Induction Of Explants Research Articles

A Transcriptomics Analysis of the Regulation of Lens Fiber Cell Differentiation in the Absence of FGFRs and PTEN.

Adding 50% vitreous humor to the media surrounding lens explants induces fiber cell differentiation and a significant immune/inflammatory response. While Fgfr loss blocks differentiation in lens epithelial explants, this blockage is partially reversed by deleting Pten. To investigate the functions of the Fgfrs and Pten during lens fiber cell differentiation, we utilized a lens epithelial explant system and conducted RNA sequencing on vitreous humor-exposed explants lacking Fgfrs, or Pten or both Fgfrs and Pten. We found that Fgfr loss impairs both vitreous-induced differentiation and inflammation while the additional loss of Pten restores these responses. Furthermore, transcriptomic analysis suggested that PDGFR-signaling in FGFR-deficient explants is required to mediate the rescue of vitreous-induced fiber differentiation in explants lacking both Fgfrs and Pten. The blockage of β-crystallin induction in explants lacking both Fgfrs and Pten in the presence of a PDGFR inhibitor supports this hypothesis. Our findings demonstrate that a wide array of genes associated with fiber cell differentiation are downstream of FGFR-signaling and that the vitreous-induced immune responses also depend on FGFR-signaling. Our data also demonstrate that many of the vitreous-induced gene-expression changes in Fgfr-deficient explants are rescued in explants lacking both Fgfrs and Pten.

Effects of a New Plant Growth Regulator on Callus Induction from Immature Embryo Explants of Korean Pine (Pinus koraiensis)

Somatic embryogenesis is currently the most promising technique for the large-scale production and breeding of conifer species. Nonetheless, the low explant induction rate in this process hampers the development of improved materials. In this study, three immature Korean pine (Pinus koraiensis) embryos capable of induction were used as experimental materials. Various concentrations of brassinolide (BL), melatonin (Mel), salicylic acid (SA), glutathione (GSH), and ascorbic acid (AsA) were added to induce embryogenic callus tissue. The results show that BL had the most significant positive effect on promoting induction and increasing explant survival. Mel was also found to slightly increase the induction and survival rates of explants. When 1.00 mg·L−1 BL was added to the explants derived from stock tree I, which had strong self-induction ability, for 30 days, the callus induction rate rose to 400% compared to the control group. Following the addition of 25 mg·L−1 Mel to stock tree I for 30 days, the callus induction rate further increased to 450% of the control group. The effect of GSH on callus induction was insignificant. The addition of 0.10 g·L−1 to stock tree I for 30 days resulted in only a 150% higher induction rate compared to the control group. When 1.00 mg·L−1 BL was applied to explants with weak self-induction ability (stock tree III) for 15 days, the callus induction rate increased to 600% of that of the control group. The callus induction rate further increased to 800% of the control group after 50 mg·L−1 BL were added to stock tree III for 15 days. This study presents a method to improve the induction of embryogenic callus tissue in Korean pine.

Multiple bud clumps induction of banana cv. Barangan Merah (Musa acuminate Colla.) under in vitro treatment of benzyl amino purine and indole-3 acetic acid

Cultivar Barangan is one of the famous banana plants that has good taste and price. The disadvantages of this banana is susceptible to Fusarium disease. Tissue culture techniques is one ways of plants propagation for this plant. This study is aimed to determine the effective concentration of benzyl amino purines (BAP) and indole-3 acetic acid (IAA) for optimal multiple bud clumps (MBC) induction in explants of Barangan Merah banana. The Research was carried out at the Laboratory of Tissue Culture, Faculty of Agriculture, Universitas Syiah Kuala. The research design was a completely randomized design, 3x2 factorial. BAP concentration was the first factor, i.e. 5, 10 and 15 mg L−1. The second factor was the concentration of IAA, i.e. 0.1 and 0.5 mg L−1. The results of this research showed that 96,29% explants of all treatments survived. Explant contamination was 11,11% occurred by bacteria and brownish exudate that spread to the medium. MBC are significantly increased at 16 days after induction at concentration of BAP 5 mg L−1 + IAA 0.5 mg L−1. All combinations of BAP and IAA concentrations have produced MBC explants (100%) at eighth weeks after induction. The concentration of 5 mg L−1 BAP + 0.5 mg L−1 IAA resulted the highest shoots number. Analysis of variance and Least Significant Different further test showed that the longest of shoot length was founding the concentration of 5 mg L−1 BAP compared to other treatments. Increasing concentrations of BAP 10 mg L−1and 15 mg L−1 resulted in significantly lower shoot length. It can be concluded that taken together the concentration of 5 mg L−1 BAP + 0.5 mg L−1 IAA is the best to effectively multiple bud clumps induction in explants of Barangan Merah banana through tissue culture technique.

Improvement of the technology of obtaining stable (di)haploid regenerants from embryonic culture of apomictic sugar beet (Beta vulgaris) breeding material without the use of colchicine

Aim. To evaluate the effi ciency of inducing generative, reduced parthenogenesis and to better use the differentiating potential of the embryo culture under apomictic seed production in selection materials of sugar beet with cytoplasmic male sterility (CMS), and B) to isolate homozygous lines (dihaploids) without the use of polyploidizing substances. Methods. Apomictic (agamosper- mous) seed production in apocarpous pollen sterile lines from B. vulgaris subsp. vulgaris var. altissima (sugar beet) using classi- cal so-called Owen sterile cytoplasm and sterile cytoplasm from Beta maritimа and Beta patula as sources, was conducted under pollen free conditions and spatial isolation in the greenhouse breeding complex of the Yaltushkivska experimental breeding station (Yaltushki, Ukraine). The specifi cities of embryonic development of apomictic embryos were studied with the purpose of effi cient regulation of the induction of explants in vitro as donors of the culture of immature embryos. Fluorescent fl ow cytophotometry in combination with the computer program of the Partec Ploidy Analyser PA-2 (Partec GmbH, Germany, now Sysmex), were used to determine the degree of ploidy, enabling the selection of haploid and dihaploid lines in vitro. A genetic method was developed using the expression of morphological marker indices of nuclear genes of anthocyanin coloring (R+ r–) of regenerant plants in vitro and ploidy determination for differentiation by generative (reduced) parthenogenesis. The sampling technique that took into account the hormonal composition of cultural media and the level of genome ploidy, sample frequency and statistical analysis of the results was determined using the appropriate statistics; the percentage of regenerants, induced by different types of morpho- genesis and ploidy in vitro, was determined along with the measurement error to control the accuracy of the selected sampling (number of seed embryos). Results. The selected cultural medium No. 3, based on the basal medium according to Gamberg et al., 1968 (21), contained 6 BAP – 2 mg/l, 2.4 D – 0.5 mg/l, gibberellic acid – 0.1 mg/l, which ensured a success rate of 4.4 to 23.3 % of direct regeneration of shoots from the embryo culture, depending on the genotype of donors, and 4–10 % for induction and proliferation of callus. In ten experimental numbers of alloplasmic lines of sugar beet, the incidence of haploids and mixoploids among the regenerants from the embryo culture fl uctuated within the wide range of 14.8 – 62.2 % and exceeded the indices, ob- tained by other known methods of haploid parthenogamy, which had the values of 3.79 – 6.25 %. Conclusions. The homozygous lines and dihaploids were determined and set apart/stabilized in the process of micropropagation, where the differentiation of clones was made on the basis of total DNA content in interphase nuclei, using information of histograms generated in fl uorescent fl ow cytometry with the Partec Ploidy Analyser PA-II instrumentation. The medium, based on macro- and microsalts according to Gamberg et al., 1968 (21) was found to be the most effi cient; it ensured at least partially successful direct regeneration in the culture of embryos within the range of 4.40 ± 1.29 to 23.3 ± 3.45 %. The success of direct regeneration of apomictic material depended on the composition of the cultural medium used fi rst and foremost, and to a lesser extent on the stages of embryogenesis from day 12 till day 32, differentiated by the fi xation period for seed embryos starting from the beginning of fl owering. Homozygous lines were created without polyploid-inducing substances due to spontaneous transfer of some cells of haploid regenerant plants to a higher level of ploidy, that can be used in the breeding of sugar beet. Genetic determination of apomictic seed reproduction in alloplasmic lines and pollen free lines of sugar beet and the technologies of inducing dihaploids allow reducing the period of inzucht-crossing considerably to obtain homozygous lines, creating unique material for chromosome engineering and marker-oriented selection with target combinations of genes in homozygous state.

The role of nanosilver in improving frequency of single cell formation of Limonium sinuatum (L.) Mill

In this research, nanosilver with different concentrations and treatment time was used to sterilize infection agents and induce initial explants which were used as materials for cell suspension culture of Limonium sinuatum. The result showed that leaf explants sterilized with 0.2 g/L nanosilver for 20 minutes had highest effect (live rate 73.33%) comparing to HgCl2and Ca(ClO)2(56.66% and 64.44%, respectively). In addition, the leaf explants which were treated with nanosilver and cultured in ½ MS medium supplemented with 20 g/L sucrose, 1 mg/L picloram and 2.5 g/L gelrite also induced calli (friable calli, milk white color, embryogenic callus structure). Moreover, cell suspension formation process from these calli was also observed highest on the 20thday (49,088 cells/mL) in liquid shaking culture condition comparing to control treatment (19,361 cells/mL) when they were cultured on similar medium combined with 1.2 mg/Lnanosilver. These cells had the best growth, development and regeneration from the 16thday to the 20thday. The ability of shoot and callus regeneration was highest (67.77%) in ½ MS medium which was supplemented with 20 g/L sucrose, 1 mg/L zeatin, 2.5 g/L gelrite and 1.6 mg/L nanosilver compared with control treatment having no nanosilver (40.00%). This research showed that in micropropagation of Limonium sinuatum, nanosilver was proved to be an effective agent for sterilization, explant induction, cell suspension origination, and callus and shoot regeneration. Beside that, nanosilver had negative impact on the development of explants when it was used with high concentration for extended period.

Evaluation of Rasna panchaka (indigenous drug) as oxidative stress down-regulator using serum-free explant culture system.

CONTEXT:The importance of phytochemicals/natural products as potential therapeutic agents in the present context is gaining a lot of importance. India with a rich heritage of such preparations needs evaluation as potent drugs. Explant culture system is a method, which is sensitive, reliable, reproducible and is capable of mimicking the in situ conditions maintaining the tissue in sufficiently high level of integration.AIM:The current study aimed to test the antioxidant activity of test compounds, namely, traditional aqueous (4212) and aqueous-methanolic (4308) extracts of Rasna panchaka using liver explant cultures.MATERIALS AND METHODS:Dose-response optima of extracts (0.2–10 μg/mL) were determined using mouse liver explant culture system up to 48 h. The antioxidant property of extracts was assessed by primary oxidative defense parameters, namely, superoxide-dismutase (SOD), catalase, reduced glutathione (GSH), and malondialdehyde (MDA).RESULTS:The results indicated that the cellular architecture of the cultured tissue was well conserved in the first 6 h with a gradual display of specific changes in the next 24 h. There was a significant increase in MDA levels in experimental groups indicating the oxidative stress induction in explants. A dose of 2.0 μg/mL extracts have shown statistically significant (P < 0.05) protection against oxidative stress. MDA levels, a measure of lipid peroxidation, were significantly (P < 0.01) reduced by 50% in extract treated explants compared to control. This effect was accompanied by the increase in the first defense enzymes SOD (50%) and catalase (18%) with no change in reduced GSH levels.CONCLUSION:The study enforces the importance of “explant culture system,” as it not only reduces the use of nonclinical/animal model but also is rapid and sensitive. Further, results of the current study also suggest that aqueous-methanolic extract of Rasna panchaka is having superior antioxidant activity compared to traditional water extract.

Cryotherapy: A Novel Method for Virus Eradication in Economically Important Plant Species.

Virus diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.

Morphological Characterization and Identification of Coffea liberica Callus of Somatic Embryogenesis Propagation.

Compared with other types of coffee, Liberica coffee is more difficult to be propagates using clonal methods. Meanwhile, demand for planting materials and consumption of this type of coffee is increasing lately. The objective of this paper is to present results of the work on morpological characterization of Liberica coffee (Coffea liberica) callus produced by somatic embryogenesis propagation. This research used C. liberica Arruminensis clone. This clone was one of Liberica coffee clones which had superior taste. Main activitis carried out in this experiment were explant sterilization, explant induction and histological analysis on the callus produced. The result of this research showed that non embryogenic callus was higher (72%) than embryogenic callus (28%). The callus description can be used to identify type and characteristic of the callus. Therefore, it can be a parameter to choose type of callus for propagation material. This is important because choosing the right callus is determine of the succesfully process of Liberica somatic embryogenesis. Keywords: somatic embryogenesis, Liberica, embryogenic, non-embryogenic

An Efficient Regeneration System and Optimization of the Transformation from the Cotyledonary Node of Jute (Corchorus capsularis L.)

The purpose of this study was to use cotyledonary nodes as explants to establish an efficient regeneration protocol for jute (Corchorus capsularis L.) on Murashige and Skoog (MS) basal medium. Research into the different growth regulators using the orthogonal design L16 (45) revealed that the best shoot induction medium is MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6BA and 0.25 mg/L NAA. The average number of shoots per explant and the explant induction rate were 9.8 and 100%. After 3 weeks, 2–3 cm shoots were rooting on 1/2 MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6-BA + 0.2 mg/L NAA. Moreover, we optimized Agrobacterium-mediated transformation using the GUS gene transient expression system. The best condition for obtaining higher transformation rate consisted of the use of fresh explants to which 100 μM acetosyringone was added for a co-culture time of 10 min, the OD value of Agrobacterium liquid is 0.5 at 600 nm. These data provide an important basis for the application of other trait gene in the improvement of jute fiber quality.

Enhancing plant regeneration in tissue culture

Micropropagation is used for commercial purposes worldwide, but the capacity to undergo somatic organogenesis and plant regeneration varies greatly among species. The plant hormones auxin and cytokinin are critical for plant regeneration in tissue culture, with cytokinin playing an instrumental role in shoot organogenesis. Type-B response regulators govern the transcriptional output in response to cytokinin and are required for plant regeneration. In our paper published in Plant Physiology, we explored the functional redundancy among the 11 type-B Arabidopsis response regulators (ARRs). Interestingly, we discovered that the enhanced expression of one family member, ARR10, induced hypersensitivity to cytokinin in multiple assays, including callus greening and shoot induction of explants. Here we 1) discuss the hormone dependence for in vitro plant regeneration, 2) how manipulation of the cytokinin response has been used to enhance plant regeneration, and 3) the potential of the ARR10 transgene as a tool to increase the regeneration capacity of agriculturally important crop plants. The efficacy of ARR10 for enhancing plant regeneration likely arises from its ability to transcriptionally regulate key cytokinin responsive genes combined with an enhanced protein stability of ARR10 compared with other type-B ARRs. By increasing the capacity of key tissues and cell types to respond to cytokinin, ARR10, or other type-B response regulators with similar properties, could be used as a tool to combat the recalcitrance of some crop species to tissue culture techniques.

Regeneration of plantlets under NaCl stress from NaN3 treated sugarcane explants

In this experiment, mutation induction in explants using NaN3 and subsequently, callus growth and plant regeneration under NaCl stressed conditions was assessed in some sugarcane ( Saccharum officinarum L.) cultivars (Thatta-10, CPF-237 and SPHS-19). Immature bases of leaf tips were cultured on MS 2n (MS salts, 3.0 mg L -1 ; 2,4-D, 0.5% NaN 3 ) for 6 days then sub-cultured on MS 2 (MS salts, 3.0 mg L -1 2,4-D), MS 2a (MS 2 , 25 mol m -3 NaCl), MS 2b (MS 2 , 50 mol m -3 NaCl) and MS 2c (MS 2 , 75 mol m -3 NaCl) media in dark condition. After 6 weeks, callus growth was observed to be significantly higher (72.34 ± 3.70%) in CPF-237 in the control (MS 2 ), and lowest (57.66 ± 4.34%) in SPHS-19 in MS 2d culture. Somatic embryos were induced in proliferated calluses on MS 3 (MS salts, 0.5 mg L -1 ; BAP, 0.4 mg L -1 ; kin, 0.3 g L -1 ; casein hydrolysate, 3% sucrose) medium under dark condition for 2 weeks. These calluses were sub-cultured on MS 4 (MS, 0.3 mg L -1 ; BAP, 0.2 mg L -1 ; kin, 3% glucose), MS 4a (MS 4 , 25 mol m -3 NaCl), MS 4b (MS 4 , 50 mol m -3 NaCl) and MS 4c (MS 4 , 75 mol m -3 NaCl) media. Maximum of 8.41 ± 0.36 plantlets callus -1 were regenerated in MS 4 (control) culture of Thatta-10, and 4.94 ± 0.05 plantlets of CPF-237 in 25 mol m -3 NaCl stressed plant regeneration (MS 4a ) medium. Plant regeneration on MS 4b (2.21 ± 0.17 plantlets callus -1 ) was observed in CPF-237 only. Regenerated plantlets were rooted and considered as salt tolerant in comparison to its parent cultivars. Key words: Kinetin, somatic embryos, regenerated plantlets, Saccharum officinarum.

Encapsulation of cauliflower (Brassica oleracea var botrytis) microshoots as artificial seeds and their conversion and growth in commercial substrates

An effective protocol for the mass production of cauliflower microshoots was refined using the meristematic layer of cauliflower curd. After the meristematic layer was surface sterilized and shaved off, a commercial blender was used for homogenization and several blending treatments were tested in the range 15–120 s and 30 s was found to be optimal in terms of the amount explants produced and their subsequent growth ability. Explants were cultivated in S23 liquid medium (4.4 g L−1 MS (Murashige and Skoog) and 3% v/w sucrose) supplemented with several combinations of plant growth regulators (PGRs) including 1 and 2 mg L−1 of Kinetin in combination with three types of auxins (indole butyric acid (IBA), Naphthaleneacetic acid (NAA) and Indole-3-acetic acid (IAA)), each at 1 and 2 mg L−1 concentration. The use of 2 mg L−1 Kinetin and 1 mg L−1 IBA gave the best results in terms of its effects on explant induction. Microshoots of different sizes were encapsulated in a sodium alginate matrix and the optimal stage suitable for the production of artificial seeds was assessed in terms of both subsequent conversion and plantlet viability. The feasibility of cultivating cauliflower artificial seeds in commercial substrates (compost, vermiculite, perlite and sand) irrigated with different solution mixtures including sterilized distilled water (SDW), PGRs-free S23 medium and S23 medium supplemented with Kinetin (1 and 2 mg L−1) and IBA or NAA at (1 and 2 mg L−1) was investigated. The use of 2 mg L−1 Kinetin and 2 mg L−1 NAA applied with S23 gave the optimal response with both perlite and compost. This study showed high growth capacity of cauliflower artificial seeds in commercial substrates which is considered a promising step for their direct use in vivo.

Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family) for lycopene production

Lycopene is present in a range of fresh fruits and vegetables, especially in the leaves of Barringtonia racemosa. The traditional lycopene extraction from the plant is being employed instead of an easy propagation technique like cell culture process from the leaf explants. We intend to assess how lycopene could be extracted via tissue culture under light (illuminance: 8,200 lux under white fluorescent lamps, photoperiod 16 h per day at 25ºC) and dark. Leaf explants of Barringtonia racemosa were cultured on modified Murashige and Skoog (MS), Woody Plant Medium (WPM) and B5 media, supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). Optimal conditions for callus induction and maintenance under both dark and light were investigated, and growth and lycopene accumulation were evaluated. Among media with different concentrations of 2,4-D, fast growing, friable callus initiated within three weeks after culturing on WPM basal medium supplemented with 2.0 mg L-1 (weight per volume) of 2,4-D, whereas callus induction in explants cultured on all other media started only after five weeks. Calli were subcultured once every fortnight. Pale yellow and green calli developed under conditions of dark and light respectively were then selected for evaluation of their lycopene contents. An improved reversed phase of high performance liquid chromatography (HPLC) method was used for a selective chemical determination of the lycopene content. Light induced lycopene production; and likewise maximum lycopene level incubated in light was higher than those incubated in darkness. The best growth rates of callus and cell suspension were achieved in WPM and B5 media respectively. The production of lycopene was growth-dependent through analysis of growth and lycopene content of both callus and cell suspension cultures.

Heme Oxygenase Expression in Human Placental Villous Tissue in Response to Exposure to in vitro Ischemia-Reperfusion Injury

Heme oxygenases (HO-1 and HO-2) are responsible for the production of carbon monoxide, a vasodilator. The products of heme oxygenase are also anti-oxidants. HO is expressed within the placenta and is important in controlling placental blood flow. HO can be sensitive to oxygen, with responses differing depending on the cell type. Recent studies have suggested that in preeclampsia, the placenta would be subjected to fluctuations in oxygen tension analogous to an ischemia-reperfusion injury. Thus the present study tested the hypothesis that HO-1 and or HO-2 expression in placental villous explants would be altered by an ischemic-reperfusion insult. Human term placental explants were exposed to hypoxia then re-oxygenation in 5% or 20% O2 or repeated cycles of hypoxia-re-oxygenation. HO protein concentrations were assessed by Western blotting. No changes in HO-1 or HO-2 were found with any treatment protocol. Chemical induction of HO-1 was possible in explants showing that HO-1 induction in explants is possible. The results suggest that cells in term placental villous tissue do not respond to hypoxia-re-oxygenation by altering the amount of HO-1 or HO-2 protein.

An FGF Response Pathway that Mediates Hepatic Gene Induction in Embryonic Endoderm Cells

While particular combinations of mesodermal signals are known to induce distinct tissue-specific programs in the endoderm, there is little information about the response pathways within endoderm cells that control their specification. We have used signaling inhibitors on embryo tissue explants and whole-embryo cultures as well as genetic approaches to reveal part of an intracellular network by which FGF signaling helps induce hepatic genes and stabilize nascent hepatic cells within the endodermal epithelium. Specifically, we found that hepatic gene induction is elicited by an FGF/MAPK pathway. Although the PI3K pathway is activated in foregut endoderm cells, its inhibition does not block hepatic gene induction in explants; however, it does block tissue growth. We also found that at the onset of hepatogenesis, the FGF/MAPK and PI3K pathways do not crossregulate in the endoderm. The finding of separate pathways for endoderm tissue specification and growth provides insights for guiding cellular regeneration and stem cell differentiation.

Shoot tissue culture ofRobinia pseudoacacia f.decaisneana

Robinia pseudoacacia f.decaisneana is a transfiguration ofRobinia pseudoacacia. For enhancing propagation co-efficient of the species, the experiment of shoot tissue culture ofRobinia pseudoacacia f. decaisneana was conducted in Forestry College of Shenyang Agricultural University from July 1999 to July 2001. The experiment included medium selection of explant induction survival, initial culture, subculture as well as rooting culture, and forming seedling with callus. The results showed that shoot segmentin vitro survive rate is larger in spring than in autumn, and green dense callus could form plantiet. The best medium for initial culture was SH+0.5mg/L BA+0.05mg/L NAA, with a propagation coefficient of 4.1 (per micro-cutting in a month), and for subculture it was B5+0.5mg/L BA+0.05mg/L NAA+10mg/L Glu., with a propagation coefficient of 4.7. The best rooting medium was 1/2MS+0.5mg/L NAA+10mg/L Glu., with a rooting rate of 84.4%. These results provide reference data for reproduction of superior individuals ofRobinia pseudoacacia f. decaisneana.

BMP regulates vegetal pole induction centres in early xenopus development.

Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.

Regeneration and frequency of tetraploid variants of Cucumis metuliferus are affected by explant induction on semi-solid medium versus the liquid/ membrane system.

Ninety-eight percent of Cucumis metuliferus (PI 482439) cotyledons regenerated shoot buds after 5 weeks on basal Murashige and Skoog medium amended with 10 µM benzyladenine. Regeneration rates of explants maintained only on agar-gelled medium versus explants induced first on a liquid/membrane system were compared after weekly transfers of tissue from liquid/membrane to agar during the first 5 weeks of regeneration. The number of regenerant buds increased from six per cotyledon (on agar-gelled medium) to nine per cotyledon when explants induced on the liquid/membrane system for 3 or 4 weeks were transferred to agar-gelled medium. Shoot development, rooting and survival in the greenhouse were adequate, regardless of whether regeneration was initiated on the agar or liquid/membrane system. Tetraploid regenerants accounted for 9% of the 391 regenerants screened. Pollen morphology was a reliable screening technique to identify tetraploid plants. The frequency of tetraploid plants varied from 13.5% to 6.9% after 5 weeks of induction on the agar or liquid/membrane system, respectively. This frequency decreased to 1.5% if the explants were transferred from the liquid/membrane system to agar after the first week of induction. Transfers during this period play a critical role in eliminating tetraploid variants.

The Role of Intracellular Alkalinization in the Establishment of Anterior Neural Fate inXenopus

Our previous work demonstrated thatXenopusectoderm cells undergo an alkalinization in response to planar inductive signals during neural induction in explants. We have examined the role of intracellular alkalinization in the establishment of anterior neural fate. First, RT-PCR was used to examine neural-specific gene expression in planar explants in which the alkalinization is prevented by treatment with 4,4′-dihydrodiisothiocyanatostilbene-2,2′-disulfonate (H2DIDS). In explants cultured in the presence of H2DIDS, expression ofNCAMand the anterior neural geneotx2is greatly reduced or absent. Second, neural-specific gene expression was examined in isolates of uninduced animal cap ectoderm cultured in the presence of either methylamine or ammonium chloride.NCAM, otx2,and the anterior neural inducernogginwere expressed in alkalinized ectoderm, while the more posterior neural markerskrox-20andHox B9were undetectable. Expression ofNCAM, otx2,andnogginwas observed at stage 11 in both alkalinized ectoderm and the newly induced neural plate, suggesting that intracellular alkalinization could contribute to propagation of noggin signaling through the dorsal ectoderm. Alkalinization of uninduced ectoderm at stage 10.5 led to an upregulation ofotx2within 15 min. Activation ofNCAMexpression in alkalinized dissociated cells was identical to that observed in intact animal caps, indicating that alkalinization-mediated changes in gene expression do not require cell–cell contact. Finally, the effects of intracellular alkalinization on protein tyrosine phosphorylation were investigated using 2D gel electrophoresis and immunoblots probed with an anti-phosphotyrosine antibody. Several phosphorylated proteins detected in induced and alkalinized ectoderm were greatly reduced or absent in uninduced ectoderm, indicating that alkalinization elicits alterations in tyrosine phosphorylation similar to some of those observed during neural inductionin vivo.Our results indicate that intracellular alkalinization plays a critical role in the activation of anterior neural-specific gene expression and that alkalinization may act by regulating the activity of a tyrosine kinase or phosphatase.

Regeneration and Frequency of Tetraploid Variants of Cucumis metuliferus are Affected by Explant Induction on Liquid/Membrane System

Ninety-eight percent of cotyledons of Cucumis metuliferus (PI 482439) regenerated shoot buds in 5 weeks on MS medium with 10 μm BA. Regeneration rates on agar gelled medium and liquid/membrane system were compared after weekly transfers of tissue from liquid/membrane to agar during the 5 weeks of regeneration culture. Number of shoots and buds increased from six (on agargelled medium) to nine per cotyledon when explant on liquid/membrane system for 3 or 4 weeks was transferred to agar gelled medium. Shoot development, rooting, and survival in greenhouse was adequate regardless of whether regeneration was initiated on agar or liquid/membrane system. Tetraploid regenerants were found to be about 9% of the almost 400 regenerants screened. Pollen morphology was a quick and definitive screening technique for tetraploid plants. Frequency of tetraploid plants was similar after 5 weeks of induction on either agar or liquid/membrane system. This frequency decreased by a factor of 10 following transfer of explants from membrane to agar after the first week of induction. Timing of this transfer plays a critical role in eliminating tetraploid variants.