Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Electroporation of intact embryonic leaflets of peanut: Gene transfer and stimulation of regeneration capacity

Summary Direct gene transfer into peanut intact embryonic leaflets was performed through electroporation. In transient β-glucuronidase expression assays, maximal expression was obtained by using pulses of 625 V cm⁻¹ in EPRm (modified electroporation) buffer supplemented with 75 μM NaCl. Kanamycin-resistant plants were obtained, and the presence of the nptII gene was demonstrated by PCR analysis. The positive effect of electroporation on the efficiency of in vitro regeneration was demonstrated. Explants submitted to field strengths between 500 and 625 V cm⁻¹ displayed a significantly increased number of shoots and originated faster growing calluses relative to control explants. Whereas in control explants callus formation occurred only at the petiolule, electroporated leaflets developed additional organogenic calluses on the foliar lamina. These authors have contributed equally to this work.     

Patterns of morphogenesis from cotyledon explants of pigeonpea

Summary Cotyledons excised from seedlings of Cajanus cajan (pigeonpea) were grown on media containing cytokinins (6-benzyladenine, zeatin, and zeatin riboside) and an allied compound, thidiazuron. With the exception of zeatin riboside, initial response in terms of induction of organized structures was very high. However, subsequent regeneration of shoots from cotyledon explants was very poor. Anatomical studies on the regenerating explants were undertaken to study the pattern of morphogenesis. Cytokinins and thidiazuron induced divisions in the epidermal and sub-epidermal cell layers leading to the formation of primary protrusions on the surface. This was followed by the development of foci of high meristematic activity either on the surface or within the primary protrusions. These foci differentiated into embryo-like structures or shoot meristem-like structures. Mostly aberrant shoots, with poorly developed apical meristems, regenerated from these structures.     

Changes in protein profile of pigeonpea genotypes in response to NaCl and boron stress

Two pigeonpea [Cajanus cajan (L.) Millsp.] genotypes, a salt tolerant Manak and a salt sensitive ICPL 88039 were subjected to stress treatment of 3 mM boron, 60 mM NaCl and boron + NaCl at the seedling stage. Radicle and plumule proteins were analyzed by SDS-PAGE. Boron treatment increased 28.3 kDa proteins in plumule and 38.3 and 51.9 kDa proteins in radicle of Manak, however, there was no specific protein in ICPL 88039 either in plumule or in radicle. In NaCl treatment 95.6 kDa proteins appeared in plumule and 67.5 kDa proteins in radicle of Manak. Conversely content of some proteins decreased by boron treatment alone or in combination with NaCl although they were present in the controls. Thus, 54.3 kDa protein disappeared in ICPL 88039 plumule, 68.4 kDa in Manak radicle and 28.1 kDa in ICPL 88039 radicle.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Variability in nuclear DNA content within pigeonpea,Cajanus cajan (Fabaceae)

4C DNA values have been estimated in 16 cultivars ofCajanus cajan by cytophotometry. The values range between 6.19 pg to 7.97 pg, a 23.6% variation. The cultivars form four groups which differ significantly from each other but have insignificant difference within them. The implications of this variability with respect to the heterogeneity and origin of this legume crop are discussed.     

Jasmonic acid induced changes in protein pattern, antioxidative enzyme activities and peroxidase isozymes in peanut seedlings

Protein pattern, ammonia content, glutamine synthetase activity, lipid peroxidation, superoxide dismutase, catalase, peroxidase and peroxidase isoforms were studied in the leaves and roots of 7-d-old peanut (Arachis hypogaea L. cv. JL-24) seedlings treated by 25, 100 and 250 μM jasmonic acid (JA). SDS-PAGE protein profile of leaves and roots after JA application showed a significant increase in 18, 21, 30, 45, 47 and 97.4 kDa proteins and significant decrease in 22 and 36 kDa proteins. Pathogenesis related PR-18 was specific in leaves at 250 μM JA and PR-21 have cross reacted differently with 21 and 30 kDa proteins in leaves and roots treated by all JA concentrations. Further, the immunoblot analysis with glutamine synthetase, GS-45 antibodies revealed a specific cross reaction with 45 and 47 kDa proteins of both control and JA treated leaves, however, higher at 100 and 250 μM JA treated leaves than control ones. Further, the malondialdehyde (MDA) content significantly increased in leaves and roots treated with JA, indicated membrane damage with JA treatments that led to the generation of peroxidation products. The peroxidase isozymic pattern showed two specific isoforms. Besides, the activities of SOD and catalase were significantly elevated in JA treated leaves.     

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 M 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.Abbreviations EA zygotic embryo axes - MS Murashige and Skoog (1962) medium - picloram 4-amino-3,5 - 6 trichloropicolinic acid     

Tolerance of peanut to excess boron

The tolerance ofArachis hypogaea cv. Shulamit to high concentrations of B in nutrient solution, [B]o, was determined under greenhouse conditions that promoted the production of vegetative dry matter. Plants grew in large containers in which a root zone of nutrient solution was separated from a pod zone of soil. Grain yield was reduced at a calculated [B]o-threshold of 0.29 mM, which was associated with a concentration of B in the vegetative shoots that was approximately four times larger than the control. Symptoms of B toxicity occurred on leaves as young as the third unfolded leaf from the shoot apex before the [B]o-threshold. Excess B caused a relatively larger decrease in pod number than in vegetative shoot weight, which was high in all treatments (78 g d.wt/plant) and it did not decrease single grain weight. It was suggested that the tolerance of grain development to excess B was a consequence of the high ratio of vegetative matter to pod number.     

Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.)

Summary Embryogenic masses were obtained from immature leaves of peanut (Arachis hypogaea L.) cultured on a medium containing 20 mg/l 2,4-D. Somatic embryos developed from these masses following transfer to a medium containing 3 mg/l 2,4-D. The embryo morphology was quite variable. Following transfer to hormone-free medium, these embryos germinated. Shoot elongation was obtained in 25% of the embryos following transfer to a medium supplemented with 0.5 mg/l each of BAP and Kn. The plants grown in vitro by this method survived in sand:soil mixture and were grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzyl amino purine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - Kn kinetin - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - Z zeatin     

Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana

In order to test an alternative selectable marker system for the production of transgenic peanut plants (Arachis hypogaea), the bacterial mercuric ion reductase gene, merA, was introduced into embryogenic cultures via microprojectile bombardment. MerA reduces toxic Hg(II) to the volatile and less toxic metallic mercury molecule, Hg(0), and renders its source Gram-negative bacterium mercury resistant. A codon-modified version of the merA gene, MerApe9, was cloned into a plant expression cassette containing the ACT2 promoter from Arabidopsis thaliana and the NOS terminator. The expression cassette also was inserted into a second vector containing the hygromycin resistance gene driven by the UBI3 promoter from potato. Stable transgenic plants were recovered through hygromycin-based selection from somatic embryo tissues bombarded with the plasmid containing both genes. However, no transgenic somatic embryos were recovered from selection on 50-100 micromol/L HgCl2. Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of transgenic peanut, but not in somatic embryos. Western blot analysis showed the production of the mercuric ion reductase protein in leaf tissues. Differential responses to HgCl2 of embryo-derived explants from segregating R1 seeds of one transgenic line also were observed.     

In vitro culture provides additional variation for pigeonpea

Summary The present study is an attempt to exploit somaclonal variation for the varietal improvement of pigeonpea [Cajanus cajan (L) Millsp.]. The pigeonpea plants were regenerated from cotyledon explants, and their progeny was screened for variability. The regenerated R1 plants exhibited a spectrum of alterations in floral morphology and architecture that were absent in the control population. The field-sown R2 plants segregated for traits such as flower color, leaf shape, seed size, color and strophiolation, flowering habit, and fertility. Tissue culture produced different mutational events resulting in both dominant and recessive alleles. Significant variation was observed for plant height, seed mass, and damage due to the insect pest Helicoverpa armigera. The R3 plants, obtained from seed of R2 generation selected for traits such as white seed coat, strophiolation, reduced plant height, seed mass and low damage due to Helicoverpa, maintained the traits when compared with the seed-derived control populations. The results indicate a definite gene for white seed coat and the possibility of additional genes for pest tolerance and high seed mass in an adapted background. Submitted as Journal Article No. 1906 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Comparison of anionic with cationic peroxidase from cultured peanut cells

A cationic and an anionic peanut peroxidase were isolated to purity as shown by 2D electrophoresis. Amino acid analysis offered evidence for differences. Variations between the isozymes were also noted in a slight difference in the heme absorption maxima, specific enzyme activity and particularly in the relative amount of each in the suspension medium measured by the heme absorption. In contrast the two isozymes were at least partially similar in their structure as demonstrated by the crossreaction with the antisera. The percent crossreactions were used in turn to amend the calculation for the synthetic rate of each isozyme. In spite of the difference in amount secreted in the suspension medium, the in vivo biosynthetic rate of the two isozyme measured cellularly is much the same.     

Exploratory integration of peanut genetic and physical maps and possible contributions from Arabidopsis

Arachis hypogaea is a widely cultivated crop both as an oilseed and protein source. The genomic analysis of Arachis species hitherto has been limited to the construction of genetic maps; the most comprehensive one contains 370 loci over 2,210 cM in length. However, no attempt has been made to analyze the physical structure of the peanut genome. To investigate the practicality of physical mapping in peanut, we applied a total of 117 oligonucleotide-based probes (overgos) derived from genetically mapped RFLP probes onto peanut BAC filters containing 182,784 peanut large-insert DNA clones in a multiplex experimental design; 91.5% of the overgos identified at least one BAC clone. In order to gain insights into the potential value of Arabidopsis genome sequence for studies in divergent species with complex genomes such as peanut, we employed 576 Arabidopsis-derived overgos selected on the basis of maximum homology to orthologous sequences in other plant taxa to screen the peanut BAC library. A total of 353 (61.3%) overgos detected at least one peanut BAC clone. This experiment represents the first steps toward the creation of a physical map in peanut and illustrates the potential value of leveraging information from distantly related species such as Arabidopsis for both practical applications such as comparative map-based cloning and shedding light on evolutionary relationships. We also evaluated the possible correlation between functional categories of Arabidopsis overgos and their success rates in hybridization to the peanut BAC library.Electronic Supplementary Material Supplementary material is available for this article at     

Plant regeneration from immature embryos of peanut

Plant regeneration from immature embryos of peanut (Arachis hypogaea L.) can be accomplished through somatic embryogenesis. The highest frequency of somatic embryo formation occurred on B5 medium plus 0.5–1.0 mg/l picloram. Shoots and plants developed from the somatic embryos only after extended culture on basal medium. Shoots were excised from thick embryonic roots and rerooted on Murashige and Skoog medium containing half the normal concentration of inorganic salts. This technique should be useful for the production of interspecific hybrid plants from immatureArachis embryos.     

Soil amelioration effects on peanut growth,yield and quality

Summary In order to study the reasons for poor peanut (Arachis hypogaea L.) performance on an Avalon medium sandy loam, a three year field study was undertaken to investigate the effects of lime and gypsum applications on growth, yield and quality. Rates of up to 2,400 kg agricultural lime/ha/annum significantly increased soil pH (1N KCl) and exch. Ca and decreased levels of exch. Al and Al saturation in the soil (0–150 mm). The effect of the same rates of gypsum was much less marked, only exch. Ca increasing and Al saturation decreasing to any substantial extent. In the absence of lime (i.e. even where gypsum was applied). nodulation was poor and the plants developed a general chlorosisc. 90 days after planting. Liming markedly improved nodulation whereas annual applications of gypsum had the opposite effect. Liming significantly increased the hay, pod and kernel yields by up to 73, 105 and 117%, respectively. On average, gypsum applications had no significant effects. Liming increased shelling percentage, the percentage mature pods, 100-kernel mass and protein concentration in the kernel, and decreased the incidence of pops and kernels with black plumule. Applications of gypsum had little effect on quality except for a decreased incidence of black plumule. It appeared that the improved crop performance with liming resulted from a reduction in Al toxicity which improved nodulation. Calcium deficiency did not appear to be a major cause of poor peanut growth and quality in the unameliorated soil.     

Comparative identification by fatty acid analysis of soil,rhizosphere, and geocarposphere bacteria of peanut (Arachis hypogaea L.)

Bacterial isolates were collected from the geocarposphere, rhizosphere, and root-free soil of field grown peanut (Arachis hypogaea L.) at three sample dates, and the isolates were identified by analysis of fatty acid methyl-esters to determine if qualitative differences exist among the bacterial microflora of these zones. Five bacterial genera were associated with isolates from soil, while pod and root isolates constituted 16 and 13 genera, respectively, indicating that bacterial diversity was higher in the rhizosphere and geocarposphere than in soil. The dominant (most frequently identified) genus across all three samples dates was Flavobacterium, for pods, Pseudomonas for roots, and Bacillus, for root-free soil. Sixteen bacterial taxa were only isolated from the geocarposphere, 7 only from the rhizosphere, and 5 only from soil. These results show that specific bacterial taxa are preferentially adapted to colonization of the geocarposphere and suggest that the soil, rhizosphere, and geocarposphere constitute three distinct ecological niches. Bacteria which colonize the geocarposphere should be examined as potential biological control agents for pod-invading fungi such as the toxigenic strains of Aspergillus flavus and A. parasiticus.     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid