Peanut Embryos Research Articles

Identification of Key Gene Networks and Deciphering Transcriptional Regulators Associated With Peanut Embryo Abortion Mediated by Calcium Deficiency.

Peanut embryo development is easily affected by a variety of nutrient elements in the soil, especially the calcium level. Peanut produces abortive embryos in calcium-deficient soil, but underlying mechanism remains unclear. Thus, identifying key transcriptional regulators and their associated regulatory networks promises to contribute to a better understanding of this process. In this study, cellular biology and gene expression analyses were performed to investigate peanut embryo development with the aim to discern the global architecture of gene regulatory networks underlying peanut embryo abortion under calcium deficiency conditions. The endomembrane systems tended to disintegrate, impairing cell growth and starch, protein and lipid body accumulation, resulting in aborted seeds. RNA-seq analysis showed that the gene expression profile in peanut embryos was significantly changed under calcium deficiency. Further analysis indicated that multiple signal pathways were involved in the peanut embryo abortion. Differential expressed genes (DEGs) related to cytoplasmic free Ca2+ were significantly altered. DEGs in plant hormone signaling pathways tended to be associated with increased IAA and ethylene but with decreased ABA, gibberellin, cytokinin, and brassinosteroid levels. Certain vital genes, including apoptosis-inducing factor, WRKYs and ethylene-responsive transcription factors, were up-regulated, while key regulators of embryo development, such as TCP4, WRI1, FUS3, ABI3, and GLK1 were down-regulated. Weighted gene co-expression network analysis (WGCNA) identified 16 significant modules associated with the plant hormone signaling, MAPK signaling, ubiquitin mediated proteolysis, reserve substance biosynthesis and metabolism pathways to decipher regulatory network. The most significant module was darkolivegreen2 and FUS3 (AH06G23930) had the highest connectivity among this module. Importantly, key transcription factors involved in embryogenesis or ovule development including TCP4, GLK1, ABI3, bHLH115, MYC2, etc., were also present in this module and down regulated under calcium deficiency. This study presents the first global view of the gene regulatory network involved in peanut embryo abortion under calcium deficiency conditions and lays foundation for improving peanut tolerances to calcium deficiency by a targeted manipulation of molecular breeding.

Integrated microRNA and transcriptome profiling reveals a miRNA-mediated regulatory network of embryo abortion under calcium deficiency in peanut (Arachis hypogaea L.)

BackgroundPeanut embryo development is a complex process involving a series of gene regulatory pathways and is easily affected by various elements in the soil. Calcium deficiency in the soil induces early embryo abortion in peanut, which provides an opportunity to determine the mechanism underlying this important event. MicroRNA (miRNA)-guided target gene regulation is vital to a wide variety of biological processes. However, whether miRNAs participate in peanut embryo abortion under calcium deficiency has yet to be explored.ResultsIn this study, with the assistance of a recently established platform for genome sequences of wild peanut species, we analyzed small RNAs (sRNAs) in early peanut embryos. A total of 29 known and 132 potential novel miRNAs were discovered in 12 peanut-specific miRNA families. Among the identified miRNAs, 87 were differentially expressed during early embryo development under calcium deficiency and sufficiency conditions, and 117 target genes of the differentially expressed miRNAs were identified. Integrated analysis of miRNAs and transcriptome expression revealed 52 differentially expressed target genes of 20 miRNAs. The expression profiles for some differentially expressed targets by gene chip analysis were consistent with the transcriptome sequencing results. Together, our results demonstrate that seed/embryo development-related genes such as TCP3, AP2, EMB2750, and GRFs; cell division and proliferation-related genes such as HsfB4 and DIVARICATA; plant hormone signaling pathway-related genes such as CYP707A1 and CYP707A3, with which abscisic acid (ABA) is involved; and BR1, with which brassinosteroids (BRs) are involved, were actively modulated by miRNAs during early embryo development.ConclusionsBoth a number of miRNAs and corresponding target genes likely playing key roles in the regulation of peanut embryo abortion under calcium deficiency were identified. These findings provide for the first time new insights into miRNA-mediated regulatory pathways involved in peanut embryo abortion under calcium deficiency.

Peroxidase characterization isolated from germinated peanut embryos (GPE) and application of the freeze-dried GPE powder as enzyme source for biomimetic production of δ-viniferin

Novel enzyme source is essential in biomimetic synthesis. Use of peanut peroxidase in mediation of oxidative polymerization of phenolic compounds is meager. In this study, the 5 day-germinated peanut embryos were homogenized, centrifuged to collect supernatant and (NH4)2SO4 fractionated, the 40–75% saturated fraction exhibited most peroxidase activities. After UF-concentration between MWCO 300 and 10 kDa, Sephacryl S300 gel filtration and DEAE-Sephadex A25 ion-exchange, a 50.6-fold-purified peroxidase with 32 kDa in SDS-PAGE analysis was obtained. The freeze-dried and pulverized into powder (GPEP) bearing potent peroxidase activities was used in biomimetic resveratrol dimerization. In a semi-continuous system mediated by 0.5 g GPEP suspended in phosphate buffer (pH 7.4) with continuous feedings of resveratrol dissolved in acetone and aqueous H2O2 solution for 150 min, all added resveratrol reacted. After subjecting the reactant to SPE C18 cartridge clean-up, semi-HPLC purification and NMR 1- and 2D spectroscopic elucidation, a dimeric product was identified as δ-viniferin with ca. 60% of recovery. The δ-viniferin exhibited potent trolox equivalent antioxidant capacity as did BHT. It is of merit to demonstrate GPEP as a potential peroxidase source enabling scalable biomimetic production of δ-viniferin.

Small RNA profiling and degradome analysis reveal regulation of microRNA in peanut embryogenesis and early pod development

BackgroundAs a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored.ResultsIn this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM 5′-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively.ConclusionsLarge numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

Erratum to: Comparative transcriptome analysis of basal and zygote-located tip regions of peanut ovaries provides insight into the mechanism of light regulation in peanut embryo and pod development.

Erratum to: Comparative transcriptome analysis of basal and zygote-located tip regions of peanut ovaries provides insight into the mechanism of light regulation in peanut embryo and pod development.

Comparative transcriptome analysis of basal and zygote-located tip regions of peanut ovaries provides insight into the mechanism of light regulation in peanut embryo and pod development

BackgroundPeanut zygotes typically divide a few times to form a pre-embryo before further embryonic development halts under normal day/night photoperiods. Ovary elongation, however, continuesforming a downward growing peg-like structure. When the peg is buried in the soil, embryo development resumes in the darkness. The embryo-located region (ER) of the peg begins to enlarge and form a pod, while the basal region (BR) of the peg has a distinct fate. The molecular mechanisms governing these unique embryo development processes are unknown.ResultsIn this study, histological analysis demonstrated that from 4 days after pollination to 3 days after soil penetration, the peanut pre-embryo remained morphologically similar. By 9 days after soil penetration, the embryo had changed to a globular embryo. Transcriptome analysis revealed differentially expressed genes in the ER and BR before and after peg soil penetration. In addition to light signaling and plant hormone metabolism genes, we identified differentially expressed genes in the ER that contribute to embryo development and pod formation processes, including MADS-box transcription factors, xyloglucan endotransglucosylase/hydrolase protein, cellulose synthase, homeobox-leucine zipper (HD-Zip) protein family genes, amino acid permease, and seed growth and embryo morphogenesis regulators (DA1, TCP3, and YABBY).ConclusionsA large number of genes were found to be differentially expressed in the ER and BR across three developmental peg stages. Exact changes in gene expression were also identified in the ER during early embryo and pod development. This information provides an expanded knowledgebase for understanding the mechanisms of early peanut pod formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2857-1) contains supplementary material, which is available to authorized users.

The Seed Biotinylated Protein of Soybean (Glycine max): A Boiling-Resistant New Allergen (Gly m 7) with the Capacity To Induce IgE-Mediated Allergic Responses.

Soybean is a common allergenic food; thus, a comprehensive characterization of all the proteins that cause allergy is crucial to the development of effective diagnostic and immunotherapeutic strategies. A cDNA library was constructed from seven stages of developing soybean seeds to investigate candidate allergens. We searched the library for cDNAs encoding a seed-specific biotinylated protein (SBP) based on its allergenicity in boiled lentils. A full-length cDNA clone was retrieved and expressed as a 75.6-kDa His-tagged recombinant protein (rSBP) in Escherichia coli. Western immunoblotting of boiled bacterial extracts demonstrated specific IgE binding to rSBP, which was further purified by metal affinity and anion exchange chromatographies. Of the 23 allergic sera screened by ELISA, 12 contained IgEs specific to the purified rSBP. Circular dichroism spectroscopy revealed a predominantly unordered structure consistent with SBP's heat stability. The natural homologues (nSBP) were the main proteins isolated from soybean and peanut embryos after streptavidin affinity purification, yet they remained low-abundance proteins in the seed as confirmed by LC-MS/MS. Using capture ELISAs, the soybean and peanut nSBPs were bound by IgEs in 78 and 87% of the allergic sera tested. The soybean nSBP was purified to homogeneity and treatments with different denaturing agents before immunoblotting highlighted the diversity of its IgE epitopes. In vitro activation of basophils was assessed by flow cytometry in a cohort of peanut-allergic children sensitized to soybean. Stronger and more frequent (38%) activations were induced by nSBP-soy compared to the major soybean allergen, Glym5. SBPs may represent a novel class of biologically active legume allergens with the structural resilience to withstand many food-manufacturing processes.

Isolation and Characterization of Inositol Tetraphosphate 1-Kinase (AhITPK1) and Inositol 1,4,5-Tris-Phosphate Kinase (AhIPK2) Gene in Peanut

In the current study, partial cDNA clones of inositol tetraphosphate 1-kinase (ITPK1) and inositol 1,4,5-trisphosphate kinase/inositol polyphosphate multikinase (IPK2), were isolated from embryo using RT-PCR and designated as AhITPK1 and AhIPK2 isoforms of the gene. The partial cDNA sequence of AhITPK1 and AhIPK2 genes have an open reading frame (ORF) of 1146 and 891bp respectively and showed high similarity to other plant genes. AhITPK1 shared high homology with Aradu. Q95MC of Arachis duranensis, had a single exon with no introns and belonged to ATP-grasp family of proteins. AhIPK2 shared high similarity with Aradu.24V9G of A. duranensis and contained three exons with 5’ and 3’ UTR’s on either side. Unlike other IPK2 genes, AhIPK2 possessed conserved domains such as PxxxDxKxG and [L/M][I/V]D[F/L][A/G][H/K]. Phylogenetic analysis grouped AhITPK1 with A. duranensis, A. ipinensis and Oryza brachyantha into one cluster, whereas AhIPK2 was grouped along with Cucumis melo and C. sativus. Evolutionarily, AhITPK1 and AhIPK2 were genetically distinct from other plant genera. Furthermore, real-time PCR analysis revealed high expression of AhITPK1 and AhIPK2 genes in the peanut embryo and flower bud. For the first time AhITPK1 (KR778986) and AhIPK2 (KR778988) genes belonging to phytic acid pathway from Arachis hypogaea were identified and characterized the expression pattern of these two isoforms on different tissues. These genes were found to be abundant in flower bud and embryo. Results suggest that embryo development significantly influences the expression of the two AhIPK isoforms in peanut. Evolutionarily they were found to be distinct from their parental species. This study is an important step toward understanding the role of these two AhIPK isoforms in phytic acid synthesis. However, future research involving RNAi-based functional characterization is warranted to establish their link to embryo development in peanut.

Identification of low Ca(2+) stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL).

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca(2+) deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up-regulated cell wall hydrolases and down-regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down-regulated under Ca(2+) -deficient conditions from 15days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8'-hydroxylases, key enzymes for ABA catabolism, were up-regulated by 21-fold under Ca(2+) -deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over-expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca(2+) deficiency-induced embryo abortion via ABA-mediated apoptosis. The results elucidated the mechanism of low Ca(2+) -induced embryo abortion and described the method for other fields of study.

Transgenic Peanut (Arachis hypogaea L.) Expressing the Urease Subunit B Gene of Helicobacter pylori

Helicobacter pylori (H. pylori) has been identified as the main pathogenic factors of chronic gastritis and peptic ulcer, and the Class I carcinogen of gastric cancer by WHO. Vaccine has become the most effective measure to prevent and cure H. pylori infection. The UreB is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. UreB antigen gene was cloned into the binary vector pBI121 which contains a seed-specific promoter Oleosin of peanut and a kanamycin resistance gene, and then UreB gene was transformed into peanut embryo leaflets by Agrobacter-mediated method. The putative transgenic plants were examined for the presence of UreB in the nuclear genome of peanut plants by PCR analysis. Expression of UreB gene in plants was identified by RT-PCR and Western blot analysis. These results suggest that the UreB transgenic peanut can be potentially used as an edible vaccine for controlling H. pylori.

Regulation of oleosin expression in developing peanut (Arachis hypogaea L.) embryos through nucleosome loss and histone modifications

Nucleosome loss and histone modifications are important mechanisms for transcriptional regulation. Concomitant changes in chromatin structures of two peanut (Arachis hypogaea L.) oleosin genes, AhOleo17.8 and AhOleo18.5, were examined in relation to transcriptional activity. Spatial and temporal expression analyses showed that both AhOleo17.8 and AhOleo18.5 promoters can adopt three conformational states, an inactive state (in vegetative tissues), a basal activated state (in early maturation embryos), and a fully activated state (in late maturation embryos). Chromatin immunoprecipitation assays revealed an increase of histone H3 acetylation levels at the proximal promoters and coding regions of AhOleo17.8 and AhOleo18.5 associated with basal transcription in early maturation embryos. Meanwhile, a decrease of histone H3K9 dimethylation levels at coding regions of oleosins was observed in early maturation embryos. However, a dramatic decrease in the histone acetylation signal was observed at the core promoters and the coding regions of the two oleosins in the fully activated condition in late maturation embryos. Although a small decrease of histone H3 levels of oleosins chromatin was detected in early maturation embryos, a significant loss of histone H3 levels occurred in late maturation embryos. These analyses indicate that the histone eviction from the proximal promoters and coding regions is associated with the high expression of oleosin genes during late embryos maturation. Moreover, the basal expression of oleosins in early maturation embryos is accompanied by the increase of histone H3 acetylation and decrease of histone H3K9me2.

Varian Kualitatif Kacang Tanah Hasil Kultur in Vitro dan Hasil Seleksi in Vitro (Qualitative Variants of Peanut Plants Obtained from in-Vitro Culture and in-Vitro Selection)

issue cultures that have passed callus phase can induce somaclonal variation, of which the intensity was influenced by the addition of selective agents to culture media. Somaclonal variations of peanut plant obtained from in-vitro cultured and in-vitro selected somatic embryos using PEG was not yet understood. The objectives of this research were 1) to identify the qualitative variants of peanut plant var. Kelinci obtained from in-vitro cultured and in-vitro selected somatic embryos using PEG, 2) to estimate the factors that control the qualitative variants. The non-selected and the selected variant somatic embryos of peanut were germinated and cultured. From fertile R0 lines, sufficient number of R1 and R2 progenies were grown for evaluation. The results showed that phenotypic variations of qualitative characters can be observed among R0, R1 and R2 generations of somaclonal lines. Variant phenotypes of qualitative characters can be observed, these include wide branching, excessive branching, leaf variegation, leaflet number abnormality, leaf pointed tip, ‘rosette’ leaf, complete sterility and partial sterility. The data indicated that wide branching, excessive branching, leaflet number abnormality, partial sterility and complete sterility were genetically controlled, while variant phenotypes of ‘rosette‘ leaf, leaf variegation, and leaf pointed tip were epigenetically controlled. Key words: somaclonal variant, qualitative characters, in vitro selection, in vitro culture

Corrigendum

Plant Biotechnology JournalVolume 6, Issue 2 p. 210-211 Open Access Corrigendum First published: 03 January 2008 https://doi.org/10.1111/j.1467-7652.2007.00312.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Dodo H. W., Konan K. N., Chen F. C., Egnin M. and Viquez O. M. (2007) Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. The corresponding author's email should be hortense.dodo@aamu.edu. Figure 2 and its caption should be as follows: Figure 2Open in figure viewerPowerPoint Steps involved in the production of fertile transgenic peanut plants using pDK28, an Ara h 2 RNAi transformation vector. (a) Map of pDK28 binary plasmid mobilized into A. tumefaciens EHA105. (b) Germinated peanut embryos for preparation and co-culture of hypocotyl explants. (c) Selection of Kanamycin-resistant (Kr) shoots. (d) Rooting of regenerated Kr plants. (e) Transfer to soil and flowering. (f) Seeds from Kr peanut plants. In the section under ‘Regeneration of fertile transgenic peanut’, Figure 2 is cross-referenced incorrectly on page 137. The correct cross-reference should be ‘Kanamycin-sensitive plants turned white and died within 4 weeks, while kanamycin-resistant plants remained green and grew healthy (Figure 2c). A total of 59 kanamycin-resistant plants (Ara h 2-T0) were successfully rooted (Figure 2d) and transferred to pots in the Enconair growth chamber. Flowering occurred within 6 weeks following the transfer to the growth chamber (Figure 2e). Flowers were produced on both controls obtained by (i) direct peanut seed planting in soil (wild type) and (ii) tissue culture without Agrobacterium infection. The phenotype, plant growth and reproduction of the putative transgenic plants were indistinguishable from that of wild-type control. Peg elongated from the plants, and peanut pods were produced within 3–4 months (Figure 2f).’ In the section under ‘PCR and Southern hybridization confirmed the stable transformation of kanamycin-resistant peanut plants’, the second last sentence should read, ‘Analyses of the single digest (S) reveal that all the selected transgenic plants [plant #12 (19 pods), plant #23 (9 pods), plant #32 (4 pods), plant #39 (11 pods), plant #45 (7 pods), plant #55 (31 pods), plant #66 (3 pods) and plant #6 (25 pods)] carried each a single insert with a similar pattern of gene integration.’ Figure 5 should be labelled as follows: Figure 5Open in figure viewerPowerPoint Determination of Ara h 2 in peanut samples. (a) Sandwich ELISA shows the percentage of Ara h 2 in peanut total protein extracts. Numbers followed by the same letters are not statistically different at P < 0.05. (b) SDS-PAGE shows the protein profiles and (c) Western immunoblot analysis in crude peanut extracts to detect the presence of the Ara h 2 protein. Hybridization was performed using an Ara h 2 specific monoclonal antibody. About 0.5 µg purified Ara h 2 was loaded. WT, wild-type control sample; TCc, tissue culture non-transgenic control sample; (–), sample from a Southern negative plant; NT, not tested. Reference Dodo H. W., Konan K. N., Chen F. C., Egnin M. and Viquez O. M. (2007 ) Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnol. J. 6, 135– 145. Google Scholar Volume6, Issue2February 2008Pages 210-211 FiguresReferencesRelatedInformation

Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method

After pre-culture and treatment of osmosis, zygotic embryos of peanut (Arachis hypogaea L.) were transformed via particle bombardment with a plasmid containing a Bluetongue VP2 gene (BTVP2) comprising neutralizing epitopes. Selection for Kanamycin resistant calluses and somatic embryos was initiated at 12th day post-bombardment on medium containing 25 mg/L Kanamycin. Under continuous selection, 12.38% Kanamycin resistant plantlets were regenerated from bombarded somatic embryos. The presence and integration of BTVP2 DNA in regenerated Kanamycin resistant plants were confirmed by southern hybridization assay using non-radioactive Digoxiginin BTVP2 probe. β-Glucuronidase (GUS) enzyme activity was detected in transgenic somatic embryos but not from control, non-transformed embryos. The expression of the BTVP2 protein was confirmed through RT-PCR (reverse transcription polymerase chain reaction) using the RNA isolated from the transgenic callus employing BTVP2-specific primers. The production of transgenic peanut was mainly focused on evaluating a newly improved somatic embryogenesis regeneration system as well as the gene transfer method and to produce the Bluetongue outer coat protein that comprises the neutralizing epitopes.

A genetic engineering strategy to eliminate peanut allergy

Peanut allergy is an IgE-mediated hypersensitivity reaction with an increasing prevalence worldwide. Despite its seriousness, to date, there is no cure. Genetic engineering strategies can provide a solution. The post-transcriptional gene silencing (PTGS) model can be used effectively to knock out the production of allergenic proteins in peanut by specific degradation of the endogenous target messenger RNA (mRNA). Ara h 2, the most potent peanut allergenic protein, was selected as a model to demonstrate the feasibility of this concept. Transgenic peanut plants were produced via microprojectile-mediated transformation of peanut embryos using a plasmid construct, which contains a fragment of the coding region of Ara h 2 linked to an enhanced CaMV 35S constitutive promoter. Molecular analyses, including polymerase chain reaction and Southern blots, confirmed the presence of the stable integration of the Ara h 2 transgene into the peanut genome. Northern hybridization showed the expression of the Ara h 2 transgene in all vegetative tissues of the mature transgenic peanut plants, indicating the stable expression of the truncated Ara h 2 transgene throughout the development of the plants. It is, therefore, reasonable to expect that the truncated Ara h 2 transgene transcripts will be synthesized in the seeds and will trigger the specific degradation of endogenous Ara h 2 mRNA. The next step will be to grow the transgenic peanut plants to full maturity for seed production and to determine the level of allergen Ara h 2.

Cloning and sequencing of the gene Ahy-β encoding a subunit of peanut conarachin

A cDNA expression library of mid-maturation cotyledons of peanut ( Arachis hypogaea L. Shanyou 523) was constructed to clone the genes of peanut seed storage proteins. Six clones of cDNA were obtained by immunoscreening with a polyclonal antibody against a 65 kD subunit of conarachin from peanut seeds. The six cDNA clones could be divided into two groups, Ahy- α and Ahy-β. Furthermore, a complete cDNA sequence of Ahy- β was determined by using rapid amplification of cDNA ends (RACE). Northern blot analysis showed that Ahy- β was expressed only in developing peanut embryos. Sequence analysis revealed that the Ahy- β gene with 2507 bp was composed of four exons with 703, 176, 81 and 882 bp and three introns with 71, 72 and 73 bp. The deduced amino acid encoded a protein with 614 residues. Several well characterized elements for promoter were observed in the 342 bp promoter region of Ahy-β and included two TATA-boxes, four CAAT-boxes, a vicilin-box, a G-box, a RY-1 element, two RY-2 elements and a P-element, while a poly-A signal and two additional stop codons existed in the 3′UTR.

Temporal and Spatial Distribution of Hormones in Peanut Ovules During Early Development

Abstract Study of hormonal regulation of fruit development requires quantitative measurement of reproductive tissues at different developmental stages. This study reports the application of ELISA (enzyme-linked immunosorbent assay) for the measurement of free indole-3-acetic acid (IAA), dihydrozeatin riboside (DHZR) and abscisic acid (ABA) in ovules of peanut (Arachis hypogaea L., cv. NC 6). IAA levels in apical and basal ovules gradually increased with aerial peg elongation from 4 to 10 d after pollination (DAP), but decreased when peg elongation ceased by 14 DAP. Higher DHZR levels were observed in aerial ovules at 10 DAP, and the ABA level was highest at 1 DAP. Aerial apical ovules contained higher IAA and DHZR levels than basal ovules. At day 1 after soil penetration (DASP), IAA and ABA levels in both ovules decreased, whereas DHZR levels increased. Peg tip swelling and ovary enlargement at 5 DASP were accompanied by increased levels of all three hormones. The highest levels of the hormones were observed in both ovules at 7 DASP, i.e., when embryos were at the globular stage and ovaries were rapidly enlarging to form pods. Basal ovules also had higher hormonal levels than apical ovules after soil penetration. Progressive changes in hormone levels during peanut ovule and embryo development suggest a physiological relationship between the two events.

In vitro regulation of morphogenesis in peanut ( Arachis hypogaea L.)

Callusing, caulogenesis, in vitro flowering and somatic embryogenesis were induced from the base of leaflets derived from mature embryos of peanut ( Arachis hypogaea) by altering the hormonal composition of the Murashige and Skoog's (MS) basal medium. A combination of 4 mg/l alpha napththaleneacetic acid (NAA) and 5 mg/l 6-benylaminopurine (BAP) was optimum for inducing caulogenic buds. The caulogenic buds proliferated in medium with 3 mg/l BAP. Differentiation of these buds to shoots was achieved in MS basal medium with 0.5 mg/l each of BAP and kinetin (KN). Shoot buds and flower buds were produced when caulogenic buds were cultured on medium containing 1 mg/l BAP and 1 mg/l KN, prior to elongation. Clonally propagated plantlets derived from axillary buds elongated, formed roots and were grown to maturity in soil. Embryogenic mass formation was induced from the leaf base in the presence of 20 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos developed upon reducing 2,4-D to 3 mg/l.

Effect of genotype on somatic embryogenesis from axes of mature peanut embryos

Genotypes representing the three botanical varieties of peanut (Arachis hypogaea L.) were assessed for somatic embryogenesis and subsequent plant conversion from mature zygotic embryo axes. Explants were initially cultured on Murashige and Skoog medium supplemented with 12.42 μM 4-amino-3,5,6-trichloropicolinic acid. Individual somatic embryos wer isolated from explant tissue and used to initiate repetitive liquid cultures. There were significant differences among genotypes and varieties for somatic embryo formation and plant regeneration using a single media sequence. Botanical variety fastigiata had a lower embryogenic frequency and produced significantly fewer embryos than either hypogaea or vulgaris, which were similar in response.

Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants

Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T1 progeny contained T-DNA. Molecular characterization of one primary transformant and its T1 and T2 progeny plants established that T-DNA was integrated into the host genome.