Effect of the desiccation tolerance and cryopreservation methods on the viability of Citrus limon L. Burm cv. Eureka seeds

Cryopreservation of the germplasm for long-term periods is of great importance to maintain the genetic resource. Argentina is one of the world's highest lemon producing country. The performance of different cooling/warming rates in the cryopreservation method of Citrus limon L. Burm cv. Eureka seeds and their influence on the interval of optimal moisture content in the desiccation stage were analyzed. Water sorption isotherm was determined and modeled using D'Arcy & Watt equation; it provided important information concerning the amounts of water associated to strong, weak and multimolecular binding sites along the sorption isotherm. Seeds tolerated a wide range of desiccation conditions (0.1<a_w<0.85) showing a high viability (>80%), however desiccation to 0.0526 g H₂O g⁻¹ d.b. (a_w = 0.0901) produced a significant loss of viability. Differential Scanning Calorimetry was used to identify the thermal transitions of lipids and water in the seed; enthalpies were used to calculate the unfrozen water fraction (0.19 g H₂O g⁻¹ d.b. corresponding to a_w = 0.64). Two cooling/warming rates were tested on desiccated seeds (0.11<a_w<0.85): i) 200 °C min⁻¹ (reached with seeds placed inside a closed cryogenic vial); ii) 1000 °C min⁻¹ (reached with aluminum-foiled seeds placed in a perforated cryogenic vial). For both methods, viability was maximum (83.3%) at a_w = 0.64. Lethal ice formation was responsible for the loss of viability at a_w>0.64 corresponding to the unfrozen water fraction. The use of higher cooling/warming rates enables a wider range of desiccation conditions (0.33<a_w<0.76) in cryopreservation procedures. This work contributes to the optimization of cryopreservation methods of economically important germplasm.     

Sugars and desiccation tolerance in seeds

Soluble sugars have been shown to protect liposomes and lobster microsomes from desiccation damage, and a protective role has been proposed for them in several anhydrous systems. We have studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating soybean (Glycine max L. Merr. cv Williams), pea (Pisum sativum L. cv Alaska), and corn (Zea mays L. cv Merit) axes. The loss of desiccation tolerance during imbibition was monitored by following the ability of seeds to germinate after desiccation following various periods of preimbibition and by following the rates of electrolyte leakage from dried, then rehydrated axes. Finally, we analyzed the soluble sugar contents of the axes throughout the transition from desiccation tolerance to intolerance. These analyses show that sucrose and larger oligosaccharides were consistently present during the tolerant stage, and that desiccation tolerance disappeared as the oligosaccharides were lost. The results support the idea that sucrose may serve as the principal agent of desiccation tolerance in these seeds, with the larger oligosaccharides serving to keep the sucrose from crystallizing.     

Glass formation and desiccation tolerance in seeds

The formation of intracellular glass may help protect embryos from damage due to desiccation. Soluble sugars similar to those found in desiccation tolerant embryos were studied with differential scanning calorimetry. Those sugars from desiccation tolerant embryos can form glasses at ambient temperatures, whereas those from embryos that do not tolerate desiccation only form glasses at subzero temperatures. It is concluded that tolerant embryo cells probably contain sugar glasses at storage temperatures and water contents, but intolerant embryo cells probably do not.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Cytological changes related with salt tolerance in embryogenic callus of Citrus limon

Electron microscopy observations of salt-tolerant embrogenic calli of Citrus limon [(L.) Burm. f.] showed several changes in cell ultrastructure when compared with control calli. Both types of calli comprised clusters of meristematic cells, but salt-tolerant calli had several structural differences: thick cell walls, ring-shaped mitochondria, an increased content of lipid bodies, microbodies and parallel accumulation of rough endoplasmatic reticulum. These structural features seem to be related with salt tolerance in Citrus limon cells.     

Induction of desiccation tolerance in microspore-derived embryos ofBrassica napus

Summary A method was developed to induce desiccation tolerance in microspore-derived embryos ofBrassica napus. Treatment of 14- to 20-day-old embryos with 1&#x00D7;10^&#x2212;4 M abscisic acid, in the light, induced tolerance to slow desiccation over a 6-day period. Under these conditions 88 to 100% of embryos of the five cultivars tested survived (as measured by moisture uptake, greening, and growth of the shoot and root meristem) after storage for 1 wk at tissue water content levels of less than 20%. The response was found to be dependent on the abscisic acid concentration in the culture medium and time of exposure of the embryos to the abscisic acid-containing medium, with exposure times of as little as 1 day having a beneficial effect. Exposure times to abscisic acid (ABA) of 5&#x2013;7 days resulted in the highest survival rates. Embryo age and size at the time of ABA exposure also affected the subsequent survival and development of embryos, with older and larger embryos exhibiting the best responses.     

Maturation proteins and sugars in desiccation tolerance of developing soybean seeds

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 μg/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 μg/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.     

钙对吸胀的绿豆种子脱水耐性的影响

以绿豆种子为材料,研究了预吸胀种子脱水耐性的变化,以及Ca^2 处理对种子脱水耐性的影响。结果表明：绿豆种子的脱水耐性随预吸胀时间的延长而下降;Ca^2 预吸胀处理能提高种子的脱水耐性,适宜的Ca^2 浓度为20mmol／L;Ca^2 能修复预吸胀种子的脱水伤害,适宜的Ca^2 浓度为2．5～5mmol／L。     

Dry artificial seeds and desiccation tolerance induction in microspore-derived embryos of broccoli

Desiccation tolerance of broccoli microspore-derived embryos was induced by exogenous application of abscisic acid (ABA). Embryos, which were desiccated to about 10% water content, were estimated for viability after rehydration. Survival was dependent on the ABA concentration and the development stage of embryo, but not on the length of exposure period to ABA or genotype. Cotyledonary stage embryos acquired the highest desiccation tolerance when treated with 1×10^-4M ABA. Under this condition, on average 27–48% of the desiccated embryos could convert into plants. Embryos treated with 1×10^-6M ABA or no ABA or earlier development-staged embryos, such as globular and heart stages, lost viability after desiccation. A one day exposure to ABA had the similar effect on the induction of desiccation tolerance as a 7-day treatment. The dried embryos maintained their ability of plant conversion after three months of storage under room conditions. The plants derived from the desiccated embryos were not different in the morphology or ploidy level from those from non-desiccated ones.Abbreviations ABA abscisic acid - RH relative humidity     

假槟榔种子催芽技术和脱水耐性的研究

为提高假槟榔的人工种植技术,对其种子做了不同的化学催芽处理,以寻求种子的有效催芽方法,并对种子脱水耐性进行了探讨。结果表明:20%过氧化氢和98%浓硫酸浸泡5min,0.3%亚硝酸钠和0.2%硝酸钾溶液浸种24h后,发芽率显著升高,速度显著加快,尤以浓硫酸和硝酸钾处理效果为好;200～1000mg/L赤霉素和20～100mg/L激动素溶液浸泡24h也显著促进种子萌发,但催芽效果与溶液浓度有关。成熟种子轻度脱水,发芽率有所上升,但含水量下降至17%以下,发芽率急剧下降,当含水量下降10%以下,发芽力完全丧失。由此可见,种子很可能是中间型种子。     

Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 μM) and bergaptol (140 μM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.     

Phytohormone bioregulation of nomilin biosynthesis in Citrus limon seedlings

Auxins such as indoleacetic acid, indolebutyric acid, naphthaleneacetic acid and 2,4,5-trichlorophenoxyacetic acid were found to be potent inhibitors of nomilin biosynthesis in young seedlings of Citrus limon. Up to 97 % of the biosynthesis was inhibited. The auxins used were all effective and inhibited the biosynthesis of nomilin selectively. Abscisic acid was also a potent inhibitor of nomilin biosynthesis and the inhibition was reversed with a cytokinin. Gibberellic acid (GA₃) had no effect on nomilin biosynthesis.     

The role of recovery of mitochondrial structure and function in desiccation tolerance of pea seeds

Mitochondrial repair is of fundamental importance for seed germination. When mature orthodox seeds are imbibed and germinated, they lose their desiccation tolerance in parallel. To gain a better understanding of this process, we studied the recovery of mitochondrial structure and function in pea (Pisum sativum cv. Jizhuang) seeds with different tolerance to desiccation. Mitochondria were isolated and purified from the embryo axes of control and imbibed-dehydrated pea seeds after (re-)imbibition for various times. Recovery of mitochondrial structure and function occurred both in control and imbibed-dehydrated seed embryo axes, but at different rates and to different maximum levels. The integrity of the outer mitochondrial membrane reached 96% in all treatments. However, only the seeds imbibed for 12 h and then dehydrated recovered the integrity of the inner mitochondrial membrane (IMM) and State 3 (respiratory state in which substrate and ADP are present) respiration (with NADH and succinate as substrate) to the control level after re-imbibition. With increasing imbibition time, the degree to which each parameter recovered decreased in parallel with the decrease in desiccation tolerance. The tolerance of imbibed seeds to desiccation increased and decreased when imbibed in CaCl(2) and methylviologen solution, respectively, and the recovery of the IMM integrity similarly improved and weakened in these two treatments, respectively. Survival of seeds after imbibition-dehydration linearly increased with the increase in ability to recover the integrity of IMM and State 3 respiration, which indicates that recovery of mitochondrial structure and function during germination has an important role in seed desiccation tolerance.