Sinclair H. Mantell

Effects of photoperiod, mineral medium strength, inorganic ammonium, sucrose and cytokinin on root, shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.

In vitro shoot culture and microtuber induction in the steroid yam Dioscorea composita Hemsl

The individual effects of sucrose, plant growth regulators and basal salt media formulations were investigated on microtuber induction and development in shoot cultures of the steroid yam Dioscorea composita. Sucrose at 8% (w/v) was the single most significant medium constituent for microtuber induction. Of the four cytokinins tested, 6-benzyladenine at 1.25 and 2.5 μM showed strong inhibitory effects on microtuber induction. By contrast, the auxins α-naphthaleneacetic acid and indole-3-butyric acid at 5.0 μM showed striking promotive effects on microtuber induction and growth. In the presence of either one of these auxins at 5.0 μM shoot cultures produced microtubers weighing 300–400 mg fresh weight whilst kinetin, 6-(γ,γ-dimethylallylamino)-purine, 6-benzyladenine and abscisic acid failed to promote microtuber growth (microtubers weighed generally <200 mg). Media formulations Lloyd and MacCown and White supported the lowest frequencies of microtuber induction when kinetin was present at 2.5 μM. Anderson Rhododendron was as effective as Murashige and Skoog overall in promoting both microtuber induction and growth. When removed from cultures and planted in sterilized moist sand, microtubers sprouted readily (60–87% within 2 weeks) and produced vigorous shoot growth and after 5–7 months minitubers of sizes (30–80 g) suitable for direct field planting.

In vitro multiplication of cashew (Anacardium occidentale L.) using shoot node explants of glasshouse-raised plants

Abstract Using glasshouse-raised plants (1 month, 1 year and 5 years old), factors affecting shoot development from shoot nodes of two Brazilian and one Tanzanian elite selections of cashew (Anacardium occidentale L.) were assessed. Sprouting of buds decreased strongly with increasing age of mother plants. Solidified media, mainly when purified agar was used, gave better results than liquid medium. Murashige and Skoog salts containing 1/2-strength macroelements were the most suitable for bud sprouting and shoot elongation. Vitamins and sucrose concentration did not have a significant effect but by replacing 20 g/l sucrose with glucose or maltose all estimated parameters were improved. Gibberellins supported bud sprouting and shoot elongation but blocked rooting. Shoots developed in the presence of cytokinins were short and produced axillary branches. Activated charcoal, cultivation of explants in darkness for the first 7 days and superoptimal temperature (35 °C) decreased bud sprouting and supported shoot elongation. Microshoots rooted in vitro at a frequency of 42% when cultured for 5 days with 100 μμ indole-3-butyric acid. Over 40% of rooted microshoots survived weaning.

ISOLATION AND CHARACTERISATION OF CDNA CLONES REPRESENTING THE GENES ENCODING THE MAJOR TUBER STORAGE PROTEIN (DIOSCORIN) OF YAM (DIOSCOREA CAYENENSIS LAM.)

AbstractcDNA clones encoding dioscorins, the major tuber storage proteins (Mr 32000) of yam (Dioscorea cayenensis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of Mr 30015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

Use of random amplified polymorphic DNA (RAPD) markers to reveal genetic diversity within and between populations of cashew (Anacardium occidentale L.)

Summary RAPD (random amplified polymorphic DNA) markers were used to determine genetic diversity within and between populations of cashew (Anacardium occidentale L.). Efficient and novel procedures for extracting PCR-amplifiable, high molecular weight DNA from young cashew leaves were developed and the PCR conditions for RAPD analysis of cashew DNA using random 10.bp primers were defined. Optimized PCR-reaction conditions were then used to analyse differences in RAPD profiles within and between a selection of cashew varieties obtained from diverse geographical locations around the world and within a pool of twenty elite Tanazanian cashew lines. RAPD polymorphisms were obtained among the Tanzanian lines and between and within the geographically diverse lines. The relatively uniform RAPD profiles for the selection of random primers tested suggested a high degree of DNA level similarity between the Tanzanian accessions. Accessions from India, Mozambique and Tanzania showed the closest relationship, with accessions from Brazil being the most distinct from the other provenances. A specific RAPD-PCR product was linked with cashew accessions from the Cook Islands.

Endophytic bacteria expressing β-glucuronidase cause false positives in transformation of Dioscorea species

SummaryFalse positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express β-glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.

In vitro plantlet regeneration of Ocotea catharinensis, an endangered Brazilian hardwood forest tree

A successful system of somatic embryogenesis is described for the forest tree Ocotea catharinensis Mez., which used mature zygotic embryo explants cultured on a modified Murashige and Skoog (MS) medium with activated charcoal, at 25°C in the dark. A medium composed of MS supplemented with 2% (w/v) sucrose, 0.3% (w/v) activated charcoal (AC), 362 μM 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.8% (w/v) Technical Agar Grade III was used for multiplication of embryogenic cultures. Development up to the globular-stage was achieved using Lloyd and McCown woody plant medium (WPM) with 2.0% sucrose, 0.3% AC, 181 μM 2,4-d and 0.8% Technical Agar Grade III. Significant effects of media pH on differentiation of early pro-embryogenic Ocotea cell aggregates were found. Low pH of media (ca. 3–4) appeared to prevent differentiation of proembryogenic cell aggregates whereas higher pH levels (ca. 5–5.5) favoured the formation of globular structures. Once globular structures formed, they developed further to form cotyledonary somatic embryos, under the same set of culture conditions. Successful conversion of these somatic embryos to plantlets was achieved after culture on a medium composed of 1/2-strength WPM (minerals only) with 2% sucrose, 0.3% AC, 0.8% Technical Agar Grade III and 90.5 μM 2,4-d, followed by transfer to a medium composed of 1/2-strength WPM (minerals only) with 2% sucrose, 0.8% Technical Agar Grade III and 0.905 μM 2,4-d and 1.4 μM gibberellic acid, in a 16-h photoperiod regime.

Effects of jasmonic acid and its methylester on in vitro microtuberisation of three food yam (Dioscorea) species.

Abstract The effects of jasmonic acid (JA) applied in the medium and its methylester (MeJA) applied either in the medium or as a vapour, on shoot growth and microtuber formation were evaluated in three important food yam species (Dioscorea alata, D. cayenensis, and D. rotundata). Single nodes with leaves, derived from in vitro-multiplied material, were used as explants. When delivered at higher concentrations (10 or 50 μm) both JA and MeJA suppressed node formation. Microtuberisation was supported in all three species by adding either JA or MeJA to the medium. Significant promotory effects were observed only when photoperiod, salt compositions and sucrose concentrations known to favour microtuberisation processes in yams were used. MeJA applied as a vapour strongly inhibited microtuber differentiation in D. alata on all media tested but in D. rotundata and D. cayenensis yams MeJA, also applied in the vapour phase, exhibited slight promotory effects on microtuberisation.

Micrografting of pistachio (Pistacia vera L. cv. Mateur)

A successful micrografting technique was developed for Pistacia vera. High levels of graft union were achieved when shoots from Stage II cultures of four-year-old P. vera. cv. Mateur were grafted onto in vitro-raised seedling rootstocks. Light and fluorescence microscopy investigations revealed that vascular continuity was established across grafts by three weeks.

Callus induction and plant regeneration from excised zygotic embryos of the seed-propagated yams Dioscorea composita Hemsl. and D. cayenensis Lam.

A tissue culture method for regeneration of plantlets from calluses of Dioscorea composita Hemsl. and Dioscorea cayenensis L. is described. Zygotic embryos were used as initial explants. Calluses were obtained on Murashige & Skoog basal medium supplemented with 18 μM 2,4-D and plantlets were regenerated on media containing 0.1 μM zeatin and 3.3 mM glutamine according to previously described protocols [3]. Inclusion of 0.3% (w/v) activated charcoal in media did not increase callusing. Regeneration of plantlets from D. cayenensis calluses occurred only at low levels of 2,4-D (2.25 μM) contained in the media tested. The results indicated that there were genotype-dependent differences between the yam species in their ability to regenerate plantlets in vitro.