Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

Expression of Polarity during early Development of Microspore-derived and Zygotic Embryos of Brassica napus L. cv. Topas

Microspore derived (MS-)embryogenesis and zygotic embryogenesis of Brassica napus L. cv. Topas were investigated by light and scanning electron microscopy to reveal the expression of polarity during the transition phase from globular to heart and torpedo shape. During the first 5 days of MS-embryo formation, the cell wall of the former microspores remained intact and a globular mass of cells developed within. Pollen walls ruptured after 5 days of culture; embryos proceeded through heart-shape and torpedo-shape stages within 15 days in a way comparable to, but faster than observed during zygotic embryogenesis. Expression of polarity in globular and elongating MS-embryos was analyzed by detection of the distribution of activated calmodulin as well as of free cytosolic calcium by using confocal scanning laser microscopy, and by the detection of starch. Calmodulin was evenly distributed in globular embryos and only exhibited clear polar distribution in elongated embryos. Free cytosolic Ca²⁺ accumulated in the protoderm of globular embryos and in the central cylinder of torpedo shaped embryos, but never showed polar distribution. Accumulation of starch granules at the root poles of both sexual as well as MS-embryos, however, indicated polar distribution before the transition from globular to heart shape stage. Since the local rupture of the pollen wall of 6-day-old MS-embryos was never preceded by the decrease of starch at that site, it is likely that the rupture of the pollen wall plays an important role in the local activation of cell metabolism and thus in the determination of the polarity axis in MS-embryos.     

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Individual buds of Brassica napus cv. Topas, near the first pollen mitosis, were used for microspore culture. Bud and petal lengths were recorded. Microspores isolated from the individual buds were plated and small samples were fixed for cytology. Following embryo induction and three weeks of culturing, numbers of embryos were scored. Bud and petal lengths did not accurately indicate which buds would supply microspores that would form embryos at high frequencies. Fluorescence microscopy was used to examine nuclei stained with Hoechst 33258 and vacuolar morphology of microspores was revealed by the weaker fluorescence due to glutaraldehyde fixation. Following isolation, nuclear and vacuolar characteristics were used to stage the microspores as miduninucleate, late uninucleate vacuolate, late uninucleate, mitotic, or binucleate. The relationship of developmental stage to the frequency of microspore-derived embryos was evaluated. A classification scheme was developed which uses the relative proportions of microspores at each of the stages to identify microspore isolations that would form embryos at high frequencies. It was found that when 1 to 87% of the isolated microspores were binucleate, 21.4 ± 3.0% of the viable microspores developed into embryos. This was a significant ( P < 0.001) increase over the other 3 classes. The ability to select highly embryogenic microspore isolations is of great advantage for developmental cell biology studies.     

Cytological Characterization of Multicellular Structures in Embryogenic Microspore Cultures of Brassica napus L.

We investigated an embryogenic microspore culture from Brassica napus L. cv. “Topas”, 3 days after induction of embryogenesis, using light and electron microscopical techniques. According to our observations, 6 groups of uni- or multicellular structures could be distinguished by differences in size, wall structure, structure and distribution of organelles and the degree of vacuolisation. Only one multicellular group represents real proembryos which are able to form embryos and to regenerate plants. These 6 groups could be detected in living cultures using an inverted microscope. The cell size and the degree of vacuolization are especially useful markers to distinguish the groups. Separate cultivation of the multicellular complexes of the 6 groups in culture plate inserts from the third day of culture proved that only one group contains real proembryos.     

Effects of culture density,conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas

Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.     

油菜小孢子胚发生的超微结构和胚状体形态

应用透射电镜和扫描电镜分别研究油菜游离小了包子培养后细胞的超微结构和胚状体的形态。单核晚期小孢子经培养后,具有胚状体发生能力的细胞中央液泡消失,积累淀粉,含丰富细胞器。     

Identification of potentially embryogenic microspores in Brassica napus

Studies were undertaken with Brassica napus L. cv. Topas to identify buds containing microspores predisposed to embryogenesis in vitro and to investigate bud and microspore development in relation to this process. No significant correlation was found between the final embryo number and bud components. There appears to be a developmental window of less than 8 h duration during which microspores are very likely to form embryos: over 70% of the microspores can undergo division and up to 70% of these can form embryos. Embryos were mainly obtained from late uninuucleate to early binucleate microspores: the former contained mainly a G2 or M phase nucleus located at the microspore periphery and the latter a generative nucleus (associated with the intine) and a vegetative nucleus. Observations indicated that only the vegetative nucleus contributed to embryo formation. The first embryogenic division occurred between 8 and 16 h for uninucleate- and between 8 and 48 h for binucleate-derived embryos.     

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl- p -toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18-h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μ M trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.     

一种快速生长的甘蓝型油菜DH系的获得和鉴定

从快速生长的甘蓝型油菜的小孢子培养中共获得23个再生植株。经倍性鉴定,其中自发加倍成二倍体的有10株,单倍体13株。单倍体再用秋水仙碱处理后获得DH系,所得材料对油菜功能基因组学的研究可能有一定的价值。     

秋水仙碱对甘蓝型油菜离体小孢子胚胎发生的影响

研究了秋水仙碱不同浓度和处理时间对甘蓝型油菜23个基因型离体小孢子胚胎发生的影响.3个基因型的小孢子被10、50和100mg/L秋水仙碱处理24h或48h,胚产量是2.55～14.75胚/蕾,10～50mg/L处理72h则是0.94～2.43胚/蕾.这表明处理72h对小孢子胚发生有抑制作用.用200、400、500和800mg/L处理2个基因型小孢子16～48h,胚产量为0.6～1.33胚/蕾,未处理对照是6.25和9.36胚/蕾.可见200～800mg/L浓度对胚再生有不同程度的阻碍效应.结果还证明,小孢子对秋水仙碱的反应与其基因型有关.当用10、20、50和100mg/L处理48h时,22B5-6和903-3小孢子的胚产量为37.09～69.47胚/蕾,而F1-29、W592和SF10-12是0.28～1.45胚/蕾,相互之间差异很大.秋水仙碱处理小孢子的目的是使其再生植株的染色体高频率加倍,因此应根据胚产量和染色体加倍率来确定秋水仙碱浓度和处理时间.本试验中,采用10～50mg/L处理48h或者用100mg/L处理24h,约80%基因型的小孢子胚产量在5胚/蕾以上,约70%基因型的再生植株加倍率达60%以上,可有效地用于油菜遗传和育种研究等领域.     

甘蓝型油菜小孢子培养技术的几项改进

本研究在NLN-16和NLN-13的培养基中分别加入0.1mg/L 6-BA和0.05%的活性碳,结果表明6-BA对小孢子再生胚有明显地促进作用,再生胚的频率比对照增加26枚/皿,经分析达到显著水平;而0.05%的活性碳对小孢子再生胚促进作用不显著。对甘蓝型油菜小孢子培养再生植株的染色体加倍及移栽的研究结果表明,在小孢子培养初期加50mg/L秋水仙碱加倍效率最佳,加倍率达到67.6%。小孢子培养的再生苗移栽至大田后,采用遮阳网覆盖小苗,移栽成活率达到87.6%。Abstract:The application of microspore culture technique was restricted because of its low frequency of embryogenesis and chromosome doubling.Two methods of enhancing the frequency of embryogenesis were employed in the study,namely,activated charcoal treatment in NLN-13 media and 6-BA treatment in NLN-16 media.The treatment with 0.05% activated charcoal produced 24 embryos per plate,which increased 1.7 embryos per plate,as compared with the treatment without activated charcoal.However,the analysis of T-test showed that it was not significant.After adding 0.1mg/L 6-BA in NLN-16 media,the frequency of embryogeny was 38.3 embryos per plate,and it was 26 embryos more per plate than that of CK.Analysis of T-test is significant.This indicates that 6-BA promotes embryogeny in microspore culture.Adding 50mg/L colchicines in NLN-16 media,the doubling frequency was 67.6%.The plantlets transplanted into field with two methods of light-covered net and plastic films were investigated.A survival rate of 87.6% was obtained using light-covered method whereas 57.7% survived using plastic film method.     

油菜雄性核不育系及其等位可育系小孢子发育过程的比较研究

用Olympus BH2型光学显微镜对甘蓝型油菜单显性核不育系(GMS)及其等位可育系小孢子发育过程进行解剖学观察,发现在正常小孢子发育过程中,绒毡层在小孢子发育的四分体前后开始解体,为小孢子继续发育提供营养,而不育系小孢子的败育在减数分裂前就已经发生,并且不能形成四分体,小孢子逐渐解体,且小孢子解体在绒毡层解体之前发生,最后花药成为干瘪的空壳,导致不育。     

Very long chain and hydroxylated fatty acids in offspring of somatic hybrids between Brassica napus and Lesquerella fendleri

 Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F₂). Furthermore, crosses between the F₂ plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F₃ seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F₆ generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F₆ plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F₆ generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F₆ plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F₂ generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis. Received: 12 December 1998 / Accepted: 4 January 1999     

Somatic embryogenesis and plantlet regeneration in semul (<Emphasis Type="Italic">Bombax ceiba</Emphasis>)

Somatic embryogenesis in semul, Bombax ceiba L. (Bombacaceae) was achieved from immature zygotic embryo explants on MS medium. The cytokinin BA induced somatic embryogenesis at higher frequencies than the auxin 2,4-d. The rate of somatic embryogenesis was inversely related to the concentration of BA. A constant supply of 0.44 M BA was necessary for a high multiplication rate. Conversion of somatic embryos into plantlets is reported.     

Free Amino Acid, Protein and Water Content Changes Associated with Seed Development in Araucaria angustifolia

The free amino acid, protein, water and dry matter contents were determined during the seed development of Araucaria angustifolia. Soluble and insoluble proteins in the mature seed represent 4.2 % of the fresh matter. The embryonic axis stored the greatest amount of soluble proteins, while cotyledons both with the embryonic axis showed the largest quantities of insoluble proteins in the mature seed. The greatest concentration of free amino acids was detected during the stage when cotyledons start to develop. Glutamic acid, aspartic acid, alanine and serine were predominant in the whole seed while arginine, lysine and γ-aminobutyric acid were present in great amounts only in cotyledons and embryonic axis. Although megagametophyte was important as a source of free amino acids, it was not the major protein storage organ in the mature seed. In the embryogenetic process, the rise of cotyledons is closely related to physiological and biochemical changes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid-cycling Brassica napus

 A simple method to induce somatic embryogenesis from seeds of rapid-cycling Brassica napus is described. Seedlings cultured on Murashige and Skoog (MS) basal medium produced somatic embryos directly on hypocotyls and cotyledons after 2 to 3 subcultures onto the same medium. A low pH of the medium (3.5–5) was more conducive to somatic embryogenesis than a higher pH (6 and 7). Embryogenic potential of the seeds was inversely correlated to seed age: about 41–68% of immature seeds between the ages of 14 and 28 days after pollination (DAP) formed somatic embryos compared to 0–11% of the seeds obtained 29–37 DAP. About 54% of the somatic embryos produced secondary embryos after subculturing onto the same medium. The embryogenic potential of the cultures has been maintained on MS basal medium for 2 years (12 generations) without diminution. Up to 75% of the secondary embryos developed into plantlets on MS medium enriched with 10^–6  M zeatin, and 40% of these produced flowers when transferred to an optimised flower-induction medium. Viable seeds were produced in self-pollinated in vitro flowers. Received: 15 February 2000 / Revision received: 18 July 2000 / Accepted: 19 July 2000     

Fatty acid desaturation in monogalactosyldiacylglycerol of Brassica napus leaves during low temperature acclimation

Brassica napus plants grown at 5°C have the potential to desaturate fatty acids in the major membrane diacylglycerols of leaves at rates much higher than those of plants grown at 30°C. This low temperature-induced desaturation (LTD) is rapidly deactivated if plants grown at 5°C are transferred to 30°C for several hours. By exposure to ¹⁴CO₂ and a computer simulation program, we estimated the rate of desaturation of monogalactosyldiacylglycerol by (ω9-, ω6- and ω3-desaturases of plants grown at 5, 10, 20 and 30°C. Data show that LTD can be induced in mature leaves of plants grown at 20 and 30°C after transfer to 5°C. Full activation of LTD takes several weeks and occurs more rapidly in plants grown at 20°C than 30°C. This activation is largely due to the increased activity of ω9- and ω6-desaturases on C16 fatty acids and ω6-desaturase on C18 fatty acids.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.     

Oil Body Biogenesis during Brassica napus Embryogenesis

Although the oil body is known to be an important membrane enclosed compartment for oil storage in seeds, we have little understanding about its biogenesis during embryogenesis. In the present study we investigated the oil body emergence and variations in Brassica napus cv. Topas. The results demonstrate that the oil bodies could be detected already at the heart stage, at the same time as the embryos began to turn green, and the starch grains accumulated in the chloroplast stroma. In comparison, we have studied the development of oil bodies between Arabidopsis thaliana wild type （Col） and the low-seed-oil mutant wrinkled1-3. We observed that the oil body development in the embryos of Col is similar to that of B. napus cv. Topas, and that the size of the oil bodies was obviously smaller in the embryos of wrinkled1-3. Our results suggest that the oil body biogenesis might be coupled with the embryo chloroplast.