Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

Plant regeneration from suspension cells induced from hypocotyls derived from interspecific cross Alstroemeria pelegrina × A. magenta and transformation with Agrobacterium tumefaciens

Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 days after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram). Adventitious embryos developed from the suspension cells on half-strength MS medium supplemented with 0.5 mg l⁻¹ of both α-naphthaleneacetic acid and N⁶-benzylaminopurine; they grew into plantlets on the same medium. The plantlets formed rhizomes following transfer to half-strength MS medium without PGRs, and acclimatized plants were easily established. Subsequently, Agrobacterium-mediated transformation system was applied. The suspension cells were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which contain neomycin phosphotransferase II, hygromycin phosphotransferase and intron-containing ?-glucuronidase (intron-GUS) genes. Seven days after co-cultivation, the cells were subjected to GUS assay; staining was most pronounced in the cells subcultured in a picloram-containing liquid medium and co-cultivated with EHA101/pIG121Hm. The co-cultivated cells were transferred to the MS medium containing picloram and 20 mg l⁻¹ hygromycin; 1 month later, several hygromycin-resistant callus lines showing GUS activity were obtained. Transgenic plants were obtained through our plant regeneration system, and foreign gene insertion into the regenerated plants was confirmed by polymerase chain reaction.     

Improved growth performance of the mangrove <Emphasis Type="Italic">Avicennia marina</Emphasis> seedlings using a 1-aminocyclopropane-1-carboxylic acid deaminase-producing isolate of <Emphasis Type="Italic">Pseudoalteromonas maricaloris</Emphasis>

Out of 62 bacterial isolates obtained from the mangrove Avicennia marina rhizosphere that grows along the Abu Dhabi coast, United Arab Emirates (UAE), an isolate of Pseudoalteromonas maricaloris (Wild type strain) (WT) produced relatively high levels of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in vitro. Application of this WT strain under greenhouse conditions to A. marina seedlings significantly (P < 0.05), reduced endogenous levels of ACC in the roots and shoots, and significantly (P < 0.05) increased the levels of in planta endogenous plant growth regulators (PGRs) including indole-3-acetic acid (IAA), indole-3-pyruvic acid (IPYA), putrescine (Put), spermidine (Spd) and spermine (Spm) in roots and shoots compared with control mangrove seedlings. WT application has also significantly (P < 0.05) increased photosynthetic pigment contents, photosynthetic carbon assimilation, plant water use efficiency and promoted mangrove seedlings growth characteristics including increased dry weight and length of roots and shoots, total leaf area and the number of the side branches compared with control mangrove seedlings. In comparison, an ACC deaminase non-producing mutant strain (NPM) failed to reduce endogenous levels of ACC in the roots and shoots and also failed to increase endogenous PGRs and photosynthetic pigments and did not promote seedling growth. Both WT and NPM strains were incapable of producing in vitro detectable levels of IAA, IPYA, Gibberellic acid (GA₃), zeatin (Z), Put, Spd and Spm in the culture filtrates. This study demonstrated for the first time the ability of ACC deaminase-producing bacteria to promote mangrove growth under greenhouse conditions. P. maricaloris has potential as biological inoculants to promote the growth of mangrove seedlings in afforestation programs in nutrient impoverished sediments in hyper-saline coastal areas in the UAE.     

Inhibition of darkness-induced stomatal closure by ethylene involves a removal of hydrogen peroxide from guard cells of <Emphasis Type="Italic">Vicia faba</Emphasis>

Ethylene regulates many aspects of plant growth and development; however, its effect on the behavior of the stomata is still largely obscure. Here, the association between ethylene inhibition of darkness-induced stomatal closure and hydrogen peroxide (H₂O₂) levels in Vicia faba guard cells was studied. Like ascorbic acid (ASA), the most important reducing substrate for H₂O₂ removal, catalase (CAT), one of H₂O₂-scavenging enzymes, and diphenylene iodonium (DPI), an inhibitor of the H₂O₂-generating enzyme NADPH-oxidase, both ethylene-releasing compound 2-chloroethylene phosphonic acid (ethephon, ETH) and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, were found to inhibit stomatal closure by darkness and to reduce H₂O₂ levels in guard cells, indicating that ethylene-caused inhibition of darkness-induced stomatal closure involves reduction in the H₂O₂ level in guard cells. Additionally, similar to ASA and CAT, ACC/ETH not only suppressed H₂O₂-induced stomatal closure and H₂O₂ level in guard cells treated with exogenous H₂O₂ in the light, but also reopened the stomata, which had been closed by darkness, and reduced H₂O₂ level that had been generated by darkness, showing that ethylene causes H₂O₂ removal from guard cells. However, the above-mentioned effect of ACC/ETH was dissimilar from that of DPI, which not only was incapable to reduce H₂O₂ level induced by exogenous H₂O₂ but also could not abolish H₂O₂ that had been generated by darkness. Thus, we suggest that ethylene probably induces H₂O₂ removal and reduces H₂O₂ level in guard cells and finally inhibits stomatal closure induced by darkness. Furthermore, the mechanism of H₂O₂ removal caused by ethylene was also discussed.     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

A clonal propagation system for Atlantic white cedar (<Emphasis Type="Italic">Chamaecyparis thyoides</Emphasis>) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l^?1 producing more embryos than 0 g l^?1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l^?1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.     

Effect of plant growth regulators on ethylene production, 1-aminocyclopropane-1-carboxylic acid oxidase activity, and initiation of inflorescence development of pineapple

With the development of pineapple [Ananas comosus (L.) Merr.] as a fresh fruit crop, it became common to force inflorescence development with ethephon [(2-chloroethyl)phosphonic acid] or ethylene throughout the year. Environmental induction (EI) of inflorescence development disrupts scheduling of fruit harvest and may cause significant losses if small plants are induced, resulting in fruits that are too small to be marketable. Our objective was to identify plant growth regulators (PGRs) that could inhibit EI. Because circumstantial evidence indicates that EI occurs in response to naturally produced ethylene or changes in plant sensitivity to it, most work was done with PGRs that inhibit ethylene biosynthesis or block ethylene action. The synthetic auxin 2-(3-chlorophenoxy)propionic acid (CPA) was included because in one study it reduced the percentage of EI. GA₃, aminooxyacetic acid (AOA), aminoethoxyvinylglycine (AVG), daminozide [butanedioic acid mono-(2,2-dimethylhydrazide)], and silver thiosulfate (STS) had no effect on EL CPA, paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)methyl-4,4-dimethyl-2(1h-1,2,4-triazol-1-yl)penten-3-ol], and uniconazole [(E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol] delayed or inhibited EI of pot-grown pineapple plants. Uniconazole and paclobutrazol inhibited growth and ethylene production by leaf basal-white tissue, and either or both effects could account for the inhibition of EI. Production of 1-aminocyclopropane-1-carboxylic acid (ACC) was unaffected by these compounds, but the activity of ACC oxidase, which converts ACC to ethylene, was inhibited and probably accounts for the reduced ethylene production by leaf basal-white tissue. CPA stimulated ethylene production by stem apical tissue approximately fourfold relative to the control. ACC oxidase activity and the malonyl-ACC (MACC) content in stem apical tissue were also greater than in the control, indicating that CPA greatly stimulated the production of ACC and its sequestration into MACC. The mechanism by which CPA delayed or inhibited EI is not known. CPA, paclobutrazol, and uniconazole appear to have some potential for inhibiting EI of pineapple. Their effect on yield needs to be determined.Abbreviations ACC oxidase 1-aminocyclopropane-1-carboxylic acid oxidase - CPA 2-(3-chlorophenoxy)propionic acid - AOA aminooxyacetic acid - AVG aminoethoxyvinylglycine - daminozide butanedioic acid mono-(2,2-dimethylhydrazide) - DM dry mass - ethephon [(2-chloroethyl)phosphonic acid] - FM fresh mass - GA gibberellin - EI environmental induction of inflorescence development - IA inflorescence appearance - LSD Fisher's protected least significant difference - MACC malonyl-ACC - NAA naphthaleneacetic acid - PGR plant growth regulator - paclobutrazol (2RS,3RS)-1-(4-chlorophenyl)methyl-4,4-dimethyl-2-(1h-1,2,4-triazol-1-yl)penten-3-ol] - uniconazole (E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol - STS silver thiosulfate - M-leaf fourth leaf - Ml-L first leaf younger than M-leaf     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis>

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm⁻³ kinetin and 0.2, 0.5 mg dm⁻³ indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.     

Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene

Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h^–1g^–1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh^–1 g^–1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO₃and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.     

Enhancing Phytoremediation Potential of Pennisetum clandestinum Hochst in Cadmium-Contaminated Soil Using Smoke-Water and Smoke-Isolated Karrikinolide

The use of plant growth regulators (PGRs) and biostimulants to enhance phytoextraction is gaining popularity in phytoremediation technology. This study investigated the stimulatory effects of smoke-water (SW), a smoke-derived compound karrikinolide (KAR₁) and other known plant growth regulators (PGRs) [gibberellic acid (GA₃), kinetin (Kin) and indole-3-butyric acid (IBA)] to enhance the phytoextraction potential of Pennisetum clandestinum. Pennisetum clandestinum seedlings were grown for 10 weeks in vermiculite using Hoagland's nutrient solution and were treated with cadmium (Cd) (2, 5, and 10 mg L^?1) and SW, KAR₁ and PGRs. KAR₁ exhibited positive effects on shoot and root dry weight (140 and 137 mg respectively) at the highest concentration of Cd (10 mg L^?1) compared to all the other treatments. KAR₁ and SW treatments used in the present study significantly improved the phytoextraction potential of P. clandestinum (602 and 575 mg kg^?1 respectively) compared to the other tested PGRs. This is the first report on the use of SW and KAR₁ to enhance phytoremediation potential in P. clandestinum. Further studies are needed to elucidate the exact mechanisms of smoke constituents involved in phytoextraction potential of plant species.     

Repetitive somatic embryogenesis and plant regeneration in Zizyphus jujuba Mill

Summary A procedure for regenerating Zizyphus jujuba Mill. (Chinese date) plants through repetitive somatic embryogenesis (RSE) was developed. Primary somatic embryos were produced from cotyledon-derived cultures of germinated plants in vitro. The highest induction frequency of primary somatic embryogenesis (PSE) was obtained with a combination of 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.49 μM indole-3-butyric acid (IBA), and 0.44 μM benzyladenine (BA) (17.4%). These primary somatic embryos were multiplied by RSE on media with different plant growth regulator (PGR) combinations. The highest RSE frequency (51.3%), was obtained with 0.58 μM gibberellic acid (GA₃). However, the highest number (4.4 per primary somatic embryo) of repetitive somatic embryos was obtained with 0.98 μM 6-r-r-dimethylallylaminopurine (2-iP). For germination of somatic embryos, different PGRs, cold and desiccation treatment were tested. Desiccation of somatic embryos at 25±1°C for 2 wk was the best treatment for germination with epicotyl elongation and root development. Of over 256 plants regenerated, 237 (92.6%) survived.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Autopolyploid induction via somatic embryogenesis in Lilium distichum Nakai and Lilium cernuum Komar

New ornamental varieties of high quality can be created via artificial polyploid induction. In the present study, the first system of polyploid induction with somatic embryogenesis of Lilium distichum Nakai and Lilium cernuum Komar. was developed. Somatic embryos were cultured on MS with 0.41 μmol L^?1 picloram and 1.07 μmol L^?1 NAA by scales (5 mm²). After 40 days, somatic embryos were transferred to MS with 2.21 μmol L^?1 BA for somatic embryogenesis. As determined from observations of paraffin sections, embryonic cells of L. distichum originated from outer cells at first, and somatic embryogenesis occurred through an indirect pathway. In L. cernuum, embryonic cells originated from inner cells at first, and somatic embryogenesis occurred through a direct pathway. Polyploids were successfully formed from somatic embryos and scales by the soaking and mixed culture methods with different colchicine concentrations (0.01%, 0.05%, and 0.1%; v/v) and durations (24, 48, and 72 h). The polyploid induction rate reached 57.14% and 46.15% with 0.05% colchicine treatment in L. distichum (48 h) and L. cernuum (24 h), respectively. Tetraploids (28.57% and 23.08%) and aneuploids without chimeras among the obtained polyploid plantlets were identified by chromosome counts of root-tip tissue squashes in L. distichum and L. cernuum. Tetraploid plantlets of L. distichum exhibited broader leaves, longer guard cells, larger stomata and higher stomatal conductance than diploid plantlets. Tetraploid plantlets of L. cernuum showed 1.76?×?higher chlorophyll content, significantly more leaves, longer guard cells, larger stomata and lower stomatal conductance than diploid plantlets.

     

Nitric oxide,hydrogen cyanide and ethylene are required in the control of germination and undisturbed development of young apple seedlings

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are emerging as important regulators of plant development (germination, flowering, senescence), acting as secondary messengers in cooperation with classical phytohormones. Apple seeds are dormant, unless they undergo a 3 month long cold stratification. Deep dormancy of isolated apple embryos can also be broken by short pre-treatment with HCN or NO with the effect associated with enhanced ethylene synthesis. Non-dormant embryos germinate well and young seedlings grown from non-dormant embryos do not exhibit any morphological anomalies, such as asymmetric growth and greening of cotyledons. One of the aims of this work was to investigate the correlation between RNS- mediated (HCN- and NO-dependent) dormancy removal and ROS (H₂O₂ and O₂^−•) accumulation in the embryos. The beneficial effect of NO and HCN on germination of dormant apple embryos has been associated with marked increases in H₂O₂ and O₂^−• concentration in the embryos at early germination stages. We also analyzed growth of young seedlings developed from embryos pre-treatment with HCN or NO or exposed to ethylene (ethephone) and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC). ACC and ethephone removed all morphological anomalies of the seedlings (asymmetric growth and greening of cotyledons) but the radicle growth was rather slight. We propose that accumulation of ROS provoked by HCN and NO pre-treatment is required for embryo germination “sensu stricto”, while ethylene is required for post-germination seedling growth.     

An anatomical study of secondary embryogenesis inCamellia reticulata

Summary An anatomical study was carried out during the sequences of events which lead to the differentiation of secondary embryos ofCamellia reticulata cv ‘Mouchang’. Secondary embryogenesis can be induced by culturing somatic embryos on a modified Murashige and Skoog medium supplemented with 0.5 mg·liter⁻¹ 6-benzylaminopurine and 0.1 mg·liter⁻¹ indole-3-butyric acid. After about 12 days of culture, globular-shaped secondary embryos became apparent, and by 18 to 20 days of culture cotyledonary stages were formed. Embryos developed mainly on the hypocotyl of primary embryos without an intermediate callus. Histologic monitoring revealed that secondary embryos apparently had a multicellular origin from embryogenic areas originating in both epidermal and subepidermal layers of the hypocotyl region. This morphogenetic competence is related to the presence, at the time of culture, of relatively undifferentiated cells in superfical layers of the primary embryo hypocotyl. Microcomputer image analysis was applied for quantifying cytological events associated with somatic embryogenesis. This method showed an increasing gradient in the nucleus-to-cell area ratio from differentiated cells passing through preembryogenic cells to embryogenic cells. The formation of embryogenic areas was preceded by accumulation of starch in the surrounding cortical cells. The cells underlying globular secondary embryos still contained abundant starch, but it declined as the secondary embryos developed.     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M _r 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the K _m for S-adenosyl-L-methionine of 1.74 mM and k _cat of 0.56 s^-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M _r 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a K _m for ACC of 4.8 mM and k _cat of 3.52 s^-1. The presence of 7 mM Cu²⁺ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts