PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

Microspore embryogenesis and the development of a double haploidy protocol for cow cockle (Saponaria vaccaria)

Factors affecting microspore embryogenesis of cow cockle (Saponaria vaccaria) were evaluated including donor plant growing conditions, genotype, bud size, density, medium composition, and culture conditions. Of the two donor plant (day/night) temperature regimes evaluated (10/5°C and 20/15°C), plants grown at 20/15°C were the most embryogenic. An embryogenic frequency of greater than 350 embryos/100 buds was observed in the most embryogenic genotype, cv. ‘White Beauty’. Buds from 3–9 mm in length were evaluated for their embryogenic potential; buds that were 4–7.9 mm produced the most embryos/100 buds. Of all the media compositions evaluated, NLN medium with 15% sucrose resulted in the most embryos. Cow cockle microspores required an initial period of 32°C for 3 days for production of microspore-derived embryos (MDEs).     

Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern and Coss)

Summary The influence of donor plant growth environment, microspore development stage, culture media and incubation conditions on microspore embryogenesis was studied in three Indian B. juncea varieties. The donor plants were grown under varying environments: field conditions, controlled conditions, or a combination of the two. The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same does not hold true for the field-grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive with genotypic variability ranging from 10 to 35 embryos per Petri dish, irrespective of the other factors. NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis The fortification of this medium with activated charcoal, polyvinylpyrrolidone, colchicine, or growth regulators (6-benzylaminopurine and 1-naphthaleneacetic acid) was observed to be antagonistic for microspore embryogenesis, while silver nitrate (10 μM) had a significant synergistic effect. A post-culture high-temperature incubation of microspores at 32.5±1°C for 10–15 d was found most suitable for high-frequency production of microspore embryos. The highest frequency of microspore embryogenesis (78 embryos per Petri dish) was observed from the late uninucleate microspores (contained in bud sizes 3.1–3.5 nm irrespective of genotype) cultured on NLN medium containing 13% sucrose and silver nitrate (10 μM), and incubated at 32.5°C for 10–15 d.     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

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     

Microspore embryogenesis in Apiaceae

Haploid technology is used to develop uniform, true-breeding lines, as well as to accelerate crop improvement programs. Among 20 Apiaceae species screened for response to doubled haploidy, 11 generated microspore-derived embryos, and all but one of the latter yielded doubled haploid plants. Donor plant conditions, basal media, and culture conditions were evaluated for their efficacy on inducing microspore-derived embryos. Growing donor plants at temperature conditions of 10/5 or 15/10°C promoted microspore embryogenesis in fennel (Foeniculum vulgare Mill.), whereas, growing donor plants at a temperature regime of 10/5°C, along with the use of a cold extraction method, enhanced embryogenesis in dill (Anethum graveolens L.) and anise (Pimpinella anisum L.). The culture of fennel and dill microspores in an NLN basal medium and caraway (Carum carvi L.) in AT-3 basal medium promoted the highest frequencies of embryo induction.     

Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis)

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.     

Influence of stock plant pretreatment on gynogenic embryo induction from flower buds of onion

The gynogenic response of a range of onion genotypes to flower bud culture was compared using a two-step culture system. Embryogenic cultures and plantlets were produced from unpollinated ovules in whole flower bud explants 6 to 19 weeks after culture initiation. Preconditioning stock plants significantly influenced gynogenic embryogenesis. A ten-fold increase in embryogenesis was obtained when flower buds were cultured from stock plants maintained at 15 °C compared to 10 °C or the ambient temperature conditions of a glasshouse (maximum-minimum of 25–12.7 °C). A total of 49 embryos was obtained from 2660 cultured flower buds and 45% of plantlets were successfully acclimatised to glasshouse conditions. The majority of acclimatised plantlets were haploid (68%) but spontaneous double haploid plants (23%) were obtained from three genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of somatic embryogenesis for plant propagation in cassava

In cassava, somatic embryogenesis starts with the culture of leaf explants on solid Murashige and Skoog-based medium supplemented with auxins. Mature somatic embryos are formed within 6 wk. The cotyledons of the primary somatic embryos are used as explants for a new cycle of somatic embryogenesis. The cotyledons undergo secondary somatic embryogenesis on both liquid and solid Murashige and Skoog-based medium supplemented with auxins. Depending on the auxin, new somatic embryos are formed after 14–30 d after which they can be used for a new cycle of somatic embryogenesis. In liquid medium, more than 20 secondary somatic embryos are formed per initial cultured embryo. In both primary and secondary somatic embryogenesis, the somatic embryos originate directly from the explants. Transfer of clumps of somatic embryos to a Greshoff and Doy-based medium supplemented with auxins results in indirect somatic embryogenesis. The direct form of somatic embryogenesis has a high potential for use in plant propagation, whereas the indirect has a high potential for use in genetic modification of cassava. Mature somatic embryos germinate into plants after desiccation and culture on a Murashige and Skoog-based medium supplemented with benzylaminopurine (BA). Depending on the used BA concentration, plants can either be transferred either directly to the greenhouse or after using standard multiplication protocols.     

Common Leafspot of Alfalfa: Ascospore Germination and Disease Development in Relation to Moisture and Temperature

In a moist chamber Pseudopeziza medicaginis ascospores infected alfalfa (Medi sativa L.) moderately to abundantly within 6–10 h at 10–20 °C and within a longer time-span outside this temperature range. Approximate limits of the range were 2.5 and 28 °C; no infection took place at 30 °C. At 14°C ascospores infected alfalfa abundantly at 98 %relative humidity (RH) and above, moderately at 97%, sparsely at 95 and 96%, but not at 94% and below. Ascospores were hydrophilic, germinating best at or near 100%, RH but did not germinate at or below 93 % RH. After infection was established, tiny leafspots became visible within 6–7 days at constant temperatures of 15–25°, 10 days of 10°C, 13 days of 5 °C, and 25 days of 2.5 °C. They failed to develop into normal size spots within 4 weeks at constant temperatures near 30 °C, or near 10 °C and lower. Temporary exposure of incipiently diseased plants 1–6 days to 30–38 °C adversely affected subsequent leafspot development at 20–24°C. Inhibition depended on temperature and on the extent of post-infection disease development.     

Influence of post-flowering temperature on seed development, and subsequent performance of crisp lettuce

Crisp lettuce plants cv. Saladin were grown from the time they started flowering, at 20/10°C (16 h day, 8 h night), 25/15°C and 30/20°C in glasshouses on two occasions in 1985. Yields of seed increased from, on average, 15 g to 27 g and then fell to 20 g per plant with progressive increases in temperature. The number of mature florets per plant increased with temperature but the number of seeds per mature floret was lower at 20/10°C and 30/20°C than at 25/15°C. An increase in temperature reduced mean seed weight by up to 45%, seed volume by 15%, cell numerical volume density (N_v) by 27% and the number of cells per seed by 39%. Percentage seed germination reached a maximum early in seed development at the stage when the pappus appeared through the involucral bracts. Differences in percentage germination and vigour of seeds (slope test) from different temperatures were accounted for largely by the effects on mean seed weight. However, when germinated at 30°C seeds produced at 30/20°C germinated more readily than those produced at 25/15°C or 20/10°C. Seed vigour gradually increased with an increase in the length of storage after harvest, reaching a maximum after 260 days. In general, seeds produced at 25/15°C exhibited a greater variation in numbers of seeds per floret, N_v, seed weight, times of seedling emergence, seedling and mature head weight than seeds produced at lower or higher temperatures.     

Differing effects of thermal stress on coral fertilization and early embryogenesis in four Indo Pacific species

Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. The fertilization and early embryogenesis of four reef-building coral species representing three Indo-Pacific families were examined in a series of laboratory experiments where temperatures were increased up to 5–6°C at ambient. High levels of fertilization and normal embryogenesis were observed for Favites abdita, Favites chinensis and Mycedium elephantotus at temperatures to 32°C (+5°C) and embryos developed normally until the 5th cell cleavage. Acropora millepora was the only species to be affected by higher temperatures, exhibiting significantly reduced fertilization and a higher frequency of embryonic abnormalities at 32°C (+4°C), and fertilization ceased altogether at 34°C (+6°C). Early cell cleavage rates increased with temperature up to 32°C for all species.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

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.     

The development of Puccinia hordei on barley cv. Zephyr

Germination of uredospores of Puccinia hordei was similar on cover-slips and on the first leaves of barley seedlings (cv. Zephyr) at 100 % r.h. over the range 5–25 °C, being greatest at 20 °C. At 15, 20 and 25 °C maximum germination was attained in 6 h. No uredospores germinated on coverslips in humidities below saturation. The numbers of pustules which subsequently developed on plants incubated at 5, 10, 15 or 18 °C and 100 % r.h. for varying periods up to 24 h, were directly related to rise in temperature and length of incubation. The time from inoculation to eruption of pustules (generation time) was 6 days at 25 °C, 8 days at 20 °C, 10 days at 15 °C, 15 days at 10 °C and 60 days at 5 °C. Pustule production on inoculated plants which had been kept at 5 °C was rapidly accelerated when they were transferred to 20 °C. Data obtained at constant temperatures were used to predict generation times of the fungus in the field. The productivity of pustules, determined as weight of uredospores, was examined at 10, 15 and 20 °C. Significantly more spores were produced at 15 than at 10 °C and most were produced at 20 °C. The results are discussed in relation to those obtained by other workers and to the development of brown rust in the field.     

Quercus suber L. Somatic embryo germination and plant conversion: Pretreatments and germination conditions

Summary Osmoticum (high sucrose concentration) and chilling (4°C) treatments were assayed in order to promote germination (radicle elongation) and plant conversion of Quercus suber somatic embryos. Those treatments were applied before embryos were set to germinate on Murashige and Skoog (MS) liquid medium. Chilling for 2 mo. improved germination rate (best results close to 100%). On the other hand, that treatment increased plant conversion, especially when high sucrose concentration was used. The best results were obtained with 150 g l⁻¹ sucrose (75% of cultured embryos converted to plants). Incubation conditions during germination were also studied. Although there were no significant differences on final results, warmer temperatures and darkness increased germination rate. The influence of the different treatments on shoot morphology was also studied. The highest percentages of shoots with normal leaves obtained were close to 25%.     

Salinity and Temperature Effects on Germination for Three Salt-resistant Euhalophytes, Halostachys caspica, Kalidium foliatum and Halocnemum strobilaceum

The effects of three salinities (0, 100 and 500 mM NaCl) and four constant temperatures (10, 20, 30 and 35 °C) on seed germination of Halostachys caspica (M. B.) C. A. Mey., Kalidium foliatum (Pall.) Mop. and Halocnemum strobilaceum (Pall.) Bieb. were investigated. After seeds were treated with different concentrations of NaCl at constant temperatures of 10–35 °C for 16 days, ungerminated seeds were transferred to distilled water for 10 days to investigate the total germination; after this time, the ungerminated seeds from the 10 and 20 °C treatments were then moved to 35 °C for another 5 days to determine the final germination. The three plant species in the present experiment are salt-resistant euhalophytes growing in high saline soils in the Zhungur Basin in Xinjiang, a northwest province of China.Compared with germination under control conditions, germination percentages of all three species were not affected by 100 mM NaCl at 10–35 °C, while severely inhibited by 500 mM NaCl; germination percentages were very low at 10 °C up to 100 mM NaCl for all species; the optimum temperature for germination of H. caspica and K. foliatum was 20–30 °C, while 35 °C for H. strobilaceum, up to 100 mM NaCl; seeds did not suffer ion toxicity for all species, as evidenced by the high total germination after ungerminated seeds pretreated with 500 mM NaCl were transferred to distilled water at constant temperatures of 10–35 °C for 10 days, and the high final germination after the ungerminated seeds from the 10 and 20 °C treatments were subsequently moved to 35 °C for another 5 days; Halostachys caspica had greater sensitivity to increasing temperatures from 10 and 20 °C to 35 °C compared with the other two species.     

Uscana lariophaga, egg parasitoid of bruchid beetle storage pests of cowpea in West Africa: the effect of temperature and humidity

The stored-product bruchid pests,Callosobruchus maculatus (Fabricius) andBruchidius atrolineatus (Pic) cause considerable production losses in cowpea in West Africa.Uscana lariophage Steffan parasitizes the eggs of the bruchids both in the field and in storage. As chemical control of bruchids in traditional granaries is not appropriate for poor farmers, enhancement of the efficacy of the parasitoid by environmental manipulation has been investigated. The effect of temperature on the capacity ofU. lariophaga to parasitize eggs has been studied at eleven constant and three fluctuating temperatures within the range 10 to 45°C. Longevity of the female wasp decreased with increasing temperature. The rate of development increased linearly at temperatures from 17.5 to 35°C, but decreased from 35 to 40°C. Mortality of the developing wasp remained below 20% from 20 to 37.5°C, but outside this range, mortality reached 100% at 15 at 42.5°C. Most parasitization occurred at temperatures of 25 and 30°C. Sex ratio (percentage females) increased with temperature in the high temperature range. The intrinsic rate of increase (r_m) forU. lariophaga was highest in the temperature range from 30 to 37.5°C and was higher than that ofC. maculatus at all temperatures. While the r_m value ofC. maculatus did not vary much at temperatures from 25 to 35°C, the r_m value of the wasp doubled. Relative humidity did not effect longevity, egg-laying capacity, mortality, development time and sex ratio of the wasps withC. maculatus as host. However, withB. atrolineatus as the host, development time and mortality increased at lower relative humidity levels. The results indicate that temperature is the major regulating factor on the parasitoid. As the type of storage structure and its location (sun or shade) affects the temperature inside the store, ways are being investigated of manipulating the storage environment through temperature regulation to increase the impact of the parasitoid.     

In vitro differentiation of embryogenic pollen,control by cold treatment and embryo formation inab initio pollen cultures ofNicotiana tabacum var. badischer burley

Summary InNicotiana cold treatment causes differentiation of embryogenic pollen. This differentiation initiates on the plant and is completed in culture. Differentiation on the plant results in pollen dimorphism and differentiation in culture leads to pollen embryogenesis. An increased number of pollen capable of embryogenesis is possible on plants induced to flower in short days and low temperature (8 hours light, 18 °C). These embryogenic pollen on selective isolation, from buds at a petal length of 3.4±0.1 cm, fail to form embryos but do so in the cultures which receive cold treatment at 10 °C for 10 days. To some extent the differentiation of embryogenic pollen can be completed on plants induced to flower at 15 °C and embryogenic pollen from such plants form embryos at a low frequency which can be substantially increased on giving the cultures a cold treatment. The frequency of embryogenesis is higher in cultures of 15 °C plants than those of 18 °C plants. Low temperature requirements at two stages—to the plant and to the culture—are essential and complimentary for embryogenesis inab initio pollen cultures.Cold treatment causes repression of gametophytic differentiation and this results in the differentiation of embryogenic potential. The embryogenic pollen, unlike gametophytic pollen, are not fully differentiated structures. They are unable to divide and form embryos in presence of metabolic inhibitors such as actinomycin-D and cycloheximide.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.