years, still contained the rolC gene from the A. rhizogenes Ri plasmid. HPLC revealed the HR line possessing a certain capacity of synthesizing gentiopicroside (0.11 mg/g dry wt).