Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

Quantification of the geocarposphere and rhizosphere effect of peanut (Arachis hypogaea L.)

Roots and pods of field-grown peanut (groundnut) (Arachis hypogaea L.) were sampled at the R3, R5, and R7 developmental stages and examined in comparison to root- and pod-free soil for microbial population densities to assess the geocarposphere and rhizosphere effects. G/ S (no. geocarposphere microorganisms/no. soil microorganisms) and R/S (no. rhizosphere microorganisms/no. soil microorganisms) ratios were calculated for total fungi,Asperigillus flavus, spore-forming bacilli, coryneform bacteria, fluorescent pseudomonads, and total bacteria isolated on low- and high-nutrient media. A clear geocarposphere effect was evidenced by increased population densities of bacteria and fungi associated with developing pods compared to soil. G/S and R/S ratios were generally greater than 1.0 for all groups of microorganisms except bacilli. G/S ratios were greater for total bacteria than for total fungi at two of the three sample times, suggesting that bacteria were stimulated more than fungi in the zone around developing pods. In contrast, R/S ratios, were higher for total fungi than for total bacteria at two of three sample times. The preferential association of fungi and bacteria with early developmental stages of the pod indicates that some microorganisms are particularly well adapted for colonization of the peanut geocarposphere. These microorganisms are logical candidates for evaluation as biological control candiates forA. flavus.     

Utilization of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Synthetic amphidiploids were established in 32 combinations involving 8 diploid wild species representing both A and B genomes of section Arachis. Bivalent and multivalent associations in the amphidiploids of 7 A genome species confirm that these species have identical genomes. Contrastingly, high bivalent frequencies in amphidiploids involving the A and B genome species suggest that A. batizocoi has a distinct B genome that is partially homologous to the other genome A represented in the rest of the species. Crossability, chromosome pairing and pollen and pod fertility in hybrids between A. hypogaea and amphidiploids have revealed that these amphidiploids can be used as a genetic bridge for the transfer of genes from the wild species into the cultivated groundnut.Submitted as Journal Article No. 530 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Light, dark and growth regulator involvement in groundnut (Arachis hypogaea L.) pod development

Gynophore elongation and pod formation were studied in peanut plants (Arachis hypogaea L.) under light and dark conditions in vivo. The gynophores elongated until pod formation was initiated. Pod (3–20 mm length) development could be totally controlled by alternating dark (switched on) and light (switched off) conditions, repeatedly. Gynophore elongation responded conversely to light/dark conditions, compared to pods. In this study we aimed to correlate the light/dark effects with endogenous growth substances. The levels of endogenous growth substances were determined in the different stags of pod development. Gynophores shortly after penetration into the soil, white gynophores, released twice the amount of ethylene as compared to the aerial green ones, or to gynophores bearing pods. Ethylene inhibitors had no effect on the percent of gynophores that developed pods, but affected pod size which were smaller compared to the control. A similar level of IAA was extracted from gynophore tips of green gynophores, white gynophores and pods. ABA levels differed between the three stages and were highest in the green gynophores and lowest in the pods.Abbreviations ABA abscisic acid - AOA aminooxyacetic acid - ELISA enzyme linked immunosorbent assay - Ethrel 2-chloroethanephosphonic acid - GC gas chromatography - HPLC High Performance Liquid Chromatography - IAA indole-3-acetic acid - NAA naphthalene acetic acid - RIA radioimmunoassay - STS silver thiosulfhate - TIBA 2,3,6-triiodobenzoic acid     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

Utilization of wild relatives in genetic improvement of Arachis hypogaea L.

Summary The chromosome complements of 12 taxa in section Arachis were karyotypically and meiotically analysed. In taxa with 2n=20 the arm ratio of the respective pair of chromosomes was taken as an independent quantitative character and statistically analysed by Mahalanobis D². Two clusters were formed, one represented solely by A. batizocoi and the other consisting of the remaining 11 taxa. This grouping was confirmed by canonical analysis. In the larger group of species, A villosa and A. correntina were closely related karyotypically and on D² distance, while A. cardenasii forms a distinct subgroup. A. cardenasii lacks the short A chromosome recorded in other species of this group, and A. batizocoi is no longer the only species to have a pair of chromosomes with a secondary constriction. The taxa with 2n=40, A. monticola and A. hypogaea, are karyotypically very similar, though there is a difference in the number of chromosome pairs with a secondary constriction. On the basis of karyomorphological affinity, especially in relation to marker chromosomes, A. cardenasii is probably one of the ancestors of the tetraploid species studied.Approved as ICRISAT Journal Article No. 169 and released for publication     

Uptake and partitioning of cadmium by cultivars of peanut (Arachis hypogaea L.)

Cadmium has been found to accumulate in peanut (Arachis hypogaea) kernels to levels exceeding the current maximum permitted concentration in Australia of 0.1 mg kg^-1. Little is known of the mechanisms of Cd uptake into kernels by cultivars of peanut, so the aims of the experiments reported here were to determine if Cd is absorbed directly through the pod wall or via the main root system, and if differences exist between cultivars in this respect. Split-pot soil and sand/nutrient solution experiments were performed with two cultivars of peanut (cv. NC7 and Streeton) known to accumulate Cd to different levels in the kernel. The growth medium was separated into pod and root zones with Cd concentrations in each zone varied. In confirmation of previous field trial results, cv. NC7 had higher concentrations of Cd in kernels, given the same Cd levels in the external medium (solution or soil). Despite total Cd uptake by cv. NC7 being similar to cv. Streeton, cv. NC7 appeared to retain more Cd in the roots and translocate less Cd to shoots. Results from both soil and sand/solution culture indicated that the dominant path of Cd uptake by peanut was via the main root system, with direct pod uptake contributing less than 5% of the total Cd in the kernel. There was little difference between cultivars in this characteristic. This indicates that unlike Ca nutrition of peanuts, agronomic techniques to manage Cd uptake will require modification of soil to the full depth of root exploration, rather than just the surface strata where pods develop. Cadmium concentrations in testa were up to an order of magnitude higher than in the kernel, indicating that blanching of kernels would be effective in reducing Cd in the marketed product. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression of a synthetic cry1EC gene for resistance against Spodoptera litura in transgenic peanut (Arachis hypogaea L.)

The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). S. litura is susceptible to the chimeric delta-endotoxin Cry1EC reported earlier. De-embryonated cotyledon explants of peanut were transformed using Agrobacterium tumefaciens strain EHA101 harboring a synthetic cry1EC gene driven by the CaMV 35S promoter. Transgenic plants of peanut with a single copy insertion of cry1EC were selected in the T(0) generation by Southern blot hybridization. Real-time PCR, Western blot and ELISA analysis indicated that expression of the cry1EC gene was higher in single copy T(1) plants. Immunoassay showed expression of Cry1EC up to 0.13% of total soluble protein in T(1) plants. Leaf feeding bioassay on highly expressing transgenic lines showed 100% killing of larvae at the 2(nd) instar stage of S. litura. This is the first report of transgenic peanut plants with resistance to S. litura.     

Utilisation of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Cross-compatibility of species in section Arachis Krap. et Greg. nom. nud., and chromosome pairing and pollen fertility in their interspecific F₁ hybrids were studied to further understand the phylogenetic relationships among these species. Except those with A. batizocoi Krap. et Greg. nom. nud., hybrids between diploid species have near normal bivalent frequency (9.1–9.8) and moderate to high pollen fertility (60–91%). Hybrids between A. batizocoi and other species have low bivalent frequency (5.2–6.9) and very low pollen fertility (3–7%). These results confirm the earlier separation of these species into two groups based on karyomorphology and Mahalanobis D² calculated on arm ratios. These studies also provide a picture of relative affinities between A. batizocoi, the lone member of one cluster, and the other species, and among the rest of the species. They also indicate that the basic chromosome complement in the two groups of species is the same. Chromosome pairing in triploid hybrids, (A. hypogaea L. X diploid wild species), suggests that A. batizocoi is the closest diploid relative of A. hypogaea. It is closer to A. hypogaea subspecies fastigiata Waldron than to A. hypogaea subspecies hypogaea Krap. et. Rig. Other diploid species of the section Arachis are equidistant from A. hypogaea, and have the same genome which has strong homology to one of the genomes of A. hypogaea. Based on the present results, the two tetraploid species, A. monticola Krap. et Rig. and A. hypogaea can be recognised as two forms of the same species. Breeding implications have been discussed in the light of chromosome behaviour observed in hybrids of A. hypogaea X diploid species, and on the presumptions that A. hypogaea has an AABB genomic constitution, and that among the diploid species, the B genome is present in A. batizocoi while the A genome is common to the other diploid species of section Arachis.Submitted as Journal Article No. 328 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Diallel analysis in groundnut (Arachis hypogaea L.)

Summary An 8 × 8 full diallel experiment based on 4 bunch plus 4 spreading types of groundnut (Arachis hypogaea L.) was conducted over three environments. For both number of pods and pod yield, additive, nonadditive and reciprocal cross effects were detected and these were also influenced by changes in environments. For number of pods additive genetic variance was predominant whereas it was approximately equal to non-additive genetic variance for pod yield. Graphical analysis revealed the presence of strong non-allelic interaction for number of pods whereas for pod yield absence of dominance and/or presence of non-allelic interaction was evident.Part of Ph.D. Thesis of the first author     

The influence of zinc and sulphur deficiency on oil-filling in peanut (Arachis hypogaea L.) kernels

In the developing peanut (Arachis hypogaea L.) kernels, the period between 15 and 35 days after podding (DAP) was identified as the active period of oil-filling. The period of active oil-filling was associated with a decrease in the starch, soluble sugars and proteins so as to make available the energy and carbon skeleton for the synthesis of oil. The oil content in the mature kernels decreased by 11, 12 and 25 per cent with Zn, S and Zn+S deficiency, respectively. In addition, proteins and starch content decreased significantly while that of soluble sugars increased slightly. The activity of malate dehydrogenase and glucose-6-phosphate dehydrogenase also decreased due to Zn as well as S deficiency. The deficiency treatments resulted in a decrease in phospholipids, free fatty acids and triacylglycerols in mature kernels. Further the proportion of 16∶0 and 18∶2 decreased while that of 18∶1 increased in developing kernels.     

Boron mobility in peanut (Arachis hypogaea L.)

In most plant families, boron (B) is phloem immobile. For plants such as peanut which bury their fruit, the mechanism for B delivery and the B source for fruit and seed growth remains enigmatic. Therefore, this study aimed to establish evidence of B retranslocation in peanut and to identify its importance in plant development. In a sand culture experiment, the increase in B contents in new organs after B withdrawal and the corresponding decline in B contents in older organs was evidence of B redistribution. In a foliar ¹⁰B experiment, the ¹⁰B abundance of treated-leaves decreased and ¹⁰B was detected in leaves and flowers formed after the application of foliar B. Application of ¹⁰B to the roots for a period also provided evidence for the retranslocation of ¹⁰B accumulated during the first growth period. The ¹⁰B abundance in older plant parts declined and ¹⁰B appeared in new organs (flowers, pegs, leaves) that had developed after the ¹⁰B supply had been replaced by ¹¹B. In the fourth experiment, foliar application of B reduced hollow heart, a symptom of B deficiency in seeds, in cv. TAG 24 from 39 to 8% and in Tainan 9 from 63 to 18%. These experiments all provide evidence for B retranslocation in peanut, but further work on the relative importance of the xylem and phloem pathways for B loading into the fruit is needed.     

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.     

Evaluation of peanut (Arachis hypogaea L.) leaflets from mature zygotic embryos as recipient tissue for biolostic gene transfer

Leaflets from mature peanut embryos are a useful recipient tissue for biolistic DNA transfer. Fertile plants were regenerated from leaflets from genotypes representing all botanical types of peanut. Regeneration frequency was strongly influenced by genotype. NPT II and GUS chimaeric gene fusions, driven by the CaMV 35S promoter, were expressed transiently following biolistic delivery to unexpanded leaflets. Bombardment conditions affecting transient expression frequency were determined using a prototype of the Bio Rad PDS 1000/He helium-powered particle acceleration apparatus. Stably transformed calli were derived routinely from leaflet tissue bombarded with the NPT II gene and subsequently cultured on kanamycin. Several plants have been regenerated from treated explants under kanamycin selection. Thus far, none of these has been stably transformed. The occurrence of escapes suggests that kanamycin is an inefficient selective agent for the recovery of transgenic peanuts from this explant. Experiments designed to regenerate plants using published regeneration protocols from stably transformed calli, devoid of primary explant tissue, have been unsuccessful.     

Early generation intermating for yield improvement in groundnut (Arachis hypogaea L.)

Summary Seeds of 4 crosses of groundnut (Arachis hypogaea L.), Robut 33-1 x Chico, Robut 33-1 x NC Ac 17090, Robut 33-1 x PI 298115 and MK 374 x GAUG 1, were irradiated with 30 kR. In the F₁, some branches of each plant were intermated with other plants at random and others selfed in each cross to produce S₂ and F₂ seeds. They were evaluated for pod yield, shelling percentage and 100-kernel weight. The frequency of plants superior to F[in1] was much higher in S₂ than in F₂, which was, in general, true for the values of yield and its components. The S₂ and F₂ were advanced to third generation by selfing. The families descending from S₂ showed clear superiority over those from F₂. The reason for such superiority was suggested to be the recombination of genes from the upper and lower ends of the genotypic distribution under intermating.     

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     

Influence of salinity on mineral nutrition of peanut (Arachis hypogea L.)

Summary Effect of sodium chloride and sodium sulphate salinities on growth and mineral nutrition of peanut (A. hypogea L.) variety TMV-10 has been studied. Both salts suppressed growth of the plants. The inorganic analysis revealed that NaCl and Na₂SO₄ caused accumulation of Na, P, Fe and Mn in root, stem, leaf and gynophore. NaCl treatment caused accumulation of Cl in these parts. The uptake of K was hampered by both salts whereas Ca uptake was retarded mainly by Na₂SO₄. The results are discussed in relation to the salt tolerance capacity of the plant.