Teramnus labialis (L.f.) Spreng is a legume that plays an important role in agriculture, due to its use as animal feed and its role as an enhancer of soil physicochemical conditions. However, given previous reports on the effects of seed cryopreservation on seedling vigor and biochemistry in a number of species, the present study looked at the effects of T. labialis seed cryopreservation on subsequent seed and seedling [0–28 days post seed exposure to liquid nitrogen (LN)], growth (germination, seedling length and fresh and dry mass) and biochemistry (chlorophyll, aldehyde, phenolic and protein levels). The seeds were intact in terms of macrostructure after exposure to LN, however, there was a significant (3.5-fold) increase in electrolyte leakage during imbibition. Seedling emergence was also improved by cryostorage during the 1st week of culture (4.1-fold increase) and at 28 days (2.5-fold increase). Consequently, seedling growth (in terms of plantlet length, and fresh and dry mass) was superior in seedlings arising from cryopreserved seed, but this stimulatory effect was more evident at 14 than 28 days of culture. An increase in malondialdehyde levels in cryopreserved seeds is most likely a consequence of damage to the external seed structures following cooling and rewarming, while the rise in cell wall-linked phenolics and aldehydes in roots of seedlings produced from cryopreserved seeds could be linked to water and nutrient stress brought about by greater root growth.

Biojas® is a fermented broth of Lasiodiplodia theobromae, a jasmonic acid–producing fungus characterized as a plant growth regulator and as biological control of phytopathogenic microorganisms and pests. The present work describes the use of Biojas® in in vitro culture of pineapple. On the other hand, plant scientists usually record multiple indicators in their experiments. The common statistical data evaluations involve univariate analyses such as t test, Mann-Whitney, and analysis of variance (ANOVA) followed by Tukey’s HSD. Such analyses do not evaluate integrally the effects of the experimental treatments because each indicator is analyzed independently. For this reason, we explored in this study the Euclidean distance combined with the data of the Biojas® treatment (0–2 mg l−1) on pineapple in vitro plantlets as an integrating indicator. Plant height; number of leaves; D leaf length, width, and area; diameter of the plant base; fresh and dry weights of the plant; levels of chlorophylls; transpiration rate; CO2 assimilation; and water use efficiency were recorded. Several statistically significant differences among Biojas® treatments were recorded. However, the most significant effects of Biojas® treatments were only noted in the plant height, length and area of D leaf, and water use efficiency. Variables mentioned above increased until 1.0 mg l−1 Biojas® and decreased with high levels of Biojas®. Calculation of the Euclidean distance from each Biojas® level to the ideal physiological status of the pineapple plantlets revealed that 1.0 mg l−1 Biojas® produced the pineapple plantlets with the best physiological status.

Globally, production of pineapple fruits reached more than 25million tons in 2013. Therefore, many scientists are searching for new varieties and ways for the conservation of pineapple genetic resources. Cryoconservation in liquid nitrogen has been described as a suitable technology in many plant species. However, its potential effects in the subsequent plant growth in the field should be studied before large-scale implementation of cryopreserved germplasm banks. This short communication describes the field performance of cryopreserved shoot tip-derived pineapple adult plants grown in the field for 14months. Three genetic materials [cv. MD-2; cv. Red Spanish Florencia; Hybrid 54 (Smooth Cayenne/Red Spanish)] were compared. The following treatments were established in the field: (1) conventional micropropagation-derived plants; (2) plants from shoot tips never exposed to liquid nitrogen but submitted to pre-cryostorage conditioning treatments; and (3) plants from shoot tips exposed to liquid nitrogen. Results indicated that shoot tip exposure to liquid nitrogen did not alter pineapple field performance which supports cryopreservation as an important tool for conservation of pineapple germplasm. As far as we know, this is the first publication of a detailed study of pineapple agricultural traits after cryopreservation.

Sodium azide (NaN3) is widely used to induce mutagenesis within in vitro plant systems. However, since this mutagenesis is undirected, its unintended effects demand characterization. This study investigated the mutagenic effects of sodium azide (0-0.45 mM) on selected growth (shoot multiplication rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants within temporary immersion bioreactors (TIBs). The content of soluble phenolics in the culture medium was also evaluated. Irrespective of the concentration NaN3 decreased shoot multiplication rate (by 87% relative to the control at 0.45 mM) and fresh weight (by 66% relative to the control at 0.45 mM). Levels of chlorophyll a and b, and soluble phenolics in the culture medium were also negatively correlated with NaN3 concentration. Interestingly, NaN3 application increased shoot carotenoid and soluble phenolic levels but had no significant effect on a range of established plant stress biomarkers: cell wall-linked phenolic levels, malondialdehyde and other aldehydes. Given that 0.19 mM NaN3 decreased shoot multiplication rate by 50% and resulted in propagants that displayed no morphologically abnormalities, increased levels of photoprotective pigments (relative to the control) and no significant increase in lipid peroxidation products, the mutagen can be used at this concentration to induce pineapple mutagenesis in TIB based studies aimed at producing agriculturally-useful mutants.

Climate change poses risks to both wild and crop plant biodiversity, which can be mitigated by cryopreservation (usually at -196 °C in liquid nitrogen [LN]) of crop germplasm. Cryopreservation is widely regarded as a reliable method for the ex situ conservation of plant genetic resources but its effects on subsequent field performance of popular crop species such as sorghum are largely unknown. This hampers the large-scale implementation (i.e. germplasm banks) of cryostorage for such species. This short communication describes the early stages of germination and field performance of plants derived from cryopreserved sorghum seed. Compared with the control, cryopreservation significantly increased seed electrolyte leakage and from 24 to 120 hours, percentage of germination of the control was ~2.6 folds higher than cryopreserved seeds. At 0 days, chlorophyll a/b rate was ~1.7 folds higher in the control and at 7 and 14 days, chlorophyll a level (~1.5 folds) and chlorophyll a/b rate (~1.8-1.9 folds) were higher in the control. Contrastingly, at 7 days, seedlings derived from cryopreserved seeds (treatment seedlings) showed ~1.5 folds more superoxide dismutase activity and ~1.9 folds more peroxidase activity. In contrast, treatment and control adult plants were statistically comparable in terms of chlorophylls, proteins, superoxide and peroxidase activities, plant architecture, and yield components. The fact that differences in biochemical indicators observed between control and treatment seedlings did not persist in adult plants validates the use of seed cryopreservation for the conservation of sorghum genetic resources.

In light of climate change and risks of food insecurity, it is becoming increasingly important to preserve plant germplasm in genebanks. Storage of seeds, particularly via cryopreservation, is one of the most proficient methods for ex situ plant germplasm conservation. Whilst seed cryo-banking can have little, to no, or even beneficial effects on subsequent seedling vigor in some species, it can lead to a number of plant abnormalities (morphological and physiological). This study investigated the effects of maize seed cryopreservation on seedling growth (until 14 d) and levels of selected amino acids produced in the shikimate pathway, a major link between primary and secondary metabolism. Seed cryopreservation reduced FW in recovered seedlings, reduced caffeic acid (2.5-fold decrease), and increased levels of all other shikimate pathway–related compounds assessed: phenylalanine (2.9-fold increase), tyrosine (2.6-fold increase), and shikimic (2.1-fold increase) and protocathecuic (3.1-fold increase) acids in cotyledons. Our results suggest that maize seed cryopreservation results in seedlings that exhibit signs of an ‘overly’ efficient and caffeic acid–deficient shikimate pathway, possibly related to their reduced growth during a highly vulnerable growth stage. However, these metabolic abnormalities manifested most severely in the maternal (cotyledonary), as opposed to vegetative (roots, stems, and leaves), tissues and hence are likely to disappear when the seedlings shed the cotyledons and become completely autotrophic.

Chemical mutagens such as sodium azide (NaN3) have been widely used to increase genetic variability in crops, but the undirected mutations induced can have undesirable effects, which need to be characterized. This study investigated the effects of in vitro NaN3 (0–0.45 mM) exposure (30 days) on the micropropagation of sugarcane within temporary immersion bioreactors (TIB). Shoot multiplication rate and cluster fresh weight, and aldehyde, phenolic, carotenoid, and chlorophyll levels were measured on in vitro produced shoots. The soluble phenolic content of the culture medium was also assessed. NaN3 concentration was negatively correlated with sugarcane shoot multiplication rate and fresh weight; at 0.45 mM NaN3, these parameters were only 20% and 39% that of the untreated control, respectively. Shoot multiplication rate and fresh weight, and chlorophyll a and b levels were negatively correlated with NaN3 concentration. In contrast, malondialdehyde, other aldehyde, carotenoid, and exuded phenol levels were positively correlated with NaN3 concentration. Statistical comparisons suggest that shoot multiplication rate and the biochemical parameters that were positively correlated with NaN3 concentration may be the most suitable indicators of stress when optimizing the concentration of NaN3 for sugarcane explants. An interpolated 50% reduction of multiplication rates at 0.23 mM NaN3 suggests that this concentration to be suitable for TIB-based induction of mutagenesis in shoots and eventual production of agriculturally useful mutants.

Conservation of pineapple (Ananas comosus L. Merr.) genetic resources - including cryopreservation in liquid N-2 at -196 degrees C - is essential for future breeding programmes to develop new varieties with improved agronomic performance. However, the potentially deleterious effects of cryopreservation on subsequent plant regrowth should be evaluated before large-scale development of cryobanks is implemented. This paper describes the histological analysis of pineapple plantlets regenerated from cryopreserved shoot tips. Two controls were included in the study: i) conventional micropropagation-derived plantlets, and ii) plants from shoot tips subjected to pre-cryostorage conditioning treatments but never exposed to liquid N-2. Histological studies of roots, leaves and stems were conducted after 45 days of hardening. No statistically significant differences with the controls were observed in any of the histological parameters evaluated, which supports the practical value of cryopreservation of pineapple germplasm.

Teramnus labialis is an herbaceous legume that serves as a source of carbohydrates and proteins for animals and humans, and is valued for its nitrogen contribution to soil. The benefits of this species are, however, limited by low seed availability, small seed size and low in situ seed germination levels, due to physical dormancy. Cryostorage has been shown to be beneficial for both seed storage and breaking physical dormancy in seeds of various species. However, its potential effects on subsequent seedling emergence, plant growth and seed production need to be studied before largescale implementation for T.labialis. OBJECTIVE: To record agricultural traits of T.labialis after seed exposure to liquid nitrogen. MATERIALS AND METHODS: Seeds were maintained at 5 degrees C (control) or stored in LN before sowing. Seedling emergence percentage and traits related to plant growth and seed production were evaluated for 6 months. RESULTS: Except for seed weight, all traits differed significantly between seedlings generated from cryostored and control seeds. Except for pod number, seedling emergence and plant growth traits were enhanced by cryostorage to a greater extent than seed production traits. Cryostorage resulted in cracks and breaks in the seed coat which were absent in control seeds (scanning electron microscopy), and in breaking physical dormancy may have facilitated more rapid seedling emergence than for control seeds. CONCLUSION: Seed cryostorage enhances subsequent plant productivity in terms of growth and to a lesser extent seed production in Teramnus labialis, validating its use for commercial growth of this species.

BACKGROUND Cryo-preservation of plant materials in liquid nitrogen (LN) has been described as a suitable technology to conserve genetic resources of several species. However, the potential effects of LN in the subsequent plant growth in the field should be studied before large-scale implementation of cryopreserved germplasm banks. OBJECTIVE To describe the field performance of cryopreserved seed-derived maize adult plants. MATERIALS AND METHODS Germination percentage and numbers of leaves and ears per plant, internodes in stems, middle - aged leaf length, plant height, ear traits and weight of 100 seeds were recorded. RESULTS Statistically significant differences between adult plants derived from cryopreserved seeds and the control treatment were not observed (t-test, p=0.05). CONCLUSION The results presented confirm at the phenotype level the effectiveness of maize seed cryostorage to preserve and regenerate true-to-type plants.

BACKGROUND: Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. OBJECTIVE: In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. METHODS: Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. RESULT: No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. CONCLUSION: Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.

In this work, we studied the effect of short-term liquid nitrogen storage on the germination and phenolics contents of wild tomato (Solanum lycopersicum Mill.) seeds. After storage in liquid nitrogen for different time periods (0, 7, 14, 21 and 28 days), seeds were retrieved from liquid nitrogen and set to germinate. Both control and cryopreserved seeds displayed about 60% germination without statistically significant differences. No phenotypic changes were observed visually in seedlings recovered from different treatments 7 days after onset of germination. After 7 days, levels of phenolics (free, cell-wall linked and total) were recorded in roots, stems and leaves of seedlings. When seeds were immersed in liquid nitrogen for 7, 14 and 21 days, the levels of cell wall-linked, free and total phenolics decreased significantly in roots and stems, compared to non-cryopreserved controls. However, their concentration generally increased when seeds were immersed in liquid nitrogen for 28 days. In leaves, a similar pattern to that observed in roots and stems was noted with the free phenolics content but cell wall-linked phenolics increased in leaves of seedlings derived from seeds immersed in liquid for 7 or 14 days. Further studies are required to clarify the mechanisms underlying the changes recorded here. (C) 2013 Elsevier B.V. All rights reserved.

This paper presents some of the effects of cryopreservation of wild Solanum lycopersicum Mill. seeds on the early stages of germination post liquid nitrogen exposure. Percentage of germination, conversion into plantlets and plant fresh mass were evaluated after cryostorage. Levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and proteins were determined. Peroxidase and superoxide dismutase activities were recorded. Liquid nitrogen exposure increased the percentage of seed germination at 5 days but at 7 days, the conversion into plantlets and the plant fresh mass were not statistically different between non-cryopreserved and cryopreserved samples. Several significant effects of cryopreservation were recorded at the biochemical level at 7 days of germination under controlled conditions. Highly significant effects due to liquid nitrogen exposure were observed in leaves: increased levels of peroxidase enzymatic and specific activities and cell wall-linked phenolics. Very remarkable effects were also recorded in roots: decreased contents of chlorophylls and cell wall-linked phenolics.