Transglutaminase activity during greening and growth ofHelianthus tuberosus explants in vitro

Summary Explants of dormant tubers ofHelianthus tuberosus were grown in vitro, with or without 10 M 2,4-D, for 3 weeks. The 2,4-D-treated explants grew by cell enlargement and division and formed a non-photosynthetic friable callus composed of thin-walled cells. However, untreated explants, whose cells did not divide, differentiated chloroplasts and contained intercellular spaces filled with opaque material; chloroplasts were derived from non-photosynthetic plastids with tubular complexes and secondary starch grains: both disappeared when the thylakoids began to organize and form small grana. Nuclei also changed their morphology and became invaginated. Treated and untreated explants showed differences in their protein electrophoretic patterns and transglutaminase activity. This enzyme activity, low in dormant tubers, increased in both explants; considerably in untreated greening explants but much less in 2,4-D-treated growing ones. SDS-PAGE analysis of labelled conjugates, formed by in vitro incubation with labelled putrescine, indicated that, in addition to some apparently common substrates with M_r more than 36 kDa, proteins of lower mass were also labelled in the untreated greening explants. These data are discussed in the light of the possible role of transglutaminase in plants.Abbreviations DAPI 4,6-diamidino-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid - FM fluorescence microscopy - LM light microscopy - PA polyamines - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulphate - TCA trichloroacetic acid - TEM transmission electron microscopy - TGase transglutaminase - Tris-buffer tris(hydroxymethyl)aminomethane hydrochloride and tris(hydroxymethyl)aminomethane     

Early cellular events during induction of carrot explants with 2,4-D

Summary The cellular events occurring in carrot hypocotyl explants during long-term and pulse treatment with 2,4-D were followed using different techniques (light and transmission electron microscopy, flow cytometry, PCNA staining). Different morphogenetic pathways were induced under the various experimental conditions. Nevertheless, in the explants the activated cells were the same (provascular cells) and they showed very similar structural and ultrastructural changes. The long-term treatment with 2,4-D induced rapid re-activation of the cell cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - PCNA proliferating cell nuclear antigen - ER endoplasmic reticulum - GA Golgi apparatus     

Changes in carbohydrates,amino acids and proteins in developing seed of chickpea

Developing seeds of chickpea cultivars G-130, L-550 and 850-3/27 grown under field conditions were sampled at different stages of maturity and analysed for soluble sugars, starch, soluble nitrogen, protein nitrogen and amino acids. Fr. wt of seeds of all three cultivars decreased after 28 days of flowering while the dry wt continued to increase. Rapid starch accumulation was observed between 14 and 28 days after flowering. Starch as per cent of seed dry wt started to decrease after 28 days, while starch per seed increased till maturity. The percentage of salt-soluble proteins decreased with maturation of seed. The electrophoretic pattern revealed that deposition of seed storage protein in cotyledons occurred 14 days after flowering. Most of the biochemical activity apparently occurred between 14 and 28 days after flowering.     

Hormone-like effect of vascular tissue on starch accumulation in stem explants of kale, Brassica oleracea

Explants of stem pith of kale ( Brassica oleracea L. var. medullosa cv. Krasa), cultured for several days on agar medium containing sucrose, accumulate starch. Application of streptomycin, 5-fluorouracil and other inhibitors indicates that starch accumulation depends on protein synthesis on 80 S ribosomes. If explants derived from plants grown under natural long-day conditions contained vascular tissue, including cambium, in addition to pith parenchyma, the amount of starch formed in the pith tissue increased up to seven fold when compared with explants without vascular tissue. Similar increase of starch content as caused by vascular tissue was achieved by the addition of kinetin or trans -zeatin (10 μ) in the presence of 5 μ indole-3-acetic acid or 1-naphthaleneacetic acid. A further three-fold increase in starch accumulation could be achieved by application of cytokinin and auxin to explants containing vascular tissue. When explants were derived from plants grown under natural short-day conditions cytokinins and auxins had little or no effect, but vascular tissue enhanced starch formation significantly. The spreading of starch inducing stimulus from vascular tissue (probably from its meristematic region) to the pith parenchyma up to a distance of at least 20 mm was demonstrated. It was concluded that a hormone-like factor other than cytokinin and auxin was involved in the stimulatory action of vascular tissue. The effects of this factor on protein accumulation and growth in the explants and its possible production by meristematic tissues in vivo are discussed.     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

Anthocyanin production in callus cultures of Cleome rosea: Modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 μM 2,4-D. Reddish-pink regions were observed on callus surface after 6–7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH₄⁺/NO₃⁻, 70 g L⁻¹ sucrose and supplementation with 0.90 μM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.     

Cellular changes in early development of regenerating thin cell layer-explants of rapeseed analyzed by light and electron microscopy

Cellular changes in thin cell layer (TCL) explants of stem origin of Brassica napus L. cv. Vega were studied from 0 to 15 day by light and transmission electron microscopy. Apical and basal ends of the old explants were analysed separately. Quantitative and qualitative analyses showed that during the first culture day the parenchyma cells enlarged significantly as did the cytoplasm/vacuole ratio. The cytoplasm contained increased rough endoplasmic reticulum (RER), polysomes and dictyosomes associated with both coated and uncoated vesicles. The cell enlargement continued during the first 5 days of culture. The structural organization of the cell wall became somewhat loose and inhomogeneous. Parenchyma in the basal end divided frequently, resulting in several centres of division, while cell division in apical cells was less frequent and cells there remained enlarged. Starch accumulation started on the first day and increased until the third day. i. e. until cell divisions became more frequent. The starch content of dividing cells gradually decreased and starch was almost totally lacking in 15-day-old explants. Starch grains remained numerous, however, in the large non-dividing apical cells, except in those cells adjacent to the medium. Cell divisions started close to medium in explants containing vascular tissue, but closer to the epidermis in the explants without vascular tissue.
The results show how rapid (one day) striking changes in the cells take place and suggest that optimal hormone concentration and intertissue relations between epidermis and parenchyma and between parenchyma and vascular tissues as well as intercellular relations among parenchyma cells determine the first cell division sites and planes in the explants. Although the cells change from elongated to spheroid, their original polarity remains as evidenced by the formation of more numerous basal shoot primordia than in apical shoot primordia.     

Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.     

Histological and semi-quantitative approaches to in vitro cellular responses of ovule,embryo and endosperm in sugar beet,Beta vulgaris L.

Summary Fertilized ovules from sugar beet, Beta vulgaris L., of different intra- and interspecific crosses have been grown under in situ and in vitro conditions and investigated by light microscopy. Selected anatomical parameters were observed and entered in a computer program for statistical treatment. After a few days in culture the cells of the inner integument epidermis develop reticulate wall thickenings and their content of tannins decrease. Likewise, the starch content in the outer integument decreases and no real seed coat is formed. The funiculus tissue increases its metabolic activity, i.e., abundant accumulation of protein and starch. Callus or callus-like proliferations develop in the nucellus and the suspensor, but only rarely in the embryo or endosperm. However, the embryo may show an irregular morphology. Very rapid metabolism of starch in the suspensor may be related to the ability of the embryo to survive the first days in culture. Generally, the cellular responses, most significant in the maternal sporophytic tissue and the suspensor rather than in the embryo and endosperm, can be explained as structural adaptations to alternative pathways of nutrient supply.     

Characterization of immature embryos of interior spruce by SDS-PAGE and microscopy in relation to their competence for somatic embryogenesis

SDS-PAGE analysis of total proteins from cotyledonary embryo explants reveals that their competence to form somatic embryos is limited to a specific stage of development prior to the accumulation of storage proteins. When protein profiles of embryo explants of different open pollinated families from the same collection date are compared, there is a close relationship between the absence of storage proteins and their ability to produce embryogenic callus. In addition, the appearance of storage proteins in embryos from subsequent collections is associated with their loss of competence. Light microscopy combined with staining for total protein demonstrates that competent immature embryos have cotyledons but do not contain protein bodies.Abbreviations SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis - 2,4-D 2,4 dichlorophenoxyacetic acid - ABA Abscisic acid - BA Benzyladenine - EDTA Ethylenediaminetetraacetic acid     

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

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

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.Abbreviations TGase transglutaminase - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine     

Morpho-histological study of direct somatic embryogenesis in endangered species Frittilaria meleagris

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm⁻³ 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.     

2,4-二氯苯氧乙酸(2,4-D)对雨生红球藻中虾青素积累的影响

本文初步研究了一定浓度范围内的2,4-D对雨生红球藻积累虾青素的影响.在对数生长 期的藻液中分别加入一系列不同浓度的2,4-D溶液,然后进行胁迫培养(25℃、24h、5000Lx连续光照 营养盐饥饿),诱导细胞内虾青素的合成积累.在诱导过程中,显微观察不同浓度2,4-D处理后细胞形态和虾青素积累的动态变化,并定期取样测定虾青素含量.结果表明,20.0mg/L的2,4-D能够明显促进雨生红球藻中积累虾青素.它不仅可以加快虾青素积累进程(比对照提前15 d),而且比对照能提高13.4%的虾青素产量.     

铁皮石斛愈伤组织诱导研究

研究不同培养基和光照条件对铁皮石斛愈伤组织诱导的影响。结果表明,外植体直接接种于培养基上,最适宜培养条件是MS 5.92 g·L^-1＋2,4-D 5 mg·L^-1＋IAA 1.5 mg·L^-1＋KT 0.62 mg·L^-1＋蔗糖37.5 g·L^-1＋琼脂0.8% (pH 5.9～6.0),暗培养15 d后再光培养;外植体捣碎后平铺于培养基上,最适宜培养条件是MS 4.74 g·L^-1＋2,4-D 1 mg·L^-1＋IAA 1.5 mg·L^-1＋KT 0.25 mg·L^-1＋蔗糖30 g·L^-1＋琼脂0.8% (pH 5.9～6.0),25 ℃持续光培养。     

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

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

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Atmospheric CO2 concentration, N availability, and water status affect patterns of ergastic substance deposition in longleaf pine (Pinus palustris Mill.) foliage

 Leaf chemistry alterations due to increasing atmospheric CO₂ will reflect plant physiological changes and impact ecosystem function. Longleaf pine was grown for 20 months at two levels of atmospheric CO₂ (720 and 365 μmol mol^–1), two levels of soil N (4 g m^–2 year^–1 and 40 g m^–2 year^–1), and two soil moisture levels (– 0.5 and – 1.5 MPa) in open top chambers. After 20 months of exposure, needles were collected and ergastic substances including starch grains and polyphenols were assessed using light microscopy, and calcium oxalate crystals were assessed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Polyphenol content was also determined using the Folin-Denis assay and condensed tannins were estimated by precipitation with protein. Evaluation of phenolic content histochemically was compared to results obtained using the Folin-Denis assay. Total leaf polyphenol and condensed tannin content were increased by main effects of elevated CO₂, low soil N and well-watered conditions. Elevated CO₂ and low soil N decreased crystal deposition within needle phloem. Elevated CO₂ had no effect on the percentage of cells within the mesophyll, endodermis, or transfusion tissue which contained visible starch inclusions. With respect to starch accumulation in response to N stress, mesophyll > endodermis > transfusion tissue. The opposite was true in the case of starch accumulation in response to main effects of water stress: mesophyll < endodermis < transfusion tissue. These results indicate that N and water conditions significantly affect deposition of leaf ergastic substances in longleaf pine, and that normal variability in leaf tissue quality resulting from gradients in soil resources will be magnified under conditions of elevated CO₂. Received: 5 November 1996 / Accepted: 7 March 1997     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997