Adventitious shoot regeneration from in vitro-cultured leaves of Rubus genotypes

Regeneration of adventitious shoots from leaves of several in vitro-cultured Rubus genotypes was affected by media components and incubation conditions. Leaves cultured at 20°C with a photosynthetic photon flux (PPF) of 40 mol m^-2 s^-1 had a higher regeneration frequency and more shoots per regenerating leaf than ones cultured at 25°C with a PPF of either 40 or 80 mol m^-2 s^-1. Thidiazuron (TDZ) was significantly more effective than benzyladenine. Medium containing 1 M TDZ had the highest percentage regeneration for leaves of Autumn Bliss, Canby, Summit and Sentry red raspberries, whereas leaves of MD-ETCE-1 blackberry hybrid responded more to 10 M TDZ. Higher regeneration frequencies were obtained with 0.5 M indolebutyric acid (IBA) than with 1 M, but no significant difference was observed between 0.5 M and no IBA in other experiments. More shoots per regenerating leaf formed on Murashige and Skoog (MS) and N6 media, than on half-strength MS, Anderson, or Woody Plant media for all genotypes tested. The youngest two expanding leaves near the shoot apex were the most regenerative and yielded the highest number of shoots per regenerating leaf.Abbreviations AND Anderson (1980) - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) - N6 Chu et al. (1975) - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron (N-phenyl-N'-1, 2, 3-thiadiazol-5-ylurea) - WPM Woody Plant Medium [Lloyd & McCown (1980)]     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Seedling responses of three Australian tree species to toxic concentrations of zinc in solution culture

A frequently desired outcome when rehabilitating Zn toxic sites in Australia is to establish a self-sustaining native ecosystem. Hence, it is important to understand the tolerance of Australian native plants to high concentrations of Zn. Very little is known about the responses of Australian native plants, and trees in particular, to toxic concentrations of Zn. Acacia holosericea, Eucalyptus camaldulensis and Melaleuca leucadendra plants were grown in dilute solution culture for 10 weeks. The seedlings (42 days old) were exposed to six Zn treatments viz., 0.5, 5, 10, 25, 50 and 100 M. The order of tolerance to toxic concentrations of Zn was E. camaldulensis> A. holosericea> M. leucadendra, the critical external concentrations being approximately 20, 12 and 1.5 M, respectively. Tissue Zn concentrations increased as solution Zn increased for all species. Root tissue concentrations were higher than shoot tissue concentrations at all solution Zn concentrations. The critical tissue Zn concentrations were approximately 85 and 110 g g^–1 DM for M. leucadendra, 115 and 155 g g^–1 DM for A. holosericea and 415 and 370 g g^–1 DM for E. camaldulensis for the youngest fully expanded leaf and total shoots, respectively. The results from this paper provide the first comprehensive combination of growth responses, critical external concentrations, critical tissue concentrations and plant toxicity symptoms for three important Australian genera, viz., Eucalyptus, Acacia and Melaleuca, for use in the rehabilitation of potentially Zn toxic sites.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Micropropagation of the rare species Stylidium coroniforme and other Stylidium species

The number of plants in the gazetted rare species Stylidium coroniforme was increased through micropropagation. A method was first developed using the common species S. brunonianum. It was found that for both species, rapid propagation could be obtained by excising shoots from sterile seedlings and inducing shoot proliferation on Murashige and Skoog medium supplemented with 1 M BAP. Rooting was achieved using 1 M IBA and over 100 plants of each species were successfully established in soil. Leaf pieces could also be used to initiate cultures. In media with 20–25 M BAP and 1–5 M IBA, leaf pieces of S. brunonianum, S. piliferum, S. caricifolium and S. crassifolium produced adventitious buds, thus providing another method of micropropagation.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Increased taxane accumulation in callus cultures of Taxus cuspidata and Taxus × media by some elicitors and precursors

In Taxus cuspidata callus, vanadyl sulfate (10 mg l^–1) induced a high (146 g g^–1 dry wt) production of 10-deacetylbaccatin III in comparison to 7 g g^–1 dry wt of the control. The content of paclitaxel in this species increased from 16 g g^–1 to 74 g g^–1 dry wt when 20 mg phenylalanine l^–1 was used. In T. media, p-aminobenzoic acid induced the highest content of 10-deacetylbaccatin III (481 g g^–1 dry wt) versus 181 g g^–1 in the control. Paclitaxel increased from 89 to 139 g g^–1 dry wt after adding chitosan (20 mg l^–1) to the cultures.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis

In order to maintain axenic seedstock cultures axenically of thecommercially important red seaweed, Porphyra yezoensis, aprocedure was developed for axenic isolation and culture of conchocelis andmonospores. For axenic isolation of the conchocelis, contaminated microalgaewere most effectively removed by filtering contaminated samples through a100-m mesh after sonication. Removal of bacteria and otheralgaewas accomplished using a mixture of 5 agents (0.02% chitosan, 100 gml^–1 GeO₂, 10 gml^–1 ampicillin, 40 gml^–1 kanamycin and 200 gml^–1 streptomycin). Axenic single colonies wereisolatedfrom a semi-solid medium prepared from 1% transfer gel. After collectingmonospores from the 40–50% density layer on a percoll-gradient, removalofbacteria and fungi from the monospores was accomplished using a mixture of 5antibiotics (3.5 g ml^–1 nystatin, 2 mgml^–1 ampicillin, 400 gml^–1 kanamycin, 50 gml^–1 neomycin and 800 gml^–1 streptomycin). Axenic single juvenile blades wereisolated from a semi-solid medium prepared from 0.5% transfer gel.     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Somatic embryogenesis in callus culture of Mussaenda erythrophylla cvs. Queen Sirikit and Rosea

Somatic embryogenesis was achieved from mid-rib and internodal calluses of Mussaenda erythrophylla L. cvs. Queen Sirikit and Rosea cultured on Murashige and Skoog basal medium containing 8.9 M BA+0.57 M IAA+10 mg l^-1 ascorbic acid. Clumps of somatic embryos were separated and grown into complete plantlets when transferred to 1/2 MS medium+37 M adenine sulphate with 2% (w/v) sucrose.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Resistance to Spodoptera exigua in Apium prostratum

Two Apium accessions were compared with the commercial cultivar Tall Utah 52–70R (A. graveolens [L.]) for resistance to Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae). Oviposition rate was not significantly different between the three genotypes. In all accessions, eggs were usually placed on the upper half of the plants. Implications of this oviposition pattern on S. exigua management in celery are discussed. The wild species A. prostratum ssp prostratum var filiform (A230) showed a significantly higher resistance to S. exigua than 52–70R. The levels of carcinogenic and mutagenic linear furanocoumarins in the commercial cultivar 52–70R (1.41 g/g in the petioles; 5.85 g/g in the leaves) and in the plant accession A. nodiflorum (5.40 g/g in the petioles; 2.99 g/g in the leaves) were far below the concentration reported to produce acute contact dermatitis (18.0 g/g). The levels of furanocoumarins in A. prostratum petioles (186.14 g/g) and leaves (326.45 g/g) were 10 and 18 times higher, respectively, than the concentration known to cause contact dermatitis. However, resistance in A. prostratum was primarily due to non-preference and the linear furanocoumarins did not induce non-preference. Therefore, the resistance shown by this plant accession does not appear to be furanocoumarin-based and may be suitable for transfer to commercial celery for use in S. exigua management.     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 M) or kinetin (4.7–185.9 M) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 M BA or 92.9 M kinetin in C. praetermissum, and 88.8 M BA or 116.2 M kinetin in C. annuum after 4 weeks of culture. Combining 1 M 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 M indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog's medium - TIBA 2, 3, 5-triiodobenzoic acid     

Ecotoxicity of lead to the zebra musselDreissena Polymorpha,Pallas

The effect of lead on the filtration rate of the zebra musselDreissena polymorpha was investigated, together with the accumulation of Pb in the soft tissues of the mussels. The NOEC-filtration was 116 g.l^–1 (0,56 mol.l^–1) and the EC₅₀-filtration was 370 g.l^–1 (1.79 mol.l^–1). The NOEC-accumulation was the concentration found in the control water (1.4g.l^–1). These experiments show that the EC₅₀-filtration for Pb is similar to that for Cd, higher than that for Cu and lower than that for Zn. The water quality criteria for lead allow 25 g Pb.l^–1 in surface water. This will not cause short-term effects. Long-term effects may, however, occur, since an accumulation of Pb as low as 16 g.l^–1 was recorded in this study.     

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.)

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m -naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.Abbreviations BA Benzyl adenine - IBA Indole-3-butyric acid - 2-ip N⁶-(-dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - NAA -Naphthaleneacetic acid