Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

Photosynthetic and biochemical characterization of in vitro-derived African violet (Saintpaulia ionantha H. Wendl) plants to ex vitro conditions

African violet (Saintpaulia ionantha H. Wendl) is one of the most easily and commonly tissue-cultured ornamental plants. Despite this, there are limited reports on photosynthetic capacity and its impact on the plant quality during acclimatization. Various growth, photosynthetic and biochemical parameters and activities of antioxidant enzymes and dehydrins of micropropagated plants were assessed under three light intensities (35, 70, and 100 µmol m^?2 s^?1 photosynthetic photon flux density – PPFD). Fresh and dry plant biomass, plant height, and leaf area were optimal with high irradiance (70–100 µmol m^?2 s^?1 PPFD). Chlorophyll and carotenoid contents and net photosynthesis were optimal in plants grown under 70 µmol m^?2 s^?1 PPFD. Stomatal resistance, malondialdehyde content, and F_v/F_m values were highest at low light irradiance (35 µmol m^?2 s^?1 PPFD). The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, increased as light irradiance increased, signaling that high light irradiance was an abiotic stress. The accumulation of 55, 33, and 25 kDa dehydrins was observed with all light treatments although the expression levels were highest at 35 µmol m^?2 s^?1 PPFD. Irradiance at 70 µmol m^?2 s^?1 PPFD was suitable for the acclimatization of African violet plants. Both low and high irradiance levels (35 and 100 µmol m^?2 s^?1 PPFD) induced the accumulation of antioxidants and dehydrins in plants which reveals enhanced stress levels and measures to counter it.     

Inbreeding and inbreeding depression in a threatened endemic plant,the African violet (Saintpaulia ionantha ssp. grotei), of the East Usambara Mountains,Tanzania

Mating among closely‐related individuals in small and isolated plant populations may result in reduced vigour of the inbred offspring, i.e. inbreeding depression, especially in naturally outbreeding plants. Occurrence of inbreeding and inbreeding depression was studied in Saintpaulia ionantha ssp. grotei, a threatened endemic plant species with a narrow ecological amplitude from the East Usambara Mountains. The level of inbreeding (measured as the fixation index, F) was investigated in twelve populations by analyzing variation at one microsatellite marker locus. The effect of one generation of selfing and outcrossing on the progeny fitness was studied by controlled crosses in two small patches that differ in the level isolation. The fixation index (F) across the populations was on the average 0.21 and varied among the populations from substantial inbreeding (F = 0.58) to surplus heterozygosity (F = −0.29). High inbreeding depression (δ) was observed at early and late stages of the life‐cycle. The isolated patch exhibited lower inbreeding depression than did the non‐isolated patch. The results of this study suggest that inbreeding and subsequent inbreeding depression are potential threats to the survival of Saintpaulia populations.     

Structure and Development of Plastids in Epidermal Cells of African Violet (Saintpaulia ionantha Wendl.) in Culture

The structure of plastids in epidermal cells of African violet(Saintpaulia ionantha Wendl.) ‘Marge Winters’ wasexamined using transmission electron microscopy before and afterplacement of leaf tissue in culture. The plastids from matureepidermal cells contained membrane-bound inclusion bodies andprolamellar bodies in various stages of development. Starchgrains and poorly developed granal stacks were observed in asmall number of plastids. After placement of the leaves on suitableculture medium, inclusion bodies decreased in size and the lamellarsystem became more organized. The plastids in the epidermaltissue are believed to be in an arrested state of developmentand are released from this state by placement on a medium inductiveto organogenesis. Saintpaulia ionantha Wendl., African violet, membrane-bound inclusion, tissue culture, plastid development     

Effect of chilling, heat shock, and vigor on the growth of cucumber (Cucumis sativus) radicles

Chilling at 2.5°C reduced the subsequent growth of cucumber ( Cucumis sativus L.) radicles at 25°C. The reduction in radicle growth was linear for 1–3 days of chilling at ≈10% per day of treatment, but then it increased in a non-linear pattern until subsequent radicle growth was all but eliminated by 6 days of chilling. A heat shock of 40°C for 4–12 min increased chilling tolerance such that 4 days of chilling caused only a 36% decrease in radicle growth, compared to 66% for seedlings not heat shocked. Heat shocks were only able to protect that part of radicle growth that was in excess of the linear decrease in radicle growth projected from 0–3 days. There appear to be two effects of chilling on radicle growth. The first inhibition of subsequent growth was linear and was not affected by heat shocks. The second inhibition was much more severe; it appeared after 3 days of chilling and could be prevented by heat shock. Seeds classified with different levels of vigor (i.e., different initial rates of growth) did not respond significantly different to chilling stresses following heat-shock treatments.     

Acquisition of competence for shoot regeneration in leaf discs ofSaintpaufla ionantha xconfusa hybrids (African violet) cultured in vitro

Leaf discs fromSaintpaulia ionantha xconfusa hybrids (African violet) were transferred between basal medium (BM) containing no hormones and shoot-inducing medium (SIM) containing 2.0 mg 1^–1 indole acetic acid and 0.08 mg l^–1 6-benzylaminopurine to determine whether there is a window of competence for shoot regeneration. Leaf discs precultured on BM prior to transfer to SIM formed buds 3 days earlier than the controls (leaf discs not precultured) regardless of whether the discs were placed upside down or right side up on the medium. This suggests that cultured leaf cells were not competent for shoot induction during the first 3 days of culture. Leaf discs cultured right side up (abaxial surface to the medium) did not form buds on BM alone, unlike discs cultured upside down. Leaf disc survival was affected by a delay in hormonal exposure, but surviving leaf discs produced as many shoots as control leaf discs. This suggests that in the absence of exogenous plant hormones, cellular competence to regenerate shoots is not lost in excised leaf discs of African violet.Abbreviations BM Basal medium - SIM_Sho Shoot-inducing medium     

Effect of acetaldehyde, arsenite, ethanol, and heat shock on protein synthesis and chilling sensitivity of cucumber radicles

Exposure to a chilling temperature of 2.5°C for 96 h inhibited the subsequent growth of cucumber seedling radicles at 25°C by 92%. Exposing seedling with 5 ± 1 mm long radicles to acetaldehyde vapour (275 µl l⁻¹) or to an aqueous ethanol solution (0.6  M ) for 2 h, or to 45°C for 10 min before chilling, increased chilling tolerance so that the chilling treatment reduced growth by only 47, 39 or 36%, respectively. All of these effective treatments induced the synthesis of a number of proteins, and suppressed de novo protein synthesis (i.e. the incorporation of [³⁵S]-methionine) by about 70%. In contrast, treatment for 2 h with an aqueous arsenite solution (100 µ M ) had no effect on chilling sensitivity or the incorporation of [³⁵S]-methionine, yet it induced the synthesis of a complement of proteins that were similar to that induced by the effective heat-shock treatment. A unique protein or set of proteins may be responsible for heat-shock-induced chilling tolerance, but none was detected. The ability of various abiotic stresses to suppress protein synthesis may be more important in increasing tolerance to chilling injury than their ability to induce the synthesis of specific proteins.     

Effect of heat shock on ultrastructure and calcium distribution in Lavandula pinnata L. glandular trichomes

The effects of heat shock (HS) on the ultrastructure and calcium distribution of Lavandula pinnata secretory trichomes are examined using transmission electron microscopy and potassium antimonate precipitation. After 48-h HS at 40°C, plastids become distorted and lack stroma and osmiophilic deposits, the cristae of the mitochondria become indistinct, the endoplasmic reticulum acquires a chain-like appearance with ribosomes prominently attached to the lamellae, and the plasma and organelle membranes become distorted. Heat shock is associated with a decrease in calcium precipitates in the trichomes, while the number of precipitates increases in the mesophyll cells. Prolonged exposure to elevated calcium levels may be toxic to the mesophyll cells, while the lack of calcium in the glands cell may deprive them of the normal protective advantages of elevated calcium levels. The inequality in calcium distribution may result not only from uptake from the transpiration stream, but also from redistribution of calcium from the trichomes to the mesophyll cells.     

Reproductive ecology of three endangered African violet (Saintpaulia H. Wendl.) species in the East Usambara Mountains, Tanzania

Knowledge of the reproductive biology of endangered plants is essential for their effective conservation. It also provides important information for understanding the evolutionary processes that affect speciation, thus helping the definition of proper units for conservation in endangered plants with problematic taxonomy. We studied the reproductive potential and possibility for hybridization in the endangered genus Saintpaulia (Gesneriaceae) by examining flowering phenology, flower and seed production and pollination of three sympatric cross‐compatible Saintpaulia species in the East Usambara Mts., Tanzania. The synchrony observed in flowering in S. confusa and S. difficilis may enable hybridization between these two species, whereas partial phenological separation may contribute to the integrity of S. grotei. Although the level of flower abortion is high in S. confusa, each pollinated flower yields about 1000 seeds. Saintpaulia confusa produces fruits following both self‐ and cross‐pollination but spontaneous self‐pollination seems not to occur. Thus, seed production depends on sufficient pollinator service. Floral heteromorphy (i.e. enantiostyly) and bee pollination are likely to further enhance cross‐pollination, suggesting that the genus predominantly outcrosses. Thus, Saintpaulia populations are likely to suffer from negative effects of inbreeding if they become small and isolated.     

Heat shock proteins and chilling sensitivity of mung bean hypocotyls

Excised mung bean (Vigna radiata L.) hypocotyl sections wereexposed to 40 C for up to 4 h in the presence or absence of50 µM cycloheximide (CHX) before being held at a non-chilling(20 C) or chilling (2.5 C) temperature. Mung bean hypocotyltissue is chilling sensitive, and the rate of solute leakageis highly correlated with the extent of chilling injury. A 3h heat shock at 40 C reduced chilling-induced solute leakageby up to 40%, but leakage was similar to non-heat-shocked hypocotylswhen CHX was present. Specific proteins were labelled when hypocotylswere exposed to [³⁵S] methionine during the last hour of heatshock. The nine most intense bands on the autoradiographs ofSDS-PAGE gels of extracted protein corresponded to molecularweights of 114, 79, 73, 70, 60, 56, 51, 46, and 18 kDa. The18 kDa band reached a maximum after 1 h at 40 C and then rapidlydecreased in intensity as the heat shock continued, becomingundetectable at 4 h. The four most intense bands after 3 h at40 C corresponded to molecular weights of 79, 70, 51, and 46kDa. The synthesis of these four hsps was markedly reduced whenthe hypocotyl sections were exposed to CHX during heat shock.During chilling for 6 d, the levels of hsps 79 and 70 remainedsignificantly higher in tissue that was heat shocked prior tochilling than in tissue that was not heat shocked. In contrast,the levels of hsps 51 and 46 were similar in bothheat-shockedand control tissues. Heat-shock-induced chilling tolerance waslost between 6 and 9 d ofstorage at 2.5 C; this loss coincidedwith the decay of hsps 79 and 70 to control levels. These resultssuggest that heat shock induces an increase in both chillingtolerance and the de novo synthesis of specific heat shock proteins;namely hsps 79 and 70. This is the first report showing a relationshipbetween heat-shock-induced chilling tolerance and specific heat-shock-inducedproteins. Key words: Ion leakage, protein synthesis, Vigna radiata     

Histological changes associated with acquisition of competence for shoot regeneration in leaf discs ofSaintpaufla ionantha xconfusa hybrid (African violet) cultured in vitro

In leaf discs ofSaintpaulia ionantha xconfusa hybrid (cv. Virginia) cultured on shoot-inducing medium, periclinal divisions were initiated in epidermal cells 3–5 days after explant isolation. This timing coincided with the time for competence acquisition determined in tissue-transfer experiments. Some of the daughter cells from periclinal divisions formed the target cells which divided both anticlinally and periclinally to form cell division centers (meristemoids), precursors of adventitious shoots. The target cells were not morphologically distinct from other epidermal cells at the light microscope level. It is suggested that the periclinal divisions in epidermal cells represent the dedifferentiation phase during which target (competent) cells are formed. Once the cells have acquired the ability to divide periclinally, both dedifferentiation and shoot induction occur in the presence of exogenous plant hormones.Abbreviations SIM Shoot-inducing medium     

Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (<Emphasis Type="Italic">Saintpaulia ionantha </Emphasis>Wendl.)

Regeneration via shoot organogenesis and somatic embryogenesis was observed from thidiazuron (TDZ)-treated leaf and petiole explants of greenhouse- and in vitro-grown African violet plants. The response of cultures to other growth regulators over a range of 0.5 microM to 10 microM was 50% less than that observed with TDZ. A comparative study among several cultivars of African violet indicated that "Benjamin" and "William" had the highest regeneration potential. In "Benjamin", higher frequencies of shoot organogenesis (twofold) and somatic embryogenesis (a 50% increase) were observed from in vitro- and greenhouse-grown plants, respectively. At concentrations lower than 2.5 microM, TDZ induced shoot organogenesis, whereas at higher doses (5-10 microM) somatic embryos were formed. These findings provide the first report of simultaneous shoot organogenesis and somatic embryogenesis of African violet explants in response to TDZ.     

Studies on chilling sensitivity of zebrafish (Danio rerio) oocytes

Human activity in the last few decades has had a devastating effect on the diversity of fresh water and marine fish. Further decline of fish population may have serious economic and ecological consequences. One of the most promising techniques to preserve fish population is to cryopreserve their germ cells. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryo cryopreservation and fish oocyte cryopreservation has never been studied systematically. The aim of this study is to investigate the chilling sensitivity of fish oocytes. Experiments were conducted with zebrafish stage III (vitellogenic) and stage V (mature) oocytes, which were chilled at 10, 5, 0, -5 or -10 degrees C for 15 or 60 min using a low temperature bath. Control oocytes were kept at room temperature at 22 degrees C. Oocyte viability was assessed using three different methods: trypan blue staining (TB), thiazolyl blue tetrazolium bromide (MTT) staining and observation of germinal vesicle breakdown (GVBD). The results showed that zebrafish oocyte are very sensitive to chilling and their survival decreased with decreasing temperature and increasing exposure time periods. Normalised survivals assessed with TB staining after exposure to 0, -5 or -10 degrees C for 15 or 60 min were 90.1+/-6.0, 77.8+/-7.6, and 71.2+/-9.3%, and 60.2+/-3.8, 49.6+/-6.7, and 30.4+/-3.0%, respectively. The study found that the sensitivity of viability assessment methods increase in the order of MTT < TB < GVBD. It was found that stage III oocytes were more susceptible to chilling than stage V oocytes, and that individual female had a significant influence (p < 0.0001) on oocyte chilling sensitivity. Zebrafish oocyte chilling sensitivity may also be one of the limiting factors for development of protocol of their cryopreservation.     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Effect of egg‐phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa

This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling. Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5‐carboxyfluorescein diacetate (cFDA)) at 16°C, 12°C, 4°C, and 22°C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope. The LPT center, T_m, was determined by statistical analysis. The LPT and T_m were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg‐phosphatidylcholine (EPC) liposomes at 16°C, 12°C, 4°C, and 26°C (control). The results show that the membrane integrity of spermatozoa incubated at 16°C, 12°C, and 4°C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26°C) and 10°C, with T_m at 14–16°C. The T_m for sperm incubated with liposomes or egg‐yolk extender was below the measured range (2°C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated‐to‐polyunsaturated FAs. Here we show that the protection afforded by the presence of egg yolk or liposomes in the extender is accomplished by shifting the T_m to below the 4°C point at which chilled semen is maintained for transport, or the point at which fast freezing begins to minimize cellular damage. Zoo Biol 0:1–13, 2005. © 2005 Wiley‐Liss, Inc.     

非洲紫罗兰两侧与辐射对称花中两个CYC类完整基因的分离和序列分析

在已知GCYC基因部分序列基础上, 通过改进的mTAIL-PCR方法克隆非洲紫罗兰Saintpaulia ionantha两侧对称栽培种中CYC类基因的5′未知序列, 并进而从两侧与辐射对称栽培种中分离得到苦苣苔科Gesneriaceae中第一组完整基因: SiCYC1A与SiCYC1B。对以上基因的核酸和氨基酸序列比较发现, SiCYC1A与SiCYC1B序列同源性很高, 均含有完整的功能调控区域(即TCP domain和R domain)并与模式植物金鱼草Antirrhinum majus中CYC基因同源。因此, 这两个基因应具有正常功能, 是功能上互补的冗余基因。令人意外的是在辐射对称花栽培品种中的这两个基因和两侧对称花栽培品种中对应基因的序列完全相同。经过对金鱼草以及相关类群辐射对称花突变体中CYC类基因序列的比较分析, 推论在非洲紫罗兰中, SiCYC1A与SiCYC1B基因可能受上游未知的共同调控因子调控, 该调控因子的改变是导致栽培品种中花对称性发生变化的主要原因。另外, 对改进后的TAIL-PCR(mTAIL-PCR)的方法和过程进行了详细叙述, 并对其技术特征和优势开展了简单的论述。