Interactions between seed priming treatments and nine seed lots of carrot, celery and onion. I. Laboratory germination

Samples of three seed lots of each of three cultivars of carrot, celery and onion were primed in polyethylene glycol solution for 2 weeks at 15 °C. Priming reduced the mean germination times (recorded at 15 °C) of all seed lots (compared to the untreated control) by 3–4 days in carrot, 6–10 days in celery and 3–5 days in onion. The largest reductions in mean germination time occurred in the slowest-germinating seed lots. There were highly significant interactions between priming and cultivars, and between priming and seed lots within cultivars for each species. Drying back the primed seeds at 15 °C increased the mean germination times (compared to primed seed which had not been dried) by 0·6 day in carrot and 1·4 days in celery, and there was no interaction with cultivars or seed lots. The corresponding increase for onion was either 1·0 or 1·8 days, according to the cultivar, but this variation was largely attributable to differences in time taken for the dried seeds to re-imbibe. Seeds dried back at 30 °C germinated 0·2·0·7 day (depending on the species) later than those dried at 15 °C. Percentage germination was not affected by either priming or drying back. Priming reduced the spread of germination times in all cultivars. For primed and dried-back seed, the spread of germination times was larger than that of primed seed in certain cultivars, but was always smaller than that of untreated seeds.     

A comparison of methods for evaluating seed quality in carrots (Daucus carota)

Comparisons of embryo length, ISTA (International Seed Testing Association), cold (10 ^oC), controlled-deterioration, and slope tests for estimating seedling size variability and percentage seedling emergence were made using eighteen stocks of carrot seeds. The coefficient of variation (C.V.) of seedling weight was closely correlated with the C.V. of embryo length (r = 0–87 D.F., 16) and also with the C.V. of root length from the slope test (r = 0–63 D.F., 16). Poorer estimates of percentage seedling emergence were obtained from the cold test in all three sowings (correlation coefficients were between 0–65 and 0–70 D.F., 16) compared with the ISTA, controlled deterioration, and slope tests. For these three tests the correlation coefficients varied between 0–80 and 0–87 (D.F., 16) for all sowings. The embryo test is a rapid (1 man hour per seed lot) and accurate method of assessing potential variability between seed lots. The slope test could provide a less labour demanding method of assessing potential variability than the embryo length test though the results would not be available immediately. However, the slope test could also provide as good an estimate of seedling emergence in the field as the ISTA or controlled-deterioration tests.     

The germination and emergence of seeds of winter oilseed rape stored and sown in admixture with pelleted methiocarb

The possibility that pellets of the molluscicide Draza (a.i. methiocarb) might reduce field emergence of seeds of winter oilseed rape (Brassica napus, cv. Jet Neuf) when sown admixed, laboratory germination when stored admixed, or field emergence when both stored and sown admixed was the subject of three separate investigations. Admixture at sowing did not influence the emergence of five seed lots (P > 0·05). Neither did admixture influence loss in germination or change in moisture content during 2 months' storage of one lot in four different regimes (P > 0·05). Field emergence following 2 months' storage in paper bags was not influenced by admixture (P > 0·05), whereas a 12% reduction in field emergence resulting from storage in admixture within polyethylene bags was detected (P < 0·05). Thus short-term storage within paper sacks of winter oilseed rape seeds in admixture with slug pellets containing methiocarb did not damage the seeds. The emergence of five seed lots differed considerably within and between field sowings. Tentative advice on the calculation of sowing rates for different seed lots of winter oilseed rape is provided to account for this variation.     

Effects of Stratification, Gibberellic acid and Germination Temperature on the Germination of Betula nana

Experiments were carried out with three seed lots of Betula nana collected in 1967 from different localities in Norway. Seeds were stratified for 0-20 days in dark at +2-+3 °C on filter papers moistened with distilled water, or treated with solution of GA₃ for 24 h at room temperature, and then moved into special germination boxes that were placed in different temperature conditions. All the seed lots had conditional dormancy. Quantitatively, the dormancy was different in the different seed lots (pronenances), but there were no qualitative difference in the reaction to stratification gibberellic acid and to germination temperature. Differences between seed lots may have been due to different stage of seed development. The dormancy was deepest at low temperatures(12 and 15°C) decreasing gradually with increasing temperature (to 24 °C). The dormancy was effectively broken by a short stratification (from 5 to 15 days), and by treatment with gibberellic acid. The deeper the dormancy and the lower the germination temperature the longer the stratification that was needed for maximum germination. Similarly, the concentration of GA₃ needed for maximum germination increased with decreasing temperature and with increasing dormancy.     

Further studies on the slant test as a measure of the vigour of crisp lettuce seedlot

Measurements of seed vigour, emergence and maturity characters were made on six seedlots of crisp lettuce cvs Pennlake and Saladin, vigour being determined by the measurement of root length in a slant test. There were significant differences between the root length and transplant weight of different seedlots of each cultivar. Root length was significantly rank-correlated with transplant dry weight in both cultivars (cv. Pennlake r_s= 0·94, N = 6; cv. Saladin r_s= 0·83, N = 6) but not with head weight at maturity. Percentage germination was significantly correlated with seed weight and root length. It is suggested that the slant test can be reliably used to indicate early plant vigour.     

Deep and intermediate complex morphophysiological dormancy in seeds of Ferula gummosa (Apiaceae)

We tested the hypothesis that seeds of the monocarpic perennial Ferula gummosa from the Mediterranean area and central Asia have deep complex morphophysiological dormancy. We determined the water permeability of seeds, embryo morphology, temperature requirements for embryo growth and seed germination and responses of seeds to warm and cold stratification and to different concentrations of GA₃. The embryo has differentiated organs, but it is small (underdeveloped) and must grow inside the seed, reaching a critical embryo length, seed length ratio of 0.65–0.7, before the seed can germinate. Seeds required 9 weeks of cold stratification at <10°C for embryo growth, dormancy break and germination to occur. Thus, seeds have morphophysiological dormancy (MPD). Furthermore, GA₃ improved the germination percentage and rate at 5°C and promoted 20 and 5% germination of seeds incubated at 15 and 20°C, respectively. Thus, about 20% of the seeds had intermediate complex MPD. For the other seeds in the seed lot, cold stratification (5°C) was the only requirement for dormancy break and germination and GA₃ could not substitute for cold stratification. Thus, about 80% of the seeds had deep complex MPD.     

Maturity of surface structures in northern Pinus sylvestris L. seeds: A key to improved prediction of germination potential

Prediction of germination potential prior to cone harvesting is problematic due to great variation of maturity between and within northern Scots pine (Pinus sylvestris L.) seed lots. In forest seed centres maturity is traditionally evaluated on the basis of the embryo size using X-radiography, which is a relevant method for predicting germination potential of mature seeds. Still, except for embryo growth, maturation also includes morphological changes in the surface tissues, i.e. the seed coat, the nucellar layers, and the megaspore membrane, which we explored in this study. During two growing seasons 10–30 developing cones were harvested 11 times on two trees at each of three locations, at the boreal conifer forest line (68°48′ N), at the Arctic Circle (66°53′ N), and in central Finland (62°70′ N). Seeds were detached by hand for moisture content measurements, X-radiography, germination tests, light microscopic and field emission scanning electron microscopic studies. Our results confirmed the close relationship between temperature and the embryo growth, whereas maturation of the surface structures and the embryo were partly separate processes. Microscopy proved that biochemical maturation of the sheltering surface structures was partly light-reactive and continued as well as further development of the embryo for several weeks after the embryo had reached its full size. The reliability of radiography improved when the moisture content of seeds approached 30% and the temperature sum was about 800 degree days. The simple drying test of seeds from manually opened cones presented here can supply needed additional information for early prediction of the germination capacity particularly in the northern seed lots.     

Seed Germination in Chenopodium album L: Relationships between Nitrate and the Effects of Plant Hormones

Effects of ethylene, gibberellins, and kinetin on the germination of two lots of Chenopodium album L. seeds, collected from the field in 1982 and 1983, were studied in relation to the availability of nitrate. The experiments were conducted in darkness and at temperatures ranging from 12 to 32°C. Ethylene induced over 75% germination in the 1983 seed but had little effect on the 1982 seed. Nitrate was only slightly promotive in either of the two seed lots. A combination of ethylene and nitrate, however, acted synergistically on 1982 seed, resulting in as much germination as that induced in 1983 seed by ethylene alone. In 1983 seed, a combination of ethylene and nitrate was only marginally more effective than ethylene. A similar relationship was observed in the effects of gibberellic acid₄₊₇ (GA₄₊₇) and nitrate on seeds from the two lots. The 1982 seed, which responded synergistically to combinations of nitrate with ethylene or GA₄₊₇ was found to contain an extremely low endogenous level of nitrate as compared to 1983 seed. Thus, high levels of either endogenous or applied nitrate appeared to enhance the germination response to ethylene or GA₄₊₇.

Kinetin had no effect on 1982 seed and only a small promotive effect on 1983 seed. There was no synergism between kinetin and nitrate in either of the seed lots.

     

Variation in embryo size of inbred lines of carrot

Mean embryo lengths and coefficients of variation (CVs) of embryo length of inbred carrot lines differed between years but not between the two sets of inbreds tested nor between male-sterile and maintainer lines. Estimates of CVs were of similar magnitude to those previously obtained for experimental and commercial open-pollinated seed lots, but estimates of mean embryo length were lower. Thus greater genetic uniformity did not lead to lower CVs of embryo size of the inbreds.     

Systematic overestimation of Salicaceae seed survival using radicle emergence in response to drying and storage: implications for ex situ seed banking

Biodiversity conservation programmes are underpinned by seed banking following drying to low water contents (WC), and supported by both the assessment and prediction of seed viability over time. The means of judging viability is thus crucial to the comprehension of seed vigour. We selected seeds of three species and one hybrid in the Salicaceae likely to have variation in tolerance to drying, processing and storage, including in relation to cryobanking, and compared survival growth as radicle emergence (germination) and normal seedling production. With three seed lots of Salix gracilistyla, air-drying to 8–10 % WC enhanced seed survival after 40 days’ storage at 5 °C as compared with non-treated seeds at 14–20 % WC. Four seed lots of Populus alba × P. glandulosa showed equally high germination (88–100 %) and proportions of normal seedlings (81–99 %) when stored at 5 °C for 7–10 weeks. Among seven seed lots of S. gracilistyla, two groups with different storage behaviour could be statistically distinguished with normal seedling production ranging from 0 to 45 % after storage at 5 °C for 13 weeks. Seed tolerance to WC manipulation and cryopreservation was very variable among species and seed lots. Seed lots of S. hallaisanensis and S. gracilistyla with ~80 % germination survived cryopreservation at 10 % WC, but were sensitive to lower WCs. In contrast, Populus seeds had greater desiccation tolerance combined with cryopreservation capability. With seed lots of all species and hybrids, cryopreservation had little effect on viability unless the high moisture freezing limit had been exceeded (~10–20 % WC, depending on seed lot). However, under all conditions of handling (drying, rehydration, storage at 5 °C or cryopreservation) using germination as the only indicator of viability over-estimated survival compared with normal seedling production.     

Trichoderma harzianum antagonistic activity and competition for seed colonization against seedborne pathogenic fungi of sunflower

This study investigated the antagonistic effects of Trichoderma harzianum isolate (TRIC8) on mycelial growth, hyphal alteration, conidial germination, germ tube length and seed colonization by the seedborne fungal pathogens Alternaria alternata, Bipolaris cynodontis, Fusarium culmorum and F. oxysporum, the causes of seedling rot in over 30% of sunflowers. The antagonistic effect of TRIC8 on mycelial growth of pathogens was evaluated on dual culture that included two inoculation assays: inoculation of antagonist at 48 h before pathogen (deferred inoculation) and inoculation at the same time with pathogen (simultaneous inoculation). TRIC8 inhibited mycelial growth of the fungal pathogens between 70·67 and 76·87% with the strongest inhibition seen with deferred inoculation. Alterations in hyphae were observed in all pathogens. Conidial germination of F. culmorum was inhibited by most of the fungal pathogens (38·28%) by TRIC8. Inhibition of germ tube length by the antagonist varied from 31·83 to 37·67%. In seed colonization experiments, TRIC8 was applied in combination with each pathogen to seeds of a sunflower genotype that is highly tolerant to downy mildew. Seed death was inhibited by TRIC8 and the antagonist did not allow growth of A. alternata, B. cynodontis and F. culmorum on seeds and inhibited the growth of F. oxysporum at the rate of 58·32%.     

Environmental regulation of dormancy loss in seeds of Lomatium dissectum (Apiaceae)

Background and Aims

Lomatium dissectum (Apiaceae) is a perennial, herbaceous plant of wide distribution in Western North America. At the time of dispersal, L. dissectum seeds are dormant and have under-developed embryos. The aims of this work were to determine the requirements for dormancy break and germination, to characterize the type of seed dormancy, and to determine the effect of dehydration after embryo growth on seed viability and secondary dormancy.

Methods

The temperature requirements for embryo growth and germination were investigated under growth chamber and field conditions. The effect of GA₃ on embryo growth was also analysed to determine the specific type of seed dormancy. The effect of dehydration on seed viability and induction of secondary dormancy were tested in seeds where embryos had elongated about 4-fold their initial length. Most experiments examining the nature of seed dormancy were conducted with seeds collected at one site in two different years. To characterize the degree of variation in dormancy-breaking requirements among seed populations, the stratification requirements of seeds collected at eight different sites were compared.

Key Results

Embryo growth prior to and during germination occurred at temperatures between 3 and 6 °C and was negligible at stratification temperatures of 0·5 and 9·1 °C. Seeds buried in the field and exposed to natural winter conditions showed similar trends. Interruption of the cold stratification period by 8 weeks of dehydration decreased seed viability by about 30 % and induced secondary dormancy in the remaining viable seeds. Comparison of the cold stratification requirements of different seed populations indicates that seeds collected from moist habitats have longer cold stratification requirements that those from semiarid environments.

Conclusions

Seeds of L. dissectum have deep complex morphophysiological dormancy. The requirements for dormancy break and germination reflect an adaptation to trigger germination in late winter.Key words: Apiaceae, cold stratification, Lomatium dissectum, morphophysiological dormancy, secondary dormancy, seed germination     

Effect of stimulating maize germination on cell cycle proteins

The germination process can be accelerated if seeds are stimulated either by adding cytokinins or by osmopriming. Under these conditions, cells in maize ( Zea mays ) embryo axes shorten the time at which the first round of DNA replication and mitosis takes place, thus advancing the cell cycle. Using heterologous antibodies against different cell cycle proteins, we have followed the behaviour of several markers for G1 phase (cyclin D, E2F and p53) and a marker of G2 phase (cyclin B) under either control or "accelerated" germination conditions. The results showed two classes of behaviour: either there was no variation in the amount of the protein present under control or accelerated germination conditions, represented by cyclin Band E2F‐type proteins, or the amount of the proteins was drastically reduced, more rapidly under accelerated germination, as was the case for cyclin D‐ and p53‐type proteins. Although the cyclin D‐type protein was synthesized de novo during germination, the balance was towards degradation so that there was no cyclin D detected 15 h after germination in benzyladenine‐treated and osmoprimed seeds. A Cdk4‐type protein seemed to be present in cyclin D immunoprecipitates and its kinase activity paralleled the fluctuations of the cyclin amount during germination. These data are discussed in the context of early seed germination.     

元谋干热河谷车桑子种子休眠与萌芽的空间变异特征研究

为明确车桑子[Dodonaea viscosa(L.)Jacq.]种子的休眠特性和地理变异特性,测定了云南省元谋县凉山乡(属北亚热带区)和坝区苴林乡(属南亚热带区)车桑子种子形态,观察了种子的休眠特性和萌发特性。结果表明,两地的车桑子种子都具有物理休眠特性,经热水处理的种子萌发率显著高于未处理的种子(P=0.001)。两地种子在长、宽、长/宽以及种子百粒重上十分接近,其萌发特征和幼苗生长特征没有表现出显著差异。因此,坝区苴林乡和凉山乡种子并未发生地理变异,两地均适合车桑子采种,但在播种前应进行种子休眠破除,以提高种子发芽率。     

Geographic variation in life-history traits of the endemic kite skate Dipturus chilensis (Batoidea: Rajidae), along its distribution in the fjords and channels of southern Chile

Life-history traits of kite skates Dipturus chilensis were examined from two regions (c. 2286 km apart) in the sheltered fjords and channels of southern Chile. A total of 482 and 403 specimens were collected from the southern fjords (c. 42–46° S) and the fjords of Chilean Patagonia (c. 51–54° S), from September 2003 to 2004, respectively. Vertebra marginal increment analysis indicated an annual deposition of growth rings which was completed during the winter months. For each region, von Bertalanffy growth parameters showed that females attained a larger asymptotic size, L_∞, had a lower growth coefficient, K, and lived longer than males. Growth analysis indicated that D. chilensis from the Patagonian fjords had a longer life span (females: 22 v. 21 years; males 19 v. 17 years), attained a larger L_∞ (females: 150 v. 136 cm; males: 122 v. 118 cm total length, L_T) and had a lower K value (females: 0·087 v. 0·104; males: 0·110 v. 0·116) than their counterparts in the southern fjords. Comparisons with previous studies indicated that D. chilensis from both southern and Patagonian sheltered fjords had larger L_∞, and grew more slowly than their counterparts from central-southern Chile (L_∞= 119–123 cm, K= 0·123–0·127), suggesting latitudinal variations in growth. Females attained sexual maturity later than males in both regions. For both sexes, lengths at 50% maturity (L_50%) between regions were similar (females: c. 103 cm; males: c. 87 cm L_T); however, D. chilensis from Patagonia appeared to mature 1 year earlier (females: 13 v. 14 years; males: 10 v. 11 years). Specimens from Patagonia had a lower ovarian fecundity than those from the southern fjords. An increase in the proportion of mature females and males during summer, suggests that the reproductive peak occurs in this season, and no regional differences were found. The size of the egg cases increased with maternal L_T and these were longer in Patagonia. The information provided here represents the first evidence of regional variations in life-history traits for elasmobranchs in the south-eastern Pacific.     

Hormonal and temperature regulation of seed dormancy and germination in <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Fluctuating temperature plays a critical role in determining the timing of seed germination in many plant species. However, the physiological and biochemical mechanisms underlying such a response have been paid little attention. The present study investigated the effect of plant growth regulators and cold stratification in regulating Leymus chinensis seed germination and dormancy response to temperature. Results showed that seed germination was less than 2 % at all constant temperatures while fluctuating temperature significantly increased germination percentage. The highest germination was 71 % at 20/30 °C. Removal of the embryo enclosing material of L. chinensis seed germinated to 74 %, and replaced the requirement for fluctuating temperature to germinate, by increasing embryo growth potential. Applications of GA₄₊₇ significantly increased seed germination at constant temperature. Also, inhibition of GA biosynthesis significantly decreased seed germination at fluctuating temperatures depending upon paclobutrazol concentration. This implied GA was necessary for non-dormant seed germination and played an important role in regulating seed germination response to temperature. Inhibition of ABA biosynthesis during imbibition completely released seed dormancy at 20/30 °C, but showed no effect on seed germination at constant temperature, suggesting ABA biosynthesis was important for seed dormancy maintenance but may not involve in seed germination response to temperature. Cold stratification with water or GA₃ induced seed into secondary dormancy, but this effect was reversed by exogenous FL, suggesting ABA biosynthesis during cold stratification was involved in secondary dormancy. Also, cold stratification with FL entirely replaced the requirement of fluctuating temperature for germination with seeds having 73 % germination at constant temperature. This appears to be attributed to inhibition of ABA biosynthesis and an increase of GA biosynthesis during cold stratification, leading to an increased embryo growth potential. We suggest that fluctuating temperature promotes seed germination by increasing embryo growth potential, mainly attributed to GA biosynthesis during imbibitions. ABA is important for seed dormancy maintenance and induction but showed less effect on non-dormant seed germination response to temperature.     

Seed ecology of the geophyte Conopodium majus (Apiaceae), indicator species of ancient woodland understories and oligotrophic meadows

• Conopodium majus is a geophyte with pseudomonocotyly, distributed in Atlantic Europe. It is an indicator of two declining European habitats: ancient woodland understories and oligotrophic hay meadows. Attempts to reintroduce it by seed have been hindered by scarce seedling emergence and limited knowledge of its seed biology.
• Micro‐CT scanning was used to assess pseudomonocotyly. Embryo growth and germination were studied in the laboratory and the field, using dissection and image analysis. The effects of temperature, light, nitrate and GA₃ on germination were tested. Seed desiccation tolerance was investigated by storage at different RHs and by drying seeds at different stages of embryo growth.
• Seeds possess morphological but not physiological dormancy. Embryo growth and germination were promoted by temperatures between 0 and 5 °C, arrested above 10 °C, and indifferent to alternating temperatures, light, nitrate and GA₃. Pseudomonocotyly appears to result from cotyledon fusion. While seeds tolerated drying to 15% RH and storage for 1 year at 20 °C, viability was lost when storage was at 60% RH. Seeds imbibed at 5 °C for 84 days had significant internal embryo growth but were still able to tolerate drying to 15% RH.
• Reproduction by seed in C. majus follows a strategy shared by geophytes adapted to deciduous temperate forests. The evolution of fused cotyledons may enable the radicle and the hypocotyl to reach deeper into the soil where a tuber can develop. The embryo is capable of growth within the seed at low temperatures so that germination is timed for early spring.

     

Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens

Wild adult specimens of the Peruvian anchovy Engraulis ringens were captured and reared to validate the daily periodicity of otolith microincrement formation. The postcapture stress generated spontaneous spawning, making it possible to conduct a rearing trial on larvae first in an artificial nutrient‐enriched system (ANES) for 52 days followed by an artificial feeding regime in a culture tank until day 115 post‐hatch. Microincrements of the sagittal otoliths of sacrificed juveniles [mean ± s.d. total length (L_T) = 5·13 ± 0·37 cm, range 5–6 cm; c.v. = 7·5%] showed very distinct light and dark zones. The slope of the relationship between the total number of increments after the hatch check and days elapsed after hatching was not significantly different from 1. The transfer from ANES to the artificial feeding regime induced a mark in the sagittal otoliths. The number of microincrements after this induced mark coincided with the number of days elapsed after the transfer date. In parallel experiments, adult E. ringens (mean ± s.d. L_T = 14·92 ± 0·55 cm, range 13–16 cm) were exposed to one of two fluorescent marking immersion treatments with either alizarin red S (ARS; 25 mg l^?1 per 6 h) or oxytetracycline hydrochloride (OTC; 200 mg l^?1 per 10 h). The microincrements between fluorescent bands were distinct, ranging from 0·89 to 2·75 µm (mean ± s.d. =1·43 ± 0·28 µm; c.v. = 32%) and from 0·71 to 2·89 µm (1·53 ± 0·27 µm; c.v. = 35%) for ARS and OTC, respectively. The relationship between the number of microincrements between marks and the number of elapsed days for ARS and OCT treatments indicated that there was a significant correspondence between the number of increases observed and the number of days. Hence, daily microincrements of otoliths of E. ringens are likely to be formed in juveniles and adults under natural conditions.     

Genetically determined variation in seed germination and field emergence of Brassica oleracea

The germination and field emergence levels and germination rates were measured of Brassica oleracea seed lots obtained by outcrossing the progenies of two half diallels to a single pollen parent. Analysis of variation of the seed lots classified according to their maternal grandparents showed that both general and specific combining abilities were important in accounting for the observed differences; the observed performance of many of the seed lots was satisfactorily accounted for by their grandmaternal parentage. The capacity to give high levels of germination was not necessarily related to the capacity to give high relative field emergence levels, maternal grandparents apparently differing in their ability to contribute to these two characters.     

Ageing and Osmotic Priming in Wheat Seeds: Effects Upon Certain Components of Seed Quality

Deteriorated wheat seed lots were osmotically primed and surface-dried,dried-back, then aged for two more weeks Osmotic priming didnot affect the final germination in any of the aged seed lots,but mean germination time was decreased The response of unagedseed lot to treatments was studied in the following the timecourses of germination, as well as L-[4, 5-²H]leucine and [6-³H]thymidineincorporation (as a measure of protein and DNA synthesis, respectively)into dissected embryos during the early hours of germinationIt was found that the variation in mean germination time andthe rate of synthetic reactions were related The greatest improvementin the components of seed lot quality was achieved after primingand surface drying The beneficial effects of priming were apparenteven after dehydration followed by two further weeks of ageingCumulative correlations between mean germination time and variousbiochemical tests in variously treated aged seed lots were highlysignificant The physiological processes involved in deteriorationand recovery and the potential application of biochemical testsin detecting vigour changes are discussed Triticum durum, seed, ageing, osmotic priming, mean germination time, protein DNA synthesis