Plant regeneration from microspore-derived embryos ofBrassica Napus: Effect of embryo age,culture temperature,osmotic pressure,and abscisic acid

Summary Microscope cultures ofBrassica napus cv. Topas undergo high frequency embryogenesis in vitro; however, the majority of microspore-derived embryos do not develop directly into plants but usually undergo abnormal development including the formation of secondary embryos on the hypocotyls. The present studies show that older embryos or embryos cultured at higher temperature (25° C) were more likely to undergo secondary embryogenesis whereas embryos cultured at 20° C or pretreated at 5° to 10° C for 28 days developed more readily into normal plants. Compared with embryos cultured at 25° C, those cultured at 20° C gave a threefold increase in normal plant production. Pretreatments at cooler temperatures (5° to 10° C) resulted in an additional two-to threefold increase in the recovery of normal plants. Higher osmoticum during pretreatment improved embryo survival at low temperatures but generally inhibited normal plant development. Abscisic acid was ineffective or deleterious.     

Genotype,plant, bud size and media factors affecting anther culture of cauliflowers (Brassica oleracea var. botrytis)

Summary Eleven F₁ hybrid cultivars of cauliflower, representing a range of maturity types, were examined for their responsiveness to anther culture. Embryos were produced from each of the cultivars tested, and the mean embryo yield varied from 82.2 embryos per 100 anthers cultured for cv Dova to 0.6 embryos for cv Serrano. Variation between genotypes and between plants within a genotype was significant, both in terms of embryo yield and percentage responsive anthers. Autumn and winter maturing cauliflowers were generally more responsive than summer types. Embryo yields were enhanced by culturing anthers on solid rather than on liquid media. An increase in concentration of 2,4-Dichlorophenoxyacetic acid (2,4-D) from 0.1 to 0.3 mg/l also increased embryo yield. Embryo yield was doubled when anthers were cultured on solid media containing 0.3 mg/l 2,4-D compared to liquid media containing 0.1 mg/l 2,4-D. Although bud size alone did not have a significant effect on embryo production, genotype x bud size and plant x bud size (within genotype) interactions were significant. Estimation of the variance components demonstrated that, apart from the residual plate-to-plate variation, variation between plants was the largest source of variation, accounting for approximately 30% of total variance. Plant x bud size (within genotype) interaction accounted for 18% of total variance and genotypic differences for approximately 8%.     

A study of factors affecting embryo yields from anther culture of cabbage

In cabbage (Brassica oleracea var. capitata), a thermal shock treatment of 24 h at 35 °C at the start of the culture period resulted in higher embryos per 100 anthers (30.0) compared to a treatment of 48 h. Similarly , a chilling treatment of 24 h at 4 °C resulted in a higher embryo yield (6.0) per 100 anthers compared to a treatment of 48 h. However, the embryo yields were significantly higher (p> 0.01) in thermal shock than chilling treatments in all experiments. Treatments of 6 days at either 35 °C or 4 °C gave no embryos. The most responsive cultivar was the F₁ hybrid , Hercules, in all experiments. Although anther culture was successful in the other genotypes, the open pollinated ones, the highest number of embryo yields per 100 anthers was obtained in the hybrid. High temperature treatment before culture had a beneficial effect on the embryo yields. The responsiveness of anthers to addition of increasing concentration of silver nitrate (AgN0₃) (the ethylene inhibitor) to the culture medium, showed a progressive increase in the embryo yields in all the genotypes. Since embryos were also formed in the absence of silver nitrate, probably, due to a greater genotype × medium interaction, it is noted that the presence of silver nitrate in the medium may not be essential for cabbage anther culture as reported earlier. The findings of this study may be recommended for large production of cabbage embryos in culture.     

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     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Captive hybridization of two geographically isolated pygmy angelfish species,Centropyge fisheri and Centropyge resplendens

This study documents the rearing of two pygmy angelfish species, Centropyge fisheri and Centropyge resplendens, and the early life history and reproduction of their hybrid offspring. A C. fisheri female, collected from Hawaii, and a C. resplendens male, captive‐bred from parental stock collected from Ascension Island, were maintained at the hatchery facility for 7 months. Continuous spawning was achieved at a photoperiod cycle of 14L:10D and a water temperature of 26·5° C, range ±1° C. Over the 110 day period, the C. fisheri female spawned 102 times, 57% of which resulted in embryos (fertilized eggs). The mean ±s.d. fecundity per spawn was 730 ± 459 eggs (range 52–1967). Fertility (% eggs that developed into embryos) of all eggs that were preserved was 22·4 ± 25·6%. A total of 235 hybrid juveniles were raised through metamorphosis with an average larval survival of 16·4%. Eight F₁ hybrid juveniles isolated for further study began to display signs of reproductive behaviour c. 300 days post‐hatch (dph). Spawn resulting in non‐fertile eggs were first obtained 319 dph, and fertilized eggs developing into embryos were obtained after 411 dph from at least two female individuals. While no attempt was made at rearing the F₂ larvae, embryo and larval development were normal up to 8 dph. Reproduction and development observed for all hybrid generations in this study were normal, similar to other Centropyge species and indicates a very close phylogenetic relationship between what are currently considered distinct species, e.g. C. fisheri and C. resplendens.     

Production and conversion of haploid embryos in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) anther cultures using high 2,4-D and silver nitrate containing media

Due to recalcitrant nature of chickpea (Cicer arietinum L.) to androgenesis, the production of double haploid plants has been only reported by Grewal et al. (Plant Cell Rep 28:1289–1299, 2009) using some physical stresses such as anther centrifugation and electrical shock. In the present study, we successfully obtained haploid plants from cultured anthers of two chickpea cultivars, Bivanij and Arman, using high 2,4-D and silver nitrate containing media without applying of these time and labor consuming stresses. For induction of androgenesis, different concentrations of 2, 4-D (0, 2, 5 and 10 mg/l) and silver nitrate (0, 5, 10, 15, 25 and 50 mg/l) were used in embryo development medium. In Bivanij cultivar, anther induction medium containing 10 mg/l 2,4-D and 15 mg/l silver nitrate produced the highest number of embryos (0.188) and regenerated plants (0.1) per each cultured anther, while the highest frequencies of embryos (0.1) and regenerated plants (0.075 and 0.063) were obtained from Arman cultivar when 10 mg/l 2,4-D was combined with 15 and 50 mg/l silver nitrate in anther culture medium, respectively. In second part of this study, different cold (4 °C for 4 and 7 days) and heat (30 °C for 10 days, 32 °C for 2 days and 35 °C for 8 h) pretreatments were applied on cultured anthers of Bivanij cultivar. Incubation of cultured anthers at 32 °C for 2 days significantly enhanced the rate of embryo formation up to 0.222 embryos per each anther, while the highest number of regenerated plants/anther (0.0332) was obtained when cold treated anthers at 4 °C for 7 days incubated at 30 °C for 10 days. Taken together, these results provide a good basis for large-scale generation of DH plants in this important legume species.     

Volume contents volume 38 (1985)

Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l nahpthaleneacetic acid (NAA). Embryogenic responses were observed at cultured temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7–54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium.     

Production of trigeneric (barley × wheat) × rye hybrids

Summary Rye (Secale cereale cv. Prolific 2n=14 and 2n =14 + 2B was crossed onto hybrids between barley (Hordeum vulgare 2n = 14) and wheat (Triticum aestivum 2n= 42). Pollinated florets were injected with GA₃ to promote fertilization and hybrid embryo development. At 16 days after pollination the watery caryopses were removed, embryos dissected and cultured on a modified B₅ medium. Approximately 20% of the cultured embryos produced both roots and coleoptile and developed into viable seedlings. Viable seeds were also obtained at a low frequency from the same cross combinations. The hybrids were wheat-like except for the hairy neck characteristic of rye. There were 35 chromosomes in somatic tissue; 21 wheat, 7 barley and 7 rye. The rye chromosomes were distinguishable by their larger size and terminal C-bands. A lower seed set was obtained using pollen from rye plants with 2n=14 + 2B chromosomes than from plants without B chromosomes.Contribution No. 577, Ottawa Research Station     

Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.)

Protocols were developed for the generation of haploid and doubled haploid plants from isolated microspores of carrot (Daucus carota L.). Forty-seven carrot accessions, including six inbred lines, 11 cultivars, 20 F₁s, two BC₁F₁s, four F₂s, one F₃, and three F₄s, were screened to evaluate the genotype influence on isolated microspore embryogenesis over 4 years. Twenty-eight accessions responded by producing embryos and/or calli. A cytological analysis showed that two modes of carrot microspore embryogenesis exist: an indirect route via calli (C mode), and a direct route via embryos (E mode). Eleven accessions were in the C mode, and 17 were in both modes. The highest production rates were in 10Y25 (a European Nantes cultivar) with 27 calli and 307 embryos, and 100Q6 (a semi-Nantes F₁ hybrid) with 176 calli and 114 embryos. The time period to produce embryos or calli differed significantly between 2 and 6 months. Cold and heat pretreatment generally had a negative impact on the induction of microspore embryogenesis, but a short pretreatment showed a positive influence on some accessions. Twenty-eight lines regenerated plants from the primary individual embryos or calli of three accessions were established to analyze the ploidy level. The percentage of spontaneous diploidization showed very wide differences among the accessions and lines. Differences in leaf color intensity, leaf size, and leaf dissection were found among haploid, doubled haploid, and triploid plants.     

Stimulation of androgenesis in white cabbage (Brassica oleracea var. capitata) anthers by low temperature and anther dissection

Anther culture was performed on two local cultivars, Ljubljansko and Varadinsko, and the F₁ cv. Krautman (Bejo-Zaden). The effects on androgenesis of hot and cold temperature treatments and different dissections of anthers were evaluated. In contrast to cv. Krautman, cvs. Ljubljansko and Varadinsko produced more embryos after cold pretreatment of flower buds (4°C, 48 h) than after standard treatment (35°C, 24h). Simultaneous cutting of the anther tip and removal of the filament gave the best results in comparison to other tested dissections. Microscopical observations of sectioned anthers revealed enhanced embryo development near the cut ends of the anthers. Ploidy analysis revealed the presence of haploids among embryos resulting from cold treatment (4°C, 48 h), treatment at elevated temperature (35°C, 24 h), and among embryos resulting from dissections of anther tips.     

Effects of root zone temperature and paraquat in the induction of oxidative stress in <Emphasis Type="Italic">Trichosanthes cucumerina</Emphasis> L.

We investigated the influence of root zone temperature (RZT) and the aerial application of paraquat on stress defence mechanisms of Trichosanthes cucumerina L. To achieve this objective, T. cucumerina cv Green was grown with roots at 25 and 30°C root zone temperature and maintained at 20 ± 1°C air temperature in a growth chamber. These RZT and air temperature had earlier been shown to favor growth and fruit production in T. cucumerina. Plants at each RZT were subjected to paraquat treatment (+P) and without paraquat treatment (−P). Paraquat (0.2 mmol/L) was applied as aerial spray. Results showed that the individual main effects of RZT and paraquat treatments significantly affected the chlorophyll fluorescence and gas exchange parameters, while the interaction of both treatments had no significant effect. Results showed that the total phenolics and ascorbic acid contents of T. cucumerina at 30°C were significantly higher than at 25°C. The T. cucumerina plants in +P treatment recorded significantly lower maximum photochemical efficiency (F _v/F _m), net photosynthesis (A), transpiration rate (E), intercellular CO₂ concentration (C _i) and stomatal conductance (g ₁) compared to untreated plants. Also, plants raised at 30°C recorded significantly higher F _v/F _m, A, E, C _i and g ₁ compared to plants raised at 25°C. Plants that were sampled at 48 h after paraquat treatment recorded a higher degree of oxidative damage compared to those sampled at 24 h after treatment. We showed that the degree of damage suffered by T. cucumerina, when treated with paraquat either at 25 or 30°C RZT was similar at 48 h after treatment. We concluded that either at 25 or 30°C, exposure of T. cucumerina to paraquat would impose the same degree of oxidative damage.     

Cold-induced changes of enzymes,proline, carbohydrates,and chlorophyll in wheat

Quantitative changes in total leaf soluble proteins, proline, carbohydrate content, chlorophyll fluorescence, guaiacol peroxidase (POD) and catalase (CAT) activities were determined in a less cold-hardy (LCH) spring cv. Kohdasht (LT₅₀ = −6°C), a semi cold-hardy (SCH) facultative cv. Azar 2 (LT₅₀ = −15°C), and a cold-hardy (CH) winter cv. Norstar (LT₅₀ = −26°C) of wheat (Triticum aestivum L.) exposed to 4°C for 9 weeks. Seedlings were grown in a controlled growth room for 14 days at 20°C and then transferred to 4°C (experimental day 0) for 63 days (cold treatment); otherwise they were maintained continuously at 20°C (control treatment). The samples were harvested 0, 2, 21, 28, 42, and 63 days after exposure to 4°C. The results showed significant low temperature (LT)-induced accumulation of total soluble proteins, proline, and carbohydrates and elevation in activities of CAT and POD in leaves of SCH and CH winter cultivars rather than in LCH spring cultivar. In contrast, the chlorophyll fluorescence (F _v/F _m) declined during LT treatment irrespective of cultivar. The results suggest that developmental traits such as vernalization requirement of wheat affects on cold-tolerance expression system of plants.     

RECIPROCAL DIFFERENCES IN INTRASPECIFIC CROSSES OF TOBACCO RESULT FROM EMBRYO DEATH

Tetraploid tobacco plants (Nicotiana tabacum) derived from cultured cell lines (TC) are partially cross-incompatible with their diploid progenitors (C). C × TC crosses (TC-derived tetraploids as the pollen parent) yield only 2% viable seed. The remaining seeds are normal size but lack an embryo (apoembryonic seeds). Apoembryonic seeds do not occur in the reciprocal TC × C crosses. Sections of ovules from C × TC crosses revealed that an embryo formed but that embryo growth slowed at 5 days postpollination and that by the 12th day following pollination, the embryo had disappeared although the ovule continued to develop. Endosperm degeneration occurred concurrently with embryo death. Culturing ovules from C × TC crosses has increased the yield of F₁ plants from 2% to as much as 25% indicating that embryo rescue is possible. Surviving F₁ plants from TC × C crosses have close to triploid chromosome numbers and are fertile.     

Some effects of temperature during seed development on carrot (Daucus carota) seed growth and quality

The growth and development of carrot seeds cv. Chantenay Red-cored Royal Chantenay at day/night temperatures of 20/10°C, 25/15°C and 30/20°C and subsequent seed performance were examined in 1984 and 1985. An increase in temperature from 20/10°C to 30/20°C reduced mean weight per seed by 20% in 1984 and by 13% in 1985. There were no effects of temperature on endosperm + embryo weight, or on endosperm cell number but the weight of pericarp decreased with an increase in temperature. Seeds grown at the highest temperature had the largest embryos and the highest nitrogen, DNA and rRNA content; they germinated and emerged earlier, and gave higher percentage seedling emergence than those grown at the lowest temperature. There were no effects of temperature during seed growth on the rate of imbibition of water by seeds during the germination process.     

Chilling injury and recovery in detached and attached leaves measured by chlorophyll fluorescence

Smillie, R. M., Nott, R., Hetherington, S. E. and Öyustt, G. 1987. Chilling injury and recovery in detached and attached leaves measured by chlorophyll fluorescence Chilling injury was compared in detached and attached leaves chilled at 0 or 0.5°C by measuring the decrease in induced chlorophyll fluorescence in vivo. The fluorescence parameter measured was F_R, the maximal rate of rise of induced chlorophyll fluorescence emission after irradiating dark-adapted leaves. The plants used were bean, Phaseolus vulgaris L. cv. Pioneer, and maize, Zea mays L. cvs hybrid GH 390 and Northern Belle. Leaves were detached and placed on wet paper and covered with thin polyethylene film to prevent water loss during chilling. Leaves left attached on plants were treated similarly. When chilled in this way at 100% relative humidity, the chilling-induced decrease in F_R was the same in detached and attached leaves. For the attached leaves, the same result was obtained whether just a single leaf was chilled or the whole plant. Expression of chilling injury was greatest in fully turgid leaves and comparisons can be invalid unless the water status of the detached and attached leaves are the same. Problems arising from diurnal fluctuations in water potential of plants grown in a glasshouse were circumvented by placing leaves on the wet filter paper under polyethylene film prior to chilling, which allowed high water potentials to be regained, or mist sprays in the glasshouse were employed. Determinations of the time course for changes in F_R of maize (cv. Northern Belle) during chilling at 0°C showed that F_R decreased exponentially, at the same rate (time to 50% decrease in F_R was 9.3 h) in detached and attached leaves. Chilling injury was largely reversible for the first 20 h of chilling stress as both detached and attached leaves recovered their pre-chilling values of F_R after a further 20 h at 20°C in darkness. Leaves chilled for 48 h showed partial recovery, while those chilled for 72 h did not recover. Recovery was impeded by light. Inability to recover from chilling as indicated by measurements of F_R was paralleled by the incidence of visible symptoms of injury. It is concluded that detached and attached leaves behave similarly during chilling and short-term recovery, provided a similarity in treatments is rigorously maintained.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Tentative determination of the molecular weight of substances stimulating the flowering of winter wheat

Murashige & Skoog nutrient was supplemented with substances of molecular weight (M_W) less than 5 kDa, which were separated from extract of winter wheat ears by means of Sephadex G-25 ultrafiltration. Isolated embryos of the same wheat cultivar (Grana) were vernalized in the nutrient for 0 and 7 days at 2 °C for 2 weeks and planted in a glass-house. After 150 days of growth (20/17 °C day/night) the development of the shoot apices was observed. It was found that substances of M_W<5 kDa strongly stimulated the generative development of the plants, enabling the earing of 30 % of non-vernalized plants (control=0%) and 100 % plants vernalized for 7 days (control=29 %). The substances present in the extract of both spring (cv. Jara) and winter (cv. Grana) varieties were fractionated by means of Sephadex chromatography into 300 fractions of M_W=1 to 5 kDa and each of them was added to the isolated embryos of cv. Grana. The embryos were vernalized at 2 °C for 7 days and then cultured as previously described. It was found that the differentiation of the shoot apices was stimulated by over 34 % by fractions of winter wheat extract and more than 50 % by fractions of the spring wheat extract. However, only a few of identical fractions of the extracts of both wheat varieties were able to induce the earing of the plants. These fractions were grouped in 4 continuous intervals of M_W equal to about 4.5–4.9, 3.2–3.3, 2.1–2.6 and 1.00–1.03 kDa. Within the three intervals was identified a small group of identical fractions, which affected the growth of the seedlings in similar mode i.e. inhibiting or stimulating. Thus it can be assumed that these intervals contained identical or similar substances capable of stimulating strongly the earing of winter wheat.