Isolation and characterization of microsatellite markers for the waratah,Telopea speciosissima (Proteaceae)

Thirteen microsatellite loci were developed from an (AG)‐enriched library using Telopea speciosissima, a widespread species of dry‐sclerophyll forest in New South Wales (NSW, Australia). Genotyping of 30 seeds from one population of T. speciosissima and 15 seeds from one population of the related Telopea aspera (restricted to the Gibraltar Range in northern NSW) was successfully performed. The loci were also tested across seven other related species. The microsatellites will be used to compare population dynamics across a range of taxa representing different distribution patterns.     

Relationship of embryogenic competence in maize (Zea mays L.) leaves to mitotic activity,cell cycle and nuclear DNA content

Axillary bud explants of 11 selected mature waratah clones were established in vitro on a modified Murashige & Skoog medium. Adequate proliferation of axillary shoots was achieved by optimisation of the growth regulator status of the culture medium. For the majority of clones, a three to six times rate of proliferation was achieved with 1.25 M BA and 1.0 M GA₃ without the occurrence of abnormalities. The white flowering clone did not respond favourably to the addition of GA₃ to the medium.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige & Skoog medium     

Micropropagation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees

In vitro propagation protocols were established for two leguminous trees, Bauhinia variegata and Parkinsonia aculeata. In each case axillary shoot proliferation was achieved from nodal explants from mature (6-2-8 years) trees using Murashige & Skoog's medium supplemented with 2.22–31.1 M of 6-benzyladenine. Subsequent rooting of the regenerated shoots was achieved on medium containing 2.46–14.8 M of indole-3-butyric acid. Successful transfer of the regenerants to soil has been accomplished.     

Micropropagation of the Mediterranean species Viburnum tinus

In vitro propagation of the Mediterranean species Viburnum tinus L. was established from an outdoor-grown shrub. Two standard macrosalt formulations (Margara N30K and Murashige and Skoog), a range of benzyladenine and sucrose concentrations were tested for their effect on shoot multiplication. The cytokinin concentration was the most important factor affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-node explants cultured on Murashige and Skoog medium supplemented with 4.4 M benzyladenine. Cytokinin concentration and an interaction of macrosalts and benzyladenine influenced shoot length on the multiplication stage: best shoot growth was observed on MS medium containing 1.1 M benzyladenine. In addition, sucrose concentrations of 87.6–146.0 mM gave the highest multiplication rates and improved shoot growth. Following a shoot ellongation stage, single shoots were rooted on media containing naphtaleneacetic acid (1.3–5.4 M). Although enhanced in vitro rooting was obtained on media containing 5.4 M naphtaleneacetic acid, reducing the auxin concentration to 1.3 M during the in vitro rooting stage improved acclimatisation frequency and further plant growth in a horticultural substrate.     

Micropropagation of Rollinia mucosa (JACQ.) baill

Summary A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l⁻¹ polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l⁻¹ sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.     

Micropropagation and genetic stability of a Dendrobium hybrid (Orchidaceae)

Summary Dendrobium hybrids have great economic importance in a number of countries. Asymbiotic seed germination and the conventional vegetative method have been commonly used by growers to propagate these plants. To overcome somaclonal variation, which is commonly exhibited by Dendrobium (Nobile group) when micropropagated from protocorm-like bodies, a protocol for propagating Dendrobium Second Love in vitro using axillary buds in the presence of thidiazuron was developed. Random amplified polymorphic DNA analysis was also carried out to check for possible genetic alterations in plants originating from six consecutive subcultures. The results revealed that the established protocol was efficient for the in vitro cloning of this orchid hybrid and the plants obtained from the six subcultures did not exhibit any type of polymorphism.     

Micropropagation of the endangered <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis> Baijnath

Summary The species, Kniphofia leucocephala is extant at only one location, Langepan, KwaZulu-Natal in South Africa, where the population is threatened by afforestation and possibly grazing. Consequently, a continuous culture system was established as part of a program for the propagation and re-introduction of plants into the wild. The efficiency of the system in terms of shoot multiplication and, particularly, the frequency and rate of root initiation was strongly influenced by the concentration of benzyladenine in the shoot multiplication medium. The optimum shoot multiplication medium for subsequent root initiation contained 2 mgl⁻¹ (8.9 μM) benzyladenine alone. The shoots were successfully rooted and acclimatized. Approximately 200 shoots can be produced from one shoot after five 4-wk cycles. Thus, large numbers of plantlets can be propagated in this continuous culture system, serving conservation interests.     

Rhizogenesis and root growth ofCarica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).     

Micropropagation of tree peony (<Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>)

We investigated the in vitro propagation by axillary budding of different cultivars of tree peonies, selected for cut flower production under Mediterranean conditions. Buds with expanded leaves were better to initiate cultures than just emerged ones (64%compared to 43%). The aptitude for micropropagation was genotype-dependent, and the propagation ratio ranged between 2 and 5 per cycle. Tendency to necrosis and/or hyperhydricity were also genotype dependent. Indole-3-butyric acid improved rooting but was not really necessary provided the shoots were pre-treated at 2 °C for 7days. Plantlets were successfully acclimatized under in vitro conditions. Adventitious propagation was achieved using filaments and petals as explants. They first developed callus, able to regenerate shoots after 8 weeks on media supplemented with thidiazuron.     

Micropropagation of endangered endemic Brazilian bromeliad Cryptanthus sinuosus (L.B. Smith) for in vitro preservation

An efficient liquid culture system for plant regeneration from leaflessstem–root axes of Cryptanthus sinuosus L. B. Smith(Bromeliaceae) was established. High regeneration rates (93%) were achieved inMurashige and Skoog's medium without growth regulators. Whole plants wereobtained in a single-step procedure, resulting in the production of 25.3± 3.6 plants/explant after 6 months of culture. Incubationof plant material at 35 ± 3 °C resulted in an increaseof 60% in the regeneration efficiency compared with tissues incubated at 28± 2 °C. Moreover, after 5–6 sub-cultures in thesame medium, the axes originated bud clusters that could be continuouslymultiplied and gave rise to 19.4 ± 3.2 whole plants per gram of freshmatter. It was estimated that the liquid culture system described is potentiallyable to produce about 4500 plants/explant/year. Rates of 98% acclimatizationwere achieved. The use of plants produced following this method for populationreinforcement and for in vitro preservation programs ofendangered populations is suggested.     

Micropropagation of sweet viburnum (<Emphasis Type="Italic">Viburnum odoratissimum</Emphasis>)

A micropropagation protocol for shoot culture of sweet viburnum (Viburnum odoratissimum) is described. Nodal explants, initially established on MS medium, were transferred to WPM supplemented with combinations of BA and GA₃. Maximum shoot multiplication was observed on explants cultured on medium supplemented with BA concentration higher than 1.1 μM, and 14 μM GA₃. Although Stage II medium supplemented with BA concentration higher than 1.1 μM resulted in increased shoot multiplication, it also caused a decrease in shoot length. A negative carry over effect of GA₃ on rooting was observed in subsequent Stage III cultures. The presence of GA₃ in Stage II medium promoted shoot elongation, but it also caused a decrease in microcutting rooting. For this reason, 0.5 μM BA and 14 μM GA₃ were selected for optimum Stage II shoot multiplication. Although 100% microcuttings formed roots when cultured on medium containing 6.0 μM NAA, significant callus formation was observed and ex vitro survival rate was low (49%). Rooting was achieved after 3 weeks with 82% of microcuttings on medium supplemented with 3 μM IBA. The survival rate of plantlets under ex vitro conditions was 100% after 3 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 30 plants.     

Micropropagation of Eclipta alba and Eupatorium adenophorum using a single-step nodal cutting technique

Protocols for the micropropagation of two traditional medicinal plants Eclipta alba (L.) and Eupatorium adenophorum (L.) from nodal segments were developed. Proliferated microshoots of Eclipta alba and Eupatorium adenophorum were obtained through axillary branching by culturing nodal segments in modified MS medium and half strength of MS, respectively, with minimal strength of nutritional support. Simultaneous rooting could also be induced in the same medium. Regenerated rooted plantlets were successfully acclimatized in soil where they grew normally without showing any morphological variation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation and effects of mycorrhiza and soil bacteria on acclimatization and development of lucumo (Pouteria lucuma R. and Pav.) var. La Molina

Summary A micropropagation protocol for Pouteria lucuma R. and Pav. var. La Molina, was developed. Shoots from zygotic embryos with a portion of endosperm were established in vitro on Murashige and Skoog (MS) medium with 0.47 μM kinetin (Kin) and 0.54 μM naphthaleneacetic acid (NAA). Multiplication of shoots was accomplished using subapical, shoots. The best axillary-shoot production was observed on MS basal, medium with 2.2μM, benzyladenine (BA), 0.5 μM NAA, 1.4. μM gibberellic acid (GA₃), and 40 mgl⁻¹ adenine sulfate, with the development of up to three axillary shoots per subapical shoot. One hundred percent rooting was obtained from shoots grown, for 4wk on MS medium with 246 μM indole-3-butyric acid under light conditions. Eighty percent of the microplantlets survived after acclimatization when transplanted to a substrate previously enriched with beneficial soil bacteria. This study describes, for the first time, arbuscular mycorrhizal (AM) colonization of this species. Inoculation with AM fungi improved growth and development of lucumo plants and induced changes to the root morphology.     

In vitro Micrografting of Mature Pistachio (Pistacia vera var. Siirt)

The success of various in vitro micrografting methods of shoot tips of pistachio (Pistacia vera L. var. Siirt) have been examined. Excised zygotic embryos that germinated in vitro were used as rootstocks. Current year shoot tips from mature trees of pistachio micrografted onto in vitro juvenile rootstocks, resulted in the restoration of shoot-bud proliferation. Variables tested include a size of microscion, grafting method, effects of culture medium and effects of time of the year at which shoot tips were used. The results indicate that the easiest and most successful method for grafting was slit micrografting. High levels of micrograft take were achieved with 2–4 mm (56.75%) and 4–6 mm (79.25%) long scions obtained from the regenerated shoot tips. The survival rate of the shoot tips was directly related to time of the year. The best growth of microscion was obtained with the in vitro forced shoot tips rather than with shoot tips excised from tree. Slow growth and lack of axillary shoot development on the micrografts was noticeable when the micrografts were cultured on hormone-free and germination medium. In vitro micrografted plantlets were successfully weaned and no problems were encountered with the establishment of micrografted plants in vivo.     

Nutlet morphology in Hemigenia R.Br. and Microcorys R.Br. (Lamiaceae)

Nutlets of Hemigenia R.Br. and Microcorys R.Br. were examined using SEM. Significant variation, mainly useful at the infrageneric level, was found in nutlet shape, nature of the attachment scar, nature of surface sculpturing, exocarp cell shape and sculpturing, and nature of the indumentum. Typical nutlets are ovoidal, strongly reticulate or rugose. The exocarp cells are isodiametric and convex to papillate. Also common are cylindrical nutlets, often with longitudinal ridging and papillate exocarp cells. Surface pitting and concave exocarp cells are rare. A cladistic analysis of nutlet characters suggests both Hemigenia and Microcorys are polyphyletic, and Microcorys paraphyletic with respect to Westringia Sm. Notwithstanding that, the infrageneric classification of Hemigenia was largely supported, while in Microcorys, there was support for sect. Hemigenioides, but sects Anisandra and Microcorys were not resolved as distinct.     

Effects of in vitro treatments on leaf area growth of potato transplants during acclimatisation

The importance of leaf area of in vitro propagated potato (Solanum tuberosum L.) plantlets for further growth during acclimatisation and the after-effects of in vitro treatments on growth were examined. The in vitro treatments included different levels of alar, nitrogen or mannitol or different temperatures during the last in vitro phase, the rooting phase. Leaf area or ground cover was recorded one day after planting to soil and at the end of the first phase of ex vitro growth, the acclimatisation phase. Regression analysis showed that leaf area of a transplant at the end of acclimatisation phase was positively influenced by leaf area of the same plantlet at the beginning of the phase. The relative increase in leaf area during acclimatisation (increase/early leaf area) was linearly related to the inverse of the early leaf area, indicating almost comparable relative increases for plantlets having larger early leaf areas, but more variable responses for plantlets having smaller early leaf areas. In vitro treatments mainly affected leaf area of transplants through their effects on early leaf area. Adding alar, reducing nitrogen and reducing temperature increased leaf area. Reducing mannitol increased ground cover. A lower nitrogen concentration and higher temperature in some cultivars had slight negative effects on the relative increase in leaf area after acclimatisation. For nitrogen these negative effects were less significant than the positive effects through early leaf area. Results stress the importance of manipulation of leaf area in vitro to enhance plant performance in later stages of growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of <Emphasis Type="Italic">Lavandula dentata</Emphasis> from axillary buds of field-grown adult plants

Axillary buds from adult field-grown plants of Lavandula dentata L. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growth. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with a combination of 2.2 μM of benzyladenine and 2.5 μM indole-3-butyric acid. The best condition for rooting was MS medium plus 2.5 μM naphthaleneacetic acid. Rooted plantlets were successfully transferred to soil. Short-term culture derived plants (6 month) exhibited a normal development, but a low frequency of not heritable morphological changes were detected in long term culture derived plants (more than 1 year).This work was supported by a grant from the University of Caxias do Sul and CNPq, Brazil.