Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon

The influence of light incubation during embryo germination on shoot organogenesis from cotyledons of four diploid watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai cultivars was examined. Germinating embryos in darkness significantly improved the number of explants that produced harvestable shoots during the 6 week incubation period on shoot regeneration medium under a 16-h photoperiod. The percentage of explants with shoots more than doubled for `Crimson Sweet' and was about 1.5-fold greater for `Sweet Gem' and `Yellow Doll' when embryos were germinated in darkness. The percentage of explants with shoots was not significantly improved for `Minilee' by pretreating seedlings in darkness. This study demonstrates that optimal shoot regeneration can be obtained by germinating embryos in darkness before preparing cotyledon explants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Peroxidase and catalase changes during in vitro adventitious shoot organogenesis from hypocotyls of Albizia odoratissima L.f. (Benth)

Hypocotyls of Albizia odoratissima cultured on shoot induction medium (MS medium with 7.5 μM BAP and 0.5 μM NAA) showed adventitious shoot organogenesis under light with 16 h photoperiod. Similar cultures under total darkness produced non-morphogenic calli. The changes in the specific peroxidase and catalase activity, total protein content and acidic isoperoxidase pattern were compared between the culture showing shoot organogenesis and culture producing non-morphogenic calli. It was found that in vitro shoot bud differentiation is accompanied by an increase of the specific activities of peroxidase and catalase in culture kept under light. In parallel with the above changes the total protein content reached to the maximum level and also a new isoperoxidase (P10) expressed on the 21st day in cultures kept under light. Conversely, culture producing non-morphogenic calli underwent a reverse change in specific peroxidase activity. This change in antioxidant enzyme activities corresponds to the histological observation of shoot bud differentiation in cultures kept under light.     

Enhancement of shoot regeneration by treatment with inhibitors of auxin biosynthesis and transport during callus induction in tissue culture of Arabidopsis thaliana

In two-step culture systems for efficient shoot regeneration, explants are first cultured on auxin-rich callus-inducing medium (CIM), where cells are activated to proliferate and form calli containing root-apical meristem (RAM)-type stem cells and stem cell niche, and then cultured on cytokinin-rich shoot-inducing medium (SIM), where stem cells and stem cell niche of the shoot apical meristem (SAM) are established eventually leading to shoot regeneration. In the present study, we examined the effects of inhibitors of auxin biosynthesis and polar transport in the two-step shoot regeneration culture of Arabidopsis and found that, when they were applied during CIM culture, although callus growth was repressed, shoot regeneration in the subsequent SIM culture was significantly increased. The regeneration-stimulating effect of the auxin biosynthesis inhibitor was not linked with the reduction in the endogenous indole-3-acetic acid (IAA) level. Expression of the auxin-responsive reporter indicated that auxin response was more uniform and even stronger in the explants cultured on CIM with the inhibitors than in the control explants. These results suggested that the shoot regeneration competence of calli was enhanced somehow by the perturbation of the endogenous auxin dynamics, which we discuss in terms of the transformability between RAM and SAM stem cell niches.     

Effect of culture medium and light conditions on the morphological characteristics and carbohydrate contents of Medicago strasseri calli

Using 6 culture media (12, 12D, 12G, 11, A and B) made up of MS medium (Murashige-Skoog, 1962) supplemented or not with glycerine, with different cytokinins, and/or 2,4-D, the morphological characteristics and contents in total carbohydrates, reducing sugars, sucrose and starch were studied in calli induced from explants (cotyledon, petiole, hypocotyl and leaf) obtained from Medicago strasseri seedlings. Callus formation was induced under photoperiod (16h light/8h darkness) conditions or in the absence of light. Considerable variability in the calli was observed, depending on the explants and media used. Under photoperiod conditions, medium A with KIN (1 mg/l) and 2,4-D (3 mg/l) induced many calli with the highest contents in total carbohydrates (886.1–889.3 mg/g DW), sucrose (132.1–188.2 mg/g DW) and starch (125.2–247.6 mg/g DW) and the lowest contents in reducing sugars (118.4–173.3 mg/g DW). In media 11, A and B, under conditions of darkness, calli degenerated at the start of culture. Calli developed in darkness generally had dry weights and total carbohydrate and starch contents lower than those cultured under photoperiod conditions. However, sucrose contents were greater in calli formed in darkness. At these cultures times, differentiation, in the form of organogenesis, was only seen using medium B with cotyledons, petioles and leaves as explants. It was also observed when petioles were cultured in medium A but with a less pronounced organogenic response.     

Effect of Abscisic Acid, Cold Hardening, and Photoperiod on Recovery of Cryopreserved in Vitro Shoot Tips of Silver Birch

Recovery of cryopreserved in vitro shoot tips of silver birch (Betula pendula Roth) was doubled by incorporating abscisic acid (ABA) in the culture medium during cold hardening of the mother shoots. The average recovery of shoot tips was over 40% after cold hardening for 28 days at +5 degreesC under an 8/16 light/dark photoperiod on medium containing 10(-4) M ABA. ABA was effective in combination with low temperature and short daylength only, although large genotypical differences were noted. ABA had two different effects: it enhanced cold hardening and increased callus formation during regeneration of cryopreserved shoot tips. Copyright 1998 Academic Press.     

High efficiency <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration from epicotyl explants of Ponkan Mandarin (C<Emphasis Type="Italic">itrus reticulata</Emphasis> Blanco)

Ponkan mandarin (Citrus reticulata Blanco) is one of the most important commercial cultivars of mandarin orange in China. This study reports an improved and efficient protocol for in vitro plant regeneration of Ponkan mandarin. Epicotyl segments, which were cut longitudinally into two halves, were used as explants. The shoot regeneration frequency was significantly increased by longitudinal cutting. A 100% shoot regeneration frequency and 13.2 shoots per explant were obtained when cultures were maintained in darkness for 20 d before being transferred to light conditions, with bud induction by indirect organogenesis. A 72.5% shoot regeneration frequency and 7.8 shoots per explant were obtained when explants were incubated under a 16-h light photoperiod continuously with buds differentiating directly from the cutting wound surface. The optimal medium for shoot formation was Murashige and Tucker basal medium supplemented with 2 mgL⁻¹ BA and 30 gL⁻¹ sucrose both under light conditions. The addition of the auxin NAA reduced the frequency of regeneration. A “filter-paper bridge” technique was used for rooting in this study. The basal portion of regenerated shoots was dipped into 1,000 mgL⁻¹ IBA solution for 15 min before placement on a filter-paper bridge that was maintained in 1/2 MS liquid medium supplemented with 10 gL⁻¹ sucrose. Eighty percent of the shoots rooted, and an average of 2.0 roots per shoot were achieved. Survival rate through acclimatization was 100%.     

Integrative regulation of leaf morphogenesis by gibberellic and abscisic acids in the aquatic angiosperm proserpinaca palustris L

A two-hormone system regulating leaf development in the heterophyllous amphibious angiosperm Proserpinaca palustris L. is described. Aerial shoots develop expanded, lanceolate, serrate leaves under long-day photoperiods (LD, 16 h light: 8 h dark), whereas growth under short days (SD, 10 h light: 14 h dark) induces dissected leaf formation. The photoperiodic effect on leaf development of aerial shoots involves changes in endogenous gibberellins (GAs) since plants grown under SD in the presence of GA₃ develop expanded lanceolate serrate leaves. However, when submerged, shoots develop highly dissectedaquatic leaves regardless of photoperiod or GA₃ treatment. In the present study, submerged plants exposed to 1.0 or 5.0 μM abscisic acid (ABA) developed aerial-type leaves typical of the photoperiod under which they were cultured. Both exogenous ABA (5.0 μM) and GA₃ (10 μM) treatments were required for laminar expansion to occur on submerged shoots under SD. It is suggested that (1) leaf development in Proserpinaca is regulated by both endogenous GAs and ABA, and (2) the endogenous status of these phytohormones is modulated by different environmental stimuli of photoperiod and water stress, respectively. The adaptive significance of this mechanism is discussed.     

Abscisic acid is required for somatic embryo initiation through mediating spatial auxin response in Arabidopsis

Abscisic acid (ABA) regulates many aspects of plant development, including somatic embryo (SE) initiation. However, mechanisms of ABA functions on SE initiation have remained to be investigated. In this study, we examined the endogenous ABA contents of calli in Arabidopsis during the SE inductive process. We further found that the capacity for SE initiation was strongly impaired by treatment of fluridone, a potent inhibitor of ABA biosynthesis, as well as by mutation of ABA biosynthetic gene ABA2, suggesting that ABA is required for SE initiation. Furthermore, treatment of fluridone inhibited local auxin biosynthesis and auxin polar transport in the embryonic calli, resulting in the disturbance of auxin response pattern and the decreased regeneration frequency of SEs. However, application of exogenous ABA in the medium almost recovered patterns of auxin response and SE initiation. Thus, the results suggest that ABA functions on SE initiation through mediating both auxin biosynthesis and polar transport for establishment of auxin response pattern in callus. Our study provides new information for understanding mechanisms of SE initiation.     

Plant regeneration from meristem-derived callus protoplasts of apple (Malus3domestica cv. `Fuji')

A procedure has been established for regeneration from meristem-derived callus protoplasts of scion cultivars of apple that have been difficult to regenerate from leaf protoplasts. Calli were induced from the meristem of apples, Malus×domestica cvs `Fuji' and `Jonagold' and Malus prunifolia var `ringo Asami Mo84-A', cultured on MS medium (2 mg/l 2,4-D, 1 mg/l BA, 0.8% agar) and subcultured in a liquid medium. The ability to regenerate plants from suspension calli was studied under eight different combinations with respect to IAA, ABA, and TDZ concentrations. With the materials studied here, two combinations, one with 0.1 mg/l IAA, 0.1 mg/l ABA, and 2.0 mg/l TDZ and another with 0.1 mg/l IAA, 1.0 mg/l ABA, and 2.0 mg/l TDZ, were effective for plant regeneration. Protoplasts were isolated from the above suspension cultures and then cultured in KM8P medium containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5 mM, pH 5.7). Shoot formation of protoplast-derived calli was studied in the above-mentioned regeneration media. The high concentration of Gelrite (0.5% and 0.7%) was also shown to be important for shoot formation of protoplast-derived calli. Shoot primordia were formed in the medium containing IAA (0.1 mg/l), ABA (1.0 mg/l), and TDZ (2.0 mg/l). Ultimately, five regenerants of `Fuji' protoplasts were obtained from 200 protoplast-derived calli. Received: 19 June 1998 / Revision received: 9 October 1998 / Accepted: 27 October 1998     

Interactive effect of light,temperature and TDZ on the regeneration potential of leaf discs of <Emphasis Type="Italic">Fragaria x ananassa</Emphasis> Duch

This is the first report where shoot regeneration in strawberry cultivar Chandler has been achieved simultaneously through both somatic embryogenesis and shoot bud formation. Direct somatic embryogenesis was observed in leaf discs which were cultured on medium containing MS salts + B₅ vitamins + 2% glucose + 18.16 μM thidiazuron (TDZ) and given both chilling and dark treatment for 2 wk at 4 ± 2°C followed by incubation at 25 ± 2°C under 16-h photoperiod for third wk. After 3 wk, these explants were then subcultured on medium containing MS salts + B₅ vitamins + 2% glucose and incubated under 16-h photoperiod at 25 ± 2°C for further growth and development. Direct regeneration via de novo shoot bud formation was observed in leaf disks which were given dark treatment and were cultured on medium containing MS salts + B₅ vitamins + 2% glucose supplemented with 9.08 μM TDZ. There was a synergistic effect of photoperiod, dark, and chilling treatments on somatic embryogenesis, whereas chilling treatment had an inhibitory effect on shoot organogenesis.     

Effect of calcium restriction on cardenolide accumulation in two cell lines of Digitalis thapsi grown under different light regimes

The effect of calcium-deprivation on growth and the production of cardenolides in two undifferentiated cell lines of Digitalis thapsi maintained under three different light regimes (16 h photoperiod, darkness, or continuous light) was investigated. Growth was stimulated by continuous light in both cell lines cultured in complete medium. The light regime did not affect cardenolide accumulation in the cells of the hypocotyl-derived line; by contrast, continuous light or darkness increased the production in the leaf-derived line. The elimination of calcium favoured cardenolide production independently of the origin of the suspensions and the light regime, this beneficial effect being predominantly manifested under continuous light.     

In vitro seed culture and seedling development of Calopogon tuberosus

A major obstacle to native orchid production is difficulty in seed germination. Culture media and light effects on seed germination of Calopogon tuberosus var. tuberosus, a native orchid with horticultural potential, were studied. Culture media included Knudson C, Malmgren modified terrestrial orchid, and PhytoTechnology orchid seed sowing. Effects of 8 weeks continual darkness, 8 weeks 16-h photoperiod, 2 weeks dark followed by 6 weeks 16-h photoperiod, 4 weeks dark followed by 4 weeks 16-h photoperiod, and 6 weeks dark followed by 2 weeks 16-h photoperiod were examined. Percent seed germination was highest on Knudson C after 8 weeks culture; however, seedling development was enhanced on PhytoTechnology seed sowing medium during 8 weeks culture under a 16-h photoperiod. This suggests that while KC and darkness promoted seed germination, P723 and light enhanced further seedling development. Seedlings of C. tuberosus readily acclimated to greenhouse conditions.     

Apparent endogenous circadian rhythm inent-kaurene biosynthesis

Net synthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid was assayed in cell-free enzyme extracts prepared from Alaska pea (Pisum sativum L.) seedlings throughout 44 h of a regimen consisting of a 16-h day and an 8-h night. Activities generally followed an upward trend during the dark period and a downward trend during the photoperiod. Activity was also assayed in enzyme extracts prepared at intervals during a 12-h photoperiod and a following, continuous 36-h dark period after entrainment of plants to a regimen of 12-h days and 12-h nights.Ent-kaurene synthesis activity again followed an upward trend in enzyme extracts prepared during what would have been the entrainment dark period, and a downward trend during the entrainment photoperiod. The apparent endogenous rhythm ofent-kaurene biosynthesis may have implications for the regulation of gibberellin biosynthesis.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

Role of phytochrome B in the development of cold tolerance in cucumber plants under light and in darkness

Cold tolerance of cucumber (Cucumis sativus L.) seedlings was investigated using wild-type plants and the phytochrome B-deficient mutant (lh-mutant). Plants were subjected for 6 days to intermittent short-term cooling (12°C for 2 h per day) and to continuous chilling under conditions of 16-h photoperiod (day/night = 16/8 h) and permanent illumination. “Dehardening” process was initiated by the transfer of plants to either light or dark conditions at 23°C. It was concluded that phytochrome B participates in the development of cold tolerance in cucumber plants under stress conditions, i.e., under short-term intermittent chilling at nights and during dehardening in continuous darkness.     

Vindoline formation in shoot cultures of Catharanthus roseus is synchronously activated with morphogenesis through the last biosynthetic step

Background and Aims

The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here.

Methods

Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle.

Key Results

In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins.

Conclusions

The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus.Key words: Catharanthus roseus, deacetylvindoline acetyl CoA acetyltransferase, DAT, in vitro shoot cultures, morphogenesis, vindoline     

In vitro shoot regeneration of Centranthus ruber DC from hairy roots induced by Agrobacterium rhizogenes,and determination of valepotriate content

ABSTRACT

Hairy roots were induced by infection with Agrobacterium rhizogenes strain LBA 9402 containing the plasmid 1855 from the valepotriate-producing medicinal plant Centranthus ruber. Plants were regenerated from callus derived from the hairy roots. The induction of shoot domes was obtained when hairy root calli, after a period of 3 months of dark incubation in MS (Murashige & Skoog, 1962) medium without growth regulators, were transferred for one week on the same medium under a 16-h light / 8-h dark photoperiod. Shoot regeneration increased with benzyladenine alone applied monthly at 2.5 µM concentration after the 3 months period of dark incubation. No bud formation was observed in untransformed tissues grown under the same light and hormonal conditions. Callus cultures of non-transformed plants was obtained by using leaves and roots as source material in the presence of 10.7 µM NAA and 1 µM kinetin as growth regulators, and 9 µM BA and 2.6 µM NAA for the next phase of shoot regeneration. BA and NAA together were not used for plant regeneration from transformed tissue. The morphological characteristics of the transgenic plants were analysed during two years of ex vitro growth. In the greenhouse, the transgenic plants showed pale pink flowers, heterostyly, leaves smaller than those of wild type plants, and a larger amount of roots. Roots of transgenic plants continued to produce valepotriates.     

Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of <Emphasis Type="Italic">Oncidium flexuosum</Emphasis> Sims (Orchidaceae)

The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 μM TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulator-free medium under a 16-h photoperiod.     

Regulation of dormancy in barley by blue light and after-ripening: effects on abscisic acid and gibberellin metabolism

White light strongly promotes dormancy in freshly harvested cereal grains, whereas dark and after-ripening have the opposite effect. We have analyzed the interaction of light and after-ripening on abscisic acid (ABA) and gibberellin (GA) metabolism genes and dormancy in barley (Hordeum vulgare 'Betzes'). Analysis of gene expression in imbibed barley grains shows that different ABA metabolism genes are targeted by white light and after-ripening. Of the genes examined, white light promotes the expression of an ABA biosynthetic gene, HvNCED1, in embryos. Consistent with this result, enzyme-linked immunosorbent assays show that dormant grains imbibed under white light have higher embryo ABA content than grains imbibed in the dark. After-ripening has no effect on expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene (HvABA8'OH1), a GA biosynthetic gene (HvGA3ox2), and a GA catabolic gene (HvGA2ox3) following imbibition. Blue light mimics the effects of white light on germination, ABA levels, and expression of GA and ABA metabolism genes. Red and far-red light have no effect on germination, ABA levels, or HvNCED1. RNA interference experiments in transgenic barley plants support a role of HvABA8'OH1 in dormancy release. Reduced HvABA8'OH1 expression in transgenic HvABA8'OH1 RNAi grains results in higher levels of ABA and increased dormancy compared to nontransgenic grains.     

Growth and shoot formation in protoplast-derived calli of Brassica oleracea ssp. acephala and ssp. capitata

The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.