Isolation and identification of substances inducing formation of tracheary elements in cultured carrot-root slices

Data are presented on the cytokinin status of seeds and seed components, at different stages of development in Phaseolus coccineus L., as determined with the soybean callus growth bioassay: A change in cytokinin types according to developmental stage occurred: from biologically very active less polar types (zeatin=Z) at early stages to more polar types (zeatin glucoside=Z₉G and zeatin riboside=Zr), with relatively low biological activity, at intermediate and late stages of seed development: When cytokinins were analyzed separately in embryos (embryo proper) and suspensors at two embryonic stages: heart-shaped (A) and middle cotyledonary embryos (stage B) respectively, it was found that: i) at stage A, the suspensor showed cytokinin activity at the level of Z, 2iPA (2-isopentenyladenosine) and Zr, whereas more polar cytokinins (Z₉G, Zr) were present in the embryo; ii) at stage B, when the embryo seems to become autonomous for cytokinin supply, there was a relative abundance of active cytokinins (Z, 2iPA) in the embryo to which Z₉G activity in the suspensor corresponded. It is concluded that the suspensor plays an essential role in embryogenesis by acting as a hormone source to the early embryo.Abbreviations GA gibberellic acid - 2iPA 2-isopentenyladenosine - Stage A heart-shaped embryo - siage B middle cotyledonary embryo - Z zeatin - Z₉G zeatin glucoside - Zr Zeatin riboside     

Suspensor,gibberellin and in vitro development of Phaseolus coccineus embryos

Summary Embryos of Phaseolus coccineus in different stages of development (from 0.5 to 5 mm in length) were grown in vitro. Both intact embryos (with suspensor) and embryos deprived of suspensor were studied. It was found that removal of the suspensor has no effect on the development of embryos which have reached a length of 5 mm. With younger embryos, removal of the suspensor reduces embryo development, the negative effect being the greater the younger the embryo. It was shown that gibberellic acid (GA₃) concentrations of 10^-8 to 10^-6M can replace the suspensor in heart-shaped and early cotyledonary embryos (0.5 to 1.5 mm in length), whereas they reduce the development of suspensor-deprived embryos of later stages (embryos 2 to 3 mm in length) as compared with intact embryos of similar size grown on hormone-free medium. GA₃ concentrations of 10^-5 and 10^-4M are generally inhibitory and may stimulate callus formation in some embryos. The present data and those of Alpi et al. (1975) concur in ascribing a major role to gibberellins in characterizing the physiological function of the suspensor in early embryogenesis in Phaseolus coccineus.Abbreviation GA gibberellic aid     

Characterization and systematic implications of the diversity in timing of programmed cell death of the suspensors in Leguminosae

? Premise of the study: In angiosperm seeds, the developing embryo acquires nutrients via a suspensor that typically undergoes programmed cell death (PCD) at the early cotyledon stage. However, in Leguminosae (the third largest angiosperm family), the suspensors can disappear at the heart-shaped stage (i.e., prior to the cotyledon stage) or still persist at the cotyledon stage. Here, in a comprehensive survey of legume suspensors and embryos, the variation and the evolutionary direction of timing of suspensor PCD in Leguminosae were characterized, and systematic implications were evaluated. ? Methods: Suspensor development and morphology for 66 leguminous species from 49 genera, 21 tribes, and 3 subfamilies were comparatively studied using standard paraffin sectioning and light microscopy. ? Key results: Three patterns of suspensor PCD were observed at the early cotyledon stage. (A) The suspensor persisted. (B) The suspensor separated from the wall of the embryo sac and persisted as a vestige at the radicle apex. (C) The suspensor disappeared completely, and the absorption of nutrients by embryo was carried out via a "contact zone" between the embryo and the endosperm. Pattern C of early suspensor PCD was found only in the tribe Fabeae. An ancestral character reconstruction revealed that the long-lived suspensors of pattern A represented a plesiomorphic condition in Leguminosae and that the suspensors of pattern C evolved only once in the common ancestor of Fabeae. ? Conclusions: In Leguminosae, short-lived suspensors have thus evolved multiple times from long-lived suspensors. It remains largely unknown, however, how the embryo acquires nutrients after the early suspensor PCD.     

Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development

Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C₁₉-GAs, GA₁, GA₄, GA₅, GA₆, and one C₂₀ GA, GA₄₄, were identified. From the embryo, five C₁₉-GAs GA₁, GA₄, GA₅, GA₆, GA₆₀ and two C₂₀ GAs, GA₁₉ and GA₄₄ were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development.     

Identification of gibberellin A1 in the embryo suspensor of Phaseolus coccineus

By combined gas chromatography-mass spectrometry the gibberellin present in suspensors of heart-shaped embryos of Phaseolus coccineus has been identified as Gibberellin A₁ (GA₁). The amount of GA₁ in 2000 suspensors (452 mg), as estimated by gas chromatography. was 4g. The presence of GA₁ in suspensors of P. coccineus is discussed in relation to our present knowledge of the occurrence of many gibberellins in developing seeds and immature fruits of the same species.Abbreviations FID flame ionization detector - GA gibberellin - GC gas chromatography - MS mass spectrometry - PGC preparative gas chromatography - Stage A heart-shaped embryo - Stage B cotytedonary embryo - TMS trimethylsilyl     

Activity of Some Hydrolytic Enzymes in Autolysis of the Embryo Suspensor in Tropaeolum majus L.

Assay of amino-peptidase, acid and alkaline phosphatase, esteraseand ß-glucosidase activity in the haustoria and suspensorof Tropaeolum majus embryos at different stages of developmentis made to evaluate the function of hydrolases in autolysisof the suspensor. Enzyme activities rise to a maximum sometimebetween heart-shaped and cotyledonary phases of embryo development. Tropaeolum majus L. nasturtium, embryology, suspensor, autolysis, hydrolytic enzymes     

Chlorophyll and cutin in early embryogenesis in Capsella,Arabidopsis, and Stellaria investigated by fluorescence microscopy

Advanced globular embryos of Capsella and early heart-shaped embryos of Arabidopsis begin to show chlorophyll fluorescence. It is not present in the suspensor, epiphysis, radicle and embryo of Stellaria. Cutin fluorescence appears on the protoderm of all advanced globular embryos. Fluorescence disappears during the early torpedo stage. It is not present on suspensors.     

Embryo-suspensor of Tropaeolum majus: Identification of gibberellin A63

Embryos and suspensors of Tropaeolum majus at the same stages of seed development were analysed for gibberellins by GC-MS. GA₆₃ was the only gibberellin present in both tissues. The amount of GA₆₃ in the suspensor is higher than in the embryo.     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein synthesis

The role of the suspensor in the early development of the dicot embryo has been described as merely an anchor or, conversely, as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor we have examined protein synthesis in early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos in tissue culture. Protein synthesis was examined in embryos and suspensors. Our results showed that in 0.2-mm embryos virtually all protein synthesis was dependent on an attached suspensor. Maximum protein synthesis in 0.5-mm embryos was observed when embryos were cultured attached to the suspensor. The levels were moderately decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M elicited the same protein diversity and greater [35S]methionine incorporation than did the attached suspensor in 0.2-mm embryos. Embryos of 0.5 mm did not appear to be differentially responsive to various gibberellin concentrations.     

Role of enzymes and identification of stage-specific proteins in developing somatic embryos of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Accumulation of proline, activities of peroxidase (POX), catalase (CAT), phenylalanine ammonia lyase (PAL) and malate dehydrogenase (MDH) were studied during different developmental stages of somatic embryos in chickpea. Callus cultures that did not form somatic embryos served as control. While increased levels of proline and POX activity were noticed in globular stages of embryos, CAT activity increased during early and late heart-shaped embryo formation indicating tissue-specific activation of these enzymes. The activity of PAL reached a peak during torpedo and cotyledonary stages of embryo development. On the other hand, MDH activity enhanced during the germination of somatic embryos inferring more requirement of energy during this stage. Electrophoretic (sodium dodecyl sulfate polyacrylamide gel electrophoresis) pattern of proteins revealed that ten bands are associated with non-embryogenic tissues, whereas 11 bands with globular, heart, torpedo and cotyledonary stages of embryo development and nine bands during the germination stage of embryos. Two extra stage-specific protein bands with molecular masses of 16 and 18 kDa appeared during globular, heart, torpedo, and cotyledonary stages. But, these bands disappeared during germination of embryos and are absent in non-embryogenic cultures. This study thus may help in the identification of proteins and the role of above enzymes during different developmental stages of somatic embryo induction and their maturation in a recalcitrant leguminous crop plant chickpea.     

Cytokinins and in vitro development of Phaseolus coccineus embryos

Phaseolus coccineus embryos at the heartshaped and the middle cotyledonary stages were cultured in vitro on media added with different concentrations of zeatin (Z) or zeatin riboside (Zr). Growth of early embryos was clearly favored by concentrations of Z from 10^-8 M to 10^-5 M, lower concentrations having no effect. Zr also promoted in vitro growth of early embryos, but in concentrations from 10^-12 M to 10^-10 M, higher concentrations being inhibitory. More developed embryos were scarcely sensitive to the presence in the culture medium of either Z or Zr at any concentration.Abbreviations Stage A heart-shaped embryo - stage B middle cotyledonary embryo - Z zeatin - Zr zeatin riboside     

DNA content of Phaseolus coccineus x P. vulgaris suspensors

Summary At the globular stage of embryo development, the level of DNA, as determined from microspectrophotometric analysis of Feulgen-stained squashes, was significantly higher in the interspecific hybrid suspensor than in suspensors from self-pollination of Phaseolus coccineus, the maternal parent. However, at the early-heart and early-cotyledonary stages of development, DNA content of interspecific hybrid suspensors was significantly lower than that of suspensors formed after self-pollination of either P. coccineus or P. vulgaris. The relationship between DNA content and suspensor cell length for P. coccineus and P. vulgaris at all developmental stages and between DNA content and cell area for P. coccineus at the early-cotyledonary stage was altered in hybrid suspensor cells. Nuclei in large cells of interspecific suspensors exhibited uneven distribution of polytene chromosomes and no clear nuclear outline.Paper number 18,470 of the Scientific Journal Series, University of Minnsota Agricultural Experiment Station, St. Paul, MN. This research was funded by the USDA CRGO under grant number USDA-85-CRCR-1-1676     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content

The role of the suspensor in the early development of the dicot embryo has not yet been defined. It has been described as merely an anchor and also as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor, early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos have been examined in tissue culture. It is known that Phaseolus embryos in culture at low osmotic potential will germinate precociously and that embryos in culture at high osmotic potential will either fail to grow or form callus. Optimum sucrose concentrations for continued, normal embryonic development of 0.2 mm and 0.5 mm P. vulgaris in tissue culture with Gamborg B5 medium were determined to be 12 and 6%, respectively. Protein content was examined in embryos and suspensors. Results showed that both 0.2- and 0.5-mm embryos required an attached suspensor for maximum protein content. Protein levels were substantially decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M restored the protein content to that of freshly excised embryos.     

无籽八月桔胚囊及胚胎发育细胞学观察

运用酶解振荡压片技术和常规石蜡切片技术分别研究了无籽八月桔的胚囊育性及无籽八月桔自交和异交(无籽八月桔×台湾椪柑,无籽八月桔×有籽八月桔)的胚胎发育.结果表明:无籽八月桔胚囊可育,成熟胚囊具一个卵细胞、两个助细胞、三个反足细胞以及一个大的含二个极核的中央细胞;其自交和异交的胚胎发育均正常,授粉后2周出现球形胚和少量心形...     

Turnover of cell-wall polysaccharides during somatic embryogenesis and development of celery (Apium graveolens L.)

Non-embryogenic cells (NEC) and embryogenc cells (EC) were separated from cell clusters derived from the hypocotyl segments of celery seedlings, which had been suspension-cultured in MS medium supplemented with 10⁵ M 2,4-D. The EC formed globular embryos in medium without 2,4-D. The globular embryo developed through heart-shaped, torpedo to cotyledonary embryos within 10 days. The EC and developing embryos were fractionated into symplastic [MeOH, hot water (HW), starch (S)] and apoplastic [pectin, hemicellulose, TFA (trifluoroacetic acid)-soluble and cellulose] fractions. The EC contained lower levels of sugar in the MeOH fraction and higher levels of starch than NEC. In the apoplastic fractions, there were no differences of total sugar amounts between NEC and EC. Cellulose contents were about 10% of the wall polysaccharides. During somatic embryogenesis, total sugar contents of the MeOH and HW fractions increased till the heart-shaped embryo stage, and then decreased during the torpedo and cotyledonary embryo stages. The sugar contents of the starch, pectin, TFA-soluble, and cellulose fractions did not change during the stages mentioned above. However, the hemicellulose substances remarkably increased during embryogenesis, and then decreased as the development proceeded. The neutral sugar components of the hemicellulosic fractions were analyzed. Arabinose increased markedly in EC to the globular embryo stage, but decreased as the development proceeded. Galactose increased only at the torpedo and cotyledonary embryo stages. Xylose was present at lower levels in all stages of embryogenesis than in the differentiated hypocotyl cell walls. These results suggest that there was a high turnover of arabinogalactan polysaccharides during embryogenesis, and that xylan accumulated in the cell walls of differentiated cells     

Development of white spruce somatic embryos: I. Storage product deposition

Summary The growth and development of white spruce somatic embryos was followed from the filamentous immature to the mature cotyledonary embryo stage. Histochemical examination of the various stages of embryo development showed that lipids, proteins, and polysaccharides were produced to varying degrees during the process. During early stages (1 to 2 wk on ABA), mostly polysaccharide was produced, whereas during later stages, polysaccharides, lipids, and protein accumulated. Electron microscopy indicated that lipid deposition in somatic embryos started during the first week after transfer to ABA-containing medium. Deposition of the storage products began at the basal end of the embryonal mass and within the proximal zone of the suspensors. Accumulation continued to the peripheral regions and then inward toward the cortex of the developing embryo. In all cases, polysaccharide accumulated first, followed by lipid and lastly, protein. Quantitatively, cotyledonary stage somatic embryos had less lipid and protein and more starch when compared to zygotic embryos at the same developmental stage. Total protein profiles elucidated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the majority of proteins were similar in zygotic and somatic embryos. Prominent protein bands were found at 30, 20, 19.5, 15, 14.4, 12, and 10 Kd. However, protein bands at 40, 15, and 12 Kd in total protein from somatic embryos were either absent or highly underexpressed.     

On the Embryonic Development and Ovule Culture of Interspecific Hybrids Between Populus simonii Carr and P.pyramidalis Borkh

The embryonic development following P. simonii Cart. × P. pyramidalis Borkh. is described in the present paper. The majority of pollen grains of P. pyramidalis Borkh. may germinate on the stigma of P. simonii Cam and the pollen tubes grow normally through the style and enter the embryo sac from the micropyle. Fertilization occurs as usual 4–7 days after pollination. A lot of proembryos and heart-shaped embryos are abortive; however, the others may develop normally and grow into mature embryos. Some of the endosperms appear normal and others may degenerate at free nuclear stage or cease to develop further at cellular stage. The ovules containg immature hybrid embryos of 19 days, 22 days, 26 days and 29 days after pollination at various developmental stages (heart-shaped stage, torpedo-stage and cotyledonary elongation stage) are excised and inoculated on nutrient agar for culture. The results show that: ( 1 ) Mll0 medium ( 1/2 MS+IAA 0.01 mg/L+BA 0.1 mg/L+sucrose 2% ) is the best of all the media used; (2) immature hybrid embryos of various developmental stages contained in ovules cultured in vitro may grow into normal plantlets.     

Somatic embryogenesis in Encephalartos cycadifolius

Callus cultures of Encephalartos cycadifolius were established from zygotic embryo explants on a modified B5 medium containing 1 mg l^–1 2,4-D and 1 mg l^–1 kinetin. Callus was transferred to media containing various combinations of 2,4-D and kinetin for improvement of somatic embryogenesis. Somatic embryos were produced on media with several growth regulator combinations. The somatic embryos developed from proembryos, which developed long suspensors. A dicotyledonary embryo formed at the distal end of the suspensor. The embryos turned green in light. When transferred to a medium containing 1 mg l^–1 ABA the somatic embryos matured. The suspensors desiccated and these embryos rooted when transferred to a medium without phytohormones.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid