Variations in response to high temperature treatments in anther culture of Brussels sprouts

The level, time of application and duration of the high temperature treatment necessary for embryo production from Brussels sprouts anther culture were examined. The effects of 29, 32, 35, and 38°C given for 24 h immediately following removal of the anthers from the bud, were tested on different cultivars, on different plants within the cultivars and on different occasions for each plant. Most embryos were produced following 32 and 35°C, very few following 30°C and none following 38°C. Although there was a tendency for some cultivars to respond better to one or other of the two more favourable temperatures, this varied considerably between individual plants. Plant to plant variation was also seen in the overall level of the response, although responsiveness tended to decline with successive samplings of the same plant. Experiments with cultivars Hal and Gower suggested that high temperature was required for at least 12 h after anther removal, but beyond that time the optimum period varied from plant to plant. If the excised anthers were held at 25°C for 16 h or more with Hal or 24 h or more with Gower before being exposed to the high temperature treatment, embrogenesis tended to be reduced. It is suggested that apparent non-responsiveness in anther culture may result to a large extent from the specific conditions that are used during the anther culture process.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Haploid plants from in vitro anther culture of the leguminous tree,Peltophorum pterocarpum (DC) K. Hayne (Copper pod)

The development of haploid callus, embryos and plantlets from cultured anthers and the various factors affecting androgenesis in Peltophorum pterocarpum (Copper pod), a tropical legume tree is reported. A pretreatment of flower buds at moderate temperature of 14°C for 8 days was most effective for callus production. The colour of the anther was found to be a reliable and efficient indicator for identification of suitable stage of anther for culture. The frequency of anthers which produced callus and shoots was highest when anthers were cultured at mid or late-uninucleate stage. A high sucrose concentration of 10% is a specific media requirement for androgenesis. The haploid nature of the embryos, callus and regenerated plants (n=14) were confirmed by chromosome count.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid - BAP bezylaminopurine     

Anther culture with different genotypes of opium poppy (Papaver somniferum L.): effect of cold treatment

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.     

Pollen callus culture in Triticum aestivum

Summary Pollen shed between 4–8 d from anthers of Triticum aestivum cultured in liquid medium gave rise to calluses. Tillers were harvested at the mid-to late-unicellular pollen stages and chilled for 8 d at 4–5 °C before the anthers were dissected out. Pollen cultures gave about 6 times as many calluses on a per anther basis as anthers cultured on solid medium. With the most productive of 5 cultivars tested, pollen culture results in roughly one callus for each anther used, though the calluses formed by pollen culture were less productive for the regeneration of shoots than calluses derived from anthers cultured on solid medium. The ratio of green to albino shoots is roughly 1 1 for anther cultures but considerably less for pollen cultures.     

High regeneration rates in anther culture of interspecific sunflower hybrids

Optimization of anther culture with regard to the induction of callus formation and direct embryogenesis was obtained for interspecific hybrids ofH. annuus withH. tuberosus, H. laetiflorus, andH. resinosus by investigating six different induction media and four regeneration media. One media combination (MS-13, MS-R3 and MS-R4) used under different culture conditions (30°C / 35°C and different dark treatments) gave up to 92.7% embryogenic anthers with an average of 8.5 embryos per anther. However, direct embryogenesis as well as callus formation showed a strong genotypec and treatment specific reaction. From 5,600 anthers of the four investigated genotypes more than 2,000 plants could be regenerated. Regenerants were characterized by morphological traits and isozyme analyses to prove their androgenetic origin.     

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.     

Stimulation of embryogenesis and haploid production in Brassica campestris anther cultures by elevated temperature treatments

Summary Culture of Brassica campestris anthers at 35°C for one or three days prior to culture at 25°C significantly stimulated the yield of microspore-derived embryos. More than 100 plants were regenerated from cultured embryos and haploids were identified amongst them. The haploid frequency was greater than 70% if all small-flowered sterile plants were considered to be haploid. The yield of microspore-derived plants in B. campestris is approaching the level where anther culture may be utilized as a practical breeding tool.     

High temperature enhances ethylene promotion of anther filament growth in Brussels sprouts (Brassica oleracea var. gemmifera)

A study was made of the effect of high temperature on the growth response of Brussels sprout filaments to ethylene. Filaments with or without the anthers attached were incubated continuously at 25 °C or 35 °C for 7 days or for 2 days at 35 °C followed by 5 days at 25 °C. Growth was reduced during both 35 °C treatments compared to that of filaments at continuous 25 °C. Ethylene had little effect on filament growth at continuous 25 °C, whereas with treatment for either 2 or 7 days at 35 °C ethylene promoted filament growth considerably. Thus ethylene effectively overcame the growth inhibition induced by the 35 °C treatment.High temperature treatments reduced ethylene production from filaments alone, and from filaments with anthers attached. The ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) and the ethylene action inhibitor AgNO₃ enhanced filament growth at 25 °C but had little or no effect at 35 °C. The relevance of temperature to ethylene sensitivity is discussed in relation to filament growth and to other plant processes in general.     

Plant regeneration from rice anthers cryopreserved by an encapsulation/dehydration technique

Summary Anther-derived rice (Oryza sativa L. ssp. japonica variety Yerua P.A.) plants were obtained after cryopreservation by an encapsulation/dehydration technique. Immature anthers, excised from spikelets pretreated at 8°C for 8d, were encapsulated in calcium alginate beads. The beads were cultured on N6 medium with 11.5 μM naphthalenaecetic acid (NAA) and 2.3 μM 6-furfurylaminopurine (KIN). Fifteen percent of the encapsulated anthers formed calluses when pretreated with sucrose for 3 d in liquid medium, desiccated on silica gel, slowly cooled to −30°C, immersed in liquid nitrogen (LN), thawed, and recultured. The cryopreserved encapsulated anthers produced 1.67 shoots/callus, in contrast to the control (non-cooled encapsulated anthers), which produced 6 shoots/callus. Eighty percent of the plantlets developed into normal plants after being transferred to greenhouse conditions. Histological observations showed that the origin of the plants was not modified by the cryopreservation process.     

Effect of temperature on carbohydrate content,ADP glucose pyrophosphorylase,and ATP- and PPi-dependent phosphofructokinase activity of potato tuber callus tissue

Callus derived from Lemhi Russet and Russet Burbank tuber tissue was incubated at 20°C and 30°C on a high sucrose medium for starch-formation and subjected to simulated storage and reconditioning treatments at 5°C and 25°C after transfer of the callus to a medium without a carbon source. Percent dry weight of callus from both cultivars averaged about 21% after starch formation and 5% after storage and reconditioning treatments. Total sugars were higher in callus incubated on the starch forming treatment. Lemhi Russet callus contained predominantly reducing sugars, while Russet Burbank callus contained mostly non-reducing sugars. Total sugar content was generally lower for both cultivars after the storage and reconditioning treatment in callus after starch formation at 20°C. Starch content was generally higher in Lemhi Russet tissue. After starch formation at 20°C Lemhi Russet had higher starch after the storage and reconditioning treatment than tissue from 30°C, while the opposite trend was found in Russet Burbank tissue. Total protein declined in Russet Burbank tissue during the storage and reconditioning treatment regardless of prior incubation conditions, while this decline only occurred in Lemhi Russet tissue initially incubated at 30°C during the starch formation treatment. In tissue of both cultivars, ATP- and PPi-dependent phosphofructokinase activities were inversely proportional to total sugar concentrations, while in RB callus ADP glucose pyrophosphorylase activities were proportional to starch content.Research Paper 91B1 of the Idaho Agricultural Experiment Station.     

Influence of genotype and culture medium on microspore callus induction and green plant regeneration in anthers of Oryza sativa

Attempts to identify nutritional requirements of anthers of eight indica rice cultvars ( Oryza sativa L. spp. Indica ) for callus induction in vitro and green plant regeneration were made. The requirements varied with the genotype. Some cultivars, however, possessed a wide range of adaptability to diverse nutritional conditions. Indica rice is highly sensitive to the presence of NH+4, and MgSO4 in the medium. High sucrose concentration (11%) was deleterious for callus induction and green plant regeneration. Sucrose at 3 to 5% was optimal for growth. Auxins at high concentrations promoted regeneration of albino plants. The presence of 2,4-dichlorophenoxyacetic acid (9.0 μM) supplemented with picloram (0,3 μM) and zcatin (0.5 μM) was effective for callus induction. A combined pretreatment of 35°C for 5 min followed by 10°C for 7 days was most suitable for callus induction and green plant regeneration.     

The response of anther culture to culture temperature in Triticum aestivum

Summary The response of anther culture to culture temperature was studied in detail using many varieties, F₁ hybrids and pollen-derived lines of wheat (Triticum aestivum) as materials. The suitable culture temperature for inducing pollen callus (or embryoids) in wheat anther culture ranged from 26 °C to 30 °C, varying with genotypes. But for the great majority of wheat genotypes the suitable culture temperatures lay between 28 °C and 30°C. The most significant genotypic variation in the response to culture temperature was observed in the comparison between the culture at 33 °C for eight days followed by culture at 25 °C (or 26 °C) and the continuous culture at 25 °C (or 26 °C). This genotypic variation in the response to culture temperature is a heritable character which may be controlled by multiple genes. The effect of culture at 30 °C for eight days followed by culture at 26 °C was similar to, or in some cases, better than that of continuous culture at 28 °C, and the effect of culture at 32 °C for eight days followed by culture at 28 °C was similar to that of continuous culture at 30 °C. In the range from 26 °C to 32 °C, the overwhelming majority of pollen calli emerged before the 40th day after anther inoculation, and the higher the culture temperature, the earlier and more concentrated the emerging period of the pollen callus. The pollen callus obtained at high temperatures above 28 °C should be transferred in time onto the regeneration medium at 25°–27°C to induce shoots.     

Plant regeneration from in vitro cultures of anthers and mature seeds of rice (Oryza sativa L.) cv. Basmati-370

Pollen plants were obtained from anther-derived calli of the indica rice variety Basmati-370. Anther-response (anthers producing pollen derived calli) and plant regeneration frequency from the pollen derived calli. was very low. Donor plants which flowered at the average max/min. temperature of 34.2°/23.3°C gave a significantly higher anther-response to in vitro techniques, than did those which flowered at 29.1°/16.4°C. Somatic callus induction and subsequent plant regeneration was readily obtained from mature seed embryos. While 2,4-D or 2,4,5-T (1 or 2 mg/l) proved highly efficient for callus induction, tryptophan (50 or 100 mg/l) induced a high frequency of green plants from the calli.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - K kinetin - BA benzyladenine - Trp tryptophan - CW coconut water     

Haploid callus and regeneration of plants from anthers of Digitalis purpurea L.

Summary Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch (Science 163, 85–87; 1969) supplemented with 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct geno-type to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions. If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/l kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28° and 8 h dark at 20° but the survival was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.     

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.)

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3–5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).     

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 4days (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.     

Relationships between temperature, development and survival of different life stages of the tomato fruit fly, Neoceratitis cyanescens

The development and survival of female Neoceratitis cyanescens (Bezzi) (Diptera: Tephritidae) from egg to complete ovarian maturation were studied in the laboratory at five different constant temperatures: 15, 20, 25, 30, and 35 °C. The aim of this study was to get information on the influence of temperature on pre-mature stages, as a prerequisite to optimise rearing procedures and to understand temporal and geographical patterns of fruit fly occurrence. The developmental rate of the different life stages increased linearly with increasing temperatures up to 30 °C. The fastest development of pre-mature stages was recorded at 30 °C (22±1 days) and the slowest at 15 °C (98±3 days). The day-degrees requirements (K) to complete total development were 432.6 day-degrees. Lower temperature thresholds were 11.4, 11.9, 10.0, and 11.1 °C for egg, larval, pupal stages and ovarian maturation, respectively. The number of adults obtained from an initial batch of 100 eggs reached a maximum (64) at 25 °C. At 35 °C, no adults emerged. Larval developmental time was significantly shorter in green tomato fruits than in potato tubers at 15, 20, and 25 °C. Mortality rate of larvae was higher in green tomato fruits than in potato tubers at 25 and 30 °C.