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Teramnus labialis (L.f.) Spreng shows dormancy as a result of impermeability of the seed coat, which requires scarification treatment before sowing. Liquid nitrogen (LN) as a scarifying treatment has recently been used on this species, with excellent results. However, moisture content and immersion time on LN are factors that may affect seed germination and dormancy break. This report studies (i) the effects of dehydration on T. labialis seed viability and germination and (ii) the appropriate moisture content and extent of LN to make this scarification an effective treatment. Moisture contents of 4%, 6%, 8%, and 10% fresh weight basis (FWB) and extension to LN for 15, 30, 45, and 60 min were performed. Seed viability did not change after seed dehydration up to a moisture content of 4% FWB, whereas the percentage of germination decreased as a result of increasing the percentage of hard seeds. The seed moisture content did not affect germination after immersion in LN, but at least 30 min of exposure was required for dormancy break. The mean germination time, germination index, and time to 50% germination improved with the increasing germination from 33% to 91% and a greater duration of immersion in LN. The dormancy of T. labialis seeds with a moisture content between 4% and 10% FWB was only broken when exposed to LN for 30 to 60 min.

Medicinal plant use and trade has seen a dramatic increase over the years owing to the increased realization about its health benefitting effects. Limited commercial cultivation has forced the industries and traders to rely on the wild collections to meet the growing demand. The situation has threatened the survival of many species including those having anti-diabetic potential. These difficult to conserve species need biotechnological techniques for their long-term conservation and sustainable utilization. For non-orthodox and vegetatively propagated species, cryopreservation in liquid nitrogen (LN) at −196 °C offers the most successful and economical technique for long-term conservation. Traditional techniques based on freeze-induced dehydration and recent techniques based on vitrification have been efficiently used for cryopreservation of all types of the explants. Cryopreservation offers multiple advantages over other conservation strategies as it minimizes the risk of contamination, cost of maintenance and cost of labour. The process of cryopreservation exposes the cell or tissue to various physical, chemical and physiological stresses which may result in cryoinjury or genetic level changes sometimes. The analysis of such morphological, structural, genetic or functional changes is important to assess the genetic integrity of cryopreserved germplasm to see if they are ‘true to type’ after cryopreservation. This can be studied at the phenotypic, histological, cytological, biochemical and molecular levels. The chapter will throw light upon the importance and status of cryopreservation, different methods of cryopreservation adopted in major anti-diabetic plants along with the pre and post cryotreatments and regeneration protocols, infrastructure requirements for cryobank. The concept of root cryobanking and cryobionomics dealing with genetic stability and the reintroduction of cryopreserved plants into the environment is also covered.

In the germplasm bank of the Tobacco Research Institute of Cuba the germinative power was determined in 11 species belonging to the genus Nicotiana. All the evaluated species presented high dormancy degree which obtained less than 40% of germination. For the species Nicotianamegalosiphon Heurck and Mueller the effect of potassium nitrate (3.0 g/L), giberelic acid (0.15 g/L), stratification to 5 0C (49 days), Fitomás (1%), sulfuric acid (0.5 min) and criopreservation (49 days) on the germination of seeds was investigated. Starting from the results that they showed higher germination percentages, the influence of the combination of different levels of giberelic acid (0.00; 0.05; 010; 0.15; 0.20 and 0.25 g/L) with stratification to 5 0C (0; 7; 21; 35 and 49 days) and criopreservation (0; 7; 21; 35 and 49 days) on the germination of the seed was studied. The highest percentage in germination was observed after the exposition to 49 days to 5 0C followed by a period of 24 h in 0.20 g/L giberelic acid. When exposing the seeds from the 10 remaining species to the established procedure for the N. megalosiphon significantly superior germination percentages they were reached (of 49%to 84%) to those obtained through the traditional germination rehearsals.

BACKGROUND: It is well known that cryopreserving seeds with high water content is detrimental to survival, but biochemical and structural parameters of cryostored hydrated common bean seeds have not been published. OBJECTIVE: The objective of this work was to study the effect of liquid nitrogen exposure on selected biochemical and structural parameters of hydrated Phaseolus vulgaris seeds. MATERIALS AND METHODS: We cryopreserved seeds at various moisture contents and evaluated: germination; electrolyte leakage; fresh seed weight; levels of chlorophyll pigments, malondialdehyde, other aldehydes, phenolics and proteins; thickness of cotyledon epidermis, parenchyma, and starch storage parenchyma; and radicle and plumule lengths. RESULTS: Germination was totally inhibited when seeds were immersed in water for 50 min (moisture content of 38%, FW basis) before cryopreservation. The combined effects of seed water imbibition and cryostorage decreased phenolics (free, cell wall-linked, total), chlorophyll a and protein content. By contrast, electrolyte leakage and levels of chlorophyll b and other aldehydes increased as a result of the combination of these two experimental factors. These were the most significant effects observed during exposure of humid seed to liquid nitrogen. CONCLUSION: Further studies are still required to clarify the molecular events taking place in plant cells during eryostorage.

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 paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25 degree C, treatment with the PVS2 solution for 20-40 min at 0 degree C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55 percent.

This study compared the field performance of sugarcane plants originating from three different sources: control, non-cryopreserved embryogenic calluses, cryopreserved embryogenic calluses and macropropagated material of the same commercial hybrid. Several agronomic traits were evaluated on 100 plants per treatment over a 27-month period covering the growth of the stool and of the first ratoon. Significant differences between treatments were observed only during the first six months of field growth of sugarcane stools. Stems produced from in vitro cultured material, irrespective of their cryopreservation status, had a smaller diameter and a shorter height than those produced from macropropagated material. These differences disappeared by 12 months of stool field growth.

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.

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.To describe the field performance of cryopreserved seed-derived maize adult plants.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.Statistically significant differences between adult plants derived from cryopreserved seeds and the control treatment were not observed (t-test, p=0.05).The results presented confirm at the phenotype level the effectiveness of maize seed cryostorage to preserve and regenerate true-to-type plants.

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 large-scale implementation for T. labialis.To record agricultural traits of T. labialis after seed exposure to liquid nitrogen.Seeds were maintained at 5 degree 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.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.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.

A simplified freezing process was developed and successfully applied to embryogenic calluses of three sugarcane commercial hybrids (Saccharum sp. cv. CP 5243, C 91-301 and C 1051-73). To obtain optimal survival, the calluses were pretreated with a cryoprotective solution containing 10% DMSO and 0.3 to 0.75M sucrose. For freezing, the samples were immersed in an alcohol bath placed in a -40 degrees C freezer, thus allowing a freezing rate comprised between 0.4 and 0.6 degrees C/min. Samples were held at that temperature for 2 h before immersion in liquid nitrogen. The highest survival rates of cryopreserved calluses ranged between 20 and 94% depending on the variety, and fully developed plantlets could be obtained from regenerating calluses. Embryogenic calluses of one variety were stored for 14 months in liquid nitrogen without any effect on their survival rate and plantlet production., Comment: 6 pages, 3 tables