Isolation, purification and identification of acetylcholine in Pharbitis nil seedlings

Acetylcholine (ACh), a well known animal neurotransmitter was isolated from tissues of Pharbitis nil using five different methods. Its presence in plant extract was confirmed by infrared spectroscopy and mass spectrometry. For quantitative estimation of ACh in P. nil seedlings pyrolysis-gas chromatography was applied. The presence of ACh was found in all organs of the examined plant: seeds, shoot apex, cotyledons, leaves, shoots and roots. However, the highest level of the investigated substance was noted in the youngest growing parts. In 5-day-old etiolated seedlings they were cotyledons, whereas in 14 day-old green plants - shoot apex and young leaves.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Gibberellins are not essential for photoperiodic flower induction of Pharbitis nil

The influence on photoperiodic flowering of (2-chloroethyl)trimethylmmonium chloride (CCC), an inhibitor of gibberellin (GA) biosynthesis, was studied in the short-day plant Pharbitis nil cv. Violet. The cotyledons contained high levels of endogenous bioactive gibberellins, whereas in the plumules and first leaves the levels were low or undetectable. The first leaf responded to a single'dark treatment by inducing flowering when it was 10 mm or wider. Similar seedlings, but without cotyledons, were used as the assay plants to study the effect of CCC on photoperiodic flowering. Treatment with CCC had no effect on flowering of seedlings without cotyledons, although stem elongation was inhibited. By contrast. CCC inhibited flowering of the intact seedlings with cotyledons. Gibberellic acid applied to the shoot apex or to the first leaf promoted flowering in the CCC-treated seedlings without cotyledons. The results indicate thai gibberellins are not essential for the flower induction process in leaves, but that they promote flower initiation and/or later processes in the shoot apices.     

Flowering response of Ipomoea batatas scions grafted onto Pharbitis nil stocks

Young seedlings of Ipomoea batatas (L.) Lam. cv. Big One did not form floral buds, but were induced to flower when grafted onto Pharbitis nil Chois. cv. Violet with its cotyledons exposed to a 16 h dark period (SD). Four SD were required to induce flowering in I. batatas scions when the grafted plants were first grown under an 8 h dark period (LD) for 18 days and then exposed to SD. Transmission of the flowering stimulus across the graft union required 4 days. It was also slow in the graft combination of P. nil and P. nil , but increased greatly when the graft union was established more completely. These results suggest that the flowering stimulus of P. nil may move symplastically and its life may be between 4 and 6 days. Although the leaves of I. batatas inhibited flowering, the flowering response of P. nil grafted onto I. batatas suggested that the involvement of a transmissible flowering-inhibitor was unlikely.     

A facile procedure for efficient plantlet regeneration from self-incompatible hybrids in Antirrhinum

A rapid and efficient regeneration system has been developed for interspecific self-incompatible hybrids between Antirrhinum majus×A. hispanicum. Multiple shoots were induced from hypocotyls explants on Murashige and Skoog medium or Loblolly pine medium supplemented with a low concentration of 3-indoleacetic acid (0.05 mg l^–1) and a high concentration of 6-benzyladenine (1 mg l^–1). Rooting took place in 1/2 Murashige and Skoog medium without growth regulators. By using this procedure, it takes about 5–6 weeks from seed to plantlet. The hypocotyls also had a potential to expand and form callus, providing a source for a continuous supply of multiple shoots from the calli at periodic intervals.     

A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis

Japanese morning glory (Pharbitis nil) is a model plant characterized by a large stock of spontaneous mutants. The recessive mutant Uzukobito shows strong dwarfism with dark-green rugose leaves. The phenotype was rescued by the application of brassinolide, a bioactive brassinosteroid (BR), indicating that Uzukobito was a BR-deficient mutant. A detailed analysis of the endogenous BR levels in Uzukobito and its parental wild-type plant showed that Uzukobito had a lower level of BRs downstream of (24R)-24-methyl-5alpha-cholestan-3-one and (22S, 24R)-22-hydroxy-24-methyl-5alpha-cholestan-3-one than those in wild-type plants, while their immediate precursors (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one accumulated relatively more in Uzukobito. These results indicate that Uzukobito had a defect in the conversion of (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one to their 5alpha-reduced forms, which is catalyzed by de-etiolated2 (DET2) in Arabidopsis. The P. nil ortholog of the DET2 gene (PnDET2) was cloned and shown to have the greatest similarity to DET2 among all the putative genes in Arabidopsis. Uzukobito had one amino acid substitution from Glu62 to Val62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS-7 cells was found to be a functional steroid 5alpha-reductase (S5alphaR) converting (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5alphaR can convert an intrinsic substrate. All these results clearly demonstrate that the Uzukobito phenotype resulted from a mutation on PnDET2, and a morphological mutant has been characterized at the molecular level among a large stock of P. nil mutants.     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.     

In vitro regeneration of long spur barrenwort (Epimedium grandiflorum Morr.) from rachis explants

Summary A system for micropropagation of Epimedium grandiflorum Morr. from rachis explants was developed. Explants were cultured onto Murashige and Skoog (MS) basal salts medium supplemented with (per L) 100 mg myo-inositol, 2 mg pyridoxine-HCl, 2 mg nicotinic acid, 0.40 mg thiamine-HCl, 30 g sucrose, and 2 g Phytagel. The medium also contained 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.2, or 0.25 mg/L (0.5, 0.9, or 1.1 μM) combined with either N⁶-benzyladenine (BA) or 2-isopentenyl adenine (2ip) at 2.5, 5, or 10 mg/L (11.1, 22.2, or 44.4 μM BA or 12.3, 24.6, or 49.2 μM 2iP). Cultures were maintained at a 16-h photoperiod (40 μmol/m²/s) and 23±2° C. Callogenesis preceded shoot regeneration. Callus formation increased with higher 2,4-D concentrations. The highest percent regeneration, 83% of explants, was obtained on 10 mg BA per L (44.4 μM) combined with 0.25 mg 2,4-D per L (1.1 μM). The maximum number of shoots, 15 per explant, was obtained from explants cultured on a medium containing 0.1 mg 2,4-D per L (0.45 μM) combined with 2.5 mg BA per L (11.1 μM). Maximum shoot length, 0.4 cm, was obtained on 5 mg BA per L (22.2 μM) combined with 0.2 mg 2,4-D per L (0.9 μM). To produce whole plants, shoots were separated and rooted on hormone-free medium containing 1 g activated charcoal per L. Rachises provided an excellent source of explants for Epimedium micropropagation and proved suitable for callus production.     

Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups

In the US, soybean genotypes are classified into maturity groups (MG; total of 13) that represent areas of adaptation generally correlated with latitude bands. To determine if one regeneration procedure could regenerate representatives from diverse areas of adaptation, 18 soybean genotypes representing nine MG were compared for organogenic adventitious regeneration and plant formation from hypocotyl explants following the procedure previously tested on representatives from only three MG. Responding explants were those capable of producing shoots on the acropetal end of the explant from either the outer edge plus central region or the central region only. This enabled determination of the contribution of cotyledonary nodal tissue (outer edge) to shoot regeneration and by discounting those explants, it also enabled estimates of true adventitious regeneration. All 18 genotypes were capable of producing meristemoids/shoots solely from the central region with responses ranging from 28.5 to 64.3% after 4 weeks in culture. All genotypes were also capable of producing elongated shoots that could be successfully rooted. No morphological differences were noted among regenerants, or between them and seed-initiated plants. All regenerants produced viable seed which germinated and produced morphologically normal plants. This study confirmed the genotype- and MG-independent nature of this hypocotyl-based organogenic regeneration procedure and provided conservative estimates for responses that were truly/solely adventitious in nature.     

Plant regeneration from cocoyam callus derived from shoot tips and petioles

The effects of various growth regulators on morphogenesis from cocoyam tissues (Xanthosoma sagittifolium) were investigated. Calluses were initiated from shoot tip and petiole explants and proliferated on medium containing 1.36 μM dicamba. Callus production was significantly greater from petioles than from shoot tips. Thidiazuron (0.045 μM) enhanced callus production when dicamba (13.5 μM) was used, and was more favorable to petioles than shoot tips. Friable shoot tip callus was subcultured into liquid media containing either 1.36 μM dicamba alone, 1.35 μM 2,4-D + 0.46 μM kinetin or 1.36 μM dicamba + 0.46 μM kinetin to induce adventive regeneration. Tissues producing single or aggregated shoot buds were subcultured into media containing 0, 0.049 and 0.49 μM 2-isopentenyladenine where bud multiplication and shoot regeneration were observed. Bud aggregates were formed from callus in liquid cultures containing 1.36 μM dicamba, 1.36 μM dicamba + 0.46 μM kinetin or 1.35 μM 2,4-D + 0.46 μM kinetin. Shoot bud clumps which remained green produced shoots, daughter buds, and plantlets in stationary and agitated liquid media containing 0, 0.049 and 0.49 μM 2iP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from fragments of immature inflorescences and coleoptiles of durum wheat

Use of Hypericum perforatum L. has increased in the past few years due to the antidepressant and antiviral activities found in extracts of this plant. As a result of its potential as a pharmaceutical, a new system was developed for in vitro culture of this species. Leaf explants were inoculated onto MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 or 4.5 μM) and 6-benzyladenine (BA, 0.44 or 4.4 μM) or kinetin (0.46 or 4.6 μM). Explants were cultivated under dark or light conditions to induce callus formation. Callus initiation was observed in all media evaluated and the highest cell proliferation was obtained from explants cultivated in the presence of 4.4 μM BA and 4.5 μM 2,4-D in the dark. Shoot induction was obtained from callus induced on 4.6 μM kinetin and 0.45 μM 2,4-D 6 weeks after transferring the callus to a MS medium supplemented with 4.4 μM BA. Roots were induced from shoots on full and half-strength MS media with or without indolebutyric acid (IBA, 4.9 μM) and the highest rooting frequencies were obtained on half-strength MS medium, regardless of the presence of IBA. Regenerated plants were easily acclimated in greenhouse conditions. The procedure reported here allows the micropropagation of H. perforatum in five months of culture and the proliferation of cell masses which could be used for studies on organic compounds of pharmaceutical interest. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus induction and plant regeneration from mature embryos of sunflower

Callus development and efficient shoot and root organogenesis were obtained from five different sunflower (Helianthus annuus L.) genotypes: Trakya 80, Trakya 129, Trakya 259, Trakya 2098, and Viniimk 8931, which are commercially important for Turkey. Plant tissue culture systems were established on Murashige and Skoog (MS) media supplemented with various plant growth regulators using mature embryos of sunflower. For callus induction MS + 1 mg/l 2,4-D, for shoot regeneration MS + 1 mg/l benzyladenine and 0.5 mg/l α-naphthaleneacetic acid were used. Callus induction ratios were around 80–92% in all tested genotypes. The Trakya 259 genotype gave the best shoot regeneration response (44%). All regenerated shoots were rooted on MS medium supplemented with 1 mg/l indolyl-3-butyric acid and on MS medium without any hormones. Mature embryos could be an alternative source for indirect plant regeneration and gene transfer systems for different sunflower genotypes. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 621–624. The text was submitted by the authors in English.     

In vitro culture and plant regeneration of large flowered purslane

Culture conditions were established for callus induction from a range of Portulaca grandiflora Hook tissues. Rapidly growing calli were obtained on Murashige and Skoog medium with stem-, leaf- and sepal-derived explants. Plant regeneration via organogenesis was explant-origin dependent with hypocotyl tissues giving the highest shooting frequency. Light conditions, pH and carbon source had a pronounced effect on the percentage of explants regenerating buds and the number of buds formed. It was possible to establish stable regenerated plants in the glasshouse.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - MS Murashige and Skoog (1962) medium     

Adventitious shoot regeneration from vegetative shoot apices in pear and putative role of cytokinin accumulation in the morphogenetic process

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 M 6-benzyladenine, 1.0 M -naphthaleneacetic acid and 250 mg l^–1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.     

云南玛咖种子、块根愈伤组织的诱导培养

通过对玛咖种子和块根两种外植体愈伤组织初导培养基和增生培养基的筛选及防褐化处理试验,结果表明玛咖种子或根初导培养基最优组合分别为3/4MS+3 mg.L-16-BA+0.3 mg.L-1NAA+25 g.L-1蔗糖、MS+0.3 mg.L-16-BA+0.3mg.L-1NAA+25 g.L-1蔗糖;其种子和小穗的增生培养基最优组合分别为MS+(0.3~0.5 mg.L-1)6-BA+0.3mg.L-1NAA、MS+0.3 mg.L-16-BA+0.3 mg.L-1NAA;在特定培养条件下,对褐变有一定程度的控制。该研究为玛咖的良种选育提供技术依据。     

白刺组织培养技术的研究

试验选用唐古特白刺幼嫩茎段和叶片作为材料,研究白刺不同外植体的离体培养技术。结果表明,白刺带芽嫩茎是诱导丛生芽的良好外植体,而叶片是诱导愈伤组织的良好外植体;白刺的最适增殖、壮芽培养基是：MS BA0.5mg／L NAA1．0mg／L GA32．0mg／L;最适生根培养基是：l／2MS IBA0.5mg／L;愈伤组织诱导培养基是：MS 2,4-D0.5～1．0mg／L。     

Acylated peonidin glycosides from duskish mutant flowers of Ipomoea nil

Five acylated peonidin glycosides were isolated from the pale gray-purple flowers of a duskish mutant in the Japanese morning glory (Ipomoea nil or Pharbitis nil) as major pigments, along with a known anthocyanin, Heavenly Blue Anthocyanin (HBA). Three of these were based on peonidin 3-sophoroside and two on peonidin 3-sophoroside-5-glucoside as their deacylanthocyanins; both deacylanthocyanins were acylated with caffeic acid and/or glucosylcaffeic acids. By spectroscopic and chemical methods, the structures of the former three pigments were determined to be 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-beta-D-glucopyranoside], 3-O-[2-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-glucopyranoside], and 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside] of peonidin. The structures of the latter two pigments were also confirmed as 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside, and 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside of peonidin. The mutation affecting glycosylation and acylation in anthocyanin biosynthesis of Japanese morning glory was discussed.     

反向核不育水稻FHS组织培养高频率植株再生

以反向核不育水稻品系FHS为研究材料,对其成熟胚愈伤组织的诱导及其再生条件进行了研究。研究结果表明,通过选择合适的培养基和调节2,4-D的浓度,胚性愈伤组织的诱导率可以达到92.5%。通过调节MS培养基中KT和NAA的浓度,并加入适量的CuSO4,植株再生频率高达81.3%。由此可见,合适的激素浓度的高低与FHS植株再生频率的高低呈现出一定的相关性。