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1.
Microspore embryogenesis is an inducible pathway interesting from basic and applied perspectives. For plant breeding, it is a powerful tool to produce doubled haploids, useful as pure lines. The most efficient way to produce them is through isolated microspore culture. In eggplant, one of the most important vegetable crops, this method is still poorly explored. So far, it is possible to produce doubled haploids, but not directly from embryos, because they are converted into calli early during their development. In this work we evaluated the effect of abscisic acid, epibrassinolide, polyethylene glycol, and arabinogalactans and arabinogalactan proteins, previously described as promoters of embryo induction and development in other species. When added individually to the standard protocol, all of them significantly increased induction of microspore embryogenesis and callus cell proliferation, producing more and larger calli. Particular combinations of them further improved the efficiency of the method. In particular, gum arabic containing arabinogalactans and arabinogalactan proteins allowed embryos to progress beyond the globular stage, constituting a significant improvement in order to achieve the desired direct induction of viable, germinating embryos. We also evaluated the effect of altering the concentration and relative ratio of naphthaleneacetic acid and 6-benzylaminopurine, used in the standard protocol. Significantly better results were obtained by reducing their concentration. Together, our results shed light on the morphogenic and regulatory roles of these substances on microspore embryogenesis, opening ways to further increase the efficiency of production of androgenic doubled haploids through microspore culture in eggplant. 相似文献
2.
We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research. 相似文献
3.
Haploid mutagenesis offers several advantages over conventional (seed) approach. However, its potential has not been utilised
for Brassica juncea, an important oilseed. In this study, mutant donor plants of three Indian B. juncea genotypes, generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU), were used for microspore culture. The response
of mutant donor plants was about 100 times lower than non-mutant controls; a total of 9,411 embryos were produced from the
EMS treated donor plants, while microspores isolated from ENU treated donors did not yield any embryos. The lethality of induced
mutations demonstrated itself mainly as the induction of abnormal embryos (80%), failure of germination (70%) and failure
of plantlet development (70%). Nine doubled haploid (DH) mutant lines and three non-mutant DH lines obtained through this
approach were tested for agronomic and biochemical variation over two growing seasons. High variability was observed and stable
mutants were recovered for reduced height (125 vs. 168 cm for the control), appressed pod character, altered fatty acid composition
higher protein proportion in de-oiled meal (48%) and a lower glucosinolate content in de-oiled meal (59.5 μM/g) relative to
controls. The approach demonstrates that despite severe reduction in efficiency of the DH line production, valuable mutants
can be recovered from mutated donor plants. 相似文献
4.
Traditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid breeding. 相似文献
5.
A. M. R. Ferrie D. C. Taylor S. L. MacKenzie G. Rakow J. P. Raney W. A. Keller 《Plant Breeding》2008,127(5):501-506
A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines. 相似文献
6.
A detailed procedure for isolated microspore culture of barley is presented along with examples of response across genotypes.
Over 30 genotypes, including winter and spring growth habit and 2-row and 6-row genotypes, have shown an essentially genotype
independent response, averaging about 10,000 embryos per 5 cm petri culture plate. The regeneration frequency, checked on
samples of 500 embryos per plate ranged from 36 to 97% with most genotypes being in the range of 70 to 90%. About 70 to 80%
of the plants regenerated have been completely fertile doubled haploids, thus eliminating the need to double the chromosome
number of plants. Many little details are critical to success of the microspore procedure and while it saves much time compared
to anther culture, greater attention to details and cleanliness is essential.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
7.
Cytoplasmic effects have been occasionally implicated in the inheritance of several traits in oilseed rape (Brassica napus L.), including linolenic acid concentration (18:3) in the oil. It is important that these be considered when choosing the
direction of cross for producing new breeding populations. To study this phenomenon, a reciprocal cross was made between two
genotypes of oilseed rape, Reston and LL09, which differed for their erucic and linolenic acid concentrations in the seed
oil. Two DH populations, which were produced by microspore culture from reciprocal F1 plants, were evaluated in the growth room for one generation and in the field at two locations in Southern Ontario in 1993and
1994. Field notes were taken on days to flower, days to maturity,plant lodging, plant height and, seed quality traits. In
the growth room study, the phenotypic distribution of 18:3 differed significantly between the two reciprocal DH populations.
In the field, significant reciprocal differences between the population means were detected for 18:3,flowering date and protein
content in both years and for days to maturity and oil content in 1993 only. To further study the parental lines,chloroplast
(cp) and mitochondrial (mt) DNA from parental lines were isolated and subjected to RFLP and RAPD analysis. Several random
primers revealed reproducible DNA polymorphism (RAPD) between the parental mt DNA. It is concluded that the direction of cross
should be taken into consideration by oilseed rape breeders relying solely on doubled haploids for developing genotypes with
modified seed quality traits in Brassica napus L.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
8.
Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating
emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired
FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this
method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing
a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62%
C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were
cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos
were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested
from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The
abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the
seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection
for high oleic acid can be started already during an early stage of the in vitro culture.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
Information from previously published studies that are basic to this study is: ) Following isolated barley microspore culture around % of the resulting barley plants are completely fertile genetically homozygous doubled haploids 《分子植物育种》2007,5(2):280-281
Information from previously published studies that are basic to this study is： 1） Following isolated barley microspore culture, around 80% of the resulting barley plants are completely fertile and genetically homozygous doubled haploids （DH） （Kasha et al., 2003）. 2） Chromosome doubling occurred during early stages of microspore culture, mainly by nuclear fusion （Kasha et al., 2001）. 3） A fairly synchronous population of microspores was selected to determine the cell cycle stages （G1, S, G2） by relative DNA density measurements （Shim and Kasha, 2003a）. 4） The pretreatments of spikes used to induce microspore embryogenesis influenced the cell cycle progression during pretreatment. （i） Using a combination of cold （4℃） and 0.3 mol/L mannitol for 4 days, the microspores were held at the G1 uninucleate stage. （ii） The often used pretreatment of cold （4℃ for 21 to 28 d） permitted slow microspore stage progression so that most cells were at the G2 stage or had become binucleate by the end of the pretreatment. （iii） Pretreatment with mannitol for 4 days at 25 ℃ did not block cell cycle progression but prevented cell wall formation after the 1^st mitotic division, leading to nuclear fusion and chromosome doubling. 相似文献
10.
Production of doubled haploids (DHs) through androgenesis induction is an important biotechnological tool for plant breeding. In some species, DHs are efficiently obtained through embryogenesis from isolated microspore cultures. In eggplant, however, this process is still at its infancy, despite the economic relevance of this important agricultural crop. To date, only two studies have focused previously on this process, suggesting that in eggplant microspore cultures, the only morphogenic response is callus formation. Given the notable lack of studies on eggplant microspore cultures, in this work we explored this process with different experimental approaches. We studied the response of different cultivars and characterized the development of microspores induced to divide and proliferate. We demonstrated that microspore-derived embryos (MDEs) can be produced in eggplant; however, MDEs stopped at the globular stage, to turn into euploid and principally mixoploid calli. From these calli, 60 % of DH plants could be regenerated. In order to promote microspore induction we evaluated the effect of polyethylene glycol (PEG) and mannitol. PEG, but not mannitol, significantly increased induction of microspore embryogenesis. We also tested the ability of eight different media compositions to promote efficient plant regeneration from calli. In order to test it in a genotype-independent manner, we previously developed a method to generate clonal callus populations derived from single microspore-derived calli. Together, the results presented hereby constitute an efficient way to produce eggplant DHs through microspore culture. In addition, they contribute significant insights into the knowledge of the particularities of androgenesis induction in this species. 相似文献
11.
The reduction of saturated fats in canola oil has recently been promoted as a goal for breeders for commercial and human health
benefits. Currently, saturated fatty acids in Canadian produced canola oil are above the 7% level, and the objective of this
study was to generate canola lines with reduced major saturates (palmitic and stearic), by several percent. Mutant embryos
generated from direct ultraviolet radiation mutagenesis of microspores in vitro were subjected to heat during the maturation stage. Heat artificially elevated the saturate levels in developing mutant embryos,
allowing efficient identification of those with reduced saturates within the expanded range using HPLC fatty acid analysis
of the embryo cotyledons. Mutagenesis produced embryos with fatty acids altered in both directions. Major saturate levels
in the cotyledons of heat-treated mutant embryos ranged from 3.3 to 16.4% (heated control ca. 6–9%) and 1.3–10% (heated control ca. 2–4%) for palmitic and stearic fatty acids, respectively. Doubled haploid seed derived from embryos grown at normal temperatures
confirmed the reduction of major saturates. HPLC fatty acid analysis of DH seed identified saturate levels ranging from 3.9
to 6.5% (control ca. 5.5%) and 0.9–2.7% (control ca. 1.7%) for palmitic and stearic fatty acids, respectively. Various doubled haploids were identified with major saturate levels
below 5.5%. Concomitant positive changes in the unsaturated fatty acids (18:1, 18:2, 18:3) among the mutant lines are also
discussed. 相似文献
12.
An isolated microspore culture procedure was used to produce doubled haploid lines of Brassica juncea from F1 plants of reciprocal crosses between the cultivar‘RLM514’and a canola quality breeding line. The inheritance of two qualitative markers, seed color and leaf hairiness, was compared using traditional and microspore-derived populations from these crosses. Chi-square tests indicated that each trait is controlled by different sets of duplicate pairs of genes. Brown seeds or hairy leaves can result from the presence of either of two dominant alleles, whereas yellow seed or glabrous leaves are produced when alleles at both loci are recessive. The segregation of genes controlling seed color and leaf hairs in doubled haploid progeny did not differ significantly from that expected under random assortment, indicating that doubled haploids can be used in this species for genetic studies, and probably cultivar development as well. 相似文献
13.
Md. Masud Karim Nazmoon Naher Tonu Mohammad Shakhawat Hossain Taketo Funaki M. Bahadur Meah Delwar M. Hossain M. Asad ud-doullah Eigo Fukai Keiichi Okazaki 《Euphytica》2016,208(3):535-544
Low erucic acid (LEA) rapeseed, which has accumulated mutant fatty acid elongase genes at the BnFAE1.1 and BnFAE1.2 loci of the A- and C-genome, respectively, is an important oilseed crop. Short growing turnip rape (B. rapa) is also important as a catch crop in the continuous cropping of rice in Asia but there is no LEA B. rapa cultivar for cultivation in South Asia. In order to develop LEA turnip rape cultivars, high erucic acid turnip rape cultivars were interspecifically crossed as recurrent parents to a canola quality rapeseed. In the meantime, we monitored incorporation of the mutant bnfae1.1 (e1) gene into A-genome of turnip rape, using a dCAPS primer pair, which can amplify PCR fragment only for the mutant e1 gene from A-genome. The early backcross progenies showed poor seed set, but which was improved in advanced progenies. Finally, homozygous e1e1 genotypes were established in the selfed progenies of BC2–BC3, and their LEA content was confirmed by gas-chromatography analysis. Our results and promising lines will contribute to LEA-trait selection in turnip rape and rapeseed breeding. 相似文献
14.
The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF1 hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 °C after being transferred to solid regeneration mediumand an initial period of low temperature (2 °C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration. 相似文献
15.
Anther culture was applied as a method to develop the essential components of a cytoplasmic male sterility hybrid system in
rye (Secale cereale L.). These components are the male sterile seed parent (A line), its isogenic maintainer counterpart (B
line) and the restorer pollen parent (R line). Australian rye cultivars were crossed reciprocally to the cultivar ‘Luchs’
which carries the Pampa male sterile cytoplasm (cms-P). Anthers of the F1s in the cms-P cytoplasm (primary cross) and their
reciprocals in the normal cytoplasm (reciprocal cross) were cultured in a modified C17 medium. Male sterile and male fertile
doubled haploids were obtained from the anther culture of the F1s in the cms-P cytoplasm. Testcrosses indicated that the male
sterile doubled haploids were A lines and the male fertile doubled haploids were R lines (restorers). The anther culture of
genotypes in the normal cytoplasm (reciprocal cross) gave all male fertile doubled haploids. Testcrosses indicated that the
male fertile doubled haploids were R lines (restorers) in the normal cytoplasm. The expected maintainer B lines were not identified
because of the limited number of doubled haploids obtained from the anther culture of reciprocal crosses. Experimental single
cross hybrids between male sterile and restorer male fertile doubled haploids showed high levels of heterosis. The results
of this study have significant economic implications especially in the production of hybrids in several species.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
16.
Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus 总被引：12，自引：0，他引：12
Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process. 相似文献
17.
Anther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F2 plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F2 segregation. (3) It was possible to recover indica type recombinants among the anther-derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice. 相似文献
18.
Gang Chen Jianfeng Geng Mukhlesur Rahman Xueping Liu Jingxing Tu Tingdong Fu Gengyi Li Peter B. E. McVetty M. Tahir 《Euphytica》2010,175(2):161-174
A genetic map was constructed with 353 sequence-related amplified polymorphism and 34 simple sequence repeat markers in oilseed
rape (Brassica napus L.). The map consists of 19 linkage groups and covers 1,868 cM of the rapeseed genome. A recombinant doubled haploid (DH)
population consisting of 150 lines segregating for oil content and other agronomic traits was produced using standard microspore
culture techniques. The DH lines were phenotyped for days to flowering, oil content in the seed, and seed yield at three locations
for 3 years, generating nine environments. Data from each of the environments were analyzed separately to detect quantitative
trait loci (QTL) for these three phenotypic traits. For oil content, 27 QTL were identified on 14 linkage groups; individual
QTL for oil content explained 4.20–30.20% of the total phenotypic variance. For seed yield, 18 QTL on 11 linkage groups were
identified, and the phenotypic variance for seed yield, as explained by a single locus, ranged from 4.61 to 24.44%. Twenty-two
QTL were also detected for days to flowering, and individual loci explained 4.41–48.28% of the total phenotypic variance. 相似文献
19.
Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale. 相似文献
20.
Hong-hao Lv Qing-biao Wang Li-mei Yang Zhi-yuan Fang Yu-mei Liu Mu Zhuang Yang-yong Zhang Yu-hong Yang Bing-yan Xie Xiao-wu Wang 《Euphytica》2014,200(3):465-473
Cabbage Fusarium wilt (CFW), caused by Fusarium oxysporum f. sp. conglutinans, is one of the most devastating diseases of cabbage worldwide. In recent years, CFW has been spreading across northern Chinad. Thus, development of resistant cultivars is necessary to control CFW. Here we report microspore culture from a cross of two elite cabbage inbred lines, 96–100 (highly resistant to CFW) and 01–20 (highly susceptible to CFW), in order to obtain doubled haploids (DHs) to cultivate cabbage materials for resistance breeding. A total of 196 DHs were obtained in 2011 and 2012 and three elite DH lines were selected from them through agronomic trait evaluation as well as marker-assisted selection. These three elite DH lines were crossed with elite cabbage inbred lines to create hybrids, which were then evaluated for their resistance and agronomic traits. Three excellent hybrids were selected. The use of microspore culture and marker-assisted selection greatly shortened the time required to obtain these excellent hybrids. These resistant varieties may become an effective control of CFW in diseased areas. 相似文献