Response ofBrassica napus L. Microspore-derived embryos to exogenous abscisic acid and desiccation

Summary Microspore-derived embryos fromBrassica napus cv. Topas (low erucic acid) and Reston (high erucic acid) were subjected to treatment with abscisic acid (ABA) during late-stage embryo development and then dried under controlled relative humidities to mature dry seed levels of moisture. Exogenously medium-supplied ABA arrested growth and development, reduced moisture content, increased total fatty acids on a dry weight basis, and stimulated systhesis of proteins in microspore-derived embryos. ABA also resulted in a higher proportion of 22∶1 in cv. Reston (high 22∶1) and increased the level of fatty acid unsaturation in cv. Topas (low 22∶1). The accumulation of two proteins that co-migrated with cruciferin and napin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gels were also promoted by exposure to ABA, and the degree of accumulation was dependent on the concentration and time of application of ABA. Controlled desiccation of microspore embryos, used to simulate normal maturation and dehydration of zygotic embryos during seed development, did not seem to cause an increase of either storage proteins, total fatty acids, or 22∶1 (in cv. Reston), suggesting that dehydration is not a prerequisite for these processes, at least in culturedBrassica embryos.     

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

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

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Enhanced plant regeneration from microspore-derived embryos of Brassica napus by chilling,partial desiccation and age selection

Germination was readily induced in recalcitrant microspore-derived embryos of Brassica napus Topas when they were exposed to a period of chilling (9–12 days at 4°C) or partial desiccation (rapid or slow air drying) prior to germination. In general, embryos thirty-five days old had the highest germination rates as compared to younger or older ones. Populations of embryos were induced to germinate at a rate of over 90% under specific temperature, desiccation and age conditions. Comparisons to an embryogenic B. napus winter line, F346, are made.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Selection for salt tolerance in vitro using microspore-derived embryos ofBrassica napus cv. topas,and the characterization of putative tolerant plants

Microspore-derived embryos ofBrassica napus cv. Topas that survived salt stress, were obtained after selection against otherwise lethal doses (0.6 and 0.7%) of NaCl after mutagen treatment. A total of 10 salt-surviving embryos were obtained out of a possible 834 000 embryos that were mutagenized. One embryo out of a possible 845 000 obtained from nonmutagenized controls survived but failed to develop into a plant. Visual assessment after salt stress indicated that both the putative salt-tolerant plants and plants from control seeds behaved similarly. However, based on individual characteristics related to salt tolerance, one of the lines (PST-2) accumulated less sodium and retained more potassium, and hence was able to maintain a more favorable Na:K ratio as compared to the controls under salt stress. Also chlorophylla fluorescence induction and quenching signals indicated a high energetic state of the thylakoid membranes in PST-2 under salt stress. The other putative salt-tolerant line (PST-1) had a higher background level of proline that may have enabled it to survive salt stress during initial screening, although its later performance was no better than the control plants.     

Pyruvate-kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napus

Polyclonal antibodies against castor-oil seed cytosolic and leucoplastic pyruvate kinases (PK_c and PK_p, respectively; EC 2.7.1.40) were utilized to examine the subunit compositions and developmental profiles of canola (Brassica napus L. cv. Topas) PK_c and PK_p over 6 d of seed germination and 35 d of culture of microspore-derived embryos. The PK_c from germinating seeds appears to be composed of a single type of 56-kDa subunit, whereas the enzyme from cultured embryos contains equal proportions of immunologically related 57- and 56-kDa subunits. The PK_p was immunologically undetectable in germinating seeds, while the enzyme from cultured embryos consisted of immunologically related 64- and 58-kDa subunits in a ratio of about 12, respectively. The large increase in PK activity that occurs between the second and fourth days of seed gemination is based upon de-novo synthesis of PK_c. Between 7 and 14 d of culture of microspore-derived embryos, the levels of PK_p and PK maximal activity increased approx. 3- and 2.5-fold, respectively. These increases were coincident with an approximately fourfold rise in the in-vivo pyruvate: phosphoenolpyruvate concentration ratio. Conversely, PK_c was not only far less abundant relative to PK_p, but its level remained constant over 35 d of microspore-embryo culture. Developing non-zygotic (microspore-derived) embryos strongly resembled ripening zygotic (seed) embryos in terms of PK specific activity as well as relative amounts and subunit compositions of PK_c and PK_p. The results indicate that the synthesis of PK isoenzymes in B. napus seeds is highly regulated and that this regulation follows a preset developmental program.Abbreviations IgG immunoglobulin G - IU international unit - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PK(s) pyruvate kinase(s) - PK_c cytosolic pyruvate kinase - PK_p plastidic pyruvate kinase - PYR pyruvate Plant Research Centre contribution No. 1374We wish to thank Ms. Kathryn Hovey and Ms. Suzanne Belliveau (Agriculture Canada) for their expert assistance in the culturing and harvesting of microspore-derived embryos of canola. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

Proteomics of desiccation tolerance during development and germination of maize embryos

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos before (28N, 52N and 72HN) and after (28D, 52D and 72HD) dehydration. Nine pre-dominantly proteins, 17.4 kDa Class I heat shock protein 3, late embryogenesis abundant protein EMB564, outer membrane protein, globulin 2, TPA:putative cystatin, NBS-LRR resistance-like protein RGC456, stress responsive protein, major allergen Bet v 1.01C and proteasome subunit alpha type 1, accumulated during embryo maturation, decreased during germination and increased in desiccation-tolerant embryos during desiccation. Two proteins, Rhd6-like 2 and low-molecular-weight heat shock protein precursor, showed the inverse pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment.     

Scanning electron microscopy of desiccation-tolerant and -sensitive microspore-derived embryos of Brassica napus L.

Desiccation tolerance can be induced in microspore-derived embryos of Brassica napus L. by application of abscisic acid (ABA). Scanning electron microscopy was employed to study and compare desiccation-tolerant and -sensitive microspore-derived embryos. The external surface of those desiccated embryos in which desiccation tolerance had been induced was uniformly shriveled due to severe dehydration, and their internal tissue system was well preserved. In contrast, in desiccation-sensitive embryos, dehydration caused tearing of the epidermis and collapse of the internal tissue system. After the desiccated embryos had been rehydrated, the size and external morphology of the desiccation-tolerant ones recovered to the pre-desiccation state within 1 day, whereas the sensitive ones did not recover or remained shriveled. The effect of ABA on the induction of desiccation tolerance is discussed. Received: 25 June 1998 / Revision received: 03 September 1998 / Accepted: 24 September 1998     

Abscisic acid and proline improve desiccation tolerance and increase fatty acid content of celery somatic embryos

Desiccation tolerance of celery (Apium graveolens L.) somatic embryos was increased by supplementation of embryo-production medium with 1 M abscisic acid (ABA) or 1 mM proline, with highest survival obtained with a combination of 1 M ABA and 1 mM proline. Addition of ABA and proline increased fatty acid accumulation by somatic embryos; the effect on fatty acid composition was inconsistent. Somatic embryos capable of germination differed from mature zygotic embryos by greater size, lower fatty acid level, and substantially lower proportion of oleic acid (18:1) as compared to linoleic acid (18:2).     

Cytokinins in a genic male sterile line of Brassica napus

Endogenous cytokinin levels were analyzed in four different organs viz., root, stem, leaf and mature flower, of the wild type (cv. Westar) and a genic male sterile (GMS) line of Brassica napus . Various cytokinins viz., bases (Z, DZ), ribosides (ZR, DZR), glucosides (OGZ + OGZR, OGDZ + OGDZR) and nucleotides (ZNT, DZNT) were quantified using Amaranthus betacyanin bioassay and ELISA. The major cytokinin in all the organs was DZ. In general, leaves had the highest levels of cytokinins as compared to the other organs. Root, stem and mature flowers of the wild type plants had higher levels of cytokinins and their metabolites than the GMS line. However. levaves of the GMS line had greater amount of various cytokinins as compared to the wild type. The major cytokinin quantitatively different between the two lines was DZ. The results suggest the possible involvement of cytokinins in the expression of genic male sterility in Brassica napus .     

Role of ethylene in cotyledon development of microspore-derived embryos of Brassica napus

Ethylene production during seed development in Brassica napus occurs first at 20 d after pollination (DAP), while a second greater peak occurs at 35 DAP. Because of the inaccessible location of the embryo within the maternal tissue, microspore-derived embryos (MDEs) of B. napus were used as a model for studying the role of ethylene during embryo development. The MDEs also produced a peak in ethylene evolution at 20 DAC (i.e. the early cotyledonary stage), dropping to minimal levels by 25-30 DAC. At 20 DAC the excised cotyledon evolved 85% of the ethylene found in the whole MDE. To determine the role of ethylene, MDEs were treated with aminoethoxyvinylglycine (AVG, an inhibitor of ethylene biosynthesis), CoCl(2) (an inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase), and silver thiosulphate (STS, an inhibitor of ethylene action). An inhibition in ethylene production or action at 20 DAC resulted in diminished lateral cotyledon expansion, due to a reduction in the lateral expansion of cells within the cotyledon. Recovery to 'control-type' levels of cotyledon cell expansion was achieved by application of ACC (the metabolic precursor of ethylene) to AVG-treated MDEs. Thus, ethylene production at 20 DAP likely controls cotyledon expansion during embryo development.     

Expression of a Brassica napus heme oxygenase confers plant tolerance to mercury toxicity

Plant heme oxygenases (HOs) regulate biosynthesis of phytochrome which accounts for photo‐acceptance and ‐morphogenesis. Recent studies have demonstrated that plant HOs also regulate many other physiological processes including response to environmental stimuli. To elucidate the mechanism by which HOs regulate plant adaptation to heavy metal exposure, three novel HOs genes were isolated from rapeseed (Brassica napus) and their expression patterns were analysed. Alignment of deduced protein sequences revealed that the three BnHOs share high identity with their corresponding orthologos (AtHO1‐3) from Arabidopsis. To investigate whether the BnHO regulates plant tolerance to Hg toxicity, we constructed B. napus transgenic plants overexpressing BnHO‐1. Under Hg stress, the transgenic plants had 1.41–1.59 folds higher biomass than the untransformants. However, overexpression of BnHO‐1 resulted in less accumulation of Hg in some lines of transformants than in untransformants. The transgenic plants show lower abundance of reactive oxygen species and attenuated oxidative injury compared with the untransgenic plants. We cloned the promoter sequences of BnHO‐1 from B. napus. Analysis revealed that the 1119 bp fragment contains a conserved Cd responsive element (CdRE) and others responding to multiple environmental stimuli. Transient expression in tobacco leaves showed differential responses to heavy metals (Zn, Cu, Pb, Hg and Cd).     

Acquisition of desiccation tolerance in soybeans

The entry into a desiccation-tolerant state is a major developmental component of seed maturation. Development of desiccation tolerance of embryonic axes of soybean [Glycine max (L.) Merrill cv. Chippewa 64] was studied by measuring changes in electrolyte leakage. germination and relative growth rate after axes were rapidly air-dried to various water contents. Axes acquired the full capacity for germination at 34 days after flowering (DAF). and reached physiological maturity (maximum dry weight) at 48 DAF. When dried to water content h = 0. 08 (g water g⁻¹ dry weight). few axes germinated before 42 DAF. but more than 90% germinated after 48 DAF. However, electrolyte leakage of rehydrated axes showed a linear decline from 30 to 55 DAF. For developing axes there was a critical water content or desiccation threshold. which could be estimated by using the electrolyte leakage method. The threshold of desiccation tolerance decreased gradually from h = 1. 10 to 0. 18 as axes matured from 28 to 55 DAF. The development of desiccation tolerance continued after physiological maturity at 48 DAF. We conclude that the acquisition of desiccation tolerance of soybean axes is a gradual event, rather than an abrupt transition.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Fatty acid changes during development of zygotic and microspore-derived embryos of Brassica napus

Cultured microspores of Brassica napus L. cvs Topas and Reston initiated cell divisions within 3 to 4 days, and globular, heart and torpedo shaped embryos were prevalent after approximately 6, 8, and 10 days, respectively. Embryos with rudimentary cotyledons were evident within 2 weeks, but those that reached this stage of development represented only 1–5% of the original microspore population. The fresh weight of microspore-derived embryos at all stages of development was significantly greater than that for zygotic embryos, but the pattern of change in fresh weight and fatty acid accumulation was similar in developing zygotic and microspore embryos. In freshly isolated microspores of both Topas (low erucic acid) and Reston (high erucic acid), the predominant fatty acid was 18:3, while 18:1 comprised less than 15% of total fatty acids. During development in both zygotic and microspore embryos, the level of 18:3 declined markedly while 18:1 rapidly increased. Erucic acid (22:1) was not detected in the early stages of embryogenesis in Reston. However, small amounts of 22:1 appeared by early cotyledonary stage and the level gradually increased in both zygotic and microspore embryos through the later stages of development. The fatty acid compositions of mature embryos was nearly identical to that of dry seed, except the level of 22:1 in Reston embryos was consistently less than in the seed. Triacylglycerols comprised only 15% of total lipids in freshly isolated microspores, but increased to more than 90% by 4 weeks. The fatty acid composition of the triacylglycerol fraction was generally similar to that of total lipids at all stages of development of microspore-derived embryos.     

Induction of microspore-derived embryos of Brassica napus L. with polyethylene glycol (PEG) as osmoticum in a low sucrose medium

Isolated microspores of Brassica napus were cultured on high concentrations of mannitol or polyethylene glycol (PEG 4000), with only a very limited amount of sucrose (0.08–0.1%) provided as carbohydrate source in the medium. While microspores cultured on high mannitol yielded no embryos and no embryogenic cell divisions were observed, microspores on high PEG developed into embryos within 2 weeks, and the embryo yield appeared comparable to that of the sucrose control. When placed under light, PEG embryos quickly changed color from yellow to dark green, while sucrose embryos first remained yellowish and then slowly changed color to pale green. Three-week-old PEG embryos were strikingly similar to immature zygotic embryos developed in ovulo, dissected at 14–15 days post-anthesis (DPA), while sucrose embryos differed from the latter in the size and shape, color and morphology of their cotyledons. These results demonstrate that in microspore embryogenesis of Brassica napus: (1) the level of metabolizable carbohydrate required for microspore embryo induction and formation appears to be substantially less than commonly used amounts, (2) sucrose as an osmoticum can be replaced with high-molecular-weight PEG. With further improvement the new method described here might be suitable for other Brassica species and would have a great potential application in breeding programs. Received: 29 May 1997 / Revision received: 12 August 1997 / Accepted: 2 September 1997     

Contrasting interactions between ethylene and abscisic acid in Rumex species differing in submergence tolerance

Complete submergence of flooding-tolerant Rumex palustris plants strongly stimulates petiole elongation. This escape response is initiated by the accumulation of ethylene inside the submerged tissue. In contrast, petioles of flooding-intolerant Rumex acetosa do not increase their elongation rate under water even though ethylene also accumulates when they are submerged. Abscisic acid (ABA) was found to be a negative regulator of enhanced petiole growth in both species. In R. palustris, accumulated ethylene stimulated elongation by inhibiting biosynthesis of ABA via a reduction of RpNCED expression and enhancing degradation of ABA to phaseic acid. Externally applied ABA inhibited petiole elongation and prevented the upregulation of gibberellin A(1) normally found in submerged R. palustris. In R. acetosa submergence did not stimulate petiole elongation nor did it depress levels of ABA. However, if ABA concentrations in R. acetosa were first artificially reduced, submergence (but not ethylene) was then able to enhance petiole elongation strongly. This result suggests that in Rumex a decrease in ABA is a prerequisite for ethylene and other stimuli to promote elongation.