Changes in isoperoxidases during shoot formation in tobacco callus

Summary Shoot formation in tobacco (Nicotiana tabacum L.) callus is accompanied by an increase in peroxidase activity which takes a form similar to a sigmoid curve. The “stationary” phase coincide with the period of organ formation. Characteristic changes in isoperoxidase pattern are found in the shoot-forming part of the callus. These changes are different from those in the nonshoot-forming part or in gibberellin-treated tissue, which does not form shoots.     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

Ascorbic acid enhancement of organogenesis in tobacco callus

The effect of ascorbic acid on growth and shoot formation in callus cultures of tobacco (Nicotiana tabacum L.) was investigated, using young (4–12 subcultures) and old (more than 30 subcultures) tissue. It was found that ascorbate, at levels of 4–8×10^-4M, enhanced shoot formation in both young and old callus. Treatment with ascorbate also speeded up the shoot-forming process. In addition, ascorbate completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus.     

拟南芥不定芽发生早期的数字基因表达谱分析

目前,有关不定芽发生的研究主要集中在单基因的调控方面,缺乏转录组方面的系统研究.利用RNA-seq高通量测序技术在全基因组范围内检测了不定芽发生早期的基因表达谱,共检测到2457个差异表达基因.这些基因参与了激素代谢和信号转导、愈伤组织和侧根的形成、茎顶端分生组织的发育和光合作用等过程.进一步的途径富集分析表明,不定芽发生早期苯丙氨酸代谢和苯丙胺素合成等途径相关的基因显著富集.并且苯丙氨酸可以显著抑制不定芽的发生,暗示了苯丙氨酸代谢和苯丙胺素的合成可能在不定芽发生过程起着重要的作用.     

Hormonal requirement and tissue competency for shoot organogenesis in two cultivars of Brassica napus

An investigation of the regeneration ability of explants taken from the floral stem of Brassica napus var. oleifera was performed in the winter cultivars Darmor and Bienvenu. Our purpose was to compare the regeneration ability of the two genotypes, to compare the competence of the different tissues of the stem and then to study histologically the regeneration of shoots. A strong genotypic effect was observed between the two cvs; Bienvenu had a poorer ability to produce shoots when cultured in the presence of benzyladenine: regeneration commenced later; the percentage of explants producing shoots and the number of shoots per regenerating explant were much lower. The comparison between the regeneration ability of different explants, i.e stem segments, internal stem segments, thin cell layer and peels, showed that the superficial tissues were able to regenerate roots but not shoots. Contrariwise, internal stem segments regenerated only shoots. The origin of shoots was investigated in stem segments of cv. Darmor. A kinetic histological analysis showed the basic role played by phloem and phloem-associated cells in shoot formation.     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

Changes in water potential and its components during shoot formation in tobacco callus

Addition of plant growth regulators (5 nM NAA and 5μM BAP) to a defined basal medium stimulated adventitious bud formation of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) cotyledon explants in culture. Cytoplasmic soluble proteins synthesized during early stages of adventitious bud formation were analyzed by electrophoresis of ³H- and ¹⁴C-leucine labeled proteins on SDS polyacrylamide gels. Increased synthesis of low molecular weight proteins (16,000 to 20,000 daltons) was detected after 2 days in culture and reached a maximal level at day 4. When cotyledon explants cultured on bud medium for 2 days were transferred to callus medium (which suppressed adventitious bud formation), suppression of the synthesis of low molecular weight proteins was also observed, suggesting that these proteins may be associated with early stages of adventitious bud formation.     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

Characterization of callus formation and camptothecin production by cell lines of Camptotheca acuminata

Both incubation temperature and photosynthetic radiation affected morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis) cultured in vitro. Bud culture from nodal stem segments, regeneration of shoots and buds from internode stem segments and induction of primary callus were near optimal at incubation temperatures between 21–30°C. The optimal temperature for root formation was 27°C with temperatures above and below being clearly deleterious. Incubation in the dark or under low photosynthetic photon flux density (PPFD) was beneficial for callus induction and growth and also favored the production of rooted plantlets from bud cultures. Incubation in the dark improved considerably the regeneration of shoots and buds from internode segments and the recovery of whole plants. No off-types, as determined by protein and isoenzyme analysis, were observed among plantlets recovered from bud cultures or from regeneration of shoots from internode stem segments.     

Adventitious bud formation and shoot development from in vitro leaves of Vitis x Muscadinia hybrids

High levels of regeneration were obtained from young leaves excised from axillary shoots in proliferating nodal cultures of several Vitis x Muscadinia hybrids. Best results were obtained when the explants were cultured on solidified half-strength Murashige and Skoog medium supplemented with 8.9 M 6-benzyladenine and 0.05 M 1-naphthaleneacetic acid. Though variation was observed among the hybrids, the procedure used does not seem to be genotype-specific as all the hybrids and cultivars tested could regenerate. Scanning electron microscopy observations and histological studies carried out during the development of adventitious shoot organogenesis revealed that the promeristem initiation occurred in the outer cell layers near the wounded area of the petiolar stub.Abbreviations BA 6-benzyladenine - NAA 1-naphthaleneacetic acid     

Effects of “media osmotic agents” on the growth and morphogenesis ofActinidia deliciosa cv “hayward” callus

Summary The influence of various osmotic agents (carbohydrates) on the morphogenesis and growth of callus ofActinidia deliciosa cv Hayward was studied. Sucrose supported the highest level of growth and the lowest was supported by the sugar alcohols used in the experiments (glycerol, mannitol, sorbitol). The growth and survival of callus were evaluated with different osmotic sources in media containing glycerol, mannitol, or sorbitol at a concentration of 0.2M each for an extended period of eight subcultures (360 days). Two crucial points were identified: until the third subculture (135 days) the vitality seemed to be elevated; whereas the fifth (225 days) seemed to be a “point of no return” for tissues grown in glycerol and mannitol. Pretreatment with osmotic carbohydrates was shown to increase the magnitude of the morphogenetic events of callus subsequently transferred to sucrose-containing medium. Callus grown in the presence of mannitol and sorbitol showed a similar frequency of morphogenetic response. With respect to the media containing glycerol and sucrose, these induced more intense regeneration of shoots. When glycerol was present in the medium, however, we observed a synchronization of the morphogenetic response. Our results suggest that it is possible both to stimulate and to synchronize morphogenesis utilizing osmotic conditioning subcultures.     

Plantlet formation from embryonic tissue of chestnut grown in vitro

Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6-benzyl-aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole-3-butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin-free medium for root development.     

Efficient procedures for callus induction and adventitious shoot organogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Summary Improved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM, 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocytyl-root or hypocotyl-shoot transition zone tissue in SDM 9, 10, and HB 526 grown on PGo medium supplemented with BA to induce callus, and the regeneration frequency was 25%. Adventitious shoots were also regenerated from leaf explants of SDM 3 and 9 cultured on medium containing NAA for callus induction and BA and NAA to induce shoot regeneration, and in SDM 10 and CSM 22003 cultured on medium containing BA for callus induction and to induce shoot regeneration.     

Coniferyl alcohol, sinapyl alcohol and scopoletin in tobacco callus tissue during growth of a subculture

Coniferyl and sinapyl alcohols were isolated, identified and quantitatively determined as unbound (or weakly bound) phenylpropanoids in neutral hot-water extracts of Nicotiana tabacum L. callus tissue. This is the first identification of these alcohols in cultured tobacco callus. Scopoletin was also detected in these extracts, and it was the most abundant of these three phenylpropanoids with concentrations that ranged from 50–119 μg/g dry wt. Coniferyl alcohol (17–34 μg/g dry wt.) and sinapyl alcohol (23–35 μg/g dry wt.) were present in nearly equimolar concentration ratios and at levels which were about half those determined for unbound (or weakly bound) scopoletin. The amount of scopoletin extracted increased about 10 times when 1 M HCl-50% methanol - 0.3% ascorbic acid was used as the extractant. This indicated that most scopoletin moieties were strongly bound, perhaps by acid-hydrolyzable linkage. Coniferyl alcohol and sinapyl alcohol were not found in the acid extracts, presumably because they were acid-labile. In general, the concentration of each endogenous unbound (or weakly bound) phenylpropanoid appeared to remain relatively constant throughout the growth phase of the subculture. The only exceptions to this were the relatively higher concentrations of scopoletin and coniferyl alcohol present during the initial 0–2 weeks of subculture.     

Metabolism of 14C-aspartate during shoot bud formation in cultured cotyledon explants of radiata pine

Aspartate metabolism was investigated in excised cotyledons of radiata pine ( Pinus radiata D. Don). These cotyledons were cultured under shoot-forming (plus N⁶-benzyladenine, SF), non-shoot-forming (minus N⁶-benzyladenine, NSF) and unresponsive (plus N⁶-benzyladenine, OLD) conditions, then incubated with [¹⁴C]-aspartate for 3-h pulse treatments followed by 3-h chase treatments with cold aspartate. The majority of label was recovered in the CO₂, amino acid, organic acid and pellet fractions. Uptake was greatest in all tissue types early in culture. Most (over 80%) of the [¹⁴C]-aspartate taken up by the tissues was converted to CO₂ at day 0 in SF and NSF tissues, CO₂ accounted for less than 50% of the total radioactivity in other tissues. Greater incorporation into fractions was observed in SF tissues during promeristemoid formation, while in NSF tissues the greatest incorporation was observed during a period of rapid elongation. Generally, less incorporation was observed in OLD cotyledons than in SF and NSF cotyledons. Analysis of the amino acid fraction showed that labelled aspartate was converted to other amino acids, mainly glutamate, glutamine, asparagine and 4-aminobutyric acid.     

Effects of growth regulator concentrations and explant size on shoot organogenesis from callus derived from zygotic embryos of sunflower (Helianthus annuus L.)

Effects of medium growth regulator composition and embryo size on shoot organogenesis of callus derived from globular- to torpedo-shaped zygotic embryos of five sunflower (Helianthus annuus L.) genotypes were examined. Forty growth regulator combinations composed of 0 to 5 mgl^-1 naphthaleneacetic acid (NAA) and 0 to 1 mgl^-1 6-benzylaminopurine (BA) were tested. The frequency of zygotic embryos forming shoot-regenerating callus was analysed according to categorical data modelling using a maximum-likelihood approach. Both NAA and BA must be present to induce the formation of morphogenic callus from zygotic embryos, but each growth regulator effect varied with the genotype. For four genotypes, NAA and BA effects were neither linear nor quadratic; whereas, they were linear for the fifth one. Most effective concentrations across genotypes were 0.1 mgl^-1 NAA and 0.5 mgl^-1 or 0.2 mgl^-1 BA. However, the optimal growth regulator combination depended on the genotype and an interaction between the two growth regulators. The frequency of shoot-regenerating callus also varied with the size of the embryo explant. For all five genotypes, 0.4 to 1.2 mm long heart-shaped zygotic embryos formed morphogenic callus more frequently than smaller less-developed ones.     

Effects of 3,4-dihydroxybenzoic acid on tobacco (Nicotiana tabacum L.) cultured in vitro. Growth regulation in callus and organ cultures

ABSTRACT

The influence of 3,4-dihydroxybenzoic acid (protocatechuic acid), a naturally occurring benzoic acid derivative, on tobacco (Nicotiana tabacum L.) cell and tissue cultures was examined. The response to 0.1, 10 and 1000 µM 3,4-dihydroxybenzoic acid was tested with regards to cell proliferation in leaf explants, callus growth and shoot formation. Effects on shoot and root growth in micropropagated plants were also analysed. The highest concentration of 3,4-dihydroxybenzoic acid strongly inhibited the proliferation of leaf tissues, callus growth, shoot regeneration and root growth in micropropagated plants. On the contrary, the lowest concentration (0.1 µM) showed auxin-like activity by stimulating cell dedifferentiation, callus induction and rooting of leaf tissues. The presence of auxins and cytokinins in the media contrasted 3,4-dihydroxybenzoic acid inhibition of callus growth at all tested concentrations.     

Flower formation in vitro in a quantitative short-day tobacco: interrelation between photoperiod and infructescence development

The flowering response of thin layers excised from branch internodes of Nicotiana tabacum cv. Maryland Catterton (quantitative short-day plant for induction) was studied under three photoperiodic treatments. The explants were excised from inflorescences bearing flowers only, flowers and green fruits, or from infructescences with green fruits only. The aim of the study was to investigate the post-inductive photoperiodic effects on in vitro flower bud formation in a quantitative short-day tobacco and the relation with infructescence development. Short days quantitatively enhanced the flower bud regeneration capacities of explants in all stages of development, both as number of explants induced to produce flowers and as mean number of flowers per explant. There was no significant difference in flower bud formation on explants of the first two stages, which produced much more flowers than those of the third stage. Observations in planta showed that, during the 20 days separating the second stage from the first stage, there was no significant difference in the number of floral buds and flowers present on the inflorescence; however, the branch internodes lengthened, as did the floral buds and flowers. During the 10 days leading to the third stage, the number of capsules did not change significantly, but a high rate of floral abscission occurred. The present results show that in Nicotiana tabacum cv. Maryland Catterton short day quantitatively controls not only the inductive step of the flowering process, but also affects the capacity to regenerate flower buds during the late post-inductive phases. The responsiveness to the photoperiodic signal decreases only when the plant exhibits only fruits.     

甘肃黄花烟草愈伤组织在生长与衰老期间呼吸途径的动态变化

实验结果表明：烟划愈伤组织在生长和衰老期间,总呼吸速率（Ｖｔ）分别在１１ｄ和１９ｄ出现２次呼吸跃升;细胞色素途径的运行（ρ＇Ｖｃｙｔ）与Ｖｔ的变化几乎一致,表明细胞色素途径仍组织主要的电子传递途径;交替途径容量（Ｖａｌｔ）及其与Ｖｔ的比值（Ｖａｌｔ／Ｖｔ）：在１５ｄ前不断上升,而在１５－１９ｄ之间处于稳定水平后下降。交替途径运行（ρＶａｌｔ）及其对Ｖｔ的贡献（ρＶａｌｔ／Ｖｔ）却与Ｖａｌｔ变化趋势