The effect of photoperiod on flower formation in vitro in a quantitative short-day cultivar of Nicotiana tabacum

Superficial cell layers of a quantitative short-day tobacco plant ( Nicotiana tabacum L. cv. White Burley) were excised from different parts of the inflorescence (i.e. pedicels, branch internodes, rachises), and cultured in continuous darkness, continuous light or 8 h light/16 h dark daily. The flowering response in vitro of the different types of explants was investigated with respect to the effect of light on the post-evocation phases of the flowering process and explant commitment. Treatment effect was qualitatively and quantitatively influenced by explant origin. Three morphogenic features were observed: flower neoformation, caulogenesis and rhizogenesis (the latter on rachis explants only). Under all treatments, the highest flowering potential was shown by pedicels, while the highest vegetative potential was shown by rachises. Branch internodes showed an intermediate response, but with a tendency towards caulogenesis, which probably reflects their phylogenetic origin. Thus, opposite gradients of the neoformation of flowering and vegetative buds on explants were observed under all treatments. Pedicels formed new single flowers rather than inflorescences, while rachises regenerated mainly inflorescences. In darkness, flowering was limited mostly to pedicels. Vegetative bud formation was higher than floral bud regeneration in all types of explant. Continuous light enhanced the flowering response mostly in pedicel and branch internode explants. Short days enhanced flower bud formation in vitro on all types of explant. Results with respect to microsporogenesis, flower and inflorescence anomalies observed under darkness also seem to support the existence of a quantitative photoperiodic control on floral neoformation in vitro in this plant. These results suggest that in Nicotiana tabacum cv. White Burley in vivo floral induction, initiation and development are governed by the same photoperiodic requirements.     

The effects of storage condition on starch metabolism and regeneration potentials of twin-scales and inflorescence stem explants of <Emphasis Type="Italic">Narcissus tazetta</Emphasis>

Summary The natural propagation rate of Narcissus is very slow. In vitro micropropagation of Narcissus is more efficient than conventional propagation for rapidly building up aseptic stocks of varieties, especially for the establishment of new cvs. and the production of pathogen-free stock material. In the present study, Narcissus tazetta cv. ‘Ziva’ bulbs were used as the source of mother plants. The bulbs were kept at 30°C in a dark chamber until the start of the experiments. Prior to explant preparation, the bulbs were subjected to a cold treatment at 15°C in the dark for 6 wk to break dormancy. Twin-scales and inflorescence stem discs were isolated from aseptic bulb parts and were used as the initial explants. The polar orientation affected the regeneration of the inflorescence stem. Storage duration at 30°C followed by cold treatment were found to affect starch levels, adenosine diphosphate glucose pyrophosphorylase (AGPase) activities, and regeneration potentials. Starch levels were reduced significantly during a 10 mo. storage period at 30°C in both twin-scales and inflorescence stem disc explants. Regeneration was followed by an efficient acclimatization system with 98–99% survival. More than 500 highly uniform young bulbs were propagated from one mother bulb in a 12 mo. period.     

Efficient in vitro regeneration of leek (Allium ampeloprasum L.) via flower stalk segments

Summary A new simple, efficient and rapid in vitro method for mass clonal propagation of leek (Allium ampeloprasum L.) plants, using small (5 mm) flower stalk (peduncle) explants, was established. Adventitious shoots were produced from single subepidermal cells. A wide variation in the percentage of regenerating explants and number of regenerated shoots per explant between individual plants within one cultivar was observed. The concentration of the growth regulators 6-benzylaminopurine and -naphthalene-acetic acid influenced the percentage of regenerating explants and the average number of regenerated shoots per explant. A combination of 10 mg.l^–1 6-benzylaminopurine and 10 mg.l^–1 -naphthalene-acetic acid, resulted in a maximum percentage of regenerating explants and a high average number of regenerated shoots per explant. The percentage of regenerating explants and the average number of regenerated shoots per explant decreased with increasing flower stalk length (age). The basal explants gave both the highest percentage of regenerating explants and average number of regenerated shoots per explant. An average of 300 shoots per flower stalk was obtained for all plants, making this new in vitro method a powerful tool in hybrid leek breeding.     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.     

Factors influencing direct shoot regeneration from ovary explants of saffron

Direct adventitious shoot regeneration from ovary explants of Crocus sativus L. was influenced by media components, incubation conditions, and age of the explant. The best response towards caulogenesis (28%) with highest shoot numbers per ovary was observed when full strength Murashige and Skoog (1962) medium was supplemented with naphthaleneacetic acid and benzyladenine. Incubation in the dark at 20 °C was beneficial for induction of shoot buds. Ovaries of different growth stages having stigmas of pale yellow, pale orange and bright orange regenerated a maximum mean number (3.8 – 4.2) of shoots per ovary. Further development of ovary-derived shoots was influenced by the composition of basal salts in the culture medium where full strength Murashige and Skoog salts gave the best response of those tested. Regenerated shoots produced normal photosynthetic leaves and corms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polyp morphogenesis in a scyphozoan: Evidence for a head inhibitor from the presumptive foot end in vegetative buds ofCassiopeia andromeda

Summary Buds ofCassiopeia andromeda have been transected into fragments of various sizes. Depending on their original position in the organism, on their size and on the age of the dissected buds, the fragments either regenerated or developed to a solitary polyp's head without stalk and peduncle. Generally, basal fragments tended to regenerate complete buds, young apical parts mostly differenciated polyp heads whereas apical and middle parts of progressively older buds regenerated buds with increasing frequency. To explain the alteration of the developmental capacities a head inhibitor is postulated which originates from the basal end of the buds and which expands towards the apical pole with increasing age of the buds.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.     

Plant regeneration from seedling explants of green bean (Phaseolus vulgaris L) via organogenesis

Green bean (Phaseolus vulgaris L.) plants were regenerated from 3-day old seedling explants via organogenesis. The explants contained a cotyledon and a small portion (2–3 mm) of embryonic axis split in half. Explants were cultured on a defined medium containing glutamine as the sole nitrogen source. A ring of meristematic tissue was produced at the base of the axillary bud located at the cotyledonary node. The meristematic tissue was produced only if the axillary bud was present together with the cotyledon in the explant. Buds and shoots developed from the meristematic ring. Selected shoots produced roots when excised from the cluster of buds and transferred to root induction medium. Rooted shoots (plantlets) grew well and produced viable seeds when grown in the greenhouse. Histological studies revealed the origin of buds from the peripheral layers of the meristematic ring.Production of buds and shoots was a continuous process, so that new shoots could be removed from the explant for plantlet production every 10–14 days. With the cultivar Dark Red Kidney, an average of 49 buds and 8 shoots were regenerated per explant by 30 days after culture initiation. Sixty-seven percent of the shoots produced roots, and 90–95% of the plantlets survived greenhouse acclimatization to produce healthy plants.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media

 The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10^–2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed. Received: 24 January 2000 / Revision received: 6 March 2000 / Accepted: 7 March 2000     

Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation

Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.     

Source, etiolation and orientation of explants affect in vitro regeneration of Venus fly-trap (Dionaea muscipula)

In vitro culture of Venus fly-trap (Dionaea muscipula) was initiated using flower stalk explants. Activated charcoal was required for bud initiation, but omitted in the subculture of regenerated plantlets. Regenerated plants were subsequently used as explant source for investigations concerning effects of source of tissue, etiolation, orientation and illumination of leaf explants on plant regeneration. Etiolation of source plantlets increased the rate of regeneration from explants and decreased explant failure. Generally, adventitious buds developed at the adaxial side and proximal end of an explant. However, when explants were incubated in the dark, 20–30% of bud initiation occurred at the distal end. The site of shoot regeneration on a leaf explant was affected by both illumination and orientation of explants. Placing an explant adaxial side up resulted in the highest rate of regeneration. The most effective condition for plantlet regeneration was found with etiolated petioles incubated with the adaxial side facing the light. Received: 18 March 1998 / Revision received: 12 August 1998 / Accepted: 7 September 1998     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

In vitro regeneration via caulogenesis and brassin-induced shoot conversion of dormant buds from plumular explants of peanut (Arachis hypogaea L. cv `Okrun')

Shoot buds were induced from plumular explants of peanut (Arachis hypogaea L., cv `Okrun') preconditioned on medium containing 2,4-dichlorophenoxyacetic acid and kinetin and then transferred to regeneration medium containing benzylaminopurine and β-naphthoxyacetic acid. Buds differentiated 25 days following transfer to regeneration medium. Each explant produced 30 to 40 buds, but only 4 shoots. The remaining buds were dormant and did not produce shoots when maintained on regeneration medium. Shoots were regenerated continuously, however, when explants were subsequently transferred to shoot conversion medium containing 1 μM brassin, benzylaminopurine and β-naphthoxyacetic acid, respectively. Approximately 5 shoots were harvested every 30 days after transfer to shoot conversion medium for up to 7 months. No further shoot production was observed from explants maintained on regeneration medium without brassin. Regenerated shoots could be rooted and produced viable seeds. This procedure provides an efficient and reliable system for regeneration and transformation studies using cv `Okrun'. Received: 9 April 1997 / Revision received: 27 August 1997 / Accepted: 20 September 1997     

In-vitro flower formation in leaf explants of tomato: effect of NaCl

Leaf explants of 24 cultivars and 2 F1 hybrids of the common tomato (Lycopersicon esculentum Mill.) and ofL. pimpinellifolium Brezh. were cultured on Murashige-Skoog medium containing different concentrations of NaCl. The cultures of 11 genotypes formed flower buds when cultured on medium containing 0.5% NaCl. Flower formation occurred either by direct differentiation from the leaf cultures or by transition of the apices of regenerated shoots from the vegetative state to floral buds. No flower formation occurred on medium without NaCl or media with 1.0% NaCl or more. There existed great differences in the capacity of in-vitro flower formation in the tomato leaf explants among the genotypes tested. The genotypes whose explants did form flowers were all of determinate growth habit.     

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.     

Micropropagation of Sesbania sesban from the Mature Tree-Derived Explants

The nodal and internodal explants excised from the orthotropic shoots of Sesbania sesban var. bicolor elicited the development of shoots directly from the explants as well as via an intervening callus phase on Nitsch (N) medium. On benzyladenine (BA) supplemented media, the adventitious shoot buds developed involving a callus phase. An average of 8.9 ± 4.1 shoots developed per nodal explant on N medium containing 0.5 mg dm⁻³ BA in 95 % cultures, whereas 65 % cultures of internodal explants developed shoots with an average of 5.9 ± 3.6 shoots per explant on N medium supplemented with 1.0 mg dm⁻³ BA. On kinetin (Kn) supplemented medium shoots developed directly from the surface of both the explants at all the concentrations tried. Nodal explants on N medium supplemented with 1.5 mg dm⁻³ Kn developed an average of 12.5 ± 7.9 shoots per explant in 100 % cultures, while internodal explants induced an average of 11.6 ± 7.4 shoots per explant in 75 % explants at 0.5 mg dm⁻³ Kn. The in vitro regenerated shoots developed roots when implanted on N medium supplemented with 2 mg dm⁻³ indole-3-butyric acid (IBA), after 30 d of inoculation. The in vitro developed plantlets were initially acclimatized under controlled conditions for four months, prior to their transfer to the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

Field performance of micropropagated forestry species

Summary Micropropagated plants both from angiosperms (bamboo, birch, eucalyptus, tamarind, teak, willow) and gymnosperms (Douglas-fir, loblolly pine, Monterey pine, and redwood) have been established in the field. Plantlets were regenerated from juvenile explants (via adventitious or axillary buds) as well as explants from mature trees [apical and axillary buds (nodal segments)]. Plantlets regenerated from adventitious buds tend to show early maturation traits (Douglas fir, loblolly pine, Monterey pine). A population of selected clones showed superior performance and yield over seedlings derived from the same trees. Increased biomass production was obtained with plantlets derived from tissue culture ofEucalyptus spp. when compared to seedlings. No morphologic variation was observed in micropropagated plants. Plantlets derived from tissue culture grew very uniformly. Early flowering was observed with plantlets derived from tissue culture (tamarind, teak). Based on a presentation at an International Training Course on the Application of Biotechnology in Forest Trees held in Caracas, Venezuela, May 1991.     

Correlation between nuclear DNA values and differing optimal ploidy levels inNarcissus,Hyacinthus andTulipa cultivars

4C nuclear DNA amounts were determined in 16 large decorative cultivars ofNarcissus (Amaryllidaceae), 13 ofHyacinthus (Hyacinthaceae) and 12 ofTulipa (Liliaceae) at different levels of ploidy. Within each genus, nuclear DNA amounts and ploidy levels are positively correlated, with no DNA loss in polyploids.Based on wide surveys of chromosome numbers, the maximum numbers of cultivars, interpreted as the optimum levels of selective success or horticultural fitness, were found to be at the tetraploid level inNarcissus (2n=4x=28), the triploid inHyacinthus (2n=3x=24) and the diploid inTulipa (2n=2x=24). All these ploidy optima were shown to correspond to a small range of nuclear DNA amounts (4C=96-139 pg), which could suggest the existence of a single DNA value optimal for the three biologically similar but unrelated genera. In each case the optimum is at an equilibrium reached between enhanced size and other morphological characteristics on one hand and reduced growth rate on the other, both resulting from increase in ploidy and nuclear DNA amounts.     

Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments

An efficient system for in vitro plant regeneration from thin transversal stem sections explants (1–2 mm) using mature tissues of sweet orange cv. Pera was developed. Explants were cultured in different media to evaluate the frequency of regeneration and size of buds. A high percentage of explants (54% with 3.1 buds/explant) producing large buds (1–4 mm) was observed when the explants were cultivated for 2 weeks on Murashige and Skoog medium and then transferred to Woody Plant medium (WPM). Both media were supplemented with 1.8 M 6-benzylaminopurine and 0.7 M gibberellic acid. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting. Regenerated plants started to flower after 12 months in the greenhouse, confirming their mature nature.