The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

Genetic control of domestication traits in pearl millet (Pennisetum glaucum L., Poaceae)

Quantitative trait loci (QTLs) controlling the morphological differences between pearl millet (Pennisetum glaucum ssp. glaucum) and its wild ancestor (Pennisetum glaucum ssp. monodii, form mollissimum) were investigated in a cultivated/wild F₂ population by means of RFLP markers. The most critical adaptive changes resulting from the domestication process involved the spikelet structure: non-shedding seeds with reduced bracts and bristles and long involucral pedicel. Major differences also concerned characters describing the plant architecture, phenology and spike sizes. Many morphological differences could be attributed to the effect of a small number of loci with relatively large effects. These loci are mainly concentrated on four linkage groups (2, 5, 6 and 7). The loss of shedding ability, due to the absence of a functional abscission layer, is controlled by a single locus on linkage group 6 (al6). Genetic control of the other spikelet traits involved factors with large effects which are located in the region of linkage group 6 close to al6 and to an esterase gene, Esterase-E. Moreover, QTLs with large effects on plant and spike morphology traits such as plant height, number of spikes and weight of the spike were also mapped on linkage groups 6 and 7. This strong linkage of factors in the domestication syndrome may be involved in the maintenance of the phenotypic identity of wild and cultivated populations in sympatry. This result also brings new arguments in the understanding of the domestication process of this allogamous crop. Received: 8 March 1999 / Accepted: 29 April 1999     

Longevity of pearl millet (Pennisetum glaucum) seeds harvested at different stages of maturity

The storage potential of seeds harvested at weekly intervals after controlled pollination was studied in three diverse cytoplasmic male sterile pearl millet (Pennisetum glaucum) lines. In the first experiment in 1989, a comparison of p₅₀ (time for viability to decline to 50% during storage) among seeds of the line DSA 105A harvested 14, 21, 28, 35 and 42 days after pollination (DAP), and then stored at 35°C with 15% moisture content or 40°C with 13% moisture content, showed that those harvested 35 DAP had the greatest longevity. In the second experiment in 1990, a comparison of p₅₀ within the lines 5141A and L 67A harvested 28, 35 and 42 DAP, and then stored at 40°C with 13% moisture content, showed that seeds of both lines harvested 42 DAP had the greatest longevity. In both the seasons, and in all three lines, maximum seed longevity (p₅₀) was attained one week after physiological maturity (defined as the end of the grain filling period), which is therefore the optimum time of harvest to obtain good quality seeds for conservation.     

Genetic control of weediness traits and the maintenance of sympatric crop-weed polymorphism in pearl millet (Pennisetum glaucum)

Pearl millet (Pennisetum glaucum), a diploid outcrossing crop widely grown in semiarid tropics, provides a unique extant material for the study of crop-weed interactive evolution. Co-occurrence of a weedy, shattering type of pearl millet with the cultivated one is the rule in the traditional agro-ecosystem in the Sahel zone of Africa. Selfed progeny of weed-type plants invariably segregated into distinct weed and crop types in an approximately 3:1 ratio. Genetic analysis using a cleaved amplified polymorphic sequence (CAPS) marker strongly suggested that a series of differences between the crop and the weed types are determined by a single putative supergene that has two allelic types, C and W. The crop-type plants are CC homozygotes, and the weed-type plants are CW heterozygotes. WW homozygotes are sterile and rare in the field. Thus, the CW weed plants recurrently arise from crosses between the crop and the weed, as well as from crosses among the weed-type plants. The weed type appears to have a sufficiently high fitness to maintain the W allele in the pearl millet population, resulting in the perpetuation of this unique crop-weed polymorphism.     

Genotype identification and assessment of genetic relationships in pearl millet [Pennisetum glaucum (L.) R. Br] using microsatellites and RAPDs

 The potential of DNA markers such as microsatellites, minisatellites and RAPDs was investigated in pearl millet [Pennisetum glaucum (L.) R. Br] with respect to their abundance and variability. Southern analysis, using 22 different di-, tri-, tetra- and penta-oligonucleotide probes and five minisatellite probes, identified (GATA)₄ as the most useful probe for the detection of multiple polymorphic fragments among pearl millet cultivars and landraces from India. The clustering patterns of pearl millet cultivars and landraces based on (GATA)₄ and RAPD (randomly amplified polymorphic DNA) markers differed. The landraces, representing eight states in India, could not be grouped based on their geographical distribution with the DNA markers. RAPD analysis revealed a high degree of genetic diversity among the cultivars and landraces employed in this study. The probability of an identical match by chance for any two genotypes using (GATA)₄ and RAPDs was 3.02×10^-20 for cultivars and 5.2×10^-9 for landraces. The microsatellite (GATA)₄ and RAPDs provide useful tools for genotype identification and for the assessment of genetic relationships in pearl millet. Received: 19 October 1997 / Accepted: 9 December 1997     

The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet)

Summary Genetic analysis was conducted on the qualitative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pennisetum glaucum (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23 BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (e₁) and yellow stripe (ys). Tissue culture regenerant populations (R₀) and progeny populations (R₁) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R₀, R₁, and R₂ progeny of the genetic marker line. In a population of 1,911 plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expression of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flowering time. The incidence of chlorophyll deficiency increased with time in culture, 51 % of the progeny regenerated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also observed in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmentation and a total of 6% showed chlorophyll variation in the first generation (S₀). The presence of qualitative variation in controls suggests that the inherent variation present in the original explant was expressed and perpetuated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R₁) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic.     

Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers

Sex as a factor affecting recovered recombination in plant gametes was investigated in pearl millet, Pennisetum glaucum, by using reciprocal three-way crosses [(AxB)xCvCx(A x B)]. The two populations were mapped at 42 loci pre-selected to cover the majority of the genome. No differences in recombination distances were observed at the whole-genome level and only a few individual linkage intervals were found to differ, all in favour of increased recombination through the male. Distorted segregations found in the three-way crosses provide evidence of post-gametic selection for particular gene(s) or chromosome regions. The significance of these results for the design of pearl millet breeding programmes and inheritance experiments, as well as for other experimental strategies, is discussed.     

Quantitative analysis of ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.)

Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R.Br.]: efficient production of multiple shoots and inflorescences from shoot apices

 This report presents a procedure for high-frequency multiple shoot production from cultured shoot apical meristems of pearl millet [Pennisetum glaucum (L.) R. Br.]. Shoot apices from 1-week-old aseptically germinated seedlings were cultured in vitro on MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) with biweekly subculture. A low concentration of 2,4-D coupled with four different concentrations of BA induced the production of adventitious shoots from the enlarged shoot apical meristems. Somatic embryogenesis was also observed at higher concentrations of BA. The use of higher levels of 2,4-D resulted in callusing of shoot apical meristems, while the shoot tips produced many leaves and in vitro flowering in 2,4-D-free media containing BA. All four pearl millet genotypes produced similar results. Fertile pearl millet plants were produced from in vitro-produced multiple shoots. Received: 1 April 1999 / Revision received: 8 July 1999 / Accepted: 17 August 1999     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Somatic embryogenesis in pea (Pisum sativum L.): effect of explant, genotype and culture conditions

In this study 16 cultivars of pea (Pisum sativum L.) were screened in vitro for the formation of somatic embryos which were dependent on the genotype, culture conditions and explant source used. The cultivars Stehgolt, Maro and Progreta showed the highest tendency to form somatic embryos (c. 25%) while Alaska, Rondo and Ascona did not show any embryo production. Using the cultivar Belman, the highest embryo production was achieved by using nodal explants of shoots (10.6%) and a cotyledon-free embryo as explant source (14.1%) in the light (15.8%) compared to using apices as explants (1.8%) and a seedling as the explant source (9.4%) in the dark (3.3%). Media containing picloram (0.75 mg/litre) followed by BA (1 mg/litre) or kinetin (1 mg/litre), each for four weeks gave the highest somatic embryo production. The development of embryos to whole plants was unreliable and some 90% of the embryos induced did not develop any further, died, recallused or formed secondary embryos. The size of the embryo at separation and the timing of the separation from the original callus were important factors determining success for complete development to whole plant. Regeneration of 184 plants was achieved with ensuing flowering, pod formation and viable seed production from the techniques described.     

Somatic embryogenesis and plant regeneration in Cyphomandra betacea (Cav.) Sendt

Callus cultures with globular proembryogenic structures were induced from zygotic embryos and hypocotyl segments of Cyphomandra betacea on MS medium supplemented with 2,4-D. Proembryogenic structures produced somatic embryos and plantlets on regulator-free basal medium. Pieces of embryogenic callus subcultured on medium with the same original composition gave rise to new globular structures and the potential for plantlet regeneration has been maintained for over a year. The histological examination of these proembryogenic structures suggested that somatic embryos arise from single cells. Regenerated plants are phenotypically normal, having diploid chromosome numbers (2n = 24).     

Somatic embryogenesis from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Japanese larch (Larix leptolepis)

Embryogenic tissue was initiated using LM, LP and MS media from open-pollinated immature embryos of Larix leptolepis. The initiation frequency varied with collection dates. The highest frequencies of embryogenic tissue initiation (60, 67 and 59% on LM, LP and MS media, respectively) were observed from cones collected on July 30. At this time, all the excised embryos were at the cotyledonary stage. ABA over a wide concentration and length of exposure range did not promote maturation, but was beneficial in reducing precocious germination. Of over 400 plants regenerated, 72 were transplanted into soil mixtures and to date, 69 of these (95%) have survived. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from cultured mature caryopses of bahiagrass (Paspalum notatum Flugge)

Callus cultures were initiated from mature excised caryopses of bahiagrass (Paspalum notatum Flugge) on Murashige & Skoog medium supplemented with 20 gl^–1 sucrose and 2 mg l^–1 2,4-D. Excised mature caryopses readily germinated and callus developed at the base of coleoptiles. There was considerable variation in the amount of non-embryogenic callus among the cultures. Most of the explants produced non-embryogenic translucent callus consisting of thin-walled cells and unorganized tissue. Some of these calli gave rise only to roots. Other explants formed embryogenic calli which were distinguished morphologically as white, globular and friable. Somatic embryos developed and germinated precociously when embryogenic calli were transferred to a 2,4-D-free medium. Somatic embryogenesis was confirmed by histological sections and scanning electron microscopy. Of the 300 cultures, 35 were embryogenic but only 10 produced plants that were successfully grown to maturity.     

Somatic embryogenesis and plant regeneration in olive (Olea europaea L.)

Leaf discs from olive (Olea europaea L.) grown in vitro and immature zygotic embryos collected at 50, 75, 90 and 105 days after full bloom were tested for their somatic embryogenic capacity. The embryos were grown in half-strength MS medium and half-strength OM medium with BAP combinated with either 2,4-D or NAA. Incubation was either in an initial dark period followed by 16h daylight or in 16h daylight throughout. Somatic embryogenesis, approx. 40%, mostly directly from the embryos, was observed only in 75-day-old embryos in medium containing low cytokinin and auxin concentrations. Differentiation was inhibited by 2,4-D whereas NAA did not. In leaf discs and younger and older zygotic embryos, only callus and root formation was observed. Somatic embryos were germinated and then potted-up to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphtaleneacetic acid     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.     

Hybrid performance and genetic distance as revealed by the (GATA)4 microsatellite and RAPD markers in pearl millet

 Genetic diversity in five cytoplasmic male-sterile and seven restorer lines of pearl millet was determined by DNA fingerprinting using a (GATA)₄ microsatellite and randomly amplified polymorphic DNAs (RAPDs). A total of 160 polymorphic loci were generated and, based on the polymorphism data, similarity index values ranged from 0.81 to 0.50. Cluster analysis was performed and relationships among these lines revealed that they were not in agreement with the available pedigree data. The per se performance of parents and hybrids was analyzed for days-to-50% flowering, plant height, productive tillers, ear length, ear width, 1000-grain weight and grain yield per plot. Path co-efficient analysis revealed that productive tillers, ear width and days-to-50% flowering had a relatively large positive effect. The correlation values were mostly not significant with respect to genetic distance, except for days-to-50% flowering, ear length and ear width. Our results have indicated that genetic-distance measures based on the (GATA)₄ microsatellite and RAPDs may be useful for the grouping of parents, but not for predicting heterotic combinations, in pearl millet. Received: 3 January 1998 / Accepted: 25 February 1998