This study contributes to understanding the whole process of somatic embryogenesis, but two specific questions remain to be answered: what factors are involved in the reactivation of the somatic cells at the beginning of the initial callogenesis, and why do the somatic embryos not accumulate proteins in their tissues during maturation?

While callogenesis in date palm can be initiated by culturing immature leaf segments on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), it is difficult to obtain callus from certain genotypes. To better understand the mechanisms of callogenesis in date palm, we studied the developmental events underlying this process at the cellular level. The callogenic capacity of leaf segments depends on the state of cell differentiation. The most callogenic segments were within the leaf elongation zone, required polar auxin transport to initiate callogenesis and contained the highest quantities of free endogenous indole-3-acetic acid. At the cellular level, callus induction involves two spatially and temporally separated events. The first event involves cells from the fascicular parenchyma in an equatorial plane perpendicular to the vascular axis that within 2 days of culture in the presence of 2,4-D, acquire structural and ultrastructural features typically observed in meristematic cells. The second event occurs 3 days later and is characterised by the modification of adjacent perivascular sheath cells. The latter cells become callogenic in that they reinitiated their cell cycles and undergo cell division leading to callus formation. These data provide evidence that callus initiation in leaf vascular tissue of date palm involves a sequential response of two distinct cell types to auxin and requires polar auxin transport.

Cultivated peanut and synthetics are allotetraploids (2n = 4x = 40) with two homeologous sets of chromosomes. Meiosis in allotetraploid peanut is generally thought to show diploid-like behavior. However, a recent study pointed out the occurrence of recombination between homeologous chromosomes, especially when synthetic allotetraploids are used, challenging the view of disomic inheritance in peanut. In this study, we investigated the meiotic behavior of allotetraploid peanut using 380 SSR markers and 90 F2 progeny derived from the cross between Arachis hypogaea cv Fleur 11 (AABB) and ISATGR278-18 (AAKK), a synthetic allotetraploid that harbors a K-genome that was reported to pair with the cultivated B-genome during meiosis. Segregation analysis of SSR markers showed 42 codominant SSRs with unexpected null bands among some progeny. Chi-square tests for these loci deviate from the expected 1:2:1 Mendelian ratio under disomic inheritance. A linkage map of 357 codominant loci aligned on 20 linkage groups (LGs) with a total length of 1728 cM, averaging 5.1 cM between markers, was developed. Among the 10 homeologous sets of LGs, one set consisted of markers that all segregated in a polysomic-like pattern, six in a likely disomic pattern and the three remaining in a mixed pattern with disomic and polysomic loci clustered on the same LG. Moreover, we reported a substitution of homeologous chromosomes in some progeny. Our results suggest that the homeologous recombination events occurred between the A and K genomes in the newly synthesized allotetraploid and have been highlighted in the progeny. Homeologous exchanges are rarely observed in tetraploid peanut and have not yet been reported for AAKK and AABB genomes. The implications of these results on peanut breeding are discussed.

Fruit and seed size are important yield component traits that have been selected during crop domestication. In previous studies, Advanced Backcross Quantitative Trait Loci (AB-QTL) and Chromosome Segment Substitution Line (CSSL) populations were developed in peanut by crossing the cultivated variety Fleur11 and a synthetic wild allotetraploid (Arachis. ipaensis × Arachis. duranensis)4x. In the AB-QTL population, a major QTL for pod and seed size was detected in a ~5 Mb interval in the proximal region of chromosome A07. In the CSSL population, the line 12CS_091, which carries the QTL region and that produces smaller pods and seeds than Fleur11, was identified. In this study, we used a two-step strategy to fine-map the seed size QTL region on chromosome A07. We developed new SSR and SNP markers, as well as near-isogenic lines (NILs) in the target QTL region. We first located the QTL in ~1 Mb region between two SSR markers, thanks to the genotyping of a large F2 population of 2172 individuals and a single marker analysis approach. We then used nine new SNP markers evenly distributed in the refined QTL region to genotype 490 F3 plants derived from 88 F2, and we selected 10 NILs. The phenotyping of the NILs and marker/trait association allowed us to narrowing down the QTL region to a 168.37 kb chromosome segment, between the SNPs Aradu_A07_1148327 and Aradu_A07_1316694. This region contains 22 predicted genes. Among these genes, Aradu.DN3DB and Aradu.RLZ61, which encode a transcriptional regulator STERILE APETALA-like (SAP) and an F-box SNEEZY (SNE), respectively, were of particular interest. The function of these genes in regulating the variation of fruit and seed size is discussed. This study will contribute to a better knowledge of genes that have been targeted during peanut domestication.

In date palm (Phoenix dactylifera L. cv. Ahmar, Arecaceae), as for many monocotyledons, callogenesis is a prerequisite for the initiation of somatic embryogenesis, and requires the presence of auxin in the medium. Immature leaf explants were cultivated in medium supplemented with either 1 or 54 lM 1-naphtaleneacetic acid in order to induce either rhizogenesis or callogenesis. Histological studies performed throughout the culture period established that precocious cell reactivation is similar in both morphogenetic pathways. Early cytological modifications are associated with cell reactivation and are observed in the pluripotent cells of perivascular sheaths. Divergence between the callogenesis and rhizogenesis pathways is observed later, during the subsequent determination and morphological differentiation phases. We established that in date palm, the rhizogenesis and callogenesis pathways are initiated from the same cell type, the ultimate developmental fate depending upon auxin concentration.

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This study provides a physiological analysis of somatic embryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80%) and Amsekhsi (76%) appeared highly callogenic, whereas Tijib (10%) and Amaside (2%) produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2 mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5 mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings.

Cultivated peanut is an allotetraploid (2n = 4× = 40) with narrow genetic diversity. In previous studies, we developed an advanced backcross quantitative trait loci (AB-QTL) population from the cross between the synthetic allotetraploid ((Arachis ipaensis × Arachis duranensis)4×) and the cultivated variety Fleur11, and mapped several quantitative trait loci (QTLs) involved in yield and yield components. We also developed a chromosome segment substitution line (CSSL) population as a way to mendelize the QTLs and analyzing their effects. In this study, 16 CSSLs were used for assessing the contribution of wild alleles in yield performance and stability across environments, as well as validating QTLs for pod and seed size. The CSSLs and the recurrent parent Fleur11, used as a check, were assessed using an alpha lattice design in three locations during two consecutive rainy seasons in Senegal, totaling six environments. Our results showed that the chromosome segments from the wild species, in general, have no yield disadvantage and contributed positive variation to yield-related traits. Most of the QTLs detected for pod and seed size in the AB-QTL on linkage groups A07, A08, A09, and B06 were also found in the CSSLs, showing that the CSSLs used in this study are accurate material for QTL validation. Several new QTLs have also been identified. Two CSSLs (12CS_031 and 12CS_069) showed consistently higher pod and seed size than Fleur11 in all environments, suggesting that the QTLs were consistent and stable. Our study opens the way for pyramiding these QTLs for peanut improvement.