Effect of maturation duration on desiccation tolerance in hybrid larch (Larix×leptoeuropaea dengler) somatic embryos

Summary Cotyledonary somatic embryos ofLarix × leptoeuropaea that developed after various maturation times on media containing abscisic acid showed different frequencies of conversion into plants. Drying of these somatic embryos under high relative humidity (RH) before germination improved plantlet recovery and eliminated differences in the performance of somatic embryos matured for different times. However, dehydration of somatic embryos under 98% RH to a water content below that of zygotic embryos excised from mature seeds (0.97 and 1.36 g H₂O/g dry weight, respectively) showed a strong positive correlation between longer maturation time and desiccation tolerance. Drying somatic embryos at 4° C under 59% RH for 1 wk resulted in desiccation to a water content of 0.30 g H₂O/g dry weight, which was the closest to the hydration state of zygotic embryos in dried, stored seeds (0.20 g H₂O/g dry weight). Under this condition, only somatic embryos matured for 5 wk germinated and produced plantlets at a relatively high frequency (73 and 41%, respectively).     

Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha

Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H₂O g⁻¹ dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H₂O g⁻¹ DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H₂O g⁻¹ DW, desiccation-tolerant cells that had been precultured were vitrified above 0°C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.     

Metabolic profiling of the resurrection plant Haberlea rhodopensis during desiccation and recovery

Desiccation tolerance is among the most important parameters for crop improvement under changing environments. Resurrection plants are useful models for both theoretical and practical studies. We performed metabolite profiling via gas chromatography coupled with mass spectrometry (GC‐MS) and high‐performance liquid chromatography (HPLC) and analyzed the antioxidant capacity of the endemic resurrection plant Haberlea rhodopensis at desiccation and recovery. More than 100 compounds were evaluated. Stress response included changes in both primary and secondary metabolic pathways. The high amounts of the specific glycoside myconoside and some phenolic acids – e.g. syringic and dihydrocaffeic acid under normal conditions tend to show their importance for the priming of H. rhodopensis to withstand severe desiccation and oxidative stress. The accumulation of sucrose (resulting from starch breakdown), total phenols, β‐aminoisobutyric acid, β‐sitosterol and α‐tocopherol increased up to several times at later stages of desiccation. Extracts of H. rhodopensis showed high antioxidant capacity at stress and normal conditions. Myconoside was with the highest antioxidant properties among tested phenolic compounds. Probably, the evolution of resurrection plants under various local environments has resulted in unique desiccation tolerance with specific metabolic background. In our case, it includes the accumulation of a relatively rare compound (myconoside) that contributes alone and together with other common metabolites. Further systems biology studies on the involvement of carbohydrates, phenolic acids and glycosides in the desiccation tolerance and antioxidant capacity of H. rhodopensis will definitely help in achieving the final goal – improving crop drought tolerance.     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

Effects of light, CO2 and humidity on carnation growth, hyperhydration and cuticular wax development in a mist reactor

Summary Plant survival ex vitro requires functioning stomata, adequate cuticular wax composition and deposition, and normal morphological development. Light intensity, CO₂ and relative humidity were altered inside an acoustic window mist reactor to study their effects on carnation (Dianthus caryophyllus) growth, stomata development, hyperhydration and epicuticular wax content. Increasing the light intensity from 65 to 145 μmol m⁻² s⁻¹ and enrichment of the gas phase with CO₂ (1350 ppm) reduced the number of hyperhydrated plants from 75 to 25% and increased the percentage dry weight of normal (healthy) plants from 17 to 25%. Lowering the relative humidity (≈70% RH) surrounding the plants during the mist-off phase for the last 2 wk of culture reduced the number of hyperhydrated plants from 70 to 9% and also increased the percentage of dry weight of normal plants from 16 to 25%. The stomata on plants grown in conditions of high light or low humidity had smaller apertures and appeared sunken when compared to stomata from plants grown in low light and high relative humidity. The epicuticular wax profiles of plants from the greenhouse or Magenta boxes showed a distinct shift in wax compounds with developmental age, plant type (hyperhydrated or normal), and type of box that was used (vented or not). In addition, very different wax profiles were observed from plants grown in reactors with altered CO₂ and light intensities.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Dry artificial seeds and desiccation tolerance induction in microspore-derived embryos of broccoli

Desiccation tolerance of broccoli microspore-derived embryos was induced by exogenous application of abscisic acid (ABA). Embryos, which were desiccated to about 10% water content, were estimated for viability after rehydration. Survival was dependent on the ABA concentration and the development stage of embryo, but not on the length of exposure period to ABA or genotype. Cotyledonary stage embryos acquired the highest desiccation tolerance when treated with 1×10^-4M ABA. Under this condition, on average 27–48% of the desiccated embryos could convert into plants. Embryos treated with 1×10^-6M ABA or no ABA or earlier development-staged embryos, such as globular and heart stages, lost viability after desiccation. A one day exposure to ABA had the similar effect on the induction of desiccation tolerance as a 7-day treatment. The dried embryos maintained their ability of plant conversion after three months of storage under room conditions. The plants derived from the desiccated embryos were not different in the morphology or ploidy level from those from non-desiccated ones.Abbreviations ABA abscisic acid - RH relative humidity     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

In vitro propagation of Trichosanthes kirilowii Maxim. through nodal segment shoot proliferation

Trichosanthes kirilowii Maxim. is a vital traditional herbal medicinal plant found in northeastern Asia. Its roots, fruits, and seeds are used as food and medicine. Roots harvested for medicinal use take over 3 yr to mature when the plant is grown in a traditional way through cultivation in the field. This coupled with uncertainty in identification of the plant when collected from the wild calls for a standard in vitro propagation system to meet the increasing demand for it. The purpose of this study was to develop a standard protocol for the in vitro micropropagation of T. kirilowii. Ten different media supplemented with different concentrations of plant growth regulators were evaluated. At 5 wk, De Greef and Jacobs medium supplemented with 0.1 mg L⁻¹ kinetin led to optimal shoot growth, while the same medium supplemented with 0.5 mg L⁻¹ indole 3-butyric acid induced optimal root growth, also at 5 wk. The micropropagated plants that were acclimatized for 8 wk in the greenhouse produced mature root tubers after planted in the field for 3 mo. Therefore, these findings provide a basis for future large-scale in vitro propagation of T. kirilowii.

     

Scaling-up production and field performance of micropropagated medicinal herb ‘Safed Musli’ (<Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>)

Summary Chlorophytum borivilianum Sant. et Fernand. (‘Safed Musli’), an endangered Indian medicinal herb, is valued for its tuberous roots reputed to have aphrodisiac properties. Farmers in India cultivate this medicinal herb on a commercial scale because of its high economic value. To cater to the growing demand for planting material, a highly reproducible field-tested and cost-effective micropropagation scheme has been developed. Best shoot multiplication was achieved on agargelled Murashige and Skoog medium containing 22.2 μM 6-benzylaminopurine (BA) and 3% sucrose. Phytagel^™ at 0.2% showed slightly better response than BDH agar in terms of shoot multiplication, but the use of BDH agar was preferred due to its low cost. With the optimized conditions, more than 15 000 plantlets could be produced in 20 wk. Plantlets subjected to hardening under agro-shadenet conditions during the monsoon months of high humidity showed better survival rate and growth compared to plantlets hardened in vitro and subsequently transferred to the greenhouse for acclimatization. Rate of plantlet survival was 87 and 90% under open field and agro-shadenet conditions, respectively. Plantlets grown ex vitro under agro-shadenet and field conditions produced tuberous roots which could be grown in the next season as a secondary propagule. We concluded that in vitro production of ‘Safed Musli’ was cost-effective compared to conventional propagation and holds great potential for commercial production.     

The effect of slow-growth strategy on a production of Petunia × hybrida Vilm. microcuttings

Petunia × hybrida Vilm. is a fast-growing ornamental plant that was cultured under varying storage conditions to address seasonal fluctuations in microcutting demand. The effects of storage period (16 to 32 wk), temperature (15 to 23°C), low and high light intensity, sucrose (1 to 5% w/v), and mannitol (0 to 4% w/v) in factorial arrangements were analyzed. Stored and non-stored shoots were compared for microcutting production, harvested twice at 3-wk intervals, and were subsequently transferred to the greenhouse for 17 d. Nearly all plants from 16-wk storage survived well at all treatment conditions and the quality and quantity of microcuttings were enhanced from shoots stored at 15°C and 3% (w/v) sucrose, without mannitol for all storage periods. Another experiment tested 11- to 25-wk storage period with Petunia hybrida ‘Ragtime’ and ‘Suncatcher’ at 12°C and low light intensity. Repeated cycles of microcutting at 2-wk intervals were extended with ex vitro rooting in the greenhouse for 15 d. More and better quality microcuttings were obtained from the second and third cutting cycles than from the first or fourth cycles. By reducing temperature and light intensity, Petunia hybrida was successfully stored for 32 wk (without mannitol). A seasonal schedule with a short production window, followed by cutting large numbers of high-quality shoot-tips, could be affected and efficiently managed through storage. The value of stored shoots was enhanced by extending the number of times a shoot could be cut over repeated cutting cycles with a gain in microcutting quality.

     

兰科菌根真菌研究方法的概述

兰科植物资源在全球分布广泛,其中有许多是重要的药用植物和名贵的珍稀花卉,由于具有较高的商业价值,受到各界人士的广泛关注。兰科植物生长习性的特殊性导致其在自然状态下繁殖率极低,因此难以满足市场的广泛需求。近年研究表明,几乎所有兰科植物都能与相应的菌根真菌建立共生关系,并且必须依赖于这些内生真菌才能完成其整个生活史。因而对菌根真菌在提高兰科植物生长速度和繁殖能力过程中机制的研究以及将研究成果运用于工业化育苗中将是缓解兰科植物市场供求紧张问题的关键。通过对近几年有关天麻和铁皮石斛等兰科植物的问题研究中所采用的研究方法加以阐述,以期对今后兰科菌根真菌的研究提供一定的参考。     

The Role of Abscisic Acid in Acclimation of Plants Cultivated in vitro to ex vitro Conditions

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of air humidity on the development of functional stomatal apparatus

Phaseolus vulgaris L. seedlings were grown under different air humidities simulating conditions during micropropagation (very high humidity duringin vitro cultivation and low air humidity after transferex vitro). The functional stomatal apparatus developed after a short period of growth at low air humidity at the beginning of plant ontogeny or after transfer from high to low air humidity, but not in plants grown steadily under high air humidity. The ability of stomata to regulate gas exchange was not persistent and disappeared after transfer of plants from low to high humidity. The author thanks Mrs. L. Kolčabová for her skilled assistance. The paper is a part of the project supported by the grant No. 501/95/1303 of the Grant Agency of the Czech Republic.     

Alterations in growth and crassulacean acid metabolism (CAM) activity of in vitro cultured cactus

Unlike C-3 plants, cacti possess a crassulacean acid metabolism (CAM) physiology that can alter the pattern of carbon uptake and affect plant growth under artificial environmental conditions, especially in tissue culture. In vitro-derived plantlets of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions. Growth regulators incorporated into the culture media during shoot proliferation stage of micropropagation had a strong influence on this increased growth. Other important factors that contributed to increased growth under in vitro conditions were high relative humidity and sugar in the culture medium. An analysis of gas exchange and daily fluctuations of malic acid levels revealed an increase in net photosynthetic rate, in terms of carbon assimilation, by in vitro plants compared with that of ex vitro plants. This stimulated photosynthesis in the presence of an external carbon source was unexpected but apparently true for cacti exhibiting CAM physiology. Unlike CAM plants grown in ex vitro conditions, net CO₂ uptake by in vitro-cultured cacti occurred continuously in the light as well as the dark. Once regenerated, cacti were transferred to ex vitro conditions where the normal CAM pathway resumed with a concomitant reduction in growth and CO₂ uptake. These results showed that growth of cacti can be considerably accelerated by in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient in vitro propagation,antioxidant and antimicrobial activities of Aphyllorchis montana Rchb.f.

An in vitro plant regeneration protocol was successfully established in Aphyllorchis montana, a saprophytic achlorophyllous orchid by culturing immature seeds. Among the six basal media evaluated for seed germination, BM-1-Terrestrial Orchid medium was found to be the best followed by Knudson C medium. Half-strength BM-1-Terrestrial Orchid medium was supplemented with different growth regulators either individually or in combinations for multiplication of shoots induction. Of the five cytokinins tested, thidiazuron at 6.8 μM was found to be most effective for multiple shoot induction yielding 17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration of α-Naphthaleneacetic acid (1.3 μM) in MS medium supplemented with the cytokinin thidiazuron (6.8 μM) favored shoot multiplication. The addition of organic additives to the medium containing thidiazuron enhanced the number of regenerated shoots. The plantlets were acclimatized, and the survival rate was 100%. Screening of the antioxidant and antimicrobial activities and estimation of total phenolics and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolics and flavonoid content than in vitro propagated plants.     

Evaluation of the predatory mite <Emphasis Type="Italic">Phytoseiulus macropilis</Emphasis> (Acari: Phytoseiidae) as a biological control agent of the two-spotted spider mite on strawberry plants under greenhouse conditions

The predatory mite Phytoseiulus macropilis is a potential biological control agent of the two-spotted spider mite (TSSM) Tetranychus urticae on strawberry plants. Its ability to control TSSM was recently assessed under laboratory conditions, but its ability to locate and control TSSM under greenhouse conditions has not been tested so far. We evaluated whether P. macropilis is able to control TSSM on strawberry plants and to locate strawberry plants infested with TSSM under greenhouse conditions. Additionally, we tested, in an olfactometer, whether odours play a role in prey-finding by P. macropilis. The predatory mite P. macropilis required about 20 days to achive reduction of the TSSM population on strawberry plants initially infested with 100 TSSM females per plant. TSSM-infested plants attract an average of 27.5 ± 1.0% of the predators recaptured per plant and uninfested plants attracted only 5.8 ± 1.0% per plant. The predatory mites were able to suppress TSSM populations on a single strawberry plant and were able to use odours from TSSM-infested strawberry plants to locate prey in both olfactometer and arena experiments. Hence, it is concluded that P. macropilis can locate and reduce TSSM population on strawberry plants under greenhouse conditions.     

A novel case of autogamy and cleistogamy in Dendrobium wangliangii: A rare orchid distributed in the dry‐hot valley

Dendrobium wangliangii is an epiphytic orchid distributed in the Jinshajiang dry‐hot valley in Luquan County, Yunnan Province, China. Most Dendrobium spp. typically have a low fruit set, but this orchid shows a higher fruit set under natural conditions despite the lack of effective pollinators. The pollination biology of the critically endangered D. wangliangii was investigated in this study. A fruit set rate of 33.33 ± 4.71% was observed after bagging treatment in 2017 and a high fruit set rate (65.72 ± 4.44% in 2011; 50.79 ± 5.44% in 2017) was observed under natural conditions, indicating that D. wangliangii is characterized by spontaneous self‐pollination. The anther cap blocked the growing pollinium; thus, the pollinium slid down and reached the stigmatic cavity, leading to autogamous self‐pollination. Specifically, 51.50% of 162 unopened flowers (total 257 flowers) of this Dendrobium species under extreme water‐deficit conditions developed into fruits, suggesting the presence of cleistogamy in D. wangliangii. Here, cleistogamy may represent the primary mode of pollination for this orchid. Spontaneous self‐pollination and specific cleistogamous autogamy could represent major adaptions to the drought and pollinator‐scarce habitat in the Jinshajiang dry‐hot valley.     

Improvement of <Emphasis Type="Italic">ex vitro</Emphasis> transfer of tobacco plantlets by addition of abscisic acid to the last subculture

Tobacco (Nicotiana tabacum L.) plantlets were grown on Murashige and Skoog medium in ventilated Magenta boxes and for the last subculture 10 μM ABA was added to the medium. After three weeks plantlets were transferred into pots with Perlite moistened with water and grown in controlled conditions (16-h photoperiod, day/night temperature 25/20 °C, air humidity about 45 %) either under low or high irradiance of 150 (LI) and 700 (HI) μmol m⁻² s⁻¹, respectively. Content of endogenous ABA was 271.7 pmol g⁻¹(f.m.) in ABA treated plantlets, while in control plantlets it was only 53.3 pmol g⁻¹(f.m.). After ex vitro transfer, stomatal conductance and transpiration rate decreased considerably in comparison with in vitro grown plantlets and remained lower also 7 d after ex vitro transfer, especially in ABA-treated plants and so wilting of plants was practically eliminated. Net photosynthetic rate also decreased 1 d after ex vitro transfer but after 7 d it was mostly higher than that of in vitro grown plantlets. Water use efficiency significantly increased in ABA-treated plants. Chlorophyll a+b content did not change immediately after ex vitro transfer, nevertheless, after 7 d chlorophyll content was higher in ABA-treated plants. Pool of xanthophyll cycle pigments (XCP) and the degree of their deepoxidation (DEPS), which are connected with harmless dissipation of light energy, increased under high irradiance. Contents of XCP and ABA precursors (neoxanthin and violaxanthin) were lower in ABA-treated plants than in control plants indicating less stress in these plants. Most chlorophyll a fluorescence parameters did not change considerably after ex vitro transfer and so the photoinhibition was not observed even under HI. Slight increase in non-photochemical quenching under HI in ABA-treated plants suggested their better photoprotection. Thus application of ABA to the last subculture can improve acclimatization of in vitro grown plants to ex vitro conditions