The apparent loss of tissue culture competence during leaf differentiation in yams (Dioscorea bulbifera L.)

Explants taken from the leaves of yams (Dioscorea bulbifera L.) at different stages of development were cultured in vitro on a checkerboard using various combinations and/or concentrations of auxin (2,4-d) and cytokinin (6-BAP). An addition of cytokinin to the culture media was not essential for callus induction from explants derived from young leaves in the very early stages of expansion. When the leaves expanded further they required cytokinin and the requirement increased considerably during expansion. Explants taken from fully expanded leaves were no longer able to proliferate, even when extremely high concentrations of cytokinins were applied. Callus grown from highly immature leaves was able to continue proliferating in the absence of cytokinin when subcultured. Callus, that initially required cytokinin in the medium, proliferated in the absence of exogenous cytokinin when subcultured.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine - 1-NAA 1-naphthalenacetic acid     

Physical map of chloroplast DNA of aerial yam,Dioscorea bulbifera L.

Summary A physical map of chloroplast DNA (ctDNA) of aerial yam, Dioscorea bulbifera L. was constructed using three restriction endonucleases, PstI, SalI, and SmaI. In addition, a clone bank of the BamHI-digested fragments were generated, and the locations of most BamHI fragments on the map were also determined. The ctDNA of D. bulbifera was found to be a circular molecule with a total size of ca. 152 kb involving two inverted repeats of ca. 25.5 kb, and small and large single copy regions of ca. 18.5 and 83.4 kb, respectively. The genes for the large subunit of the ribulose 1,5-bisphosphate carboxylase (rbcL) and the ATP-synthase subunits and (atpB/atpE) were mapped.Contribution from the Plant Germ-plasm Institute and the Laboratory of Genetics (No. 504), Faculty of Agriculture, Kyoto University, Japan. The work was supported in part by a Grant-in-Aid (No. 60400005) from the Ministry of Education, Science and Culture, Japan     

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.     

Intraspecific variation of chloroplast DNA in Dioscorea bulbifera L.

Summary Restriction fragment length polymorphism (RFLP) analysis of chloroplast (ct) DNAs from 15 accessions of Dioscorea bulbifera collected from Africa and Asia was carried out using the Southern hybridization technique. Eight cloned ctDNA fragments of D. bulbifera and D. opposita, which cover 80% of the total chloroplast genome, were used as the probes to detect variation in ctDNA digested with nine restriction endonucleases. Ten variable sites, located in the large and small single-copy regions, were disclosed among the 15 accessions, of which six showed base substitution and four carried length mutation. Positions of the latter mutations were determined on the physical map of ctDNA. Based on these results, chloroplast genomes of the 15 accessions could be classified into nine types. Their phylogenetic relationships are assumed to be as follows: (1) African and Asian chloroplast genomes diverged from each other at the earliest point in time; (2) E-type chloroplast genome, occurring in the south-east edge of the Asian continent, appears to be the most ancient among all the Asian chloroplast genomes; and (3) four chloroplast genomes, found in Asian insular regions, are probably derived independently from the E-type genome. The discrepancy between the taxonomic relationship and the proposed chloroplast genome phylogeny of the present materials is noted.     

In vitro propagation of Typhonium flagelliforme (Lodd) blume

Summary An in vitro culture system was developed for Typhonium flagelliforme using buds from the rhizomes. The mineral salts of four media were tested. These were Murashige and Skoog (MS), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) of which MS medium was found to be the best medium for in vitro culture of T. flagelliforme. The addition of as low as 0.1 mg l⁻¹ (0.54 μM) α-naphthalene acetic acid (NAA) with the presence or absence of N⁶-benzyladenine (BA) in the MS medium caused abnormal shoot formation. The best medium for maximizing shoot number combined with normal complete plantlets from each bud was MS medium supplemented with 0.3 mg l⁻¹ (1.33 μM) BA and 0.5 mg l⁻¹ (2.46 μM) indole-3-butyric acid (IBA). The best acclimatization process was to transfer the normal plantlets, with all the leaves removed, into sand plus coconut husks substrate (1∶1) and placed in intermittent water mists house or shaded plant house with 50% light exclusion. Ninety two percent of the plantlets survived using this acclimatization method.     

Rapid propagation of agave by in vitro tissue culture

A procedure for rapid propagation of Agave (A. cantala Roxb., A. fourcroydes Lem. and A. sisalana Perrine, (Agavaceae) have been developed. The explants were excised from stolon plantlets, sterilized and cultivated on Murashige and Skoog (MS) basal medium containing 2% sucrose, 10% coconut water and 0.8% agar. The addition of following combination of growth substances—0.075 mgl^-1 naphthalenacetic acid (NAA)+0.1 mgl^-1 indolylbutyric acid (IBA)+0.5 mgl^-1 kinetin (KIN) caused an extensive proliferation of multiple shoot primordia. Subcultures of these on the same medium were successful for the multiplication with an index of 3–4 times per 4 weeks subculture period. Shoots were rooted on hormone free MS medium and then transferred into a sand bed for acclimation before field planting.     

In vitro propagation of green-foliaged Dracaena fragrans Ker.

Callus tissue was induced in young stem segments cultured on MS based media supplemented with 0.25–0.5 mg l^-1 2, 4-D. Shoots were differentiated on media containing 0.5–1.0 mg l^-1 BA and 0.5–2.0 mg l^-1 IBA or 0.1–0.2 mg l^-1 NAA. The same media were suitable for shoot multiplication. Shoot elongation and rooting were strongly inhibited by BA and stimulated by auxins IBA and NAA. Medium containing 0.5 mg l^-1 IBA was optimal for rooting. Root elongation was stimulated by light and inhibited in darkness. Transfer of rooted plantlets to outdoor conditions was feasible and special hardening procedures were not required. Among more than 5000 plants produced by this procedure only 9 off-type plants with variegated leaves were found.     

In vitro induction of multiple shoots and plant regeneration inVigna

Summary Cotyledonary nodes, excised cotyledons, and hypocotyl segments of six varieties ofVigna mungo andV. radiata have been tested for their morphogenic potential on media containing a range of hormonal combinations including benzyladenine, kinetin, thidiazuron (TDZ), and zeatin. Multiple shoots developed on cotyledonary node explants in all varieties tested on basal medium containing cytokinin. Presence of both the cotyledons, either full or half, resulted in a maximum number of shoots produced. Shoot bud regeneration was achieved via meristem formation on excised cotyledons on Murashige-skoog basal medium with B₅ vitamins supplemented with TDZ. Mature plants had normal phenotypes.V. mungo var. PS1 andV. radiata var. Pusa 105 were found to be the most responsive varieties for shoot regneration. The histology ofin vitro organogenesis was studied.     

Clonal propagation of papaya in vitro

A procedure for the rapid tissue culture propagation of papaya is being developed. Tissue culture methods using apices of nursery and orchard trees of Carica papaya cv. Sunrise Solo were evaluated. The explants were established in a modified Murashige and Tucker (1969) basal medium with half-strength inorganic salts, 0.5mgl^-1 6-benzylaminopurine (BA) and 0.2mgl^-1 naphthaleneacetic acid (NAA). Established explants were transferred to a proliferation medium consisting of Murashige and Tucker (1969) basal medium, 0.5mgl^-1 BA and 0.1mgl^-1 NAA, which caused extensive multiplication of shoots. Rooting was induced at a higher frequency by subculturing plantlets onto media with indole-3-butyric acid (IBA) than with NAA.     

Clonal propagation of Acacia catechu Willd. by shoot tip culture

A method is described for in vitromicropropagation through shoot apices of Acaciacatechu Willd., a semi-arid tree valued for Katha (atanin-like substance obtained from red heart wood of10–20 year old trees) and timber. Explants wereexcised from 15-days-old in vitro grownseedlings raised from superior seed stocks. Shoot budinduction from shoot apex explants was observed onMurashige and Skoog's (MS) [12] medium containingvarious growth regulators. A maximum of 12 shoots wasobtained on MS medium supplemented with 1.5 mg/l 6-benzylaminopurine (BAP) and 1.5 mg/l kinetin.Well-developed shoots (3–4 cm long) were rooted on strength MS medium with 3.0 mg/l indole-3-acetic acid (IAA) and sucrose 1.5%. In vitro regenerated plantlets of A. catechu were transferred to field conditions.     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

In vitro propagation of Hypoxis rooperi

The propagation of Hypoxis rooperi S. Moore in vitro, was most efficiently achieved by initially culturing corm explants on a basal medium (BM) supplemented with 1.0 mg l^-1 6-benzylaminopurine (BA). Explants which developed shoots only on this medium, could be rooted when transferred onto a hormone-free BM. Utilizing this technique, an average of 39 plants could be obtained from a mature corm, 90% of which could be established in a sandy soil.     

An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars

Fertile, green plants were regenerated from immature inflorescence explants from each of four Canadian wheat cultivars. The cultivars were representative of four classes of Canadian wheat. Explants from immature inflorescences of three size ranges were cultured on two types of media: MSI/MSR, which contains 1650 mg l^-1 NH₄NO₃and sucrose as a carbon source, and BII/BIR, which contains 250 mg l^-1 NH₄NO₃and maltose as a carbon source. Regeneration from all cultivars was significantly better on BII/BIR media than on MSI/MSR media. On BII/BIR media, `AC Karma', `Plenty', and `Fielder' gave the highest number of shoots per 10 explants, where the explants were derived from immature inflorescences 5.1 to 10.0 mm in length. 'Columbus' did not regenerate on MSI/MSR medium, and regenerated poorly on BII/BIR medium. Differences were found between cultivars with regard to the number of regenerant plants produced with the best treatments: `Plenty' produced 16.1 shoots per 10 explants, `AC Karma' 12.4, `Fielder' 6.4, and `Columbus' 2.2.     

In vitro propagation of caper (Capparis spinosa L.)

Summary A twenty fold multiplication per twenty days of caper was achieved by culturing nodal shoot segments in the presence of BAP (4 μM) plus IAA (0.3 μM) and GA₃ (0.3 μM). The use of a modified MS medium facilitated this response. Plantlet regeneration was induced on single shoots taken from proliferating clusters subcultured for 20 days on a reduced BAP (2 μM) without auxin and gibberellin Higher rooting responses (70%) were obtained after a 20-day incubation period in darkness on solid half-strength MS1 medium plus IAA (30 μM), followed by a subsequent 20 day culture period on half-strength MSI basal medium. Proliferation was mainly due to axillary shoot-bud development as revealed by histological studies. The extensive meristematic activities observed indicated the enormous morphological potential of this species.     

In vitro propagation of Notocactus magnificus

Most commercially grown cacti can be easily propagated by seed and/or cuttings. A group of rare and endangered species does not fit into this category and is therefore a good candidate for in vitro propagation productions as a tool to overcome habitat and plant-destruction. The number of rare and endangered species of Cacti goes into about 100. Many show a low production and germination of seeds and plantlets are prone to damping-off, making the in vitro propagation a feasible alternative for the multiplication and conservation of their germplasm. The aim of the present investigation is to establish a protocol for the in vitro culture and plant regeneration of Notocactus magnificus, the blue cactus, a highly ornamental species, native to Brazil. The surface sterilization of the explants was achieved with immersion for 10 min in sodium hypochlorite solution for either seeds (0.25% v/v) or ribs segments (1% v/v). Callus formation was observed when explants were cultured on MS medium supplemented with sucrose at 2% (w/v), 2,4-dichlorophenoxyacetic acid 0.5 μM, benzylaminopurine 4.4 μM, thiamine HCl 0.4 mg l⁻¹ and i-inositol 100 mg l⁻¹. The regeneration of shoots was carried out on MS medium supplemented with either different concentrations of benzylaminopurine and 1-naphthaleneacetic acid, or kinetin and indole-3-acetic acid. The highest number of shoots occurred when MS medium was supplemented with benzylaminopurine 22.2 μM, sucrose 3% (w/v) and agar 0,6% (w/v). In vitro spontaneous rooting of shoots was observed after eight months under culture on MS medium. Only in vitro rooted shoots developed into normal plants under glasshouse culture conditions. This in vitro protocol should be useful for the conservation as well as mass propagation of Notocactus magnificus.     

An improved medium for in vitro grain development from detached wheat spikelets

Wheat spikelets detached from the spike at anthesis were cultured on solidified media and successfully produced mature grains. These grains resembled normal grains and contained well-developed, embryos. Lower concentrations of glutamine favored dry weight increase in developing grains. Such grains were indistinguishable from grains from greenhouse-grown plants in germination on moist blotting sheets. The technique of individual spikelet culture can be used to study physiology and development of wheat grains and kernels and to study host-pathogen interactions in wheat floret diseases such as Karnal bunt.     

An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture

A thin section culture system for rapid regeneration of the monopodial orchid hybrid Aranda Deborah has been developed. Thin sections (0.6–0.7mm thick) obtained by transverse sectioning of a single shoot tip (6–7mm), when cultured in Vacin and Went medium enriched with coconut water (20% v/v), produced an average 13.6 protocorm-like bodies (PLB) after 45 days, compared to 2.7 PLB formed by a single 6–7 mm long shoot tip under same culture condition. Addition of -naphthaleneacetic acid to Vacin and Went medium enriched with coconut water further increased PLB production by thin sections. PLB developed into plantlets on solid Vacin and Went medium containing 10% (v/v) coconut water and 0.5 g l^–1 activated charcoal. With this procedure, more than 80,000 plantlets could be produced from thin sections obtained from a single shoot tip in a year as compared to nearly 11,000 plantlets produced by the conventional shoot tip method.Abbreviations BA 6-benzyladenine - CD callus development - CW coconut water - KC Knudson C medium - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PLB protocorm-like body - TS thin section - VW Vacin and Went medium     

An efficient in vitro shoot propagation of cranberry (Vaccinium macrocarpon ait.) by axillary bud proliferation

Summary Cultures of two eranberry (Vaccinium macrocarpon Ait.) cultivars, ‘Ben Lear’ and ‘Pilgrim’, and three eranberry clones from natural stands in Newfoundland were established in a nutrient medium containing N⁶[2-isopentenyl]adenine (2iP) from nodal and/or shoot-tip explants obtained under aseptic conditions. The cultivars differed in shoot regeneration in terms of shoot number per explant with various concentrations of 2iP over two culture periods. Best total shoot production was obtained when nodal segments were cultured in the medium supplemented with 2.5–5.0 mg 2iP l⁻¹ (12.3–24.6 μM). With higher 2iP levels, shoots did not expand and had a high mortality rate. Nodal explants of the three clones cultured in the same nutrient medium supplemented with 2.5 mg 2iP l⁻¹ (12.3 μM) produced three to five healthy axillary shoots per explant. In another experiment, nodal explants were more productive than shoot tips. In all experiments with subculture, there was an increase in shoot multiplication rate for all genotypes. Shoots were rooted in vitro in the same media used for shoot proliferation, but without any growth regulators. After their transfer to potting medium, almost all of the rooted plants survived. Cranberry genotypes can be efficiently propagated and maintained through nodal culture in a nutrient medium without auxin that contains 2.5–5 mg 2iP l⁻¹ (12–25 μM).     

The in vitro propagation of amaryllis (Hippeastrum spp. Hybrids)

Summary A new, rapid technique for the propagation of amaryllis (Hippeastrum spp. hybrids) by means of tissue culture is reported. Leaf bases, scapes, peduncles, inner bulb scales and ovaries were cultured successfully in vitro and plantlets were induced readily at various concentrations of growth regulators. Some plantlets also were produced in the absence of growth regulators. The most productive tissues for propagation were inverted scapes and peduncles, cultured in a modified Murashige and Skoog salt solution with added organic constituents and 1 mg per 1 (4.5μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg per 1 (4.4μM) 6-benzylaminopurine (BAP). Plantlets induced axenically also grew roots on the generalized shoot-inducing medium so that no special rooting medium was required. Although friable callus was obtained from ovary tissue cultured on a medium containing 2 mg per 1 (11μM) naphthaleneacetic acid and 4 mg per 1 (18μM) BAP, it produced shoots after 8 weeks of further subculture on the same medium. An average of 10 rooted plantlets was obtained from each scape or peduncle explant on the shoot-propagating medium. Thus, if 45 explants are obtained from each bulb, 450 rudimentary plantlets could be obtained from each mother bulb in 8 weeks of culture. This is a substantial increase over present propagation methods. This work was supported by a grant-in-aid of research, to Bruce G. Cumming, from the National Research Council of Canada.     

An efficient method for the in vitro management of multiple garlic accessions

Summary A simple protocol for garlic in vitro management is described. It comprises a single medium for all developmental stages and genotypes tested, and the use of immature bulbs as source of axillary buds. Although genetic variability exists among the different accessions tested, for both the multiplication rate and bulblet size, acceptable values of multiplication were reached for all the accessions, and values of bulb formation approached 100% in the shoots produced.