In vitro micropropagation of Primula scotica: a rare Scottish plant

A simple micropropagation method is reported for Primula scotica, a rare plant, endemic to the North of Scotland. The technique involves the clonal proliferation of seed-derived plantlets on either hormone free tissue culture medium or on medium containing benzyl amino purine and indole acetic acid. Average multiplication rates of 4–6 were obtained for plants grown on plant growth regulator supplemented medium. The micropropagation method was applied to four different clones of P. scotica and clonal differences were observed in relation to media type. Some plants from certain clones did display hyperhydricity, however, this was circumvented by using frequent sub-culturing intervals and transferring the plants to hormone-free medium. Plantlets rooted on both media types and displayed normal, true-to-type rosette morphology. The cultures did not callus and development proceeded via shoot and root production only. The in vitro-grown plants could be transferred to ex vitro conditions and a range of growth substrates were assessed for their efficacy in supporting ex vitro growth, with a view to developing longer-term strategies for the transfer and reintroduction of micropropagated P. scotica plants into natural habitats. The simple method described in this paper may offer the potential of being applied to other endangered Primula spp.     

In-vitro development of ovules obtained after pollination of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp) flowers with pollen from okra (<Emphasis Type="Italic">Abelmoschus esculentus L. Moench)</Emphasis>

The in vitro response of ovules obtained after pollination of cotton flowers with pollen from Abelmoschus esculentus was studied. For this, 492 cotton flowers from five G. hirsutum varieties, four G. barbadense varieties and 10 F1 interspecific hybrids, were pollinated with pollen from A. esculentus and 5,069 ovules were cultured in vitro. From the cultured ovules, 69 embryos were isolated and 16 of them grew into plants. However, only three of them survived after transplantation. Finally, one plant which originated from the interspecific cross (B403 × Acala Sindos) × A. esculentus reached maturity. The mature plant (Pa0) had no morphological traits from A. esculentus. On the contrary, traits from both cotton species were observed. The flowcytometric analysis of the Pa0 plant indicated that it was hypoaneuploid. Root tip chromosome counts of its offsprings revealed a progressive chromosome increase from the Pa1 to Pa4 generation. Plants with 52 chromosomes or hypoaneuploids with a lower level of chromosomes (46–51) could be isolated from the Pa4 generation. These plants exhibited morphological traits from both cotton species and they were fertile. No signs of A. esculentus morphological characteristics were observed in these plants. It was concluded that aneuploid partial interspecific cotton plants could be produced after pollination of cotton interspecific hybrids with pollen from A. esculentus and application of an in-ovule embryo rescue technique.     

A simple method for mass propagation of Spathiphyllum cannifolium using an airlift bioreactor

Summary A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid. NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 gl⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acelimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.     

Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas

Summary A RAPD marker specific to the dwarf off-type (hereafter known as dwarf) from micropropagation of Cavendish banana (Musa spp. AAA) cultivars New Guinea Cavendish and Williams was identified following an analysis of 57 normal (true-to-type) and 59 dwarf plants generated from several different micropropagation events. Sixty-six random decamer primers were used in the initial screen, of which 19 (28.8%) revealed polymorphisms between normal and dwarf plants. Primer OPJ-04 (5'-CCGAACACGG-3') was found to amplify an approx. 1.5 kb band which was consistently present in all normal but absent in all dwarf plants of both cultivars. Reliable detection of dwarf plants was achieved using this marker, providing the only available means ofin vitro detection of dwarfs. The use of this marker could facilitate early detection and elimination of dwarfs from batches of micropropagated bananas, and may be a useful tool in determining what factors in the tissue culture process lead to this off type production.Other micropropagation-induced RAPD polymorphisms were observed but were not associated with the dwarf trait.     

Analyzing somaclonal variation in micropropagated bananas (<Emphasis Type="Italic">Musa</Emphasis> spp.)

Summary In a micropropagation program, where it is of paramount importance to produce true-to-type planting material, somaclonal variation of any kind is undesirable. Variation among plants regenerated from tissue culture is termed ‘somaclonal variation’. In banana, somaclonal variants of different type have been reported with regard to plant morphology. This article discusses various factors due to which somaclonal variations may arise. Somaclonal variation may be detected by visual screening or by using molecular markers such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and by cytological studies. Although somaclonal variation is undesirable in the context of micropropagation, it can be used to advantage for genetic improvement of banana, as has been described.     

An improved micropropagation of <Emphasis Type="Italic">Eclipta alba</Emphasis> by in vitro priming with chlorocholine chloride

An efficient method of micropropagation for Eclipta alba from young nodal axils of shoot tip explants has been developed by giving special attention to ‘priming’ in vitro plantlets in view of increasing their hardening ability after transplantation ex vitro. Among 3 cytokinins—BAP, kinetin and TDZ, BAP was found most effective in inducing and proliferating adventitious shoots. The highest frequency of responding explants (100%) and maximum number of shoots (23.0) per explant were obtained after 60 days culture on MS medium containing 8.8 μM BAP. Cent percent shoots developed roots directly from shoot base when transferred to growth regulator-free MS medium. For priming E. alba microshoots, 6.3 μM of chlorocholine chloride (CCC) was found most effective. The major changes observed in 30 days old treated shoots were, production of increased number of root, elevation of chlorophyll level in leaves and increase in plant biomass. Furthermore, arrested undesirable shoot elongation made the plants sturdier and more suitable for acclimatization. The primed micropropagated E. alba plants were healthy and survived by higher frequency (100%) in soil in comparison to the non-treated plants (84% survival).     

Phenotypic and karyotypic status of Beta vulgaris plants regenerated from direct organogenesis in petiole culture

Summary A method for high frequency in vitro regeneration from petiole explants was tested on nine breeding lines of Beta vulgaris L. from the haploid, diploid and tetraploid levels. Regenerants could be obtained without a callus step, from excised petioles derived either from axillary buds sprouted in vitro or from field grown plants, by plating the explants on MS medium supplemented with TIBA (2,3,5-triiodobenzoïc acid) and BAP (6-Benzylaminopurine). The multiple shoots obtained were then rooted in vitro and transferred to soil. In some cases, these adventitious shoots were also used as a petiole explant source for further petiole culture cycles, and the phenotypic characteristics and ploidy status of the regenerants were investigated after one or three petiole culture cycles. Conventional shoot apex culture was used as an in vitro control. Phenotypic variations such as differences in morphology and changes in in vitro growth behaviour, were noticed. Chloroplast and chromosome counts indicated that the alterations in morphogenetic pathway could not be explained by the occurrence of gross cytogenetic abnormalities such as aneuploidy or myxoploidy. Our results suggest that the altered morphology is caused by the presence of the exogenous antiauxin (TIBA) during the in vitro phase. Following transfer to the greenhouse, none of these variations persisted and cytogenetic analyses revealed karyotypic stability in all the plants studied, even after three petiole culture cycles. An assessment of the in vitro petiole culture method as a true-to-type multiplication method for Beta vulgaris is made.     

Distribution of endogenous gibberellins in dwarf and giant off-types banana (Musa AAA,cv.‘Grand nain’) plants from in vitro propagation

GA₃ and GA₂₀ were quantified in leaf extracts from true-to-type and somaclonal variants (dwarf and giant) of Musa AAA cv. Grand nain by GC-MS-SIM after purification on reverse- and normal-phase HPLC and detection by ELISA with GA₃ antibodies and by a dwarf rice bioassay. GA₃ concentration in dwarf plants was 811 ng g^–1 dry weight. For normal and giant plants, the endogeneous GA₃ levels were respectively 3.6 and 4.6 times higher. The GA₂₀ concentration in the giant plant was 68 ng g^–1 of dry weight. This concentration was, respectively, 4.6 and 7.3 times higher than those of normal and dwarf plants. These results suggest that the somaclonal variations affecting banana plant height are associated with modifications in GA metabolism.Abbreviations HPLC High Performance Liquid Chromatography - GC-MS Gas Chromatography-Mass Spectrometry - SIM Selected Ion Monitoring - GA Gibberellin - BSA Bovine Serum Albumin - PB Phosphate Buffer     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Biosystematic study ofSedum L. subgenusAizoon (Crassulaceae)

Chromosome numbers of 114 individuals from twelve populations ofSedum aizoon L. var.aizoon (Crassulaceae) are reported. They include 37 different chromosome numbers ranging from 2n=71 to 124. Although the chromosome number variation has been found in all populations examined, no correlation with geographical distribution could not be found. Various kinds of meiotic irregularities, i.e., multivalents, univalents, chromosome lagging, and polysporous “tetrad” formation have been found. These irregularities lead to the formation of gametes with various chromosome numbers. All aneuploid plants set seeds and seem to reproduce sexually. The extensive aneuploidy in var.aizoon seems to be caused by the unequal chromosome segregation in meiosis and the subsequent fertilization of gametes with various chromosome numbers.     

How have the alpine dwarf plants in Yakushima been miniaturized? A comparative study of two alpine dwarf species in Yakushima, Blechnum niponicum (Blechnaceae) and Lysimachia japonica (Primulaceae)

Many plant species are miniaturized in the alpine region in Yakushima, Japan. To examine how these alpine dwarf plants are different from their related lowland ones of the same species, we analyzed two phylogenetically distinct species cytologically, genetically and morphologically: one is a fern species, Blechnum niponicum, and the other is an angiosperm species, Lysimachia japonica. The analysis shows that the alpine dwarf and the lowland plants in each of these species do not differ in chromosome number or genetic constitution. The organ-level comparison between the alpine dwarf and lowland plants of B. niponicum shows that the fertile leaf size correlates closely with the sterile one. By contrast, the flower size does not correlate with the leaf size in L. japonica. At the cell level, the leaf size of the alpine dwarf plants of B. niponicum consists of a smaller number of epidermal cells than that of the lowland plants of this species. On the other hand, the smaller leaf size of the alpine dwarf plants of L. japonica depends on both the smaller number and the smaller size of the epidermal cells. We conclude that plant dwarfism in Yakushima shows variation at both the organ and cell levels.     

The dynamics of aneuploidy in an induced tetraploid population of Lolium multiflorum x Lolium perenne

Summary Populations of induced polyploids invariably contain a substantial proportion of aneuploid individuals. A model is described which can predict the level of aneuploidy in successive generations of a closed population of Lolium tetraploids. The results demonstrate clearly that the proportion of aneuploid individuals increases sharply for two to three generations and then stabilizes at a level determined by the gametic output of the euploid plants. A change in the gametic output of aneuploid individuals has a relatively small effect on the final level of aneuploidy reached.     

Caryological behavior during the first phases of dedifferentiation and habituation inNicotiana bigelovii

Summary Hypocotylar tissues ofNicotiana bigelovii var.quadrivalvis seedlings were induced to dedifferentiate and to habituate after a short treatment with 2,4-D or kinetin.Investigation by cytophotometry and chromosome counts on the nuclear cytology of callus induction and development in minimal medium showed, in addition to diploid cells, an high incidence of aneuploid nuclei. Amitotic phenomena have frequently observed during the culture period. The sequence of nuclear events were not influenced by the hormonal composition of the medium.The origin of these aneuploid cells from nuclear fragmentation processes is hypothesized.The results are discussed in comparison with the cytological status of other habituated and tumorous tissues in plant and animal systems.     

Etude de l'aneuploïdie dans différents naissains d'Ostreidae (Bivalvia)

Study of aneuploidy in spats of Ostreidae (Bivalvia). Chromosomes of cells from gill tissue of juveniles (5–10 mm) from four different populations of Ostrea edulis and five different populations of Crassostrea gigas were examined in order to study aneuploidy and its significance. Mitotic chromosome counts were made for a sample of spats in each population. Cells with the normal diploid complement (2n=20) and with aneuploid complements (2n different from 20) were scored. The total percentage of aneuploid cells in the studied populations of the two species varies from 9% to 34%. Individual patterns of aneuploidy were examined in three populations of Crassostrea gigas. Some animals showed only normal diploid cells, others having both normal and aneuploid cells in variable proportions. The relationship between aneuploidy and growth rate was studied in experimental spats. Mitotic chromosome counts were made on individuals from two groups of animals: the first group was collected at a precise date at a control size of 8 mm, the second group reaching the same size of 8 mm only one month later. The percentage of animals showing aneuploid cells is much greater in the second group. There is a relationship between occurrence of aneuploid cells and growth rate. The karyotypes of 18 aneuploid sets of Ostrea edulis from different spats were analyzed. Chromosome loss was observed only in the submetacentric chromosome pairs. This loss of chromosomes could have an effect on the percentage of homozygotes observed in electrophoregrams. Thus, the percentage of homozygotes would be higher in juveniles showing the greatest number of aneuploid cells. An excess of homozygosity (=heterozygote deficiency) has been reported at a number of enzyme loci in over two dozen bivalve species (including Ostrea and Crassostrea). We suggest the hypothesis that this excess of homozygosity could be related to the occurrence of aneuploid cells. The relationship between aneuploidy and growth rate may prove to be a genetic factor of importance for oyster culturing.     

Micropropagation of Drosera peltata,a Tuberous Sundew,by Shoot Tip Culture

In order to establish and optimize an in vitro micropropagation method of Drosera peltata (a tuberous sundew), a carnivorous plant, the effects of medium type, MS medium concentration, pH and cytokinin type on shoot proliferation and tuber formation were investigated, using one month-old shoot tips. The shoot proliferation and tuber formation were most effective on 1/2 MS medium without cytokinins. The optimum pH of the media was pH 5.7. Tubers were planted in plastic pots filled with 1:1 peat moss and sand. The survival rate of the plantlets was almost 100%, and they exhibited normal development. With subculture every 12 weeks, hundreds of the plants were propagated from a single plant within a year.     

Regulation of karyotype stability in tobacco tissue cultures of normal and tumorous genotypes

Summary Callus cultures of Nicotiana glauca, N. langsdorffii and of their tumor-forming hybrid plants contained a high frequency of cells with irregular chromosome numbers and chromosome aberrations (hypo-, hyper-, polyploid, aneuploid cells; bridges, polytene, broken, fragmented chromosomes, megachromosomes, etc.). Meristematic cells of shoot tips regenerated from the same cultures contained only regular chromosome numbers with normal chromosome structures. Variability in chromosome numbers is a consequence of abnormal mitoses. The data suggest genome segregation in the cultures. Cytological instability appears to be independent of genome segregation composition, genotype, tumorous condition, hormonal requirement and level of ploidy. The karyotype stability of the cultures is only dependent on the degree of organization of tissues and is regulated by factors involved in the control mechanisms of organizational processes.     

Field evaluation of micropropagated bananas derived from plants containing Banana Bunchy-Top Virus

Micropropagated bananas derived from Banana Bunchy-Top Virus (BBTV) infected plants, but displaying no symptoms of the disease, were established in the field. They were grown for three years and produced a plant crop and ratoon crops. No disease symptoms were observed. There was uncertainty as to whether

In vitro Propagation of Cloudberry (Rubus chamaemorus)

The purpose of this study was to establish conditions for micropropagation of cloudberry (Rubus chamaemorus L.). Cultures were initiated from meristem cultures. When cultures were subcultured from clusters of 3–5 shoots, approximately 70 and 50 shoots were produced per cluster within 6 weeks at 8.9 μM BAP for the female cv. Fjellgull and the male cv. Apollen, respectively. Addition of 5.5 μM GA₃ reduced the number of shoots. Auxins (IBA, NAA) promoted root development in vitro, but inhibited formation of new shoots. However, as much as 85% of shoots rooted without auxin treatment when planted in a peat:sand (80:20 v/v) mixture. Some of the male plants regenerated from shoot tip cultures flowered in the greenhouse within a year after transfer to soil.     

Factors Influencing Somatic Embryogenesis Induction and Plant Regeneration with Particular Reference to Arabidopsis thaliana (L.) Heynh

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis.Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail.Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.     

In vitro propagation ofHolostemma annulare (Roxb.) K. Schum., a rare medicinal plant

Summary A rapid micropropagation system was established forHolostemma annulare (Roxb.) K. Schum., (H. ada-kodien R. Br. ex Schult; Asclepiadaceae), a rare medicinal plant. Shoot tips (0.5–0.8 cm) and terminal and basal nodes (1.0–1.5 cm) harvested from actively growing shoots of conventionally raised plants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Multiple shoot formation (3.8) was observed in 68% of basal nodes cultured on medium with optimum concentration of 4.43 μM BA and 0.54 μM NAA after 8 wk. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation (vertical/horizontal), all shoot tip explants responded with a single shoot in all the combinations of plant growth regulators tried. Effects of other cytokinins (kinetin and 2-isopentenyladenine) and auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] to enhance the regeneration potential of basal nodes were analyzed. Shoots were multiplied by subculture of basal nodes and stumps (the original explant tissue free of shoots, but with remnant axillary, meristem and two or three protruding buds) in a reduced concentration of BA (2.21 μM) and NAA (0.27 μM). Liquid medium for multiplication was found to be ineffective due to a high degree of hyperhydricity. To make the multiplication process cost effective, culture bottles with polypropylene, caps were used for multiplication. The best root induction (75%) and survival (80%) was achieved on 0.5 strength MS medium supplemented with 1.48 μM IBA. Field-established plants had uniform growth habit traits in terms of height of plants and number, length, and weight of the tuberous roots.