In vitro culture ofBellevalia romana (L.) Rchb. I. Plant regeneration through adventitious shoots and somatic embryos

Summary Callus induction, adventitious shoot and root formation, and somatic embryogenesis were investigated in root, cotyledon and mesocotyl cultures ofBellevalia romana (L.) Rchb. grown on a synthetic nutrient medium containing different plant hormones. The combination of naphtaleneacetic acid plus benzylaminopurine was very effective in causing callus growth and plant regeneration from mesocotyl explants. On the contrary 2,4-dichlorophenoxyacetic acid caused suppression of shoot bud development in the same type of callus. Both cotyledon and root derived calli showed a low growth rate and did not regenerate shoots but only roots. Differentiation of somatic embryos which eventually developed into plantlets was promoted by 2,4-dichlorophenoxyacetic acid in suspension cultures. The results are discussed in relation to studies on nuclear behaviour during different morphogenetic pathways.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems

Summary The chromosomal status of calli and plantlets regenerated fromPisum sativum shoot apical meristems was studied. Chromosome mosaicism (aneusomaty) occurs during callus induction and proliferation, mostly owing to nuclear fragmentation prior to mitosis in the first days of culture. Plantlets regenerated from calli are diploid or aneusomatic, but a selective advantage of diploid cells (diplontic selection) takes place with plantlet growth. The results are discussed in relation to the possibility of inducing chromosomal and/or genetic variability by using meristematic tissues as expiants.     

In vitro propagation of a Saccharum officinarum (L.) and Sclerostachya fusca (Roxb.) A. Camus hybrid

Summary Callus induction and plant differentiation were obtained in an intergeneric hybrid of Saccharum officinarum and Sclerostachya fusca. The sub clones showed morphological variation. Chromosome numerical variation was not observed but structural aberrations were noticed in some sub clones. The study indicates the use of tissue culture technique for inducing intergeneric gene transfer in Saccharum hybrids.     

In vitro culture of common mullein (Verbascum thapsus L.)

Summary Verbascum thapsus L. is a medicinal herb and has been used to treat inflammatory disease, asthma, spasmodic coughs and migraine headaches. Studies were initiated to establish an in vitro culture protocol for V. thapsus. Explants (leaf dises, petioles and roots) were cultured on Murashing and Skoog minimal organics (MSMO) medium with benzyladenine (BA) or kinetin. Best shoot proliferation was obtained from leaf dise and petiole explants at 13.32 μM BA. Leaf dises were cultured on MSMO medium with 13.32 μM BA in combination with naphthalene acetic acid (NAA) or 2,4-dichlorphenoxyacetic acid (2,4-D). More shoot development was obtained with 13.32 μM BA and 5.37 μM NAA. Shoots were transferred to rooting media containing different levels of NAA and 2,4-D. Most of the shoots formed roots on media with 5.37 μM NAA. Plants were transferred to vermiculite and subsequently to potting media and maintained in the greenhouse.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

In vitro propagation of olive (Olea europaea L.) cv. Koroneiki

Single node explants of 'Koroneiki' olive trees werecultured for one month on a modified Driver-Kuniyuki for Walnut medium, lackinggrowth regulators. The explants were subcultured once a month on a mediumsupplemented with zeatin riboside, 6-(--dimethylallylamino)purine,6-benzyladenine or thidiazuron. Zeatin riboside proved to be superior to othercytokinins in inducing shoot proliferation. The combination of olive knotextract at 25 or 50 mg l^–1 with cytokininssuppressed shoot proliferation. After two months at the proliferation stage,theexplants were cultured for one week in the dark in 1 ml liquidWoody Plant Medium supplemented with IBA, -NAA or IBA+-NAA. Theexplants were then transferred to the same solid medium lacking growthregulators, with a small layer of perlite on the surface. The combination ofthetwo auxins at 1+1 mg l^–1 resulted in almost 76%rooting. The combination of olive knot extract at 50 mgl^–1 with auxins increased the rooting percentage up toalmost 87%. Artificial infection of explants with the bacteriumPseudomonas savastanoi pv. savastanoiinhibited rhizogenesis, even in the presence of auxins. Rooted explants weresuccessfully acclimatised under a mist system, with the survival rate reachingalmost 75%.     

Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R ₀ and R ₁ transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level. Collaborator via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems     

In vitro micropropagation of the macarronesian evergreen treePersea indica (L.) K. Spreng

Summary Shoot propagation ofPersea indica (L.) K. Spreng was achieved using seedling axillary buds cultured on MS (Murashige and Skoog, 1962) medium with 1 mg/l (2.8 μM) N⁶-benzyladenine (BA). Forty percent of the obtained shoots did not elongate, but showed bud proliferation, which was maximal (three axillary buds per shoot) at the end of the seventh subculture. Sixty percent of the shoots elongated, did not show bud proliferation, and formed calluses at their base. Successful rooting (84.6%) was achieved dipping the base of each elongated shoot in 3 g/l (16.11 mM) indolebutyric acid (IBA) for 1–2 s, and transferring to half strength MS medium without growth regulators. These shoots presented an acclimatization success of 100%. Results suggest that micropropagated elongated shoots ofP. indica can be adequately used in reforestation programs.     

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.     

Callus formation from cell culture protoplasts of corn (Zea mays L.)

Summary Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the quality of the culture used for protoplast isolation.     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Induction of endophytic colonization in rice (Oryza sativa L.) tissue culture plants by Azorhizobium caulinodans

Endophytic colonization in rice was induced using rhizobia. Dehusked seeds of rice hybrid, CORH2, were used as explants for induction of calli. MS medium was modified with 2,4-D (2.5 mg l(-1)) and kinetin (0.2 mg l(-1)) for callus induction. Well-developed calli were inoculated with Azorhizobium caulinodans strains ORS 571 and AA-SK-5 by means of imbibition. All treated calli had significant increases in protein content, total nitrogen and nitrogenase activity. Imbibition of ORS 571 had significant biochemical effect on the developing calli than AA-SK-5. The crop response study from the regenerated plantlets showed a positive correlation in yield than uninoculated control. The endophytic colonization was observed in all parts of the plants analyzed. Further, colonization was also confirmed by microtome sectioning.     

In vitro clonal propagation of annatto (Bixa oreliana L.)

Summary A protocol for in vitro propagation of Bixa orellana is described. Plants were regenerated from shoot apex and nodal explants on B5 medium supplemented with 4.9 μM 2-isopentenyl adenine. The multiplication factor of shoot apex explants was higher (nine shoots per explant) than that of the nodal explants (five shoots per explant). Regardless of the position of the nodes, all the nodal explants gave similar responses. However, the size of the nodal explant was an important factor in producing multiple shoots: 0.5 cm nodal explants produced the maximum multiple shoots. Regenerated shoots from shoot apex explants rooted best on MS medium supplemented with 0.05 μM α-naphthalene acetic acid (NAA). whereas shoots regenerated from nodal explants needed 2.7 μM NAA for rooting. Eighty per cent survival of in vivo transferred plants occurred on the best potting substrate, coco peat. Since the multiplication factor was nine per explant, this protocol can be use for commercial microprogation. However, the regeneration capacity declined after 10 subcultures. Approximately, 3350 rooted plants could be generated in 10 mo. after eight subcultures, from one shoot with a shoot apex and four nodes.     

In vitro propagation of mature trees of Alnus incana (L.) Moench.

Mature trees of European grey alder (Alnus incana) were micropropagated on a modified MS medium containing 2.5 M BA, 6.2 mM (500 mg l^-1) NH₄NO₃ and 1.5% glucose. Prior to in vitro culture, mature scions were multiplied through grafting and cutting techniques. Shoot tips from cuttings were established in vitro. After six months of culture, shoots were rooted either in vitro or in vivo and plantlets were transferred to greenhouse conditions.     

Anther culture of kale (Brassica oleracea L. convar.acephala (DC.) Alef.)

The pollen development and androgenic ability of 18 kale (Brassica oleracea convar.acephala) genotypes was observed during an anther culture study. Anther culture was successful in 6 of the genotypes and the highest yield obtained was 17 embryos per 100 anthers plated. Two stages of anther development were identified as being responsive to anther culture. The first and most responsive was that corresponding to the late uninucleated stage and the second to the late binucleated stage. These stages correspond with the onset of mitotic events in the microspores. Pollen viability was studied and low viability was noted which declined to zero after 9 days of anther culture. The initial viability level however was not clearly related to androgenic ability. The significance of the production of haploid and dihaploid kale genotypes in the study and breeding of resistance to clubroot is discussed.     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

Cytogenetics of in vitro cultured somatic cells and regenerated plants of barley (Hordeum vulgare L.)

Summary Cytogenetic analysis of immature embryoderived calli and regenerated plants of barley has demonstrated high heterogeneity of callus cultures and significant differences in cytogenetic processes between different callus lines. Regenerated plants usually have a normal chromosome complement (2n=14). Tetraploid plaints occur with a frequency of 1%. No chromosome aberrations have been detected by Feulgen staining. The phenomenon of chromosome stickiness recorded from the 2nd day of culture was discovered in a majority of callus lines as well as the phenomena of chromatin hypercondensation and chromosome supercoiling. A possible contribution of cytogenetic and molecular processes to somaclonal variation is discussed.