Desiccated doubled-haploid embryos obtained from microspore culture of barley cv. Igri

 Barley microspore-derived doubled-haploid embryos have been produced in vitro. The development of embryo desiccation technology will allow long-term storage, germplasm preservation and low delivery cost. Treatment of the microspore-derived embryos was essential to induce desiccation tolerance and to arrest further development and plant regeneration. At the concentrations used, a treatment with trehalose was more efficient than with sucrose, and mannitol was harmful to the embryos. Up to 80% of the desiccated embryos produced complete green plants when transferred to regeneration medium, by the application of a 0.6 m trehalose or a 10^–5 m abscisic acid treatment to the embryos in the culture induction medium. The morphology of these plants was similar to plants produced directly from non-desiccated embryos. Received: 28 September 1998 / Revision received: 27 November 1998 / Accepted: 5 January 1999     

In vitro desiccation tolerance of the epiphytic Ghost Orchid,Dendrophylax lindenii (Lindl.) Benth x. Rolfe

The Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe, is a rare and endangered epiphytic orchid native to south Florida and Cuba. The orchid is considered difficult to propagate under greenhouse conditions, requiring high humidity and low air movement. In contrast, the orchid’s native habitat seasonally dries out with decreased precipitation and humidity. This suggests some level of desiccation tolerance. Ghost Orchid plants were assessed for potential desiccation tolerance and ability to recover from desiccation stress under in vitro conditions. In vitro-derived plants were placed into sterile baby food jars and transferred to chambers maintained at 10% relative humidity, which is extremely low compared to relative humidity levels (40–100%) recorded under natural field conditions. Plants were removed every week for 4 wk and recovered on P723 medium supplemented with banana powder for 4 wk. Data were collected at the initiation of the experiment, after the desiccation periods, and after 4 wk in vitro recovery. Ghost Orchid plants demonstrated extremely high desiccation tolerance. Even after 4 wk desiccation, plant survival was observed at 79.2% after recovery. Desiccated plants exhibited significant decreases in tissue water potential (− 18.44 MPa), fresh weight (65.5% loss), and water content (14.2%); however, high plant survival was still observed under these conditions similar to poikilohydric plants. Overall, the Ghost Orchid demonstrated high desiccation tolerance, which should be considered for future greenhouse culture and for its application in the direct field establishment of in vitro-derived plants without greenhouse acclimatization.

     

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     

The use of aeroponics to investigate antioxidant activity in the roots of <Emphasis Type="Italic">Xerophyta viscosa</Emphasis>

In order to ultimately understand the whole plant mechanism of attaining desiccation tolerance, we undertook to investigate the root tissues of the resurrection plant Xerophyta viscosa, as previous work has only been conducted on the leaf tissues of resurrection plants. An aeroponic plant growth system was designed and optimised to observe the root’s response to desiccation without the restrictions of a soil medium, allowing easy access to roots. Successful culture of both X.viscosa and the control, Zea mays, was achieved and dehydration stress was implemented through reduction of nutrient solution spraying of the roots. After drying to the air dry state (achieved after 7 days for roots and 10 days for shoots), rehydration was achieved by resumption of root spraying. X.viscosa plants survived desiccation and recovered but Z. mays did not. The activity of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase and quantities of ascorbate and glutathione were determined during root desiccation. There was an initial decline in activity in all enzymes upon drying to 80% RWC, but activity thereafter remained constant, at rates indicative of potential metabolic activity, to the air-dry state. This data suggests that these enzymes are not denatured by desiccation of the root tissue. Ascorbate and glutathione content remained constant at concentrations of 70 and 100 μM, respectively during drying. Thus root tissues appear to retain antioxidant potential during drying, for use in recovery upon rehydration, as has been reported for leaf tissues of this and other resurrection plants.     

A novel cold-inducible gene from Arabidopsis,RCI3, encodes a peroxidase that constitutes a component for stress tolerance

Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, beta-carotene, ascorbate, alpha-tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, gamma-tocopherol, lutein, neoxanthin and glucose-6-phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re-watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of alpha-tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, beta-carotene and alpha-tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.     

Comparison of thylakoid structure and organization in sun and shade Haberlea rhodopensis populations under desiccation and rehydration

The resurrection plant, Haberlea rhodopensis can survive nearly total desiccation only in its usual low irradiation environment. However, populations with similar capacity to recover were discovered recently in several sunny habitats. To reveal what kind of morphological, structural and thylakoid-level alterations play a role in the acclimation of this low-light adapted species to high-light environment and how do they contribute to the desiccation tolerance mechanisms, the structure of the photosynthetic apparatus, the most sensitive component of the chlorophyll-retaining resurrection plants, was analyzed by transmission electron microscopy, steady state low-temperature fluorescence and two-dimensional Blue-Native/SDS PAGE under desiccation and rehydration.     

Partial desiccation augments plant regeneration from irradiated embryogenic cultures of sugarcane

Partial desiccation treatment was applied to improve plant regeneration response in irradiated in vitro cultures. Embryogenic callus cultures of sugarcane cv. Co-671 were exposed to different doses of gamma radiation (0–80 Gy) and radiation effect was evaluated in terms of post-irradiation callus recovery, growth and regeneration of plants. Proliferative capacity of cultures was inversely correlated with radiation dose as the percentage surviving cultures or white proliferating clumps (WPC) decreased as the radiation dose increased up to 80 Gy. LD50 was found to be around 20–30 Gy and at higher doses, poor regeneration frequency was observed after 4–6 weeks of post-irradiation culture. To stimulate regeneration response, irradiated cultures were subjected to partial desiccation for 6 h and the treatment resulted in enhanced plant regeneration response. The study suggests that partial desiccation treatment can be useful in stimulating regeneration response of irradiated in vitro cultures.     

Desiccation-tolerant plants in dry environments

The majority of terrestrial plants are unable to survive in very dry environments. However, a small group of plants, called ‘resurrection’ plants, are extremely desiccation-tolerant and are capable of losing more than 90% of the cellular water in vegetative tissues. Resurrection plants can remain dried in an anabiotic state for several years and, upon rehydration, are able to resume normal growth and metabolism within 24 h. Vegetative desiccation tolerance is thought to have evolved independently several times within the plant kingdom from mechanisms that allow reproductive organs to survive air-dryness. Resurrection plants synthesise a range of compounds, either constitutively or in response to dehydration, that protect various components of the cell wall from damage during desiccation and/or rehydration. These include sugars and late embryogenesis abundant (LEA) proteins that are thought to act as osmoprotectants, and free radical-scavenging enzymes that limit the oxidative damage during dehydration. Changes in the cell wall composition during drying reduce the mechanical damage caused by the loss of water and the subsequent shrinking of the vacuole. These include an increase in expansin or cell wall-loosening activity during desiccation that enhances wall flexibility and promotes folding.     

Dynamics of Endogenous Phytohormones during Desiccation and Recovery of the Resurrection Plant Species Haberlea rhodopensis

Drought is one of the most significant threats to world agriculture and hampers the supply of food and energy. The mechanisms of drought responses can be studied using resurrection plants that are able to survive extreme dehydration. As plant hormones function in an intensive cross-talk, playing important regulatory roles in the perception and response to unfavorable environments, the dynamics of phytohormones was followed in the resurrection plant Haberlea rhodopensis Friv. during desiccation and subsequent recovery. Analysis of both leaves and roots revealed that jasmonic acid, along with and even earlier than abscisic acid, serves as a signal triggering the response of the resurrection plants to desiccation. The steady high levels of salicylic acid could be considered an integral part of the specific set of parameters that prime H. rhodopensis desiccation tolerance. The dynamic changes of cytokinins and auxins suggest that these hormones actively participate in the dehydration response and development of desiccation tolerance in the resurrection plants. Our data contribute to the elucidation of a global complex picture of the resurrection plant’s ability to withstand desiccation, which might be successfully utilized in crop improvement.     

Responses of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> to high irradiance

The effect of high irradiance (HI) during desiccation and subsequent rehydration of the homoiochlorophyllous desiccation-tolerant shade plant Haberlea rhodopensis was investigated. Plants were irradiated with a high quantum fluence rate (HI; 350 μmol m⁻² s⁻¹ compared to ca. 30 μmol m⁻² s⁻¹ at the natural rock habitat below trees) and subjected either to fast desiccation (tufts dehydrated with naturally occurring thin soil layers) or slow desiccation (tufts planted in pots in peat-soil dehydrated by withholding irrigation). Leaf water content was 5 % of the control after 4 d of fast and 19 d of slow desiccation. Haberlea was very sensitive to HI under all conditions. After 19 d at HI, even in well-watered plants there was a strong reduction of rates of net photosynthesis and transpiration, contents of chlorophyll (Chl) and carotenoids, as well as photosystem 2 activity (detected by the Chl fluorescence ratio R_Fd). Simultaneously, the blue/red and green/red fluorescence ratios increased considerably suggesting increased synthesis of polyphenolic compounds. Desiccation of plants in HI induced irreversible changes in the photosynthetic apparatus and leaves did not recover after rehydration regardless of fast or slow desiccation. Only young leaves survived desiccation.     

Protective mechanism of desiccation tolerance in <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was characterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then declined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(Φ_PSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dissipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than −21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon rewatering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation.     

Desiccation of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> at high temperature

Haberlea rhodopensis plants, growing under low irradiance in their natural habitat, were desiccated to air-dry state at a similar light intensity (about 30 μmol m⁻² s⁻¹) under optimal (23/20°C, day/night) or high (38/30°C) temperature. Dehydration of plants at high temperature increased the rate of water loss threefold and had a more detrimental effect than either drought or high temperature alone. Water deficit decreased the photochemical activity of PSII and PSI and the rate of photosynthetic oxygen evolution, and these effects were stronger when desiccation was carried out at 38°C. Some reduction in the amount of the main PSI and PSII proteins was observed especially in severely desiccated Haberlea leaves. The results clearly showed that desiccation of the homoiochlorophyllous poikilohydric plant Haberlea rhodopensis at high temperature had more damaging effects than desiccation at optimal temperature and in addition recovery was slower. Increased thermal energy dissipation together with higher proline and carotenoid content in the course of desiccation at 38°C compared to desiccation at 23°C probably helped in overcoming the stress.     

Unexplored dimensions of variability in vegetative desiccation tolerance

Desiccation tolerance has evolved recurrently across diverse land plant lineages as an adaptation for survival in regions where seasonal rainfall drives periodic drying of vegetative tissues. Growing interest in this phenomenon has fueled recent physiological, biochemical, and genomic insights into the mechanistic basis of desiccation tolerance. Although, desiccation tolerance is often viewed as binary and monolithic, substantial variation exists in the phenotype and underlying mechanisms across diverse lineages, heterogeneous populations, and throughout the development of individual plants. Most studies have focused on conserved responses in a subset desiccation-tolerant plants under laboratory conditions. Consequently, the variability and natural diversity of desiccation-tolerant phenotypes remains largely uncharacterized. Here, we discuss the natural variation in desiccation tolerance and argue that leveraging this diversity can improve our mechanistic understanding of desiccation tolerance. We summarize information collected from ~600 desiccation-tolerant land plants and discuss the taxonomic distribution and physiology of desiccation responses. We point out the need to quantify natural diversity of desiccation tolerance on three scales: variation across divergent lineages, intraspecific variation across populations, and variation across tissues and life stages of an individual plant. We conclude that this variability should be accounted for in experimental designs and can be leveraged for deeper insights into the intricacies of desiccation tolerance.     

Desiccation increases sucrose levels in Ramonda and Haberlea, two genera of resurrection plants in the Gesneriaceae

A few genera of angiosperms are known as 'resurrection plants' since their leaves withstand complete desiccation. In many organisms, including some resurrection plants, desiccation tolerance is associated with the accumulation of special carbohydrates. We examined whether this is also true for the two European angiosperm genera of resurrection plants, Ramonda and Haberlea in the Gesneriaceae. Using gas chromatography, non-structural carbohydrates were determined as a percentage of the dry weight in leaves of Ramonda nathaliae subjected to various desiccation regimes. Sucrose was the predominant soluble carbohydrate in all samples, and its level steadily increased from 2 to 10% during desiccation. Starch amounted to ca 2% in control leaves and disappeared completely within 8 days of desiccation. Considerable amounts (1–2.5%) of raffinose and smaller amounts of its precursor galactinol (1-a-galactosyl- myo -inositol) were present in control leaves; these carbohydrates showed only minor changes upon desiccation. Similar results were obtained when excised leaves of Ramonda nathaliae, Ramonda myconi and Haberlea rhodopensis were subjected to desiccation. These data indicate that sucrose accumulation is connected to desiccation tolerance in Gesneriaceae; the presence of raffinose may be a pre-adaptation since this sugar prevents crystallization of sucrose during drying.     

Protective mechanism of desiccation tolerance in Reaumuria soongorica: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was characterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then declined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(Φ_PSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dissipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than −21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon rewatering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation. Supported by the Program of the Research of Vegetation Restoration in Arid Areas of Lanzhou (Grant No. 03-2-27) and the National Natural Science Foundation of China (Grant No. 30270243)     

Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

Effects of proliferation,maturation, and desiccation methods on conversion of soybean somatic embryos

Summary Cermination of soybean [Glycine max (L.) Merrill] somatic embryos and conversion to whole plants are generally low. This study was conducted to investigate the effects of proliferation, maturation, and desiccation methods on conversion of soybean somatic embryos to plants. Soybean cv. Jack somatic embryos, proliferated on a solid medium containing 90.5 μM (20 mgl⁻¹) 2.4-dichlorophenoxyacetic acid (2.4-D) (MSD20), showed a regeneration rate signficantly higher than those proliferated in a liquid medium containing 45.25 μM (10mgl⁻¹) 2,4-D (FN Lite). When a liquid medium without 2,4-D and B5 vitamins (FN Superlite) was used for maturation, the duration of time necessary for embryo development could be shortened by more than a month compared to maturation on a standard solid medium (MSM6AC). An air-drying method, in which somatic embryos were desiccated in an empty sealed Petri dish for 3–5d, gave rise to the best germination efficiency among the four desiccation methods tested: fast, slow, air, and KCl methods. The final percentage of moisture seems important since embyros over-dried by the fast and slow methods did not convert well into plants.     

The discovery,scope, and puzzle of desiccation tolerance in plants

The modern scientific study of desiccation tolerance began in 1702 when Anthony von Leeuwenhoek discovered that rotifers could survive without water for months. By 1860, the controversy over whether organisms could dry up without dying had reached such a pitch that a special French commission was convened to adjudicate the dispute. In 2000, we know that a few groups of animals and a wide variety of plants can tolerate desiccation in the active, adult stages of their life cycles. Among plants, this includes many lichens and bryophytes, a few ferns, and a very few flowering plants, but no gymnosperms nor trees. Some desiccation-tolerant species can survive without water for over ten years, recover from desiccation to unmeasurably low water potentials, and, when plants are desiccated, endure temperature extremes from ?272 to 100 °C. Desiccation-tolerant plants occur on all continents but mainly in xeric habitats or microhabitats where the cover of desiccation-sensitive species is low. Two main puzzles arise from these patterns: What are the mechanisms by which plants tolerate desiccation? and Why are desiccation-tolerant plants not more ecologically widespread? Recent molecular and biochemical studies suggest that there are multiple mechanisms of tolerance, many of which involve protection from oxidants and from the loss of configuration of macromolecules during dehydration. Hypotheses to explain the restricted ecological range of desiccation-tolerance plants include inability to maintain a cumulative positive carbon balance during repeated cycles of wetting and drying and inherent trade offs between desiccation tolerance and growth rate.