Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature

Seeds of Dactylorhiza fuchsii (common spotted orchid) and Anacamptis morio (green-winged orchid) were encapsulated in alginate beads with hyphae of the basidomycete fungus Ceratobasidium cornigerum. Pre-treatment of beads for 18 h with sucrose at an optimum concentration of 0.75 M decreased the desiccation rate in a flow of sterile air (c. 23 degree C, 30% RH) and increased seed and fungal survival after up to 16 h drying. Pre-treated and 16-h dried beads were transferred to cryo-vials and subsequently stored at a range of low temperatures for up to 30 d. Neither embryo growth of both orchids nor fungal development was detrimentally affected by 1 d storage at -196 degree C when the beads were pre-dried to c. 20% moisture content. Encapsulated D. fuchsii seed and compatible fungus had < 5% and < 45% viability when beads of the same moisture content were stored for 1 d at -20 degree C and -70 degree C respectively. In contrast, viability of the seed and the fungus remained unchanged during 30 days storage at -196 degree C but was progressively lost at 16 degree C over the same interval. The results indicate opportunities for the use of simultaneous cryopreservation as a conservation tool for diverse taxa.     

Narrowing oF the critical hydration window for cryopreservation of Salix caprea seeds following ageing and a reduction in vigour

We investigated the effects of desiccation, rehydration and cryopreservation on the viability of seeds of a wild mountain species and seven clones of Salix caprea L. Seeds responded differently to all treatments depending on clone, seed initial moisture content (MC) and seed vigour. Fresh seeds of two randomly selected clones tolerated desiccation to MC 8.5-9.6 % FW (0.09-0.11 g water per g dry mass. g/gdw) without any noticeable loss in viability and were successfully cryopreserved at MCs ranging from 8.5 to 23.4 % (0.09-0.30 g/gdw). Storage at 5 degree C for approximately 10 weeks significantly reduced the viability of seed lots of a wild species and of three S. caprea clones, whilst viability of seeds of four other clones remained unaffected. Since all clones tested were genetically derived from one tree, this variation is unlikely to be of maternal origin. Most probably paternal x environmental factors have influenced seed behavior during desiccation and storage. As viability decreased due to partial ageing, seeds became more susceptible to desiccation stress. When seeds of three clones were cryopreserved, the hydration window for survival was wider for highly vigorous seeds (c. 0.05-0.28 g/gdw) than for seeds with intermediate vigour (c. 0.10-0.24 g/gdw) and low vigour (c. 0.20-0.37 g/gdw). Rehydration to MC above 0.15 g/gdw improved germination of low vigour seeds, both in controls and after cryopreservation. In contrast, cryopreservation of high vigour seeds rehydrated to MCs above 0.11 g/gdw resulted in a sharp decrease in normal seedling production. Whilst no effect of cryogenic temperature on germination and normal seedling production was observed when seeds of seven clones were cryopreserved within their hydration windows, the results indicate the need to account for seed lot vigour when designing cryopreservation protocols.     

Low temperature survival of slender naiad (Najas flexilis) seeds

The response to drying and storage at -20 degrees C or in liquid nitrogen was studied in seeds of the freshwater aquatic plant Najas flexilis. The seeds of this species show some desiccation sensitivity, although post-harvest storage in water at 16 degrees C resulted in improvements in desiccation tolerance. There was 63% germination of seeds dried to 9.5% moisture content (30% RH) following this maturation period. Optimum moisture contents for seeds stored at -20 degrees C for 3 months and in liquid nitrogen for 1 week were ~11% and ~15%, respectively.     

New determinants for tolerance of coffee (Coffea arabica L.) seeds to liquid nitrogen exposure

The present work establishes for the first time that tolerance of coffee seeds to liquid nitrogen (LN) exposure depends on the initial quality of the seedlot and on the rewarming regime employed. Seedlot quality was estimated by the parameters of a quantal response model of desiccation sensitivity developed previously. The percentage of seedlings recovered from cryopreserved seeds was very well correlated with the relative humidity (RH) at which 90 percent of the initial viability was retained, RH90, as estimated by the model. Whatever the cooling regime employed, rewarming the seeds slowly by exposing them to ambient air was highly detrimental. Slow rewarming-induced viability loss was not due to imbibitional damage since seeds pre-heated at 37 degree C after slow rewarming to 0 degree C exhibited a survival percentage lower than seeds thawed rapidly to 0 degree C before sowing. The optimal hydration status for coffee seed cryopreservation was also re-examined. Drying seeds in 81 percent RH provided survival percentages considerably higher than those obtained using the drying RH always employed until now, i.e. 78 percent. A new procedure for slowly precooling the seeds prior to immersion in LN was also established. It consisted of placing the vials containing the seeds in a dry ice-bath for 25 min. Using this procedure in combination with seed drying in 81 percent RH and rapid rewarming in a 37 degree C water-bath for 30 min ensured the highest survival percentages ever obtained with coffee seeds, i.e. 89 percent, a value which was not significantly different from the initial viability percentage.     

Acquisition of cryotolerance in maize embryos during seed development

A desiccation-based cryopreservation protocol was employed to study the development of cryotolerance and desiccation tolerance in maize embryos from 23 to 50 days after pollination (DAP). Tolerances were acquired gradually and concomitantly. Maize embryos had low desiccation tolerance at 23 DAP when assessed by survival (embryo elongation) and emergence (root and shoot growth) after dehydration. Desiccation tolerance increased progressively, reached its maximum at 38 DAP, and remained constant afterwards. Cryotolerance, assessed by survival and emergence of post-thaw embryos, however, was nil until 26 DAP. Embryos at 29 DAP withstood cryoexposure within a very narrow moisture range only. Throughout development cryotolerance increased gradually, reached a maximum at 44 DAP and then remained at this level. The correlation between moisture content and cryopreservation success was notably influenced by the maize embryo's development stage. As seeds developed, the moisture content allowing 90% dehydrated embryos to survive and to emerge decreased, while the upper moisture content allowing 50% post-thaw embryos to survive and to emerge increased. Moisture contents of c. 14% allowed no less than 50% post-thaw embryos to emerge at the later development stages (e.g. c. 44 DAP); but no embryos within the same moisture range survived cryoexposure at 29 DAP, although they could withstand desiccation to this moisture level without impairment of survival and emergence. The relationship between cryotolerance and desiccation tolerance during maize seed development is discussed.     

Cryopreservation of Citrus aurantifolia seeds and embryonic axes using a desiccation protocol

The desiccation and freezing tolerance of seeds, with and without testas, and embryonic axes of Citrus aurantifolia were investigated. Seeds were desiccated with silica gel, under the laminar air flow cabinet or by placing them on a laboratory bench. Whatever the desiccation method employed, survival before and after cryopreservation was higher for seeds without testas. When freezing intact seeds, the highest survival percentage (41.3 %) was achieved after desiccation to 7.3 % moisture content (fresh weight basis) on the laboratory bench. Survival of seeds cryopreserved without testas could reach up to 85 % after desiccation under the laminar air flow cabinet or on the laboratory bench, corresponding to moisture contents of 7.1 and 4.5 %, respectively. After desiccation with silica gel, survival reached a maximum of 60.0 %, for a seed moisture content of 3.3 %. Survival of control embryonic axes was high (80-100 %) whatever the sucrose concentration in the preculture medium and the duration of the desiccation period. After cryopreservation, no survival was noted with embryonic axes, which had not been precultured nor desiccated. Survival of non-desiccated embryonic axes after cryopreservation increased progressively in line with increasing sucrose concentrations in the preculture medium, from 7.5 % with 0.1 M sucrose to 77.5 % with 0.7 M sucrose. Survival of desiccated and cryopreserved embryos was always high, whatever the preculture treatment and desiccation period, ranging from 55.8 % to 92.5 %.     

Pollen from Glycine species survive cryogenic exposure

Pollen of 12 genotypes of the annual soybean and its wild perennial relatives were stored without pre-desiccation at low temperatures (-20 C and -196 C) and tested for their viability in vitro. The influence of cryopreserved pollen on pod set and seed production was also investigated. Cryopreserved pollen of all the genotypes showed germination in vitro. Pollen of annual soybean stored at -20 C retained their viability for 4 months, however, pollen of its wild perennial relatives at same storage conditions failed to germinate in vitro. Flowers pollinated with cryopreserved pollen had similar pod set and number of seeds/pod as those pollinated with fresh pollen. Results of this study suggest that cryopreservation of pollen can be used successfully for soybean breeding, and also offers the possibility of conserving the haploid gene pool of soybean and wild perennial species in a cryobank facility.     

Acquisition and loss of cryotolerance in Livistona chinensis embryos during seed development

Changes in desiccation tolerance and cryotolerance of chinese fan palm (Livistona chinensis [Jacq.] R. Br.) Embryos were studied during seed development from 15 to 45 weeks after flowering (WAF). Acquisition and then progressive loss in both desiccation tolerance and cryotolerance was observed within this period. Survival (apparent elongation of embryos) and emergence (formation of root and/or shoot) of embryos following dehydration increased progressively with development of seeds until 33 WAF, and then decreased up to 45 WAF. Similar changes occurred in the minimum moisture content at which 90% of embryos survived or emerged. Cryotolerance of embryos was nil at the early stages of seed development, until 21 WAF. Embryos acquired slight cryotolerance at 23 WAF and cryotolerance increased gradually from 27 to 36 WAF, then decreased by 45 WAF. Survival and emergence of post-thaw embryos were closely related to their moisture contents prior to freezing. However, this correlation between cryopreservation and moisture content was notably influenced by the embryos' developmental stage. Embryos at stages with greater cryotolerance gave higher post-thaw survival and emergence at a given moisture content, and the moisture content range allowing embryos to avoid cryo-damage was widened at both the lower and upper limits. Greater than 50% post-thaw emergence was observed only in embryos with moisture contents below 20% (fresh weight) at developmental stages between 27 and 36 WAF, although more than 90% of embryos could be dehydrated to < 20% moisture contents without loss in survival and emergence as early as 21WAF. Nearly 80% embryos could be dehydrated safely to 20% moisture content as late as 45 WAF.     

Zygotic and nucellar embryo survival following dehydration and cryopreservation of citrus intact seeds

A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16 percent (C. sinensis) or 10 percent (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50 percent after drying to 15 percent moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93 percent germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.     

Cryopreservation of tea (Camellia sinensis L.) seeds and embryonic axes

This study investigated the tolerance to desiccation and freezing of tea seeds, embryonic axes (EAs) and cotyledonary embryonic axes (CEAs, consisting of EAs with portions of cotyledons still attached). No seeds germinated after desiccation and cryopreservation. EAs extracted from seeds desiccated to 18.9% moisture content (fresh weight basis) and cryopreserved showed 20.7% survival but plantlet production from these EAs was impossible. When EAs and CEAs were extracted from seeds before being submitted to desiccation and freezing, survival of control and frozen samples was equivalent with both types of materials. However, plantlet production was significantly higher from control and cryopreserved CEAs than EAs. The maturity stage of the seeds from which CEAs were extracted had an important effect on their survival and plant production percentages, mature seeds providing better results than early mature and late mature seeds. The highest percentages of plantlet production from cryopreserved CEAs, which ranged between 75.1 and 80.4%, were achieved for EA moisture contents between 21.5 and 15.0%.     

Thermal analysis of the plant encapsulation-dehydration cryopreservation protocol using silica gel as the desiccant

The encapsulation-dehydration cryopreservation protocol is critically dependent upon the evaporative desiccation step, which must optimise survival with the retention of glass stability on sample cooling and rewarming. Desiccation is usually achieved evaporatively by drying in a sterile airflow. However, chemical desiccation using silica gel has advantages for laboratories that do not have environmental control and/or which are exposed to high relative humidities and risks of microbial contamination. This study characterised thermal profiles of silica gel-desiccated encapsulated shoot-tips of two Ribes species using Differential Scanning Calorimetry. For both species silica gel-desiccation at 16 degrees C for 5 h decreased bead water content from ca. 75 to 28% fresh weight (3.8 to 0.4 g x g(-1) dry weight); further desiccation (for 6 and 7 h) reduced the bead water content to 21% (0.3 g x g(-1) dry weight). These changes in water status altered the thermal properties of beads for both species. After 7 h desiccation over silica gel stable glass transitions were observed on both cooling and rewarming of beads containing meristems. Tg mid-point temperatures ranged from -78 to -51 degrees C (cooling) and from -88 to -54 degrees C (warming) [at cooling and warming rates of 10 and 5 degrees C min(-1), respectively] after 5 to 7 h silica gel-desiccation. Post-cryopreservation viability of both species was ca. 63%. Thermal analysis studies revealed that an encapsulation/dehydration protocol using silica gel as a desiccant should comprise a minimum 5 h drying (at 16 degrees C). This reduces bead moisture content to a critical point (ca. 0.4 g x g(-1) dry weight) at which stable glasses are formed on cooling and rewarming. It is concluded that silica gel has advantages for use as a desiccant for alginate-encapsulated plant meristems by promoting stable vitrification and is useful in laboratories and/or geographical locations where environmental conditions are not under stringent control.     

The effects of desiccation and exposure to cryogenic temperatures on embryonic axes of Landolphia kirkii

The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates on survival, assessed by the ability for both root and shoot development, of embryonic axes excised with a small portion of each cotyledon, from mature, recalcitrant seeds of Landolphia kirkii. All axes withstood rapid (flash) drying to a water content of c. 0.28 g water per g dry mass; however, the use of chemical cryoprotectants before flash drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g water per g dry mass, reducing survival to 10% and 0% after exposure to -196 degree C and -210 degree C, respectively. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent rewarming and rehydration. Cooling at 1 degree C per min facilitated survival of 70% of axes with attached cotyledonary segments at 0.28 g water per g dry mass after exposure to -70 degree C, with 45% viability retention when further cooled at 15 degree C per min to -180 degree C. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached.     

The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance

The effects of fruit maturity, at the time of natural dispersal, on subsequent desiccation tolerance and sub-zero storage was investigated in three lots of Acer pseudoplatanus (sycamore) collected from northern to southern Europe. Fruits from the native plant distribution range in Italy had significantly higher desiccation tolerance (0.16 g water per g DW) than those from England (0.30) and Norway (0.50), confirming that the maximum potential desiccation tolerance in sycamore exceeds that of the recalcitrant type. In contrast, the unfrozen water content varied only slightly between seedlots, but systematically reduced with development (0.35 to 0.27 g water per g DW). Maximum survival (60 percent fruit germination) of seven days sub-zero temperature storage coincided with drying the Italian fruit lot to c. 0.2 g water per g DW followed by holding at -20 degree C, above the onset temperature for freezing, or at -196 degree C (liquid nitrogen). Fruit survival was much lower in the Italian fruits when held at this water content and -70 degree C, and in all other combinations of water content, temperature and fruit lot provenance. As the risk of nucleation in partially dried fruits held at -20 degree C is high, we recommend sycamore fruits are cryopreserved for long-term conservation.     

High speed measurement of corn seed viability using hyperspectral imaging

Corn is one of the most cultivated crops all over world as food for humans as well as animals. Optimized agronomic practices and improved technological interventions during planting, harvesting and post-harvest handling are critical to improving the quantity and quality of corn production. Seed germination and vigor are the primary determinants of high yield notwithstanding any other factors that may play during the growth period. Seed viability may be lost during storage due to unfavorable conditions e.g. moisture content and temperatures, or physical damage during mechanical processing e.g. shelling, or over heating during drying. It is therefore vital for seed companies and farmers to test and ascertain seed viability to avoid losses of any kind. This study aimed at investigating the possibility of using hyperspectral imaging (HSI) technique to discriminate viable and nonviable corn seeds. A group of corn samples were heat treated by using microwave process while a group of seeds were kept as control group (untreated). The hyperspectral images of corn seeds of both groups were captured between 400 and 2500 nm wave range. Partial least squares discriminant analysis (PLS-DA) was built for the classification of aged (heat treated) and normal (untreated) corn seeds. The model showed highest classification accuracy of 97.6% (calibration) and 95.6% (prediction) in the SWIR region of the HSI. Furthermore, the PLS-DA and binary images were capable to provide the visual information of treated and untreated corn seeds. The overall results suggest that HSI technique is accurate for classification of viable and non-viable seeds with non-destructive manner.     

高光谱技术无损检测单粒小麦种子生活力的特征波段筛选方法研究

种子活力是种子质量的一项重要指标,高活力的种子具有较强的抗逆性、生长优势及生产潜力。而种子活力在种子生理成熟时最高,随后随着贮藏时间的延长而发生着自然不可逆的降低。因此,在播种前及时、准确地对种子活力进行检测和筛选具有重要的实践意义。针对传统种子活力检测方法存在的操作过程复杂繁琐、耗时长、重复性差且对种子有破坏性等缺点,研究尝试利用高光谱成像技术建立单粒小麦种子生活力快速、无损、精确的检测方法。以高温高湿老化后的190粒小麦种子（发芽128粒,不发芽62粒）作为研究样本,先利用可见-近红外(Vis-NIR)高光谱成像系统采集样本种子的光谱图像和进行标准发芽试验,并确保光谱采集试验和标准发芽试验的小麦种子一一对应。随后提取种子光谱图像的感兴趣区域并对其光谱数据进行平均和特征分析。分别采用一阶导数（FD）、均值中心化（MC）、正交信号校正(OSC)和多元散射校正（MSC）对原始光谱数据进行预处理,结合偏最小二乘辨别分析（PLS-DA）建立全波段PLS-DA模型,比较分析,并筛选出最适预处理方法。分别利用无信息变量消除算法（UVE）、竞争性自适应重加权算法(CARS)、连续投影算法(SPA)及耦合不同变量筛选方法对特征波段进行筛选提取,再分别基于所提取出的特征波段建立PLS-DA定性判别模型,对比分析,最终确立提取与单粒小麦种子生活力相关性最高的高光谱特征波段方法体系。结果表明：不同光谱预处理建立的模型其表现有所差异,在MC,FD,OSC和MSC中,采用MC对原始高光谱数据进行预处理,建立的全波段MC-PLS-DA判别模型,其校正集和预测集对小麦种子生活力的整体鉴别正确率分别为82.5%和83.0%,优于原始及其他预处理后建立的全波段PLS-DA判别模型,其校正集和预测集对小麦种子活种子鉴别正确率分别为94.8%和90.6%。进一步对比3种单特征波段提取方法及其耦合分析建模中,发现3种变量筛选方法耦合（UVE-CARS-SPA）的方式能够将光谱全波段的688个变量压缩至8个变量（473,492,811,829,875,880,947和969 nm）,利用所筛选出的8个变量建立的MC-UVE-CARS-SPA-PLS-DA模型获得了最优秀的鉴别效果,其校正集和预测集对小麦种子生活力的整体鉴别正确率分别为86.7%和85.1%,较全波段模型（MC-Full-PLS-DA）分别提升了4.2%和2.1%,活种子的鉴别正确率分别为93.8%和84.4%,经过此优秀模型筛选后,种子批最终发芽率可达到93.1%。实验结果表明,基于高光谱成像技术结合UVE-CARS-SPA-PLS-DA模型能够实现对单粒小麦种子生活力的定性判别。研究工作为小麦种子活力的快速、精确且无损的检测提供理论支持。     

Cryopreservation of Pistacia spp. seeds by dehydration and one-step freezing

Cryopreservation protocols by dehydration and one-step freezing were developed for seeds from three Pistacia species, i.e., P. vera, P. terebinthus and P. lentiscus, which were characterised by different initial germination percentages (100%, 17% and 81%, respectively). In P. vera, a maximum of 90% germination was obtained following 8 hours drying in silica gel (corresponding to 11.7% moisture content on a FW basis) and direct immersion in LN. In P. terebinthus and P. lentiscus, shorter periods of dehydration (1 hour and 15 min, respectively) were sufficient to reduce their moisture content to about 20%, which resulted in peak seed germination percentages from cryostorage of 16% and 47%, respectively. Following cryopreservation, the seeds germinated better on semi-solid MS medium, than on cotton wool wetted with dH(2)O or liquid MS medium. Finally, in P. vera and P. lentiscus, high and significant correlation coefficients were obtained between the TTC viability test and seed germinability after recovery from LN, provided that seeds which were considered positive in the test showed completely or partially red embryonic axes coupled to completely red cotyledons.     

蔬菜种子的干燥动力学及其活性

本文研究了初含水率、干燥周期、料层厚度相同时,不同供热方式（热风、热风与辐射、热风与辐射并加湿）的白菜种子在固定床的干燥动力学及其活性,与传统的只热风供热相比,当干球温度相同时辐射加热风干燥的种子终含水率比低１３．８％、发芽率比高０．３％,而辐射加热风并加湿干燥其终含水率比低５．８％,发芽率比高０．８％。为确保种子活力,建立了褚－杨白菜种子临界温度Ｔｃｖ方程,并已验证其正确性．该方程为蔬菜种子干燥提供了理论基础并有重要的工程意义。     

Vibro-acoustography imaging of permanent prostate brachytherapy seeds in an excised human prostate - Preliminary results and technical feasibility

Objective:

The objective in this work is to investigate the feasibility of using a new imaging tool called vibro-acoustography (VA) as a means of permanent prostate brachytherapy (PPB) seed localization to facilitate post-implant dosimetry (PID).

Methods and materials:

Twelve OncoSeed (standard) and eleven EchoSeed (echogenic) dummy seeds were implanted in a human cadaver prostate. Seventeen seeds remained after radical retropubic prostatectomy. VA imaging was conducted on the prostate that was cast in a gel phantom and placed in a tank of degassed water. 2-D magnitude and phase VA image slices were obtained at different depths within the prostate showing location and orientation of the seeds.

Results:

VA demonstrates that twelve of seventeen (71%) seeds implanted were visible in the VA image, and the remainder were obscured by intra-prostatic calcifications. Moreover, it is shown here that VA is capable of imaging and locating PPB seeds within the prostate independent of seed orientation, and the resulting images are speckle free.

Conclusion:

The results presented in this research show that VA allows seed detection within a human prostate regardless of their orientation, as well as imaging intra-prostatic calcifications.     

Desiccation sensitivity and cryopreservation of Korean ginseng seeds

Korean ginseng germplasm is maintained as clonal germplasm since there is no practical method for long-term seed conservation. The aim of this study was to establish a cryopreservation protocol for Korean ginseng seeds. Desiccation of undehisced ginseng seeds to a moisture content (MC) of 7.1 % did not decrease their dehiscence and germination. After cryopreservation, the dehiscence percentage of desiccated seeds decreased for MC above 12.5%; it was 26% for 22.6% seed MC and nil for 41.9% seed MC. Germination percentage did not decrease significantly between 12.5-22.6% seed MC, while germination percentage of dehisced seeds decreased below 7.2% MC, reaching 25.8% at 3.8% MC. After cryopreservation, the germination percentage decreased from 90.5-92.9% at 8.3-10.6% MC to 84.8% at 12.5% MC. At MCs below 8.3%, germination rapidly decreased from 85.0% at 7.2% MC to 34.9% at 5.3% MC. Therefore, the hydration window for cryopreservation of ginseng seeds is around 8-11% MC. Undehisced Korean ginseng seeds were characterized by their high lipid and protein content (lipids, 42.6% FW; proteins, 41.0% FW). When using thermal analysis, during the cooling phase, exothermic ice crystallization peaks were observed with dehisced ginseng seeds above 13.5% MCs (3.3 J/g FW). A second crystallization peak was detected following ice crystallization peaks.     

Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.)

In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M/0.75 M/1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1 degree C/min from 20 degrees C to -30 degrees C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.