Wheat anther culture: effect of genotype and environmental conditions

Twenty-two cultivars and lines of winter and spring wheat (Triticum aestivum L.) were studied, most for the first time, for their anther culture response. The response was genotype dependent. Plants grown in the field gave higher callus induction frequency than those grown in the greenhouse and the controlled environment chamber. Donor plants grown in a season of low drought stress as compared to a season of severe drought stress resulted in a higher frequency of callus induction. Spherical microcalli were observed in two wheat genotypes in some of only those anthers that were placed with only one loculus in contact with the medium. Wheat lines that were more responsive to anther culture were identified.     

Effect of plant growth conditions, plating density, and genotype on the anther culture response of soft white spring wheat hybrids

Ten soft white spring wheat (Triticum aestivum L.) F₁ hybrids were grown under three temperature regimes, and anthers were cultured at two plating densities to investigate the effect of plant growth conditions, plating density, and genotype on embryo induction and plant regeneration. Anthers from plants grown at high temperature (25 °/18 °C) or from plants transferred from low (15 °/12 °C) to high temperature generally produced more embryos and green shoots, with a lower frequency of albinos, than did anthers from plants grown at low temperature. However, plating densities of 10 versus 20 anthers per milliliter, had little effect on anther response. Four of the five hybrids with `Fielder' as the female parent produced more embryos and green shoots than did hybrids with `AC Reed' as the female parent. Received: 12 July 1996 / Revision received: 1 April 1997 / Accepted: 30 April 1997     

Induced androgenesis in tomato (Lycopersicon esculentum Mill). II. Factors affecting induction of androgenesis

The influence on androgenesis of donor plant growth conditions, anther size and developmental stage of the microspore, medium composition and different anther treatments prior to culture was investigated in L. esculentum Mill. cv Roma and its hybrids. Growth conditions of donor plants affected the induction of tomato androgenesis. Anthers isolated from plants grown in the greenhouse during winter at high humidity and in short days possessed higher androgenetic ability than those grown in the field. The physiological state and age of the donor plants also influenced the processes investigated. Regarding the developmental stage of microspores, the period from prophase to telophase II is optimal for tomato anther implantation. More then 20 culture media were tested. Two, based on Murashige and Skoog medium were selected as most favourable for callus induction, organogenesis and regeneration. The effect on callus induction of 2ip in combination with indole-3-acetic acid (IAA) was greater than that of zeatin and IAA. Zeatin promoted entire plant regeneration. A highly significant interaction between genotype and medium was observed. Temperature and gamma ray treatments of anthers enhanced callus production, shoot formation and plant regeneration. Treatments at 4 °C (48 h) and 10 °C (9 days) stimulated these processes. Combined treatment of anthers with 4 Gy and 10 °C for 9 days was the most efficient. Received: 5 September 1997 / Revision recieved: 5 June 1998 / Accepted: 15 June 1998     

The effect of 2,4-dichlorophenoxyacetic acid and donor plant environment on plant regeneration from immature inflorescence-derived callus of Lolium perenne L. and Lolium multiflorum L.

Callus cultures were obtained from immature inflorescences of perennial ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum). Inflorescence segments were cultured on Murashige and Skoog medium (MS) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The response in culture with regard to compact callus induction, embryogenesis and plant regeneration was determined for different varieties. The in vitro response was compared for explants from field-grown plants and explants from greenhouse-grown plants. The effect of different 2,4-D concentrations on the in vitro response was also investigated in one L. perenne variety and one L. multiflorum variety. The percentage of explants that formed compact callus and embryogenic callus differed strongly with the cultivar. There was no consistent effect of the growth conditions of the donor plants or the 2,4-D concentration of the medium on this response. Green plants were regenerated from all the cultivars tested. Explants from field-grown plants showed a higher tendency to form albino shoots than explants from greenhouse-grown plants. In the L. perenne variety tested higher 2,4-D concentrations (up to 15 mg/l) resulted in a lower regeneration frequency of green shoots and a higher regeneration frequency of albino shoots (up to 12.5 mg/l). In the L. multiflorum variety tested the effect of 2,4-D on regeneration was less pronounced.     

Interactive effects of temperature and light intensity on photosynthesis and antioxidant enzyme activity in Zizania latifolia Turcz. plants

We compared the interactive effects of temperature and light intensity on growth, photosynthetic performance, and antioxidant enzyme activity in Zizania latifolia Turcz. plants in this study. Plants were grown under field (average air temperature 9.6–25°C and average light intensity 177–375 W m^?2) or greenhouse (20–32°C and 106–225 W m^?2) conditions from the spring to the early summer. The results indicated that greenhouse-grown plants (GGP) had significantly higher plant height, leaf length, and leaf width, but lower leaf thickness and total shoot mass per cluster compared with field-grown plants (FGP). Tiller emergence was almost completely suppressed in GGP. Significantly higher chlorophyll (Chl) content and lower Chl a/b ratio were observed in GGP than in FGP. From 4 to 8 weeks after treatment (WAT), net photosynthetic rate (P _N) was significantly lower in FGP than in GGP. However, from 9 to 12 WAT, P _N was lower in GGP, accompanied by a decrease in stomatal conductance (g _s) and electron transport rate (ETR) compared with FGP. Suppressed P _N in GGP under high temperature combined with low light was also indicated by photosynthetic photon flux density (PPFD) response curve and its diurnal fluctuation 10 WAT. Meanwhile, ETR in GGP was also lower than in FGP according to the ETR — photosynthetically active radiation (PAR) curve. The results also revealed that GGP had a lower light saturation point (LSP) and a higher light compensation point (LCP). From 4 to 8 WAT, effective quantum yield of PSII photochemistry (Φ_PSII), photochemical quenching (q_P), and ETR were slightly lower in FGP than in GGP. The activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), superoxide dismutase (SOD), and malondialdehyde (MDA) content were significantly higher from 4 to 8 WAT, but lower from 10 to 12 WAT in FGP. However, catalase (CAT) activity was significantly lower in FGP from 4 to 8 WAT. Our results indicated that the growth and photosynthetic performance of Z. latifolia plants were substantially influenced by temperature, as well as light intensity. This is helpful to understand the physiological basis for a protected cultivation of this crop.     

Cell Wall Composition and Bioenergy Potential of Rice Straw Tissues Are Influenced by Environment,Tissue Type,and Genotype

Breeding has transformed wild plant species into modern crops, increasing the allocation of their photosynthetic assimilate into grain, fiber, and other products for human use. Despite progress in increasing the harvest index, much of the biomass of crop plants is not utilized. Potential uses for the large amounts of agricultural residues that accumulate are animal fodder or bioenergy, though these may not be economically viable without additional efforts such as targeted breeding or improved processing. We characterized leaf and stem tissue from a diverse set of rice genotypes (varieties) grown in two environments (greenhouse and field) and report bioenergy-related traits across these variables. Among the 16 traits measured, cellulose, hemicelluloses, lignin, ash, total glucose, and glucose yield changed across environments, irrespective of the genotypes. Stem and leaf tissue composition differed for most traits, consistent with their unique functional contributions and suggesting that they are under separate genetic control. Plant variety had the least influence on the measured traits. High glucose yield was associated with high total glucose and hemicelluloses, but low lignin and ash content. Bioenergy yield of greenhouse-grown biomass was higher than field-grown biomass, suggesting that greenhouse studies overestimate bioenergy potential. Nevertheless, glucose yield in the greenhouse predicts glucose yield in the field (ρ?=?0.85, p?<?0.01) and could be used to optimize greenhouse (GH) and field breeding trials. Overall, efforts to improve cell wall composition for bioenergy require consideration of production environment, tissue type, and variety.     

In vitro culture and fructan production by Vernonia herbacea (Asteraceae)

Vernonia herbacea is a native species from the Brazilian Cerrado that accumulates about 80 % of inulin-type fructans in the underground reserve organs, the rhizophores. This work aimed at establishing a protocol for in vitro culture of V. herbacea, using seeds (achenes) and leaf discs as explants. Following germination and seedling growth, stem nodes from 6-month-old in vitro germinated plants were isolated and incubated on culture medium free of growth regulators for plant propagation and rhizophore formation. Fructan content and composition were evaluated in leaves, stems, roots and rhizophores from plants grown in vitro and compared with those of greenhouse-grown plants, in order to evaluate inulin production in vitro. Fructan contents of aerial organs and roots from in vitro plants were higher, compared with greenhouse plants, while in rhizophores, the opposite was observed. High performance anion exchange chromatography/pulsed amperometric detection profiles revealed the presence of the inulin homologous series in the aerial organs exclusively for in vitro plants, while in roots and rhizophores, this series was detected in plants grown in both conditions. These results indicate a modification in the source/sink ratio, leading to changes in the distribution of carbohydrates in in vitro plants. The leaf disc cultures on medium supplemented with indole-3-butyric acid induced the formation of roots (0.24, 0.49 µM) and friable callus (2.46 µM), while 6-benzylaminopurine (from 1.1 through 4.43 µM) induced compact callus. However, no shoot formation was observed. The use of seeds allowed the establishment of a protocol for in vitro culture and provides a model system for a better understanding of fructan metabolism in V. herbacea.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Callus induction in culture of Oenothera hookeri and Oenothera picensis anthers

In the present work we try to determine optimum conditions for callus induction in anther culture of Oenothera hookeri and O. picensis. The anther callus yield was increased when the anthers were cultured on modified MS medium supplied with 2 mg dm^-3 2,4-D and 2 mg dm^-3 NAA, in both species. In O. hookeri, best results were obtained when anthers were excised from 7.2 - 9 mm buds at the stage of vacuolated microspores, then pretreated at 4 °C for 2 d and grown under 16-h photoperiod. The response to anther culture of O. picensis was generally very poor compared with that of O. hookeri. The higher yield of calli was obtained when anthers were excised from 6.2 - 8 mm buds at the stage of vacuolated microspores and grown under continuous light. The cold pretreatment of buds decreased anther response in this species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of head removal on leaf senescence of sunflower

Greenhouse and field studies examined the effect of flower or seedhead removal on leaf senescence and associated changes in sunflower (Helianthus annuus L.) plants. At intervals during seed development, selected leaves (leaves 6 through 8 from the top in the greenhouse and leaf 7 from the top in the field) were harvested and analyzed for chlorophyll, specific leaf weight, N, P, soluble protein, and electrophoretic gel profiles of soluble polypeptides. In both the greenhouse and the field, the leaves of headless plants retained or accumulated more N, P, soluble protein, and dry weight than leaves of plants with heads. Obviously, head removal affected the partitioning of these metabolites during seed development. None of the treatments resulted in the formation of new polypeptides (electrophoretic gel profiles). Comparisons of the rates and extent of loss of chlorophyll, soluble protein, and polypeptide bands (especially ribulose 1,5-bisphosphate carboxylase) from the leaves of headed and deheaded plants showed that head removal delayed the rate of development of leaf senescence for the greenhouse-grown but had much less effect on field-grown plants. These findings illustrate the variability in different parameters commonly associated with the leaf senescence processes of headed and deheaded sunflower plants grown under different environments.     

Hydroponic culture of Catharanthus roseus (L.) G. Don and studies on seed production

Under climatic conditions of Poland, Catharanthus roseus may be grown in the open field only as annual crop, using transplants raised in a greenhouse or in a plastic tunnel. An effective method of hydroponic culture has been elaborated and tested. Herbal material obtained in hydroponics showed considerable concentration of alkaloids in different plant parts. Seeds of C. roseus may also be produced under these conditions. Characteristics of seeds as well as the effect of different methods of their storage are presented. Seeds stored in paper bags at room temperature may retain germinability for 7 years and those stored in a refrigerator, at 5°C, up to 15 years. Storage in a freezer, at -10°C, resulted in a quick and sharp reduction of germination rate.     

Enzyme fingerprint analyses in tissue regenerated from anther culture of maize

The objectives of our studies were to investigate the effect of cold pre-treatment duration and the effect of two different culture media (YP and N6) on maize anther culture response in two maize genotypes (A 18 and A 19) and to identify the gametic origin of the maize regenerants. Androgenic induction and callus formation was compared in anther cultures following pre-treatment applied to both media tested and with both maize genotypes. Higher plant regeneration was observed in case of YP media independently of the genotype used. The best results were achieved when 12 days (genotype A 18) or 14 days (in case of genotype A 19) cold pre-treatment at 10°C was applied. We have tested the possibility of using enzyme isoform analyses to identify the microspore origin of calli and plants derived from anther cultures. The 11 enzymes tested in our experiments were acid phosphatase, alcohol dehydrogenase, catalase, diaphorase, β-glucosidase, glutamate oxaloacetate transaminase, isocitrate dehydrogenase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase. Analysis of malate dehydrogenase proved the gametic origin of the calli initiated and of the DH plants regenerated from anther culture, when the coleoptile of the donor plant material showed two forms of enzyme 3/6 and the analysed calli showed only one of the two forms (3 or 6).     

A study of factors affecting anther culture of cauliflower (Brassica oleracea var. botrytis)

Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l^-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis.     

Stem elongation of ornamental bromeliad in tissue culture depends on the temperature even in the presence of gibberellic acid

Acanthostachys strobilacea Link, Klotzsch, & Otto is an ornamental bromeliad native to Brazilian Atlantic Forest that naturally exhibits a rosette growth pattern. According to the temperature conditions of the in vitro culture, this species can exhibit stem elongation, facilitating the isolation of the nodal segments to be applied in its micropropagation. The rosette morphology is reestablished when this species is maintained under low temperature, thus allowing the maintenance of a germplasm collection (slow growth storage). Gibberellins (GA) are usually applied to stimulate stem elongation in micropropagated plants. Thus, our aim here was to verify the influence of temperature over the stem elongation of A. strobilacea when GA₃ is applied to the medium, thus estimating the use of this phytoregulator in slow growth cultures at low temperatures. Physiological and anatomical studies were performed on plants obtained from nodal segments maintained at 10, 15, 20, and 25 °C. Regardless of the applied treatment, no segments developed at 10 °C. Stem elongation occurred at 25 and 30 °C, and was not seen for plants grown under 15 and 20 °C. The application of 50 µM of GA₃ restored stem elongation in plants at 20 but not at 15 °C. The influence of gibberellins on stem elongation of this tropical bromeliad depends on the cultivation temperature, in which low temperature preponderates over the stem elongation effects of GA₃. In addition, the optimum temperature for the slow growth of this species depends on the starting temperature of the explant used in the micropropagation.     

Responses of guayule (Parthenium argentatum) seedlings to plant growth promoting fluorescent pseudomonads

Summary The potential of a number of fluorescent pseudomonad strains to promote growth of guayule plants in the greenhouse and in the field was studied. A number of bacterial strains collected from guayule roots and rhizospheres promoted growth of greenhouse-grown plants but not field-grown plants. Percent increase in shoot dry weight of 12-week-old, greenhouseinoculated guayule plants ranged from 17 to 75 nine weeks after inoculation compared to non-inoculated plants. The increased growth of plants in the greenhouse could reduce production cost by shortening the time required to maintain plants in the nursery prior to transplanting to the field.Journal Series Article no 3816 of the Arizona Agricultural Experiment Station.     

Establishing clones of Veratrum californicum,a native medicinal species,for micropropagation

Factors affecting the micropropagation of Veratrum californicum, a slow-growing species that is a potentially valuable source of cyclopamine, were investigated. Sterile cultures were initiated on modified Murashige and Skoog medium, and clones from individual donor plants were assigned to experimental conditions when approximately 100 shoots of each clone were available. The effects of temperature, light quality, and plant growth regulators on multiplication and survival were assessed. Four clones from which large greenhouse populations were obtained were selected for in-depth analysis. When shoots were cultured at 10°C and 16°C, multiplication ratios consistently >1 were observed from three of four clones and two of four clones, respectively, during the five-subculture cycles. None of the clones stably increased when cultured at 24°C, and plants from this treatment did not survive acclimatization in the greenhouse. Only one clone showed increased multiplication ratios in response to plant growth regulator treatments, with maximum multiplication when shoots were cultured with 9 μM benzyladenine and 0.5 μM naphthaleneacetic acid. Light quality in the laboratory did not affect multiplication ratio but did affect subsequent greenhouse survival. The size of plants derived from culture was most often equivalent (65% of 1,271) to 3-yr-old seed-derived plants. Although the growth of clones during acclimatization differed, plants derived from cultures incubated at 16°C had the best rates of overall greenhouse survival. Temperature and light treatments in vitro critical to long-term plant survival were demonstrated and will assist the establishment of a mass propagation system for V. californicum.     

Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.     

Production of microspore-derived plants by anther culture of an interspecific F1 hybrid between Cyclamen persicum and C. purpurascens

Anthers of a F1 hybrid (2n=41) between Cyclamen persicum (2n=2x=48) and C. purpurascens (2n=2x=34) were cultured to produce microspore-derived plants. Embryoids were produced when anthers, containing microspores at the early uninucleate stage of pollen development, were cultured in B5 medium containing sucrose (90 g l-1) and NAA (0.1, 1 mg l-1) or 2,4-D (0.1 mg l-1) in the dark at 5 °C for 4 days, then at 25 °C for 60 days. The embryoids usually developed into plantlets when cultured in B5 medium containing sucrose (30 g l-1) in the dark at 25 °C. At meiosis, the F1 hybrid, used as source for anther culture, formed some cells with restitution nuclei at telophase and dyads at the tetrad stage, which resulted in the production of viable pollen grains as unreduced gametes. Plants produced by anther culture were grouped into sterile plants with 2n=41 chromosomes and fertile plants with 2n=82 chromosomes. The present findings suggested that the sterile plants were polyhaploids originating from unreduced microspores (n=41) of the F1 hybrid and that the fertile plants were amphidiploids induced by a spontaneous doubling during culture of chromosomes of such unreduced microspores. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low temperature enhanced the podophyllotoxin accumulation vis-a-vis its biosynthetic pathway gene(s) expression in Dysosma versipellis (Hance) M. Cheng – A pharmaceutically important medicinal plant

Dysosma versipellis (Hance) M. Chang is an endemic and endangered Chinese medicinal herb. Podophyllotoxin, a lead compound for the semi-synthesis of clinically useful anticancer compounds, is the main active aryltetralin lignan accumulated in D. versipellis. In this study, morphology and physiology traits in response to podophyllotoxin accumulation and its biosynthetic pathway genes expression were investigated in D. versipellis plants grown at low temperature (4∼6 °C) and greenhouse (25∼30 °C) temperature. Our study found that low temperature significantly increased plant biomass (2.7-fold), chlorophyll, and carotenoid content compared to greenhouse temperature. The mRNA expression analysis of podophyllotoxin pathway genes secoisolariciresinol dehydrogenase and pinoresionol lariciresinol reductase expression levels were also up-regulated by low temperature. In addition, plants grown at low temperature markedly increased podophyllotoxin accumulation to 3.49-fold in rhizome of D. versipellis than the greenhouse temperature. These results suggest that plants at low temperature enhance the accumulation of podophyllotoxin in rhizome of D. versipellis by over-expressing podophyllotoxin pathway genes. This study finding could be useful in the producing pharmaceutically important podophyllotoxin without overexploiting this endangered species.     

Phytophthora cryptogea root rot of tomato in rock woo] nutrient culture. II. Effect of root zone temperature on infection, sporulation and symptom development

The effect of root-zone temperature on Phytophthora cryptogea root rot was studied in tomato cv. Counter grown under winter and summer conditions in rockwool culture. A nutrient temperature of 25°C resulted in increased root initiation and growth, higher in winter-grown than in summer-grown plants. Rhizosphere zoospore populations were greatly reduced at 25°C and above. Growth of P. cryptogea in vitro was optimal between 20°C and 25°C and completely suppressed at 30°C. Encystment was enhanced by increased temperatures above 20°C. Zoospore release in vitro occurred in cultures maintained at constant temperatures in the absence of the normal chilling stimulus. Optimal release was at 10°C; no zoospores were released at 30°C. Inoculated, winter-grown tomato plants maintained at 15°C developed acute aerial symptoms and died after 21 days. Comparable plants grown at a root-zone temperature of 25°C remained symptomless for the 3-months duration of the experiment. Summer-grown infected plants at the higher root temperature wilted but did not die. Enhanced temperature was ineffective as a curative treatment in summer-grown plants with established infection. Aerial symptoms of Phytophthora infection are seen as a function of the net amount of available healthy root. With high root zone temperatures this is determined by new root production and decreased inoculum and infection.