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.     

Stage-specific nitrogen metabolism in developing carrot somatic embryos

The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and ¹⁴C-labeling. ¹⁵N NMR spectroscopy was used to examine the uptake and incorporation of ¹⁵N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α-amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non-embryogenic cells and germinating embryos were glutamine and α-amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y-aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon-14 labeling indicated that labeled glutamine pools in non-embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non-embryogenic cells and germinating stage embryos. Furthermore, during early to mid-embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.     

ABA associated biochemical changes during somatic embryo development in Camellia sinensis (L.) O. Kuntze

The effect of ABA on reserve accumulation in maturing somatic embryos of tea was compared with and without ABA treatment. Changes in the levels of starch, total soluble sugars (TSS), proteins, and phenols were studied in the somatic embryos at different stages of development (globular, heart, torpedo and germinating embryos) in order to investigate whether ABA could trigger accumulation of storage reserves and thereby overcome the problem of poor germination. After ABA treatment (5.0 mg l(-1)) for 14 days, the starch and protein contents that were negligible in the untreated embryos increased by several fold with a simultaneous increase in TSS. When ABA treatment occurred at the heart stage, the germination of the embryos also improved, relative to untreated controls, after ABA treatment. ABA treatment prior to or after heart stage did not improve somatic embryo germination.     

Abortion and determination of stages for embryo rescue in crosses between sweet-potato,Ipomoea batatas Lam. (2n=6x=90) and its wild relative,I. trifida (H. B. K.) G. Don. (2n=2x=30)

Summary The frequency of aborted fruits and the changes and abnormalities that occur during the embryo development in intraspecific crosses of sweet-potato Ipomoea batatas (2n=6x=90) and interspecific crosses between I. batatas and I. trifida (2n=2x=30) were investigated in order to study the causes of the low seed production. Three genotypes of I. batatas and 18 genotypes of I. trifida were intermated. The frequency of aborted fruits was below 25% in the intraspecific crosses and over 90% in the interspecific crosses. Paraffin sections were used to examine the developmental stages of fruits and seeds. Embryos in different developmental stages were observed to determine the stage of abortion. These observations permitted the identification of developmental stages of embryo rescue in interspecific crosses. There were no significant differences in the frequency of embryo abortion before the early globular stage among female sweet-potato progenitors for the intraspecific and interspecific crosses. The frequency of the late occurrence of embryo abortion (when embryo abortion occurs after the pre-globular stage) was higher in interspecific crosses (19.1%) than in intraspecific crosses (5.5%). The frequency of the late occurrence of embryo abortion in interspecific crosses was higher at the globular stage (9.6%) than at the heart stage (4.3%). Providing that embryo rescue is conducted in interspecific crosses, the estimated number of potentially viable embryos could be increased: 30 times with embryos at the globular stage; 20 times with embryos at the heart stage; and 11 times if embryos at the torpedo stage were used for the rescue with respect to the seed set. The results suggested that the appropriate time for embryo rescue in interspecific crosses is at the globular stage. If embryos could be rescued at the globular stage, it would be possible to increase the number of surviving embryos up to 30 times in interspecific crosses and 0.02 times in intraspecific crosses with respect to natural conditions without embryo rescue.This research was initiated during sabbatical of M.I. at the Asian Vegetable Research and Development Center (AVRDC) in Taiwan     

Stage-specific protein regulation during somatic embryo development of Carica papaya L. ‘Golden’

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Organization of initial stages of somatic embryogenesis in tissue culture of Citrus sinensis cv. Tarocco at the organismal level

Four-step protocol was established for the in vitro regeneration of Citrus sinensis cv. Tarocco somatic embryos that were morphologically similar to small somatic embryos in vivo. The regeneration procedure comprises a mechanical destruction of embryogenic culture to obtain proembryogenic cell masses (PEMs) (step 1) followed by culturing on three different media (steps 2–4). The approach developed allows in vitro simulating somatic embryogenesis by dividing this process into three partially independent steps: PEM → globular somatic embryo → heart-shaped somatic embryo → somatic embryo with developed cotyledons. The highest frequency of morphogenetic stage transition was 64, 40, and 26%, respectively. It was shown that the first step (PEM → globular embryo) was associated with the formation of heterogeneous population of spherical bodies 50–500 μm in diameter, among which about 40% were somatic embryos at globular stage. The scheme is offered of alternative pathways for the development of spherical bodies in vitro, and interrelations between their sizes and ability to direct morphogenesis are discussed.     

Distribution of Cytosolic mRNAs Between Polysomal and Ribonucleoprotein Complex Fractions in Alfalfa Embryos : Stage-Specific Translational Repression of Storage Protein Synthesis during Early Somatic Embryo Development

Cell-free translational and northern blot analyses were used to examine the distribution of storage protein messages in the cytoplasmic polysomal and mRNA-protein complex (mRNP) fractions during development of somatic and zygotic embryos of alfalfa (Medicago sativa cv Rangelander RL-34). No special array of messages was identified in the mRNP fraction; however, some messages were selectively enriched in either the polysome or mRNP fractions, and their distribution pattern varied quantitatively during development of the embryos. During the earliest stages of somatic embryo development, storage protein messages already were present, but there was no detectable accumulation of the proteins. Selective enrichment of messages for the 11S, 7S, and 2S storage proteins occurred in the mRNP fraction during the globular, heart, and torpedo stages of somatic embryogenesis, but the distribution pattern was shifted toward the polysomal fraction at the beginning of cotyledon development. Thus, there was translational repression of storage protein synthesis at the early stage of somatic embryo development that was relieved later. During the cotyledonary development stages in the somatic and zygotic embryos, storage protein synthesis and distribution of the messages were similar in that these specific messages were predominantly in the polysomal fraction.     

Optimizing sweet potato [Ipomoea batatas (L.) Lam.] root and plantlet formation by selection of proper embryo developmental stage and size,and gel type for fluidized sowing

Potassium starch polyacrylamide, potassium acrylate, a copolymer of potassium acrylate and acrylamide, and hydroxyethylcellulose carrier gels were tested to find a fluid drilling material suited for synthetic seeding of sweet potato (Ipomoea batatas (L.) Lam.) somatic embryos. Somatic embryo developmental stage and size, and maturation (incubation) time were also evaluated to improve plantlet formation. All embryos suspended in the fluidized hydroxyethylcellulose gel were viable after six days and 7% developed into plantlets after two weeks. Up to 97% of the somatic embryos suspended in acrylate and/or acrylamide gels died within six days. Root development was at least 10% and plantlet development at least 30% greater when embryos were subcultured on basal medium for 16 instead of 25 days prior to placement and suspension in hydroxyethylcellulose gel. Up to 25% more plantlets were obtained from embryos at the elongated torpedo stage than those at the cotyledonary or torpedo stages of development. When suspended in hydroxyethylcellulose gel embryo length had no effect on the percentage of plantlets obtained.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium (1962) - PC copolymer of potassium acrylate and acrylamide - PSA potassium starch polyacrylamide - PA potassium acrylate - HEC hydroxyethylcellulose - ODR oxygen diffusion rate Florida Agr. Expt. Sta., Journal Series No. R-00253 Mention of proprietary products is for convenience of reader only, and does not constitute endorsement by the University of Florida     

A new approach to direct somatic embryogenesis in Medicago

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA Abscisic acid - B5 Medium of Gamborg et al.(1968) - COT Cotyledone stage embryos - 2,4-D 2,4-dichlorphenoxyacetic acid - FW Fresh weight - GA3 Gibberellin A3 - MS Medium of Murashige and Skoog (1962) - PEG Polyethylene Glycol - POLY Polyembryos     

组织透明技术在植物解剖学和植物发育生物学实验教学中的应用

整体组织透明和组织切片技术是研究植物组织和器官的结构及其发育过程的常用实验手段。该文针对本科生植物学课程的侧根、胚珠(特别是胚囊)以及胚胎发育内容设计了相应的实验教学课程。该实验以拟南芥幼苗、不同发育时期的花蕾和种子为材料,经固定、脱色和组织透明,利用正置显微镜观察侧根、胚珠以及胚胎的发育过程。根组织经透明后,在显微镜下可以清晰地观察到从侧根原基的建成到侧根发生的八个发育阶段。各个发育时期的胚珠经组织透明后,在显微镜下可以清晰地观察到大孢子母细胞、功能性大孢子(FG1期)以及FG2~FG7期的胚囊。各个发育时期的种子经透明后,在显微镜下可以清晰地观察到胚胎发育的1细胞期、8细胞期、球形期、心形期、鱼雷期、拐杖期和成熟期。这些实验结果可以使学生对拟南芥等双子叶植物的侧根、胚珠以及胚胎发育过程有较为系统的了解。该实验方法在其他较小的双子叶植物中也适用。     

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     

New Embryo Lethals in Arabidopsis thaliana: Basic Genetic and Morphological Study

Six different mutations with defects in immature seed development have been identified during screening of a T-DNA collection of Arabidopsis thaliana. The mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development, and therefore they belong to the embryo-lethal class of mutants. The genetic and morphological studies of T-DNA mutations affecting embryo development are presented. The youngest embryos with a defect were observed at the globular stage in the VIII-64 mutation. Externally located cells, precursor of the protoderm, were characterised by abnormal cell division. VIII-41 mutation with a defect at the late globular stage was arrested at the globular-heart stage transition. VIII-111 mutation showed defect at heart stage of embryogenesis with atypical development of cotyledon primordia. The defect was associated with abnormal pattern of cell division constituting the precursor of the shoot apical meristem. In VIII-82 mutation defect in torpedo stage with asymmetric cotyledons was observed. Cotyledon stage of embryos and chlorophyll defect were observed in VIII-75 mutant. Abnormal suspensor consisting of two columns of cells was observed in 280-4-4 mutation. Newly identified embryo-lethals can serve as starting material for more detailed genetic and molecular studies.     

Somatic embryogenesis in Vicia faba L.

The paper describes a method of somatic embryo induction in callus and suspension cultures of Vicia faba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichlorophenoxyacetic acid. The medium with 2.5 M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.     

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine (Vitis vinifera L.): Embryonic Organogenesis

Somatic embryos ofVitis vinifera L. ‘Grenache noir’develop abnormally and form viable plantlets at very low frequencies.They grow continuously and, after the torpedo stage, they formgiant structures which do not undergo further organogenesis.Morphological, histological and cytochemical data were usedto study development from the globular to the giant-embryo stage.Histological organization of somatic embryos until the torpedostage was similar to that of zygotic embryos. Somatic embryosformed bipolar axes, which differentiated precociously and simultaneouslya root and a shoot meristem. However, they differed from theirzygotic homologues by forming a cotyledonary crown or multiplecotyledons and by their rapid cellular differentiation. At theend of the torpedo stage and up to the giant-embryo stage, somaticembryos underwent some characteristic events of germination:the radical grew, tannins accumulated, and protodermal cellssuberized. However the shoot apex was rapidly disorganized anddisappeared. This peculiar behaviour is discussed in comparisonwith the phenomenon of precocious germination often observedfor immature zygotic embryos inin vitro culture. Vitis vinifera ; grapevine; somatic embryo development; precocious germination; histology     

Nuclear DNA content of somatic and zygotic embryos ofVitis vinifera cv. Grenache noir at the torpedo stage

Summary A flow cytometric analysis and an in situ DNA microspectrophotometric study were made concomitantly to establish why somatic grapevine (Vitis viniferacv. Grenache noir) embryos showed a low level of conversion into plantlets. In somatic embryos at the torpedo stage and in zygotic embryos at the same stage of development, ploidy level, DNA content per 2 C nucleus, and the cell-cycle state of the shoot apical meristem were examined. The frequency distribution histograms of nuclear DNA values were similar in the two types of embryos. At the torpedo stage both types of embryos had a majority of nuclei with 2 C DNA content equal to 1.6pg. In the shoot apices of somatic and zygotic embryos, DNA microspectrophotometry showed preferential blockage of the cell cycle at the G_0–1 stage; however, 20% of somatic embryo shoot apices were blocked at the G_0–2 stage. Analogies between somatic embryos and their zygotic homologues were shown. The genetical and environmental causes of the low level of conversion of grapevine somatic embryos into plantlets are discussed. Our work suggests that the in vitro culture conditions which were used could be incompatible with normal morphogenesis from the torpedo stage.     

花楸体细胞胚发生过程中抗氧化酶活性的变化

花楸体细胞胚发生过程中,胚性愈伤组织可溶性蛋白含量高于其他类型的愈伤组织,非胚性愈伤组织中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均高于其他类型的愈伤组织;SOD、POD活性均在胚性细胞向球形胚转化时下降,球形胚向心形胚发育时下降,心形胚向鱼雷形胚和鱼雷形胚向子叶形胚发育时再升高;CAT活性变化规律与SOD和POD活性变化不同,从胚性细胞到鱼雷形胚的3个发育时间内表现为下降-升高-下降的趋势,鱼雷形胚向子叶胚发育时略有回升。据此认为,SOD酶活性降低似可作为花楸胚性细胞分化以及胚胎早期发育的一个判断指标。     

Somatic embryony patterns and plant regeneration inIpomoea batatas poir

Summary Patterns of somatic embryo development from callus were elucidated inIpomoea batatas Poir. (sweet potato). Embryos at three stages of development were able to grow into plants. They included embryos arrested at the late torpedo and cotyledonary stages, and an arrested torpedo embryo that resulted from precocious hypocotyl expansion. Early torpedo-stage embryos rooted on reculture but did not form shoots. A diversity of other embryo stages were also produced which upon reculture formed only adventitious embryos and roots. Morphologic variants with similar growth potential were noted among embryos at similar stages of development. Plasticity in somatic embryo developmental patterns led to different forms that were mature enough to produce plants. This work has been funded in part by an IFAS/Gas Research Institute cooperative grant. Florida Agricultural Experiment Station Journal Series No. 8789.