Differential Effects of Cold and Heat Shock on Embryogenic Induction and Green Plant Regeneration from Wheat (Triticum aestivum L.) Microspores

Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum L.) microspores. Following a cold (4°C - 13°C) vs. heat (33°C) shock to wheat tillers, microspores were isolated and cultured in a liquid medium to obtain embryoids. Data on embryogenic microspore%, embryoid yield, plant regeneration% and green plant% were collected and analyzed. Cold pretreatment of 4°C or 10°C for a period of 6 or 10 days were more effective than other cold temperature regimes in inducing microspore embryogenesis. The heat shock of 33°C yielded the highest numbers of embryogenic microspores and embryoids. The albino-prone genotypes produced significantly higher green plant% following optimal cold shock, as compared to the standard 33°C heat shock. Results from present study suggest that cold shock may be a desirable alternative for germplasm that produce lower green plant% using heat shock. Lowered incubation temperature during embryoid development did not result in higher green plant.

Pollen development and multi-nucleate microspores of Populus bolleana Lauche

Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.

Embryogenesis and Development of Isolated Microspores in Raphanus sativus

[Objective] The aim was to observe embryogenesis and development of isolated microspores of Raphanus sativus.[Method] By using Chinese radish variety Shandong Huaye Xinlimei as the experimental materials,the caryocinesia features and development of isolated microspores in radish were studied.[Result] Two types of cell division appeared in the isolated microspore：unequal division and equational division.The unequal divided microspores had two different size nucleuses,while the equational divided microspores had two same size nucleuses.The equational divided microspores developed to 4-cell structure,multi-cell structure,ball-shaped embryos,heart-shaped embryos,torpedo-stage embryos and cotyledon-stage embryos.[Conclusion] This result will provide cytological basis for the isolated microspore culture of radish.

Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.

Developmental Dynamics of Wheat (<i>Triticum aestivum</i>L.) Microspores under Culture

Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% - 62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% - 33% terminated as multicellular structures, 16% - 19% arrested their development as pre-embryoids, and 7% - 10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).

The transcriptional landscape of the developmental switch from regular pollen maturation towards microspore-derived plant regeneration in barley

Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors.Haploids/doubled haploids(DHs)derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering.However,the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined.Here,RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis(ME)in barley cultivars Golden Promise and Igri,which exhibit contrasting responsiveness to microspore-derived plant formation.Our experiments revealed fundamental regulatory networks,specific groups of genes,and transcription factor(TF)families potentially regulating the developmental switch.We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME.Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.

甜瓜AMS基因结构分析及基因编辑载体构建

【目的】研究鉴定甜瓜雄性不育候选基因ABORTED MICROSPORES(AMS)的基因家族,分析CRISPR基因编技术并构建其敲除载体,为甜瓜雄性不育基因AMS功能验证提供依据。【方法】采用生物信息学技术鉴定甜瓜雄性不育候选基因ABORTED MICROSPORES(AMS)家族基因,分析其进化关系、保守基序及理化性质。利用CRISPR/Cas9基因编辑技术以甜瓜AMS基因外显子区设计gRNA引物,以载体pHSN401为模板,扩增获得sgRNA克隆框,使用Bsa I酶切pHSN401载体,利用DNA重组酶构建重组载体并转化农杆菌,挑选单克隆进行培养,随后进行菌液PCR鉴定。【结果】甜瓜AMS基因编码的蛋白质长度从501~605 aa不等,平均分子量约为59351 Da,等电点为5.04~6.45,甜瓜大部分AMS基因定位在细胞核;利用CRISPR/Cas9基因编辑技术,在AMS基因的CDS区设计3个靶位点,分别是AMS-pHSN401-1、AMS-pHSN401-2和AMS-pHSN401-3。【结论】AMS基因主要被预测在细胞核中表达,利用CRISPR/Cas9基因编辑技术构建甜瓜敲除重组载体AMS-pHSN401并转化农杆菌,获得阳性克隆。

Dedifferentiating initiation and embryogenesis from freshly-isolated microspores of barley

By using DNA-specific fluorescent dye and a confocal laser scanning microscope, the present study was designed to investigate the cytological characteristics of dedifferentiating initiation during pretreatment and em-bryogenesis during culture in freshly-isolated microspores of barley, and the difference in main developmental pathway between freshly-isolated and cold-treated microspores. The results revealed that ( i ) freshly-isolated microspores started the initiation within 12 h of mannitol pretreatment, whose main cytological characteristics were that: cell vol-ume was obviously extended; the volume of nuclei and nucleoli were also greatly increased; nucleoli were extremely clear and highly condensed; N/C ratio was very high; ( ii ) all the pretreatment methods led to the initiation of the mi-crospores, thus triggering the embryogenic process; ( iii ) pretreatment methods influenced the main developmental pathway of microspores by changing the pattern of the first mitosis. The cold-treated microspores formed main devel-opmental pathway via A patterns, but freshly-isolated microspores via B pattern.

Phylogeny,character evolution,and classification of Selaginellaceae(lycophytes)

Selaginella is the largest and most taxonomically complex genus in lycophytes.The fact that over 750 species are currently treated in a single genus makes Selaginellales/Selaginellaceae unique in pteridophytes.Here we assembled a dataset of six existing and newly sampled plastid and nuclear loci with a total of 684 accessions(74%increase of the earlier largest sampling)representing ca.300 species to infer a new phylogeny.The evolution of 10 morphological characters is studied in the new phylogenetic context.Our major results include:(1)the nuclear and plastid phylogenies are congruent with each other and combined analysis well resolved and strongly supported the relationships of all but two major clades;(2)the Sinensis group is resolved as sister to S.subg.Pulviniella with strong support in two of the three analyses;(3)most morphological characters are highly homoplasious but some characters alone or combinations of characters well define the major clades in the family;and(4)an infrafamilial classification of Selaginellaceae is proposed and the currently defined Selaginella s.l.is split into seven subfamilies(corresponding to the current six subgenera t the Sinensis group)and 19 genera(the major diagnosable clades)with nine new species-poor genera.We support the conservation of Selaginella with a new type,S.flabellata,to minimize nomenclatural instability.We provide a key to subfamilies and genera,images illustrating their morphology,their morphological and geographical synopses,a list of constituent species,and necessary new combinations.This new classification will hopefully facilitate communication,promote further studies,and help conservation.

Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Variations in Carbohydrate Content and Sucrose-Metabolizing Enzymes in Tomato (<i>Solanum lycopersicum</i>L.) Stamen Parts during Pollen Maturation

The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.

Embryogenesis and Development of Isolated Microspore in Chinese Cabbage

[Objective] The aim was to observe embryogenesis and development of isolated microspores in Chinese cabbage. [Method] Chinese cabbage F 1 hybrids were used as the experimental materials, and optical microspore was employed to observe the embryogenesis and development of isolated microspores. [Result] Cells swelled after heat shock treatment, which was the critical factor of embryoid induction. Three pathways equal division, unequal division and germination of microspores were discovered to lead to the embryogenesis from isolated microspores after swelling. Microspore could grow directly to embryoid through germination path way. Equally divided microspores formed the original embryos after successive multiple equal divisions. Original embryos could develop into cotyledon-shaped embryos via globular, heart-shaped and torpedo-shaped embryos. The large one of the two cells from unequally divided microspores continued to divide and finally formed a polar embryoid. [Conclusion] The study will provide cytological basis for high induction frequency and embryoid of Chinese cabbage.

Establishment on Isolated Microspore Culture Optimized System for Restorer of New Cytoplasmic Male Sterile (NER) of Brassica napus L.

[Objective]The aim was to establish optimized system for NER isolated microspore culture of Brassica napus L.. [Method]Twenty varieties of NER of Brassica napus were grown under uncontrolled temperature and light conditions,and their isolated microspore from anthers were used as explants in vitro culture. The influencing factors of microspore culture were preliminarily studied. [Result]The difference of genotypes was important influencing factors to embryoid yield. The embryoid yield increased by supplementing with 6-BA and NAA,culturing in solid-liquid double layer medium with activated charcoal; The difference was not significant of embryoid yield between culturing in medium supplemented with colchicines and the CK. The rates of cotyledonous embryoids directly developed into normal plantlets increased through enriching with 0.1-0.2 mg/L NAA and being treated on slim illumination two days before being inducted into normal plantlets. [Conclusion]The technical system of microspore culture of restorer of new cytoplasmic male sterile （NER） was established,which and lays a foundation for accelerating genotype purification of NER introgressive line.

Breeding dominant genic male sterility restorer line of Brassica napus L.

To facilate breeding process of Brassica napus,a microspore culture and molecular marker-assisted screening combined system were proposed in this research.At early flowering stage,F1 offspring of hybridized combination HY15 A×HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore.Treatment with 50 mg/L colchicine resulted in embryogenic rate of3.56 embryos/bud,which was substantially higher than control(0.78 embryos/bud).A total of 1,387 embryos and 862 single plants were obtained after induction culture.Ploidy detection was performed for the regenerated plants by flow cytometry.Diploid rates of microspores treated with 50 mg/L and 70 mg/L colchicine were 17.2%and 21.0%respectively,which was significantly higher than control(10.5%).Totally 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10.Approximately 54 of108 plants generated a 305 bp amplification product,whereas the other 54 plants showed a 398 bp band,thereby satisfying 1:1 separation ratio(x0.052=0.0093).These coincided with field identification results.Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time,thereby remarkably accelerate breeding.

Effects of different high temperatures and incubation time periods on embryogenesis in isolated microspore culture of Chinese cabbage (Brassica campestris L. ssp pekinensis)

Effects of five incubation temperatures (25℃ ,28℃ ,31℃ ,35℃ and 37℃ for 24hours) and four incubation time periods (0.4,16 and 24 hours at 35℃) on isolated microspore culture of Chinese cabbage were studied. The results showed that cultured microspores of Chinese cabbage developed into embryos at all the incubation temperatues from 28℃ to 37℃ ,but the best response to high temperature occured at 35℃. Among the four kinds of time periods, the highest yield of embryos was obtained at the 24h treatment. Therefore, the isolated microspore culture of Chinese cabbege ran be efficiently carried out at 35℃ for 24 hours.

Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

OsMAPK6 Affects Male Fertility by Reducing Microspore Number and Delaying Tapetum Degradation in Oryza sativa L.

The mitogen-activated protein kinase(MAPK)cascade is important in stress signal transduction and plant development.In the present study,we identified a rice(Oryza sativa L.)mutant with reduced fertility,Oryza sativa mitogen-activated protein kinase 6(osmapk6),which harbored a mutated MAPK gene.Scanning and transmission electron microscopy,quantitative RT-PCR analysis,TUNEL assays,RNA in situ hybridization,longitudinal and transverse histological sectioning,and map-based cloning were performed to characterize the osmapk6 mutant.The gene OsMAPK6 was expressed throughout the plant but predominantly in the microspore mother cells,tapetal cells,and microspores in the anther sac.Compared with the wild type,the total number of microspores was reduced in the osmapk6 mutant.The formation of microspore mother cells was reduced in the osmapk6 anther sac at an early stage of anther development,which was the primary reason for the decrease in the total number of microspores.Programmed cell death of some tapetal cells was delayed in osmapk6 anthers and affected exine formation in neighboring microspores.These results suggest that OsMAPK6 plays pivotal roles in microspore mother cell formation and tapetal cell degradation.

Isolated Microspore Culture and Plant Regeneration of Leaf Mustard(Brassica juncea(L.)Czern.et Coss.)

Isolated micmspore culture was carried out with Brassica juncea to study the effects of sterilization methods, culture period of embryos, hormone formula and the concentration of aetivatod carbon on embryogenesis, and the concentration of NAA on induction of roots. The results showed that the embryogenesis on medi- um subjected to filter sterilization is better than medium subjected to autoclaved sterilization. The embryos cultured for 15 d had the highest plantlat regenexafion rate （52%）. NLN-13 liquid medium including 0. 1mg/L 6-BA and 0.2 g/L activated carbon significantly improved the planflet regeneration rate. 1/2 MS inclu- ding 0.3 mg/L NAA had the highest plantlet regeneration rate （ 100% ）. Key words Brassica juncea ; Isolated micmspore culture ; Embryogenesis ; Planflet regeneration

Morphological Characteristics of Floral Organ at Different Microspore Developmental Stage in Balsam Pear （Momordica Charantia L.）

The relationship between microspore developmental stages and morphological characteristics of floral organ in balsam pear was studied. The results showed that flower buds developed a series of morphological changes at different microspore developmental stages, and morphological characteristics of floral organ were significantly different when sampling from different part or cultivar of balsam pear. Anther at late-uninucleate stage was best for culture, in which stage flower buds swelled obviously, and white floss on the surface reduced. In addition, calyxes were obvious but did not spread, and the most of anthers were light green.