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Annals of Botany 77: 591-597, 1996

Effects of Controlled Deterioration and Osmoconditioning on Germination and Nuclear Replication in Seeds of Pepper (Capsicum annuum L.)
S. LANTERPi, E. NADA*, P. BELLETTI*. L. QUAGLIOTTI* and R. J. BINOf * DLVA.P.R.A., Plant Breeding and Seed Production, University of Turin, via P. Giuria 15, 10126 Turin, Italy and t CPRO-DLO, Centre for Plant Breeding and Reproduction Research, PO Box 16, 6700 A A Wageningen, Netherlands
Received: 3 October 1995 Accepted: 24 November 1995

Unaged and controlled deteriorated (45 C for 4, 6 or 10 d) samples of a pepper seed lot were subjected to osmoconditioning in PEG at osmotic potentials of 11 and 1-5 MPa for 6, 10 or 14 d. The effect of osmoconditioning on nuclear replication activity was examined using flow cytometry. Priming of unaged seeds always induced nuclei of embryo root tips to enter the synthetic phase. In accordance with our previous findings the amount of induced nuclear replication activity was higher after priming at the lowest osmotic potential. Under the same osmotic potential the amount of priming-induced replication was correlated with the length of priming treatment and its efficiency in improving seed performance. However, the 14 d treatment at 1-5 MPa was as effective on seed performance as the 6 d treatment at 1-1 MPa, which induced higher numbers of nuclei to enter the synthetic phase. Osmoconditioning on controlled deteriorated seeds had different effects on seed germination depending on the degree of seed deterioration. Under the same osmotic treatment, the amount of priming-induced DNA synthesis was lower than in unaged seeds or was not induced at all. The activation of nuclear replication by osmoconditioning, therefore, appears influenced by the level of seed deterioration. In less deteriorated seeds (45 C for 4 d), 14 d priming at IT MPa caused shortening of mean germination time compared with unaged seeds, but was less effective in inducing nuclear replication. The effect of the length and osmotic potential of priming on nuclear replication and the role of molecular processes, other than DNA synthesis, in improving seed performance are discussed. 1996 Annals of Botany Company Key words: Controlled deterioration, Capsicum annuum, DNA replication, flow cytometry, priming.

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1990) and result in delayed germination, abnormal growth and, finally, loss of germinability (Ellis and Roberts, 1981). On the other hand, improvement in rate and uniformity Loss of seed germinability following natural ageing or controlled deterioration has been attributed to a series of of germination has been observed after osmoconditioning metabolic defects that accumulate in embryonic and non- (priming) of seeds in numerous species (Georghiou, Thanos embryonic structures (Roberts, 1973; Osborne, 1983). There and Passam, 1987; Vazquez-Ramos et al., 1988; Gray, is considerable evidence that both point mutations and Rowde and Drew, 1990; Bino et al., 1992; Fujikura and gross chromosome aberrations occur as seeds lose vigour Karssen, 1992; Ashraf and Bray, 1993). Incubation in an and viability. A close correlation between ageing and the osmotic solution prevents germination by restraining the accumulation of chromosome aberrations in surviving seeds processes which are directly involved in radicle protrusion, has been observed (Dourado and Roberts, 1984; Rao, such as cell wall extension. Other physiological and Roberts and Ellis 1987, 1988; Lanteri and Belletti, 1990) biochemical reactions may proceed, albeit often at a reduced and recently, non-random changes in the restriction pattern rate. Improved seed performance after priming has been of Triticum durum embryo DNA, probed with ribosomal explained by completion of DNA repair during priming cDNA, have been detected (Guy, Smith and Black, 1991). (Osborne, 1983) and by a more favourable metabolic Similarly, the integrity of ribosomal RNA in seeds of A Ilium balance of primed seeds at the start of germination in water porrum has been found to correlate with germination (Dell'Aquila, Savino and De Leo, 1978). During priming, performance (Bray et al., 1989). Many other physiological changes in protein patterns have also been observed processes have been linked to seed ageing; e.g. in aged seeds (Davison and Bray, 1991). membrane phospholipid content decreased and fatty acids Using flow cytometry, we previously studied nuclear levels increased, even though no extensive lipid peroxidation replication activity in Capsicum annuum seeds during occurred (Bruggink et al., 1991). Both genetic damage and germination and after priming (Lanteri et al, 1993, 1994). loss of membrane integrity may cause changes in protein In this species the quiescent embryo arrests nuclear division synthesis during germination (Gidrol, Noubhani and Drew, at the pre-synthetic Gl phase. Upon imbibition in water an increase in the amount of DNA was observed in the embryonic root tip region, before visible germination. This % For correspondence. INTRODUCTION 0305-7364/96/060591+07 $18.00/0 1996 Annals of Botany Company

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Preparation of nuclear samples Samples of nuclei for flow cytometry were prepared as reported by Lanteri et al. (1993). One millimetre of the distal part of the embryo was used for analysis. To detect DNA, 10 mg I"1 of propidium iodide (PI) was added to the isolation buffer (Saxena and King, 1988). For each sample at least 20 seeds were used and the determination was made in duplicate. Fluorescence was measured using a FACScan flow cytometer (Becton & Dickinson, Mountain View, CA, USA) equipped with a 488 nm light source (argon laser). Two filters were used to collect the red fluorescence due to PI staining of the DNA, one transmitting at 585 nm with a bandwidth of 420 nm (FL2), the other transmitting above 620 nm (FL3). FL2 and FL3 were registered on a logarithmic scale. Forward and side light scatter were measured simultaneously. The flow rate was set at about 200 nuclei s"1 and at least 104 nuclei were analysed for each sample. Debris was excluded from analysis by appropriately raising FL2 and FL3 thresholds to values selected experimentally. Data were recorded in a Hewlett Packard computer (HP 9000, model 300) using CellFit software (Becton & Dickinson). RESULTS Effect of ageing and priming treatments on fresh seeds The GP of fresh pepper seeds was 89-5 and the MGT 5 d. The reduction in GP and the increase in MGT due to controlled ageing treatments were proportional to their duration (Table 1). The most severe ageing condition (45 C for 8 d) induced a decrease in GP to 24-9 % and increased the MGT by 4-1 d. The seed water content during priming at 1-1 MPa and 1-5 MPa was respectively 37 % and 30 % on a dry weight basis. Priming decreased MGT without affecting GP (Table 1). The most effective priming treatments were those performed at the lower osmotic potential ( 1 1 MPa) for 10 and 14 d, which induced a decrease in MGT of about 3 d. There was no germination during the osmotic treatments. TABLE 1. Effect of controlled deterioration at 45 C and osmopriming in PEG solutions on germination percentage (GP) and mean germination time (MGT) of pepper seeds. Data are means+ s.e. offour replicates of 100 seeds. Within a column, means with the same superscript letter are not significantly different (P < 0-01; Tukey's HSD test)
Treatment Control Artificial ageing Artificial ageing Artificial ageing Priming at Priming at Priming at Priming at Priming at Priming at GP (%) 89-5+l-2 a 74-9+l-5 b 3 3 0 + l-6e 24-9+ l-4d 91-5 +l-3 a 86-8 + H a 86-9+ l-5a 91-5 + 1-7" 92-9+l-8 a 88-8 + l-8a MGT (d) 5-OO-3d 6-9 + 00 6 9-0 + 0-3f 9-1+0-4' 2-6 + 0-3b 2-2 + 0-2a 2-0 + 0-1* 3-4 + 0-2c 3-3 + 0-lc 2-6 + 0-16

increase was related to cells entering the synthetic phase of nuclear division, leading to doubling of the chromosomes. Priming was also found to induce DNA synthesis in the embryo root meristem, depending on the osmotic potential of the solution used and length of treatment. Nuclear replication of primed and subsequently redried pepper seed can thus be stably arrested in G2 phase. Furthermore, although the effect of a given osmotic treatment differed among seed lots, in all seed lots analysed, a significant inverse correlation was observed between the frequency of root tip cells induced to enter G2 and decrease in mean germination time (Saracco et al., 1995). In this paper we report the effect of priming on germination and nuclear replication of unaged or controlled deteriorated pepper seeds. Our aim was to gain a better understanding of the induction of nuclear replication and restoration of seed quality following priming treatments. M A T E R I A L S AND M E T H O D S Seed and seed treatment Seeds of pepper (Capsicum annuum L. cv. 'Corno di Toro') from a commercial lot were obtained from SAIS (Societa Agricola Italiana Sementi, Cesena, Italy). Their initial moisture content was 7-5% on a dry weight basis. Three samples of pepper seeds were kept at 75 % relative humidity (RH) and 20 C for 2 d, then sealed in foil packets and held at 45 C for 4, 6 or 8 d. During storage seed moisture content was 9-5% (on a dry weight basis). Deteriorated seeds, as well as unaged seeds, were osmoprimed using PEG 6000 solutions at osmotic potentials of 1-1 or 1-5 MPa, by placing them on filter paper saturated with the solutions at 20 C in the dark. All seeds were primed for 6, 10 or 14 d. After priming, the seeds were washed with running tap water and dried back to the initial seed moisture content (7-5 %) in a drum with ventilated air (Koopman, 1963) above a saturated solution of CaCl2 at 20 C (creating an atmosphere of 32 % RH). Moisture content was estimated using the oven method described by ISTA (1993). Germination tests Germination tests were carried out according to the ISTA guidelines for germination under laboratory conditions (ISTA, 1993). A solution of 0-2% KNO 3 was used to saturate the germination substrate. To determinate mean germination time (MGT) seeds were counted daily for 14 d and were considered germinated when radicles had emerged at least 1 mm. The MGT was evaluated according to the formula Ind/N (where n is the number of germinated seeds on each day, d the number of days from the beginning of the test and T the total V number of germinated seeds). To determinate germination percentage the number of normal seedlings was counted after 6, 10 and 14 d. Germination percentages were transformed to arcsin square roots before statistical analysis. Data on germination percentage (GP) and MGT were analysed by Tukey's HSD test.

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for 4 d for 6 d for 8 d MPa for MPa for MPa for MPa for MPa for MPa for

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Priming (d)

Priming (d)

FIG. 1. Mean germination time (MGT) and germination percentages (GP) of pepper seeds unaged (O) and artificially aged at 45 C for 4 (M), 6 (A) and 8 ( ) d after priming in PEG solutions for 6, 10, 14 d at the osmotic potentials of - 11 MPa (A, B) and - 1-5 MPa (C, D). Standard errors are indicated where they exceed the size of the symbols.

at channel 100 (Fig. 2B), corresponding to the diploid prereplication chromosomal configuration (Gl). DNA profiles In seeds aged for 4 d, priming for 6-14 d at 1-1 or observed in radicle meristems of seeds primed for 14 d are 1-5 MPa induced a reduction in MGT similar to that reported in Fig. 2 C-D, illustrating in both cases an increase observed in unaged seeds (Fig. 1 A, C); only a negligible in 4C nuclei although this was greater after priming at recovery of GP, proportional to the length of treatment, was -1-1 MPa. observed in both priming conditions (Fig. 1B, D). Priming at 1-1 MPa always induced nuclear replication In seeds aged for 6 d, priming slightly increased GP (Fig. in the root tips of fresh seeds, and its effect was proportional 1B, D) while it exerted a marked effect on MGT (Fig. 1 A, to the treatment duration (Fig. 3 A). Priming at 1-5 MPa C). After 10 or 14 d at -1-1 MPa, MGT was lowered to 2-9 did not induce DNA synthesis when performed for 6 d, but 4-5 and 15 % of nuclei in G2 were observed after 10 and 14 d and 2-7 d, respectively. In seeds aged for the longest duration (8 d) priming was (Fig. 3B). In aged seeds a marked decrease in nuclear replication detrimental to seed germination. After 6 d at 1-1 MPa, GP was reduced to about 14% and after 14 d only 3-5% of was observed after osmotic treatment (Fig. 3 A, B). In seeds germinated (Fig. 1B). A similar pattern was observed embryo root tips of 8 d aged seeds neither priming condition induced nuclei to enter the synthetic phase. Priming at at -1-5 MPa (Fig. ID). 11 MPa always induced DNA synthesis on seeds aged for 4 d but, in seeds aged for 6 d, only induced synthesis when performed for the duration of 10 and 14 d (Fig. 3 A). Effect of priming and ageing on nuclear replication Priming at 1-5 MPa induced nuclear replication only on 4 Flow cytometry analysis of chopped pepper leaves and 6 d aged seeds when performed for 10 or 14 d (Fig. 3B). resulted in a large peak at channel 100 and a smaller peak at channel 190 (Fig. 2 A). The large peak corresponded to DISCUSSION the 2C DNA level, from diploid nuclei at the pre-replication stage of nuclear division (Gl), the smaller peak corre- Osmotic preconditioning prevents seed germination since sponded to diploid nuclei with a 4C DNA content (G2 water content is kept below the threshold necessary for radicle growth (Bradford, 1990). Notwithstanding, during stage). Analysis of nuclear DNA content in the embryo root tips priming, DNA, RNA and proteins are synthesized (Bray et of dry, fully matured pepper seeds, revealed only one peak al., 1989; Ashraf and Bray, 1993; Cruz Garcia, Jimenez and Effect of priming treatments on aged seeds

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A 2C B 2C

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4C

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FIG. 2. Flow cytometric histograms of pepper nuclei from leaves (A), embryo root tips of dry, unprimed pepper seeds (B), embryo root tips of seeds primed in PEG solutions for 14 d at 15 MPa (C) and at 11 MPa (D). The peaks at channel 100 correspond to the pre-replicative DNA content (2C), while the peaks at channel 190 correspond to a post-replicative DNA content (4C).

Vazquez-Ramos, 1995). Our previous results (Bino et al., 1992; Lanteri et al., 1993) have shown that priming also induced nuclear replication in the root meristems of Capsicum annuum and Lycopersicon esculentum. The rate and the level of DNA synthesis depended on the osmotic potential of the solution and the duration of priming. Ashraf and Bray (1993) and Cruz Garcia et al. (1995) did not detect priming-induced nuclear replication in Allium porrum and Zea mays. Thus, apparently, seeds of different species may respond differently to osmotic treatment. Lanteri et al. (1994) found that the effect of a given osmotic treatment differs even between seed lots of the same pepper cultivar, with regard to both decrease in MGT and the percentage of nuclei induced to enter G2. During imbibition of tomato seeds, DNA synthesis was found to be concomitant with the accumulation of beta-tubulin (De Castro et al., 1995). Beta-tubulin, as a component of microtubules,

is required for completion of the cell cycle. Possibly, cellcycle related processes play an important role in seed germination, and proteins involved in the cell cycle must either be present in the dry seed or rapidly synthesized upon imbibition prior to activation of the cell cycle (Georgieva et al., 1994). The synthesis of DNA, therefore, follows the activation of other molecular processes related to the induction of the cell cycle. One of these processes may be the induction of DNA-repair to counter aberrations induced by ageing. Except for 6 d at 1-5 MPa, the priming conditions used in the present study always induced DNA replication in the embryo root tips of unaged pepper seeds. In accordance with our previous findings (Lanteri et al., 1993) the amount of DNA synthesis was higher after priming at the lower osmotic potential and was positively correlated with the duration of priming. However, there was not always a direct

Lanteri et al.Nuclear Replication in Deteriorated and Primed Pepper Seeds


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FIG. 3. Percentage of 4C nuclei in the embryo root tips of pepper seeds unaged and artificially aged at 45 C for 4, 6 or 8 d, after priming at the osmotic potential of - 11 (A) and - 1-5 (B) MPa, for 6, 10 or 14 d.

link between the amount of DNA synthesis and seed performance; e.g. the longest priming treatment at 1-5 MPa was as effective on seed performance as the shortest treatment at 1-1 MPa, which induced larger numbers of nuclei to enter G2. Both priming at 1-1 MPa for 6 d and at 1-5 MPa for 14 d, in fact, reduced MGT to 2-5 d but induced respectively about 30 and 12% of nuclei to enter G2. Controlled deterioration decreased seed performance as measured by the reduction in GP and the increase in MGT,

and the effect was proportional to the duration of the ageing treatment. The effect on germination of the subsequent priming treatments depended on the osmotic potential and length of priming as well as the degree of seed deterioration. Osmoconditioning of 4 d aged seeds completely reversed the effect of ageing and even caused enhancement of MGT up to the level of unaged osmoprimed seeds. In more deteriorated seeds (6 d ageing) priming induced a less marked effect although a partial recovery of seed performance and viability was observed. Differently, in strongly

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of seeds aged in slight or severe conditions. It seems possible that severe ageing conditions both amplify and change the type of deterioration occurring during normal storage, and alters response to following priming treatments. The results of the present work indicate that effects of ageing may be partly reversed by priming and part of priming effects is related to the induction of nuclear replication. The induction of DNA replication in aged seeds may thus serve as an indicator for the resumption of metabolic activities in the seed during imbibition in water or PEG. L I T E R A T U R E CITED
Ashraf M, Bray CM. 1993. DNA synthesis in osmoprimed leek (Allium porrum L.) seeds and evidence for repair and replication. Seed Science Research 3: 15-23. Bino RJ, De Vries JN, Kraak HL, Van Pijlen JG. 1992. Flow cytometric determination of nuclear replication stages in tomato seeds during priming and germination. Annals of Botany 69: 231-236. Bradford KJ. 1990. Water relations analysis of seed germination rates. Plant Physiology 94: 840-849. Bray CM, Davison PA, Ashraf M, Taylor RM. 1989. Biochemical changes during osmopriming of leek seeds. Annals of Botany 63: 185-193. Bruggink H, Kraak HL, Dijkema MHGE, Bekendam J. 1991. Some factors influencing electrolyte leakage from maize (Zea mays L.) kernels. Seed Science Research 1: 15-20. Coolbear P, Slater RJ, Bryant JA. 1990. Changes in nucleic acid levels associated with improved germination performance of tomato seeds after low temperature presowing treatment. Annals of Botany 65: 187-195. Cruz Garcia FC, Jimenez LF, Vazquez-Ramos JM. 1995. Biochemical and cytological studies on osmoprimed maize seeds. Seed Science Research 5: 15-33. Davison PA, Bray CM. 1991. Protein synthesis during osmopriming of leek (Allium porrum L.) seeds. Seed Science Research 1: 29-35. De Castro RD, Zheng X, Bergervoet JHW, De Vos CHR, Bino RJ. 1995. Beta-tubulin accumulation and DNA replication in imbibing tomato (Lycopersicon esculentum) seeds. Plant Physiology: 109: 499-504. Dell'Aquila A, Savino G, De Leo P. 1978. Metabolic changes induced by hydration-dehydration presowing treatment in wheat embryos. Plant and Cell Physiology 19: 348-354. Dourado AM, Roberts EH. 1984. Chromosome aberrations induced during storage in barley and pea seeds. Annals of Botany 54: 767-779. Ellis RH, Roberts EH. 1981. The quantification of ageing and survival in orthodox seeds. Seed Science and Technology 9: 379-409. Fujikura Y, Karssen C. 1992. Effects of controlled deterioration and osmopriming on protein synthesis of cauliflower seeds during early germination. Seed Science Research 2: 23-31. Georghiou K, Thanos CA, Passam HC. 1987. Osmoconditioning as a means of counteracting the ageing of pepper seeds during hightemperature storage. Annals of Botany 60: 279-285. Georgieva El, Lopez-Rodas G, Hittmair A, Feichtinger H, Brosch G, Loid, P. 1994. Maize embryo germination. I. Cell cycle analysis. Planta 192: 118-124. Gidrol X, Noubhani A, Pradet A. 1990. Biochemical changes induced by accelerated aging in sunflower seeds. II. RNA populations and protein synthesis. Physiologia Plantarum 80: 598-604. Gray D, Rowde HR, Drew RLK. 1990. A comparison of two large scale priming techniques. Annals of Applied Biology 116: 611-616. Gutierrez G, Cruz F, Moreno J, Gonzalez-Hernandez VA, VazquezRamos JM. 1993. Natural and artificial seed ageing in maize: germination and DNA synthesis. Seed Science Research 3: 279-285. Guy P, Smith S, Black M. 1991. Changes in DNA associated with vigour and viability in wheat as revealed by RFLP analysis: a preliminary study. Journal of Experimental Botany 18: 27.

deteriorated seeds (8 d ageing), priming induced a further deterioration by reducing the GP from about 25-9 to 5%. Thus, priming appeared of no benefit to seeds which are close to death and unlikely able to undertake essential cellular repair processes efficiently. This result accords with the findings of Roberts (1981) and Pandey (1989), who reported that severe damage developing in the phase preceding seed death is irreversible. The amount of priming-induced DNA synthesis in controlled deteriorated seeds was lower than in unaged seeds. This indicates that decline in seed viability is preceded by slowing down of metabolism related to germination, including DNA replication. When observed, the amount of DNA synthesis induced by a specific priming condition, was directly correlated to restoration of seed vigour. Interestingly in 4 d aged seeds, in spite of reduced DNA synthesis and the apparent lower metabolic advancement, the positive effect on germination performance of 14 d priming treatment at 11 MPa was analogous to that observed in unaged seeds. Recovery of seed performance after priming of controlled deteriorated seeds was also observed by Coolbear, Slater and Bryant (1990) and Fujikura and Karssen (1992). This result, together with the previously described results obtained in unaged seeds, shows that the length of a priming treatment greatly influences seed performance. Saracco et al. (1995) also observed that among priming treatments not inducing DNA replication, the most effective treatment is the longest one. From our previous and present results it appears that DNA synthesis is affected by the history of the seed lot and by the water potential during imbibition. As reported by Coolbear et al. (1990), nucleic acid synthesis is a prerequisite for germination but does not appear to be the only metabolic factor influencing seed germination. For instance, activation of DNA repair, whose efficiency is presumably proportional to the length of the preconditioning treatment, is of extreme importance. Hence, before DNA synthesis can start, both DNA repair mechanisms and cell cycle related processes must first be activated. During priming, other processes, not necessarily leading to DNA replication, may also be important in improving seed performance. These processes could be activated at lower osmotic potentials than those required for DNA replication, but could enable seed to improve its efficiency in synthesizing DNA during the first hours of germination. Lanteri et al. (1993) observed that priming treatments which do not induce DNA replication both accelerate and increase the amount of DNA synthesis during a subsequent period of imbibition in water, which precedes visible germination. In the present work we have investigated the effect of priming on artificially aged seeds, after exposure to high temperature to induce loss of viability. However Priestley and Leopold (1979) observed that response to priming is mainly related to deterioration during seed storage, and hypothesized that slight ageing might not necessarily cause accumulation of metabolic defects in the same way as severe ageing conditions. This aspect has been also analysed by comparing DNA activity (Gutierrez et al., 1993), phospholipid pattern (Koostra and Harrington, 1969), and chromosome damage (Rao et al., 1988; Lanteri and Belletti, 1990)

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ISTA. 1993. International rules for seed testing. Seed Science and Technology 21: Supplement, Rules. Koopman MJF. 1963. Results of a number of storage experiments conducted under controlled conditions (other than agricultural seeds). Proceedings of the International Seed Testing Association 28: 853-860. Koostra PT, Harrington JF. 1969. Biochemical effects of age on membranal lipids of Cucumis sativus L. seeds. Proceedings of the International Seed Testing Association 34: 329-340. Lanteri S, Belletti P. 1990. Frequency of chromosome aberrations induced during storage in Pinus nigra Arnold seeds. Journal of Genetics and Breeding 44: 281-290. Lanteri S, Kraak HL, De Vos CHR, Bino RJ. 1993. Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper {Capsicum annuum L.). Physiologia Plantarum 89: 433-440. Lanteri S, Saracco F, Kraak HL, Bino RJ. 1994. The effect of priming on nuclear replication activity and germination of pepper (Capsicum annuum L.) and tomato (Lycopersicon esculentum L.) seeds. Seed Science Research 4: 81-87. Osborne DJ. 1983. Biochemical control systems operating in the early hours of germination. Canadian Journal of Botany 61: 3568-3577. Pandey DK. 1989. Priming induced alleviation of the effects of natural ageing derived selective leakage of constituents in French bean seeds. Seed Science and Technology 17: 391-397.

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Priestley DA, Leopold AC. 1979. Absence of lipid oxidation during accelerated aging of soybean seeds. Plant Physiology 63: 726-729. Rao NK, Roberts EH, Ellis RH. 1987. Loss of viability in lettuce seeds and the accumulation of chromosome damage under different storage conditions. Annals of Botany 60: 85-96. Rao NK, Roberts EH, Ellis RH. 1988. A comparison of the quantitative effects of seed moisture content and temperature on the accumulation of chromosome damage and loss of seed viability in lettuce. Annals of Botany 62: 245-248. Roberts EH. 1973. Loss of seed viability: chromosomal and genetical aspects. Seed Science and Technology 1: 515-527. Roberts EH. 1981. Physiology of ageing and its application to drying and storage. Seed Science and Technology 9: 359-372. Saracco F, Bino RJ, Bergervoet JHW, Lanteri S. 1995. Influence of priming-induced nuclear replication activity on storability of pepper (Capsicum annuum L.) seed. Seed Science Research 5: 25-29. Saxena PK, King J. 1988. Isolation of nuclei and their transplantation into protoplasts. In: Bajaj, YPS, ed. Biotechnology in agriculture and forestry. Plant protoplast and genetic engineering. New York: Springer-Verlag, 328-342. Vazquez Ramos J, Lopez S, Vazquez E, Murillo E. 1988. DNA integrity and DNA polymerase activity in deteriorated maize embryo axes. Journal of Plant Physiology 133: 600-604.

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