Desiccation-induced changes in lipid peroxidation, superoxide level and antioxidant enzymes activity in neem (Azadirachta indica A. Juss) seeds

The freshly harvested mature neem seeds (42.2 % seed moisture content) with 100 % viability deteriorate when naturally desiccated to below 10.9 %. The desiccation-induced loss of viability was closely associated with over accumulation of superoxide anion and lipid peroxidation products both in the embryonic axes and cotyledons. The levels of superoxide anion and lipid peroxidation products were higher in axes compared to cotyledons. Superoxide dismutase activity was not much affected, both in the axes and cotyledons of 100 % viable seeds during desiccation from 42.2 % to 10.9 % seed moisture content. Steep rise in its activity was observed during drying below lowest safe moisture content (LSMC). Activities of catalase and peroxidase exhibited substantially higher levels in the 100 % viable seeds dehydrated up to LSMC. Their activities declined sharply in seeds with water content below LSMC. Impairment of catalase and peroxidase activities possibly lead to enhanced accumulation of reactive oxygen species. The accumulation of superoxide anion, lipid peroxidation and differential expression of superoxide dismutase and catalse/peroxidase activities in response to desiccation (below LSMC) is discussed to explain the intermediate storage physiology of neem seeds.     

Differential responses of Mimusops elengi and Manilkara zapota seeds and embryos to cryopreservation

Several dehydration protocols were evaluated for their ability to cryopreserve intact seeds and excised embryonic axes of Mimusops elengi and Manilkara zapota (Sapotaceae). Both interspecific and intraspecific variations in cryotolerance were found. M. zapota embryonic axes were more tolerant of cryopreservation than those of M. elengi, and showed higher desiccation tolerance, higher post-thawing survival and development, and a much wider range of moisture contents for cryopreservation. Maximum development rates were 94% and 27% for M. zapota and M. elengi, respectively. Intact seeds of both species tolerated desiccation to low moisture levels, but were sensitive to liquid nitrogen exposure, and cryopreserved seeds failed to germinate. Assessment of developing embryos excised from cryopreserved seeds associated nonviability of cotyledons and plumules with germination failure. Other structures survived at variable rates; most hypocotyls and radicles (up to 76% and 98% for M. elengi and M. zapota, respectively) were viable. The different cryotolerance between hypocotyls and cotyledons is a critical cause for failure in cryopreservation, contributing to the difficulty in developing protocols for such intermediate oily seeds.     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Gibberellin-like activity in cotyledons and embryonic axes during pea seed germination

Endogenous gibberellin-like activity was determined in dry pea seeds (Pisum sativum cv. Bördi), in cotyledons and axes of germinating pea seeds and also in excised cotyledons and axes. During the first two days of pea seed germination, neither the embryonic axes nor the cotyledons show a mutual influence on gibberellin activity, but this appears after 72–96 h of germination. The gibberellin-like activity m cotyledons and axes of germinating seeds increased during the same period, but it decreased in isolated axes and excised cotyledons.     

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H2O [g DW] ?1, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100℃. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100℃ for 15 min and that of lotus seeds was 13.5% following the treatment at 100℃ for 24 h. The time in which 50% of lotus and maize seeds were killed by 100℃ was about 14.5 h and 6 min, respectively. With increasing treatment time at 100℃, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100℃ was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100℃ was more than 12 h, plasmoly-sis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plas-molemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0-12 h of the treatment at 100℃ and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5-10 min of the treatment at 100℃ and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100℃ and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treat-ment time at 100℃. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100℃ and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100℃. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.     

Involvement of gibberellins in expression of a cysteine proteinase (SH-EP) in cotyledons of Vigna mungo seedlings.

The expression of a papain-type proteinase, designated SH-EP, in cotyledons of Vigna mungo seedlings has been shown to require some factors in the embryonic axes. Gibberellin A1 (GA(1)) and GA(20) were identified by GC-MS in embryonic axes of V. mungo seedlings. The level of accumulation of SH-EP in cotyledons of V. mungo seedlings was greatly reduced by treatment of the seeds with uniconazole-P, an inhibitor for GA biosynthesis. The reduced level of accumulation of SH-EP in cotyledons by uniconazole-P was recovered by exogenous application of GA(1) and GA(20) to the seedlings.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation

Active polyamine metabolism occurs in Glycine max (L.) seeds during development. Most (≥97%) of putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad) are present as free forms in the growing embryo. In the cotyledon or embryonic axis, Put decreases to a nearly undetectable level, while Spd level sharply increases as seed dry weight accumulation progresses. Spm level in the axis also increases along with the Spd level. There is little change in Spm level in the cotyledons. Maturation and dehydration results in a slight reduction of Spd level in the cotyledons. Cad is present in relatively large quantities (5.5-12 micromoles per gram dry weight) in the axes of mature soybean seeds. Only traces of Cad, as expressed on a dry weight basis, are found in the developing or mature cotyledons. The synthesis and accumulation of Cad in the axis begins at the time when the axis or the seed accumulates 30 to 50% of its maximum dry weight. The Cad accumulation (0.8 nanomole per axis per day) proceeds until the later stages of dehydration. When soybean plants are subjected to complete defoliation and shade during the midpoint of seed maturation, Cad accumulation in the axis and seed dry weight accumulation ceased almost immediately. The treatment, however, does not affect the viability of soybean seeds.     

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H₂O [g DW] ⁻¹, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100°C. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100°Cfor 15 min and that of lotus seeds was 13.5% following the treatment at 100°C for 24 h. The time in which 50% of lotus and maize seeds were killed by 100°C was about 14.5 h and 6 min, respectively. With increasing treatment time at 100°C, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100°C was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100°C was more than 12 h, plasmolysis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plasmolemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0 −12 h of the treatment at 100°C and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5–10 min of the treatment at 100°C and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100°C and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treatment time at 100°C. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100°C and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100°C. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.     

Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons

Halińska, A., Sińska, I. and Lewak, St. 1987. Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons.
Free and conjugated gibberellins (GAs) A₄₊₇ and A₉ were determined in embryonic axes and in cotyledons of seeds of apple ( Malus domestica Borb., cv. Antonó wka) during breaking of dormancy under cold stratification. In both organs, the maximum level of free GA₄₊₇ was found at day 30 of stratification, but the concentration was 700 times higher in axes than in cotyledons. Comparison of changes in free and conjugated GA₄₊₇ levels during stratification allow us to suggest that the accumulation of free hormone in axes is, at the most, to 40% due to release from conjugates already present in the axis; that maximally 20% is derived from hydrolysis of cotyledonary conjugates translocated to axes; and that at least 40% originate from the novo biosynthesis of the hormone. Free and conjugated GA₉ levels were similarly altered in axes and in cotyledons, markedly increasing at the end of afterripening. Both release of the free hormone from conjugates and biosynthesis of GA₉, appeared to be involved in that increase; no translocation of free or bound GA₉, between axes and cotyledons was demonstrated.     

Antioxidative response of ascorbate-glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds

Ascorbate–glutathione systems were studied during desiccation of recalcitrant seeds of the silver maple (Acer saccharinum L.). The desiccated seeds gradually lost their germination capacity and this was strongly correlated with an increase in electrolyte leakage from seeds. Simultaneously the increase of reactive oxygen species (ROS) (superoxide radical – O₂⁻ and hydrogen peroxide – H₂O₂) production was observed. The results indicate that remarkable changes in the concentrations and redox status of ascorbate and glutathione occur in embryo axes and cotyledons. After shedding, concentrations of ascorbic acid (ASA) and the reduced form of glutathione (GSH) are higher in embryo axes than in cotyledons and their redox status is high in both embryo parts. Cotyledons in freshly shed seeds are devoid of GSH. At the first stages of desiccation, up to a level of 43% of moisture content, ASA content in embryo axes and GSH content in cotyledons increased. Below this level of moisture content, the antioxidant contents as well as their redox status rapidly decreased. The enzymes of the ascorbate–glutathione pathway: ascorbate peroxidase (APX) (EC 1.11.1.11), monodehydroascorbate reductase (MR) (EC 1.6.5.4), dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) increased their activity during desiccation, but mainly in embryonic axes. The changes are probably required for counteracting the production of ROS during desiccation. The relationship between ascorbate and glutathione metabolism and their relevance during desiccation of recalcitrant Acer saccharinum seeds is discussed.     

Dynamics of carbohydrates in the embryo axes of horse chestnut seeds during their transition from dormancy to germination

It was shown that the content of carbohydrates and their composition in embryo axes of horse chestnut seeds changed as seeds acquired a capability of dormancy release and germination. Sucrose prevailed among carbohydrates, comprising to 150–160 mg/g dry wt. During the first half of the seed imbibition time, oligosaccharides, namely raffinose and stachyose, degraded, whereas the contents of glucose and fructose were very low. The second half of the imbibition period (until radicle protrusion) was characterized by a cessation of oligosaccharide breakdown and accumulation of monosaccharides. Carbohydrate balance showed that the contribution of oligosaccharide breakdown to sucrose and monosaccharide accumulation was rather small, and monosaccharides accumulated mostly at the expense of sucrose gradually coming from cotyledons during imbibition. The trend of carbohydrate metabolism in imbibing axial organs was similar during the entire period of a seed dormancy release in the course of stratification. A readiness for the commencement of these processes during the entire dormancy period implies that carbohydrate conversions in embryo axes are not a trigger for a dormancy release. Monosaccharide accumulation in embryo axes before radicle protrusion produces an increase in the osmotic pressure, as compared to that provided by sucrose, by approximately 20%. Recalcitrance of the horse chestnut seeds is discussed in relation to the role of carbohydrates and other endogenous osmotica in the establishment of osmotic properties.     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes

Characterization of sugar content and enzyme activity in germinating soybean (Glycine max L. Merrell) seeds led to the discovery of sorbitol accumulating in the axes during germination. The identity of sorbitol was confirmed by relative retention times on high-performance liquid chromatography and gas liquid chromatography and by mass spectra identical with authentic sorbitol. Accumulation of sorbitol in the axes started on day 1 of germination as sucrose decreased and glucose and fructose increased. Sucrose also decreased in the cotyledons, but there was no accumulation of sorbitol, glucose, or fructose. Accumulation of sorbitol and hexoses was highly correlated with increased invertase activity in the axes, but not with sucrose synthase and sucrose phosphate synthase activities. Sucrose synthase activity was relatively high in the axes, whereas the activity of sucrose phosphate synthase was relatively high in the cotyledons. Ketose reductase and aldose reductase were detected in germinating soybean axes, but not in cotyledons. Fructokinase and glucokinase were present in both axes and cotyledons. The data suggest a sorbitol pathway functioning in germinating soybean axes, which allows for the interconversion of glucose and fructose with sorbitol as an intermediate.     

The protective role of selenium in recalcitrant Acer saccharium L. seeds subjected to desiccation

Freshly harvested silver maple (Acer saccharinum L.) seeds were soaked in either sodium selenite (10 mg/L) or water for 6 h. After washing and air drying, seeds were desiccated at 22 °C at a RH of 45-50% to comparable water levels from 50 to 12%. Germination capacity was significantly higher in seeds treated with selenium and desiccated [from 50 to 40, 35 and 30% of water content (WC)] than in water-soaked seeds. At 20% WC, the seeds from both treatments had low viability (approximately 20%). The electrolyte leakage and the MDA content were significantly lower in the embryonic axes of seeds soaked in selenite than in seeds soaked in water. We also found that the activity of glutathione peroxidase (GPX) of embryonic axes from selenium-treated seeds that were not desiccated, or from seeds that were desiccated to 40 and 35% WC, was significantly higher than that of non-treated axes. No difference in GPX activity was detected in cotyledons. This was confirmed by activity staining of GPX after native PAGE of proteins extracted from embryonic axes and cotyledons. An increase in glutathione reductase (GR) activity was also observed in embryonic axes of seeds treated with selenium and dried to 35 and 30% WC compared to non-treated samples. Selenium appeared to have no such effect on cotyledons.     

Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings

Low temperature represents one of the principal limitations in species distribution and crop productivity. Responses to chilling include the accumulation of simple carbohydrates and changes in enzymes involved in their metabolism. Soluble carbohydrate levels and invertase, sucrose synthase (SS), sucrose-6-phosphate synthase (SPS) and alpha-amylase activities were analysed in cotyledons and embryonic axes of quinoa seedlings grown at 5 degrees C and 25 degrees C in the dark. Significant differences in enzyme activities and carbohydrate levels were observed. Sucrose content in cotyledons was found to be similar in both treatments, while in embryonic axes there were differences. Invertase activity was the most sensitive to temperature in both organs; however, SS and SPS activities appear to be less stress-sensitive. Results suggest that 1) metabolism in germinating perispermic seeds would be different from endospermic seeds, 2) sucrose futile cycles would be operating in cotyledons, but not in embryonic axes of quinoa seedlings under our experimental conditions, 3) low temperature might induce different regulatory mechanisms on invertase, SS and SPS enzymes in both cotyledons and embryonic axes of quinoa seedlings, and 4) low temperature rather than water uptake would be mainly responsible for the changes observed in carbohydrate and related enzyme activities.     

Ascorbic acid metabolism in ageing recalcitrant sal (Shorea robusta Gaertn. f.) seeds

Changes in ascorbate content and its enzymatic utilization pattern were studied in embryonic axes and cotyledons of sal seeds undergoing rapid loss of viability, at ambient conditions. Ascorbate levels were significantly higher initially in the embryonic axes (0.32 mg/g fresh weight) and cotyledons (0.21 mg/g fresh weight) of freshly mature, relatively hydrated (42.2% moisture content) and 100% viable sal seeds. It declined sharply as the tissues; embryonic axes and cotyledons, desiccated with absolutely no detectable amount in non-viable seeds (21% moisture content). Significantly strong correlation was obtained between desiccation of embryonic axes (r = 0.96) and cotyledon (r = 0.97) with loss of ascorbate levels and loss of germinability. Higher rates of ascorbic acid utilization (AAU) recorded in the embryonic axes of 100% viable seed declined sharply as the seed viability reduced due to desiccation below 36.8% moisture content. AAU was not detected in the cotyledons.     

Biogenesis of Protein Bodies in Embryonic Axes of Soybean Seeds (Glycine max. cv. Enrei)

Protein bodies in embryonic axes of soybean seeds have inclusion structures containing phytin globoids. Biogenesis of the protein bodies during seed development was examined by transmission electron microscopy. Protein bodies in embryonic axes originated from central vacuoles. The central vacuole in embryonic axes subdivided into smaller vacuoles with internal membranous structure. Then the subdivided vacuoles were directly associated with rough endoplasmic reticulum (rER), and were filled with proteinaceous matrix from the peripheral region. The increase of matrix was simultaneous with accumulation of β-conglycinin estimated by SDS-polyacrylamide gel electrophoresis. Glycinin-rich granules that had been found in developing cotyledons were not observed in embryonic axes. After proteinaceous matrix filled the protein bodies, electron-transparent regions presumably surrounded by a single membrane appeared in the matrix. Phytin globoids were constructed in this internal structures of protein bodies as the final step of protein body formation.     

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris)

French bean ( Phaseolus vulgaris ) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.     

ROS-induced oxidative stress is closely related to pollen deterioration following cryopreservation

Oxidative stress induced by excessive accumulation of reactive oxidative species (ROS) during cryopreservation is thought to be one factor contributing to cryodamage of biological materials. To explore the role of oxidative stress in the cryopreservation of plant pollen, germination, ROS, and malondialdehyde (MDA) levels of pollen from 20 ornamental plant species were compared before and after cryopreservation. The results showed that the germinability of cryopreserved pollen from 13 out of the 20 species was not significantly different from that of fresh pollen (group 1), only one increased significantly, while the other six declined significantly (group 2). The MDA content in cryopreserved pollen from nine species in group 1 showed no significant difference from that of fresh pollen, while four species in group 2 rose significantly. This suggested that pollen viability and MDA levels were negatively correlated. ROS generation in cryopreserved pollen from nine species in group 1 was unchanged compared to fresh pollen, while five species in group 2 increased significantly. This suggested that pollen viability was negatively correlated with ROS generation. Additionally, both ROS and MDA levels in pollen from four species in group 2 increased significantly. In conclusion, pollen from most species possesses some cryostorage tolerance, but some species are severely damaged by cryostorage. Oxidative stress induced by the cryostorage in liquid nitrogen (LN) may be a key factor for the decreased viability in pollen following cryopreservation.