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.     

A comparison of shoot regeneration from protoplasts and leaf discs of different genotypes of the cultivated tomato

Summary Shoot regeneration from leaf discs and leaf mesophyll protoplasts of 11 genotypes of Lycopersicon esculentum (the cultivated tomato), were compared. In both regeneration procedures genotypic differences were observed between inbred lines, and also between F1 hybrids and their parental lines. In the tested hybrid genotypes no heterosis effect with respect to shoot regeneration capacity was observed. A correlation between shoot regeneration from leaf discs and from leaf mesophyll protoplasts was apparent in the tested genotypes. This suggests that using the described procedure, shoot regeneration from leaf discs can be usef for rapid pre-screening for regeneration capacity from protoplasts of tomato genotypes.     

Thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.)

Summary Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N ⁶-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.     

Developmental studies on the leaf and the extra-axillary bud ofHistiopteris incisa

Anatomical and developmental studies have been made ofHistiopteris incisa in order to obtain a reasonable interpretation of the so-called extra-axillary bud. Single, or rarely two extra-axillary buds arise on the lateral side of the petiolar base. The branch trace appears to depart from the basiscopic margin of the leaf trace. At the earliest stage of the leaf initiation, the leaf apical cell is cut off in one of the prismatic cells of the shoot apical meristem. The leaf apical cell, then, cuts off segments successively to form a well-defined group of derivatives. On the other hand, a well-recognized cell group called “outer neighboring cell group”,onc, is found adjacent to the abaxial boundary of the derivatives of the leaf apical cell. This group of cells does not originate directly in the mother cell of the leaf apical cell. The primordium of the extra-axillary bud is always initiated in the superficial pillar-shaped cell layer ofonc. The leaf primordium may consist of two parts, the distal part derived from the leaf apical cell and the basal part from the adjacent cells includingonc. These facts suggest that the extra-axillary bud is of foliar nature. This study was partly supported by a Grant-in-Aid for Encouragement of Young Scientists by the Ministry of Education of Japan; no. 374222 in 1978.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

TDZ和KNO3对芋试管球茎诱导的影响

与浓度为5.0 mg·L-1的6-BA相比,浓度为0.2 mg·L-1的TDZ (thidiazuron)可以显著提高4个品种芋的单瓶试管球茎的个数.浓度为50 mmol·L-1的KNO3对芋试管球茎数量影响不大,但对鲜重有较大影响,单瓶试管球茎的鲜重提高幅度为12.9%.     

Effect of PEG-treatment on the leaf growth response and auxin content in shoots of wheat seedlings

Addition of Polyethylene glycol(PEG) to the nutrient medium of wheatseedlings led to a rapid cessation of leaf growth and shrinkage, measured withahighly sensitive growth sensor, followed by a partial restoration of extensionin the subsequent 40 min. This osmotic shock resulted in IAAaccumulation in the shoots. Leaf extensibility, measured by addition of 5g as a counterweight to an extensiometer, did not change for 20min after the start of the treatment, but then increased andbecametwice as high as the initial value. Changes in leaf extensibility coincidedwitha restoration of leaf growth. Leaf shrinkage, which was observed immediatelyafter addition of the osmoticum, suggests that the initial growth response wasdue to a water deficit, which led to a decrease in cell turgor. Howeverrestoration of growth during water stress could be due to changes in cell wallrheological properties, which could be triggered by IAA accumulation.     

TDZ对喜树愈伤组织生长及色素积累的影响

目的:研究苯基噻二唑基脲(Thidiazuron,TDZ)对喜树愈伤组织生长及叶绿素、花青素积累的影响。方法:采用TDZ单独应用及合并植物激素对喜树愈伤组织进行了继代培养。结果:单独应用TDZ时1mg/L为组织生长最适浓度。1mg/L TDZ+0.5mg/L NAA可显著促进愈伤组织的生长及叶绿素的积累,1mg/L TDZ+0.5mg/L 2,4-D培养条件下花青素含量高于单独应用TDZ培养。结论:TDZ在喜树愈伤组织培养中可替代植物激素并对细胞植物色素积累有明显影响。     

Interspecific and intraspecific differences in shoot and leaf lifespan of four Carex species which differ in maximum dry matter production

The effect of N supply on shoot and leaf lifespan was investigated in established stands of four herbaceous Carex species which differed in maximum dry matter production. These species were, in rank order of increasing maximum dry matter production (per unit ground area): Carex diandraC. rostrata. The observed patterns of shoot and leaf turnover were compared with data on leaf characteristics and nitrogen use efficiency indices of these species. There was no consistent difference in shoot production (number of shoots produced per unit ground area) between species with low production and those with high production: Carex diandra (low production) and C. lasiocarpa (high production) had high shoot production, while shoot production in c. rostrata (low production) and C. acutiformis (high production) was much lower. The rank order of the mean lifespan of shoots was: C. diandra. Thus, the lifespan of shoots increased with increasing maximum dry matter production of these Carex species. In all species, increased N supply led to a significant reduction in shoot lifespan. The reduction of shoot lifespans in response to enhanced N supply will result in increased nutrient turnover rates in these species. There was no consistent difference in the number of leaves produced per shoot between low-production and high-production species. C. diandra and C. lasiocarpa had relatively low leaf production, while C. rostrata and C. acutiformis had relatively high leaf production per shoot. Thus, this pattern is opposite to the pattern in shoot production. The rank order of the mean lifespan of leaves was: C. diandra. This implies that the high-production species had longer mean leaf lifespans than the low-production species. Mean leaf lifespan was not significantly affected by enhanced N supply, except in C. diandra, where leaf lifespan decreased in response to enhanced N supply. Shoot lifespans did not show any significant relation with the specific leaf area (SLA, leaf area per unit leaf mass) or the leaf area ratio (LAR, leaf area per unit plant mass) of the species under study. There was, however, a negative relation (r ²=0.71) with the nitrogen concentration in the leaves. Shoot lifespans were positively related (r ²=0.79) with whole-plant nitrogen use efficiency (NUE, dry matter production per unit N-loss) and with the mean residence time of nitrogen (MRT, the average time-span during which a unit of nitrogen is present in the plant) (r ²=0.78), but not with the nitrogen productivity (A, annual dry matter production per unit N in the plant). Leaf lifespan was positively related with the mean residence time of nitrogen in the plants (r ²–0.70). For all the other parameters, there were no significant relations with leaf lifespan. From these results we conclude that: (1) at the stand level, shoot and leaf lifespans are positively related with maximum dry matter production; and (2) shoot and leaf lifespan are important determinants of whole-plant nitrogen economy.     

Pattern formation of leaf veins by the positive feedback regulation between auxin flow and auxin efflux carrier

The generation of vascular pattern formation in plants is an interesting process of pattern formation in organisms. It is well known that the plant hormone auxin is involved in plant vascular differentiation and that the PIN1 protein, an auxin efflux carrier, localizes to one side of the cell membrane. Several hypotheses have been proposed to explain the formation of leaf venation. One is the canalization hypothesis that is based on the assumption that a positive feedback regulation exists between the flow of a signal molecule and the capacity of its flow. Here, we attempted to integrate the canalization hypothesis and experimental data. We investigated models of the positive feedback regulation between the auxin flow and PIN1 localization. Model 1, with conserved PIN1 amount in each cell, can generate a branching pattern similar to that of plant leaf venation. We introduced the diffusible enhancer "e" into the model as unknown factor. The obtained patterns show a quasi-periodic distribution of auxin flow paths, when the model dynamics includes domain growth. In order to understand the early initiation process that generates an inhomogeneity from an almost homogeneous distribution, we introduced model 2, a simplified version of model 1. Model 2 can generate inhomogeneity with a parameter dependency similar to that of model 1. To analyse parameter condition required for pattern development, approximated equations are obtained from model 2. The isocline analysis of the equations without spatial structure shows that the inhomogeneous distribution occurs from an almost homogeneous distribution. This parameter condition for generating inhomogeneity is consistent with the results of models 1 and 2.     

Agrobacterium-mediated transformation of `Alpine' Fragaria vesca, and transmission of transgenes to R1 progeny

Agrobacterium-mediated transformation was used to stably introduce β-glucuronidase (gus) and neomycin phosphotransferase (nptII) marker genes into `Alpine' Fragaria vesca FRA 197, a diploid (2n = 2x = 14) strawberry. R0 generation transformants derived from a single clump of kanamycin-resistant callus were vegetatively propagated. The presence of the gus and nptII genes in five clonal R0 runner plants was confirmed by PCR. Southern analysis suggested two sites of nptII insertion. When R1 generation seedlings obtained via self-fertilization of R0 plants were tested by histochemical assay, 591 were GUS positive and 39 were GUS negative. The R1 segregation data fit a 15 : 1 ratio (0.5 > P > 0.25), consistent with the independent segregation of two transgene insertion loci. These results demonstrate the suitability of `Alpine' F. vesca for transgene research in strawberry. Received: 20 March 1997 / Revision received: 29 July 1997 / Accepted: 3 September 1997     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Plantlet regeneration from leaf derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andr.

Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP). Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening, with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be produced within eight subcultures from callus obtained from leaf explant through the methods described here.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

In vitro organogenesis and plant formation in Acacia sinuata

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Shoot apex,leaf development and unifacial tip inAgave wightii drumm. et prain (agavaceae)

The shoot apex has one tunica layer enclosing a mass of corpus which is differentiated cytohistologically into central mother cell zone, flank zone, rib zone and a ‘cambium-like’ zone. Occurrence of ‘cambium-like’ zone during minimal phase is considered as an expression of nodal region. Agave wightii shows spirodistichous arrangement of leaves which have an expanded photosynthetic surface with a reduced unifacial tip. Leaves are initiated by periclinal divisions in the second layer. Vertical growth in the leaves is by subapical initials and lateral growth is by marginal and submarginal initials in their early stages of development. The unifacial tip is formed by the extension of adaxial meristematic activity. The derivatives thus formed are pushed to the abaxial side of the primordiuj. Hence the unifacial part of the leaf is regarded as equivalent to a phyllode.     

Organogenesis in the vine cactus Selenicereus megalanthus using thidiazuron

Segments of distal and proximal parts of the cotyledons, hypocotyls and epicotyls of yellow pitaya [Selenicereus megalanthus (Cactaceae)], were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). The proximal part of the cotyledons was found to be the most responsive tissue, maximal bud regeneration being achieved in the presence of 200 M thidiazuron. However, TDZ does not change the pattern of regeneration competence, which remains restricted to the proximal part of the cotyledons. The best medium for shoot elongation was found to be MS lacking plant growth regulators. Rooting was induced on MS medium supplemented with 5.3 M naphthaleneacetic acid. Rooted plantlets were transferred to a soil medium and acclimatized successfully in a greenhouse.     

Effects of growth temperature and winter duration on leaf phenology of Erythronium japonicum,a forest spring geophyte

The effects of growth temperature and winter duration on the leaf phenology of Erythronium japonicum were examined in two experiments. Bulbs wintered in the field were cultivated at 10 and 20° C and the bulbs were cultivated at 15° C after chilling treatment at 3° C for 60 and 120 days and without chilling in winter. The plants cultivated at 20° C showed significantly earlier leaf emergence, a more rapid rate of leaf extension and shorter leaf longevity than those cultivated at 10° C. The decrease in the leaf longevity at 20° C resulted from the decreases in the durations of all of the developmental, mature, and senescent phases. The bulbs without chilling treatment did not sprout leaves and those with chilling treatment sprouted leaves. The increase in the length of chilling treatment from 60 to 120 days affected leaf phenology in same manner as the increase in the growth temperature from 10 to 20° C.