Effect of abscisic acid and jasmonic acid on partial desiccation of encapsulated somatic embryos of sugarcane

Embryogenic calli of sugarcane (Saccharum sp. hybrid, clone CP52-43), with somatic embryos in the late scutelar stage, were subjected to different treatments for increasing embryo tolerance to desiccation. The medium was supplemented with abscisic acid (ABA) (3.8 μM), jasmonic acid (JA) (4.7 μM) or a combination of them. A control treatment without growth regulators was also included. The embryos were encapsulated in alginate beads and dehydrated or not in sucrose (0.5 M). Thereafter, they were further dehydrated in chambers containing silicagel until the beads reached either 60% or 30% of water content (WC). Survival of encapsulated-dehydrated embryos was achieved only in the control and ABA treatment. ABA induced an increase in protein, polyamines, free proline levels and starch levels as a response to desiccation tolerance. JA treatment showed the lowest protein and polyamines levels and increased the starch content almost two-fold compared to the ABA treatment. The JA treatment induced high levels of 4-methylcatechol and the lowest levels of gallic acid. However, the ABA treatment increased gallic acid and p-coumaric acid content in the induction medium. Some differences were found in growth regulator free-medium in relation to the induction medium. JA is not effective in these desiccation processes. The mechanisms by which these two plant growth regulators act on the induction of tolerance to stress are presumably different. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Enhanced Trehalose Accumulation and Desiccation Survival of Entomopathogenic Nematodes Through Cold Preacclimation

Limited storage stability is a major obstacle to further expansion of the use of entomopathogenic nematodes for pest control. Progress has been made that Steinernema carpocapsae can now be stored under partial anhydrobiosis for up to 6 months at 25°C and 10 months at 5°C in a water-dispersible granular (WG) formulation. However, other species have been more difficult to store in the WG formulation due to migration of nematodes out of the granules and sensitivity of some species to desiccation directly at cold temperatures. As acclimation to cold induces trehalose accumulation (a major cryo- and desiccation protectant) in many invertebrates, it was hypothesized that cold preacclimation of entomopathogenic nematodes will enhance their survival in the WG formulation at cold temperatures. This hypothesis was tested using a temperate species Steinernema feltiae , a subtropical species S. carpocapsae , and a tropical species Steinernema riobrave possessing different thermal niche breadths and reproduction temperature optima. Cold acclimation of infective juveniles increased trehalose accumulation in all three species and the amount of trehalose accumulated was both temperature and species dependent. Trehalose content reached at its peak after 6 days at 5°C in S. feltiae (82.28 μg/mg dry weight), after 10 days at 10°C in S. carpocapsae (94.16 μg/mg dry weight) and after 6 days at 15°C in S. riobrave (47.58 μg/mg dry weight). Cold preacclimation at 5°C for 2 days enhanced desiccation survival of S. feltiae in 25% glycerol (osmotic desiccation) at both 5 and 25° and of S. carpocapsae and S. riobrave only at 5°C. Non-cold acclimated S. carpocapsae and S. riobrave were extremely sensitive to desiccation directly at 5°C in 25% glycerol, resulting in over 98% mortality within 6 days, but S. feltiae was more sensitive to desiccation at 25°C than at 5°C. Cold preacclimation increased survival of all the three species in the WG formulation at both 5 and 25°C. The survival of S. riobrave at 5°C in the WG formulation was positively correlated with the length of preacclimation period at 5°C (R 2 = 0.99) and with the amount of trehalose accumulated during cold preacclimation (R 2 = 0.81). These results support the hypothesis that cold preacclimation enhances desiccation survival of entomopathogenic nematodes at cold temperatures and the increased survival correlates well with the increased trehalose accumulation. Results also demonstrate that cold preacclimation can be used as a tool to enhance survival of nematodes in the formulations with reduced water activity.     

Regulation of starch and protein accumulation in alfalfa (Medicago sativa L.) somatic embryos

Compared to seeds, somatic embryos accumulated relatively low levels and different types of storage carbohydrates. The regulation of starch accumulation was studied to determine its effects on desiccation tolerance and vigor of dry somatic embryos. Somatic embryos of Medicago sativa are routinely matured through three phases: 7 days of development; 10 days of phase I maturation, a rapid growth phase; and 10 days of phase II maturation, a phase leading to the acquisition of desiccation tolerance. The control of starch deposition was investigated in alfalfa somatic embryos by manipulating the composition of the phase I maturation medium with different levels of sucrose, abscisic acid, glutamine and different types of carbohydrates and amino acids. After phase II maturation, mature somatic embryos were collected for desiccation and subsequent conversion, or for biochemical analyses. Starch deposition occurred primarily during phase I maturation, and variations in the composition of this medium influenced embryo quality, storage protein and starch accumulation. A factorial experiment with two levels of glutamine × three levels of sucrose showed that increasing the sucrose concentration from 30 to 80 g/l increased embryo size and starch content, but had minimal effect on accumulation of storage proteins; glutamine also increased embryo size, but decreased starch content and increased accumulation of the high salt soluble S-2 (medicagin) storage proteins. ABA did not influence any of the parameters tested when included in phase I maturation at concentration up to 10 μM. Replicating sucrose with maltose, glucose, or glucose and fructose did not alter embryo size or starch accumulation (mg/g fresh weight), but replacement with fructose alone reduced embryo size, and replacement with glucose alone reduced germination. Suplementation with the amino acids, asparagine, aspartic acid and glutamine increased seedling vigor, but decreased the starch content of embryos. The data indicate that starch accumulation in somatic embryos is regulated by the relative availability of carbon versus nitrogen nutrients in the maturation medium. The quality of mature somatic embryos, determined by the rate of seedling development (conversion and vigor), correlated with embryo size, storage protein and free amino acid but not with starch. Therefore, further improvements in the quality of somatic embryo may be achieved through manipulation of the maturation medium in order to increase storage protein, but not starch deposition.     

Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation

BACKGROUND AND AIMS: Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. METHODS: In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. KEY RESULTS: During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. CONCLUSIONS: These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.     

Changes in starch and soluble sugars in relation to the acquisition of desiccation tolerance during maturation of Brassica campestris seed

Abstract. We investigated the onset of desiccation tolerance in developing embryos of Brassica campestris seeds and possible correlated ultrastructural modifications in the radiele cells. Since the acquisition of desiccation tolerance is a long asynchronous process which took 9 d to be achieved, we determined criteria allowing us to separate freshly intact harvested seeds into desiccation intolerant and desication tolerant batches that differed in age by only 2 d. No particular structural modifications were found except a strong depletion of intraplastidial starch (-90%) coincident with the appearance of stachyose and an increase of sucrose (+30%) on the acquisition of desiccation tolerance. As we did not observe an increase of lipid reserves as a consequence, we suggest that these metabolic events can be a key factor towards the acquisition of desiccation tolerance.     

Spherulitic crystallization of gelatinized maize starch and its fractions

Binary systems of polymers often display spherulitic morphologies after cooling from the melt, but these phenomena have rarely been reported among food polymers of native-size. Here we report the observation of spherulitic and other morphologies in gelatinized maize starch. The morphology could be manipulated by choosing polymer compositions and kinetic regimes. Spherulites (10 μm diameter) formed from gelatinized high-amylose maize starches and purified amylose at cooling rates of order of magnitude 100 °C/min. They were more numerous and exhibited a higher melting point the greater the ratio of amylose to amylopectin. Rapid cooling rates (150–500 °C/min) resulted in a more even distribution of smaller spherulites. Very rapid (liquid nitrogen quench) or slow (0.1–1 °C/min) cooling rates resulted in mixed morphology, as did addition of 15 or 60% (w/w) sucrose to a 10% (w/w) dispersion of high-amylose starch (HAS). Spherulites were observed in aqueous suspensions of high-amylose maize starch between 5 and 30% (w/w). Lower starch concentrations resulted in a broader size distribution and spherulites of more distinct shape. WAXS patterns of B-type were observed. Negatively birefringent spherulites predominated, but positive spherulites were found. The spherulite melting range overlapped with that for amylose–lipid complex. Evidence indicated that micro-phase separation takes place when a holding period at 95 °C follows gelatinization at 180 °C. Despite the high maximum temperature of treatment (180 °C) there was evidence for a memory effect in samples of 30% HAS. Spherulite morphology closely resembled that of native starch granules in very early stages of development.     

Physicochemical properties of endosperm and pericarp starches during maize development

Endosperm starch and pericarp starch were isolated from maize (B73) kernels at different developmental stages. Starch granules, with small size (2–4 μm diameter), were first observed in the endosperm on 5 days after pollination (DAP). The size of endosperm-starch granules remained similar until 12DAP, but the number increased extensively. A substantial increase in granule size was observed from 14DAP (diameter 4–7 μm) to 30DAP (diameter10–23 μm). The size of starch granules on 30DAP is similar to that of the mature and dried endosperm-starch granules harvested on 45DAP. The starch content of the endosperm was little before 12DAP (less than 2%) and increased rapidly from 10.7% on 14DAP to 88.9% on 30DAP. The amylose content of the endosperm starch increased from 9.2% on 14DAP to 24.2% on 30DAP and 24.4% on 45DAP (mature and dried). The average amylopectin branch chain-length of the endosperm amylopectin increased from DP23.6 on 10DAP to DP26.9 on14DAP and then decreased to DP25.4 on 30DAP and DP24.9 on 45DAP. The onset gelatinization temperature of the endosperm starch increased from 61.3 °C on 8DAP to 69.0 °C on 14DAP and then decreased to 62.8 °C on 45DAP. The results indicated that the structure of endosperm starch was not synthesized consistently through the maturation of kernel. The pericarp starch, however, showed similar granule size, starch content, amylose content, amylopectin structure and thermal properties at different developmental stages of the kernel.     

Influence of formulation additives on the desiccation tolerance and storage stability of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with 10% lactose/1% bovine serum albumin (BSA) or various compositions of Fantesk™, a starch-oil composite prepared by jet-cooking an aqueous dispersion of starch and oil. Storage stability studies with wet blastospore formulations showed that maximum blastospore survival was achieved during low-temperature storage at -20°C with lactose/BSA formulations or starch-oil formulations supplemented with sucrose, zein protein, and whole milk. Under conditions of wet storage at -20°C, the addition of whole milk to starch-oil formulations significantly improved blastospore stability while the addition of sucrose or zein protein had no effect. In freeze-drying studies, no significant differences were seen in blastospore desiccation tolerance or in stability during storage at either 4 or -20°C when blastospores of P. fumosoroseus were formulated with lactose/BSA or starch-oil formulations with sucrose, zein protein, and whole milk. Freeze-dried blastospore formulations stored at 4°C showed no loss in blastospore viability after 3 months storage and blastospore formulations stored at -20°C showed no loss in viability during the entire 12-month study. For freeze-dried, starch-oil formulations, sucrose was shown to improve blastospore survival during the freeze-drying process. The addition of whole milk to starch-oil formulations significantly improved the stability of freeze-dried blastospores stored at 4°C. Compared to unformulated blastospore suspensions that showed blastospore settling after 30 min, suspensions of blastospores formulated with lactose/BSA or starch-oil composites remained stable for up to 2 h after mixing.     

Plantlet Regeneration from Encapsulated and Non-encapsulated Desiccated Somatic Embryos of a Forest Tree: Sandalwood (Santalum album L.)

Somatic embryos obtained from embryogenic tissues of sandalwood (Santaium album) were grown on White’s medium containing abscisic acid, (ABA, 1.89, 3.78 or 18.92 μM) and various concentrations of sucrose (87.6 to 350.4 mM) to induce maturation. The embryos were isolated and desiccated for 10, 20 or 30 days: One lot of the desiccated somatic embryos was encapsulated in sodium alginate gel and the other lot was not encapsulated. Both encapsulated and nonencapsulated desiccated embryos showed revival of growth upon rehydration on White’s medium and developed into plants. The desiccation tolerance and regeneration of viable plantlets depended upon the pretreatment given to somatic embryos. Embryogenic tissue subjected to dry state for 30 days showed revival of somatic embryogenesis upon transfer to a fresh nutrient medium. Implications of maturation and desiccation of somatic embryos on its germinability are discussed.     

Evaluation of different cooling rates, equilibration periods and diluents for effects on deep-freezing, enzyme leakage and fertility of taurine bull spermatozoa

We studied the effects of 2 diluents (Tris and milk), 4 cooling rates (10°C/30°C to 5°C for 1 or 2 h), 2 equilibration periods (0 and 2 h) and their interactions on the freezability, glutamic oxaloacetic transaminase (GOT) leakage and fertility of frozen-thawed semen in 18 ejaculates from 3 Friesian bulls. The means of pre- and post-freezing motility, GOT leakage and fertility rates (52.81% based on follow up of 267 inseminated cows) were significantly (P<0.01) influenced by the bulls, cooling rates & equilibration periods, but not by diluents or the interactions studied. The mean prefreeze motility of spermatozoa following 1 h of cooling from 10°C to 5°C was significantly lower (60.38%) and that after 2 h of cooling from 30°C to 5°C was higher (72.38%) than 2 h of cooling from 10°C to 5°C (66.57%) or 1 h of cooling from 30°C to 5°C (67.96%). The mean post-thaw motility observed following 2 h of prefreeze cooling was, however, significantly greater (45%) than after 1 h of cooling (35%) for both the initial temperatures. Leakage of GOT pre- and post-freezing was significantly less following 2h of cooling from 30°C to 5°C (17.26 and 27.36 μmole/L) than after 1 h of cooling from either 10°C (19.71 and 30.13 μmole/L) or 30°C (18.95 and 29.58 μmole/L) and 2 h of cooling from 10°C to 5°C (21.43 and 34.48 μmole/L). The conception rates for semen frozen at the above cooling rates (66.13, 48.65, 56.67 and 42.25%, respectively) were inverse to GOT leakage. An equilibration period of 2 h over that of 0 h at 5°C adversely affected the prefreeze motility and GOT leakage, but it significantly improved postthaw motility (44.03 vs 35.49%) and fertility rates (57.86 vs 47.24%). These findings suggested that both Trisand milk-based diluents were equally efficacious for cryopreservation of bovine semen, and that slow cooling of semen straws over a period of 2 h from 30°C to 5°C as compared with faster cooling rates or a lower initial temperature (10°C), plus at least 2 h of equilibration time at 5°C were essential for optimal freezability, lower enzyme leakage & higher fertility rates within the tropics.     

Effects of Desiccation, Medium Osmolarity and Abscisic acid on the Maturation of Hevea brasiliensis Somatic Embryos

The effects of desiccation of Hevea somatic embryos and of sucroseand ABA concentrations in the maturation medium on their germinabilitywere investigated. Conversion into plant, water and histochemicalstatus of somatic embryos were compared systematically to thoseof the zygotic embryos used as reference. Slow desiccation ormaturation on 351 mol m^–3 sucrose supplemented with 1mmol m^–3 ABA strongly improved germinability and conversionof embryos into plants. The combination of the two treatmentswas the most effective, increasing the germination frequencyby 3·7 and plant conversion by 6·6 in clone PR107. Each of these two treatments increased the vigour of somaticembryos, stimulated the formation of root and shoot meristemsand the synthesis and accumulation of starch and protein reserves.At the end of maturation, the Hevea somatic embryos bore ananatomical and histochemical resemblance to mature zygotic embryos.Likewise, the two treatments brought the water status of somaticembryos closer to that of the mature zygotic embryos, but withoutachieving a perfect match. Optimization of the successful conversioninto plants may require full acquisition of this water status. Key words: ABA, embryo maturation, Hevea, somatic embryogenesis, water status     

Carbohydrate metabolism in the developing and maturing wheat embryo in relation to its desiccation tolerance

Embryos of wheat (Triticum aestivum L. cv. Sappo) were studiedthroughout their development and maturation to investigate therelationships among starch, sucrose and raffinose and the onsetof desiccation tolerance. Starch accumulated in axes and scutellafrom about 20 d post anthesis (dpa) to reach a maximum at approximately35 d. The starch content then declined to a very low value inlate maturation. Extractable -amylase activity increased inembryos throughout the period of starch deposition and showeda substantial rise coincident with starch breakdown. In earlymaturation (approximately 26 dpa) sucrose and raffinose appeared,and continued to increase. The rise in the amount of sucroseparalleled the accumulation of starch, but the major increasein raffinose approximated to the fall in starch content. Embryoswere desiccation intolerant prior to the age when free sucroseand raffinose accumulated: the development of desiccation tolerancewas associated with increasing raffinose: sucrose ratios. Possiblemetabolic and physiological relationships among starch, raffinose,sucrose and the onset of desiccation tolerance are discussed. Key words: Wheat embryos, development, maturation, starch, raffinose, sucrose, desiccation tolerance     

Influence of sucrose on the rheology and granule size of cross-linked waxy maize starch dispersions heated at two temperatures

Cross-linked waxy maize (CWM) starch dispersions (STDs) of concentration 50 g kg⁻¹ were heated in sucrose solutions containing 0–600 g kg⁻¹ (g sucrose/kg dispersion) at 85 °C at low shear and in intermittently agitated cans at 110 °C. The STDs heated in 0–300 g kg⁻¹ sucrose exhibited antithixotropic behavior, while those heated in 400–600 g kg⁻¹ sucrose exhibited thixotropic behavior. The mean starch granule diameter of the starch dispersions did not show strong dependence on sucrose concentration. The dispersions, especially those with high sucrose concentrations and heated at 110 °C, exhibited G′ versus frequency (ω) profiles of gels. The STDs exhibited first normal stress differences that increased in magnitude with the concentration of sucrose. Values of the first normal stress coefficient of canned dispersions calculated from dynamic rheological data plotted against ω and experimental values plotted against shear rate of some of the STDs overlapped.     

The effect of partial dehydration on the developmental capacity of mouse embryos stored in the supercooled state

The effect of partial dehydration on the ability of mouse blastocysts to withstand storage at subzero temperatures without freezing was studied. The embryos were equilibrated with a supercooling medium developed at the Centre for Food and Animal Research, containing 3% (Medium A) or 6% (Medium B) methanol and propanediol, and then with the same medium, A or B, containing 0–0.5 mol sucrose. The embryos were placed in 0.25 ml straws, cooled to −5°C or −10°C and stored for up to 3 days. After storage, the embryos were cultured for 48 h in M16 and their ability to develop into expanded blastocysts was used to gauge their survival in supercooled storage.

The maximal beneficial effect of partial dehydration occurred in media supplemented with 0.3–0.5 mol sucrose: the proportions of dehydrated embryos surviving 24 h storage at −5°C and −10°C were 84–85% and 91–100%, respectively, compared with only 58% and 52% of non-dehydrated, supercooled embryos. The corresponding figures for dehydrated embryos after 48 or 72 h storage at −5°C were 86–92% and 38–58% compared with 13% and 4% of non-dehydrated embryos. Similarly, 75–85% and 47–55% of partially dehydrated embryos survived storage for 48 h or 72 h, respectively, at −10°C, compared with 5% and 0% of non-hydrated embryos. Thus, reducing the water content of early mouse blastocysts improved their ability to withstand subzero storage.     

Abscisic acid and osmotic stress or slow drying independently induce desiccation tolerance in mutant seeds of Arabidopsis thaliana

Action of endogenous abscisic acid (ABA) is absent in the ABA-deficient and -insensitive double mutant ( aba-1abi3–1 ) seeds of Arabidopsis thaliana . Thus, responses to osmotic stress and dehydration can be studied without interference of endogenous ABA. Seeds of this double mutunt are viable hut desiceation-intolerant. However, desiccation tolerance can he induced by either (1) slow dehydration of immature seeds; (2) treatment of immature seeds with osmotica or; (31 due to the leakiness of the ABA-insensitivty mutation, by application of exogenous ABA. Consequently it is concluded that either ABA or osmotic- or dehydration-stress and related gene expression meets the minimal requirements for acquisition of desiccation tolerance in seeds of Arabidopsis thalianna .     

Differential effects of abscisic acid on desiccation tolerance and carbohydrates in three species of liverworts

Tissues of three species of in vitro grown liverworts, Riccia fluitans, Pallavicinia lyellii, and Marchantia polymorpha, were subjected to rapid drying with and without preculture for 1 week on medium containing 10 μM ABA. ABA preculture initiated total desiccation tolerance in R. fluitans, whereas control tissues were killed after 30 min of drying. Survival was also improved in P. lyellii, whereas ABA did not affect survival of M. polymorpha after rapid drying. ABA treatment did, however, reduce the rate of water loss in M. polymorpha. Total soluble carbohydrates were increased in ABA-treated R. fluitans and P. lyellii, but not in M. polymorpha, although there was no correlation between survival and changes in the percentage of these carbohydrates as reducing sugars. These differences in response to ABA and desiccation likely reflect different adaptations of these three species to conditions in situ.     

Influence of incubation temperature and time on resistant starch type III formation from autoclaved and acid-hydrolysed cassava starch

Raw cassava starch, having 74.94 and 0.44 g/100 g resistant starch type II and III (RS II and RS III), respectively, was autoclaved at 121 °C in water, 1, 10 or 100 mmol/L lactic acid. The formation of RS III was evaluated in relation to variable incubation temperature (−20 to 100 °C), incubation time (6–48 h) and autoclaving time (15–90 min). Negligible to low quantities of RS III (0.59–2.42 g/100 g) were formed from autoclaved starch suspended in 100 mmol/L lactic acid, whereas intermediate to high quantities (2.68–9.97 g/100 g) were formed from autoclaved starch suspended in water, 1 or 10 mmol/L lactic acid, except for treatments with water or 10 mmol/L lactic acid incubated at 100 °C for 6 h (1.74 g/100 g). Autoclaving times corresponding to maximum RS III contents were 15 and 45 min for water and 10 mmol/L lactic acid, respectively. Whereas, the RS III fractions from cassava starch suspended in water had melt transitions between 158 and 175 °C with low endothermic enthalpies (0.2–1.6 J/g), the thermal transitions of the acid treated samples were indistinct.