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1.
The morphoregulatory effect of FeEDTA chelating complex was verified in an androgenically more productive cultivar of tobacco White Burley and inDatura innoxia Mill. The presence of EDTA in Nitsch (1969) medium (N medium) was sufficient for the conversion of isolated embryoids and embryoids in anthers into complete plants. The androgenic development was most rapid in N medium with FeEDTA. In N medium with EDTA, the cultures developed more slowly and remained vital for a long time. In the medium without EDTA, new embryoids developed continuously. The embryoids arose and developed also in closed anthers. All embryoids which arose in anthers cultured on N medium were capable of forming complete plants. The threshold morphoregulatory concentration of FeEDTA was 40 μM FeEDTA per 1 litre of medium for embryoids in anthers and 30 μM FeEDTA per 1 litre for isolated embryoids. The dark brown colour of the anthers of tobacco andD. innoxia was due to the presence of iron (FeEDTA) in the culture medium. The morphoregulatory effect of EDTA in N medium is explained by the formation of a complex with iron present in the form of pollution of chemicals and agar. This amount of iron in complex with EDTA is sufficient for the conversion of embryoids into plants, but it is insufficient for the browning of anthers.  相似文献   

2.
In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.  相似文献   

3.
The effect of FeSO4.7H2O, Fe2(SO4)3.9H2O, disodium salt of ethylene-diaminotetraacetic acid, dihydrate (EDTA) and N-(2-acetamido) iminodiacetic acid (ADA) and their combinations on the androgenesis was studiedin vitro in tobacco (cv. White Burley) and datura (Datura innoxia Mill.). Simultaneously the reversibility and irreversibility of the morphogenic process leading to the conversion of the pollen embryoid into complete plant was followed. Complete plants developed in anthers on media with trivalent iron, chelated trivalent iron, chelated bivalent iron, bivalent iron in the presence of ADA and of media with EDTA. The number of androgenic plants in anthers increased in the following order: Fe3+ < Fe3+ EDTA ≦ ≦ EDTA < Fe2+ EDTA. The marked brown colour of cultured anthers was due to the presence of trivalent iron in the medium. The androgenic development was most rapid on the medium containing only trivalent iron, slower on media with chelated iron and slowest on medium with EDTA. The viability of cultures with complete plants decreased in the reverse order. No complete plants grew on media without trivalent iron and without EDTA and on media containing only bivalent iron whereas globular embryoids arose and developed continuously on these media. The anthers reacted in the same way on both complete and minimal media. Isolated embryoids formed complete plants in corresponding variants on complete media only. The development of pollen embryoids into complete plants was stopped by the transfer of globular and torpedo-shaped embryoids from medium with EDTA to the medium without EDTA. Isolated greenish cotyledonar embryoids continued to grow even on the medium without EDTA.  相似文献   

4.
辣椒游离小孢子细胞团培养的胚状体形成   总被引:2,自引:0,他引:2  
从预培养15天后的花药中机械游离小孢子及其细胞团,经28℃液体悬浮暗培养.30天后,获得了自球形期胚到子叶期胚发育程度不等的各类胚状体。从12个花药中可以形成高达22个胚状体,且子叶期胚的比例约为23%。显微镜检表明,这些胚状体来自游离的小孢子细胞.经核的对称分裂形成多核细胞或者早期形成多细胞团,最后经细胞的分裂分化形成。胚状体体表具毛,活力有差异。在适当培养基上,具活力的鱼雷期及子叶期胚状体均能发育成正常植株。7℃、32℃、35℃8天的胁迫处理均能诱导小孢子胚状体发生。但花药培养中7℃、35℃处理下的出胚率较32℃下高,而游离小孢子细胞团培养中以35℃、32℃下较好。7℃处理下获得的胚状体数很少.对产生这种现象的原因进行了探讨。出胚率在基因型间,不同胁迫处理温度间表现明显差异。而在温度处理的不同天数间差异不明显。流式细胞仪对再生株真叶的DNA含量分析表明.获得的再生株中具有单倍体、双单倍体以及单倍一双倍嵌合体植株。本结果为进一步开展辣椒雄性生殖途径的胚状体发育研究。提高辣椒成熟胚状体的频率提供了实验体系。  相似文献   

5.
Somatic embryogenesis and subsequent diploid plants have been obtained from anthers of Vitis vinifera Cabernet-Sauvignon, a cultivar so far considered as recalcitrant to in vitro regeneration. Anthers enclosing microspores near the first pollen mitosis were found to be the most responsive. However, from a practical point of view anther length proved to be an easier criterium for determining the optimal physiological anther stage. Calli derived from the anther somatic tissues produced embryoids only when cultured on a medium supplemented with casein hydrolysate. Glutamine and adenine were found to stimulate this embryoid production. Evidence is presented that early removal of cotyledons increases the frequency of normal development of embryoids into plantlets.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - BA 6-benzylaminopurine  相似文献   

6.
Anthers of Populus maximowiczii with microspores at the mononucleate stage were cultured at 20°C in the dark on agar-solidified Murashige and Skoog medium after 4 days of cold treatment (4°C). After 4 to 8 weeks anthers on medium supplemented with 0.5, 1.0 or 2.0 mg l-1 2,4-D in combination with 0.1 mg l-1 kinetin developed calli that were characterized by smooth surface and gel-like consistency. These calli were comprised of expanding microspores surrounded by a mucilaginous matrix. After transfer of anthers with embryogenic calli to MS medium with low hormone levels (NAA at 0, 0.1 and 0.1 mg l-1 and BA at 0, 0.1 and 1.0 mg l-1) microspores started to divide and initiated independent meristematic nests, which developed into embryoidal structures, resembling globular to bi-polar heart-shaped embryoids. The embryoids germinated precociously without developing cotyledons. After transfer to medium with a range of levels of BA (1.0, 2.5 and 5.0 mg l-1), adventitious shoots developed mainly from the roots. Shoots were rooted in half strength MS medium supplemented with 0.025 mg l-1 NAA. Via this pathway anther response in the best treatment combination was 10%.Abbreviations BA benzyladenine - MS Murashige & Skoog - NAA naphthaleneacetic acid - 2,4-D-2,4 dichlorophenoxyacetic acid  相似文献   

7.
Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid  相似文献   

8.
Summary The yield of pollen embryoids from cultured Hevea anthers was increased 4 fold by optimizing the proportion of ammonium nitrate to potassium nitrate in the dedifferentiation medium. For optimal differentiation of pollen embryoids, kinetin, 2,4-D and -naphtalene acetic acid are required. Anther culture for 50 days on the dedifferentiation medium is a prerequisite for the selective development of calli and embryoids from microspores.The determination of chromosome numbers in embryoids, plantlets and regenerated trees reveals that they originate from (poly)haploid pollen grains (n=2x=18). Aneuploid, triploid (3x=27) and tetraploid (4x=36) cells were encountered in increasing frequencies as the embryoids and plants developed. A few haploid cells with 9 chromosomes were consistently observed. Buds from shoots with mixoploid chromosome numbers can be grafted and the change in the chromosome constitution of the developing new shoots followed.  相似文献   

9.
Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.  相似文献   

10.
Summary Solatium tuberosum L. diploid strains with superior androgenetic capacity have been selected for from androgenetic progenies of unselected diploid material. The paper also demonstrates that the use of a liquid medium for culturing potato anthers, instead of the conventional solid agar plates, improves the yield of androgenetic embryoids. The new method, associated with two successive cycles of selection for superior androgenetic response, allows the induction and regeneration of microspore derived plants on a large scale. The best genotype (clone 21 in this paper) regenerates androgenetic plants with a frequency around 30 per each anther plated. Over 80% of the regenerated plants are diploid. It is suggested that the androgenetic embryoids mainly originate from unreduced microspores by a mechanism which maintains a heterozygous or a partly heterozygous genetic situation.  相似文献   

11.
Direct somatic embryoids were initiated from orchardgrass (Dactylis glomerata L.) anthers and unpollinated pistils cultured in the dark at 25 C on Schenk and Hildebrandt (SH) medium supplemented with 30 μM dicamba (3,6 dichloro-o-anisic acid). Stereoscopic and scanning electron microscopy indicated that the embryoids originated from anther walls and from ovary and style regions of pistils. Callus initiation from direct embryoids leading to secondary embryogenesis was observed in pistils cultured from 4–6 wk. The ability of these calli to proliferate and initiate new embryoids through the dedifferentiation and redifferentiation of preexisting embryoids suggests long-term totipotency.  相似文献   

12.
Summary Pollen embryogenesis and subsequent plant regeneration have been established from cultured whole barley spikes in agitated N6 liquid medium (Chu 1978) containing high levels of 2,4-D, Ficoll and potato extract. Microspore division within the anthers and subsequent embryogenic development were obtained in medium containing high amounts of reduced nitrogen with Zeatin, NAA and BAP (all at 0.5 mg/l levels, pH 6.2). Once embryoids were formed in the liquid medium, they produced secondary embryoids from the scutellum and subsequently plants on MS (Murashige and Skoog 1962) agar medium containing BAP and NAA. The ratio of green plants to albino was 18.7.  相似文献   

13.
A. Rashid  H. E. Street 《Planta》1973,113(3):263-270
Summary Development of haploid embryoids from the microspores of Atropa belladonna occurs with relatively high frequency when anthers are excised from buds in which the petals are shorter than the sepals (at this stage microspores are predominantly uninucleate) and cultured on a medium containing iron as the ferric salt of ethylenediamine-di-O-hydroxyphenylacetic acid (FeEDDHA). Additions of combinations of kinetin, auxin and casamino-acids to the culture medium induce callusing in both haploid and diploid tissues, lead to the origin of embryoids from somatic tissues of the anther and should be avoided. Simple techniques for the maintenance of haploid clones are described.Stages in early embryogenesis in the pollen grains have been observed and these indicate that embryogenesis is most frequently initiated by an equal division in the uninucleate spore. The frequency of grains showing embryoid formation is very low and it is estimated that plantlets are formed from up to 50% of these grains.  相似文献   

14.
To obtain the maximal production of pollen embryoids in culturedanthers of Datura innoxia, the critical stage of anther developmentand the effect of physical factors, such as the precise modeof implantation of the anthers in the culture medium, light,temperature, and pH, were studied. In almost all media used,anthers containing uninucleate pollen were the best for initiationof embryogenesis. Variations in light and temperature also affecteddevelopment of the embryoids significantly. The percentage ofanthers producing pollen embryoids increased almost linearlywhen the temperature was raised from 22 to 30 °C. At lowertemperatures (15 to 20 °C) no embryoids were produced. Cultureskept in darkness produced embryoids, but upon transfer of culturesto the light the percentage of responding anthers increasedconsiderably.  相似文献   

15.
《Plant science》1987,48(1):49-54
Embryogenesis in isolated microspores of wheat (Triticum aestivum L.) leading to plant regeneration has been established on modified liquid N6 medium (supplemented with 2,4-D, casein hydrolysate and Ficoll). Globular embryoids which were obtained after 6–8 weeks of culture of competent embryogenic microspores produced perfect embryoids when transferred to regeneration medium. Embryoids were differentiated to plants on other modified N6 agar medium (0.75% w/v agar, 20 g/l sucrose, 1 g/l myo-inositol, 8.8 μM 6-benzylaminopurine (BAP), 11.4 μM indoleacetic acid (IAA), 160 mg/l glutamine, 10 mg/l proline). Responses of microspores in regeneration and embryoid differentiation varied depending on the constituents of the media and genotypes used.  相似文献   

16.
An embryogenic suspension culture was established from cultured inflorescence segments of Pennisetum americanum in Murashige and Skoog's medium supplemented with 2.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 5% coconut milk. The suspension was composed of two major cell types: 1) small, richly cytoplasmic and starch-containing cells, generally found in small, compact clumps, here termed embryogenic cells; and 2) elongated, thick-walled cells with large vacuoles. By manipulating the duration of culture and dilution ratios (cell suspension: fresh medium) at the time of subculture, suspensions consisting predominantly of embryogenic cells were obtained. Suspensions grown for 2-3 wks were transferred to agar media with reduced amounts of 2,4-D. This resulted in the production of hundreds of globular and early cotyledonary embryoids. Further development of the embryoids was promoted by their transfer to a medium containing abscisic acid. Many of the embryoids germinated and produced normal green plants. Atypical embryoids, some containing many shoot meristems and a leafy scutellum, were also observed. The relevance of such atypical embryoids in the interpretation of organogenesis and embryogenesis reported in tissue cultures of cereal species is discussed. It is also suggested that somatic embryogenesis occurs in tissue cultures of most, if not all, species of cereals and grasses.  相似文献   

17.
Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.  相似文献   

18.
Abstract

Considerations about our anther cultures of cultivated plants. – One of the main activities performed at the Casaccia Nuclear Centre, in the framework of a contract between CNEN and the European Communities, centers on the induction of haploid plants by anther culture and the subsequent chromosome doubling in order to obtain completely homozygous diploid plants. In tobacco, it is now possible to obtain haploid plants from any cultivar; we perform in vitro culture of internodes from which homozygous diploid plants are regenerated, taking advantage of natural phenomenon of endopolyploidy. In order to try to generalize this method of producing haploid plants in other plant species, we are studying the mechanism involved in haploid embryogenesis which occurs in vitro in the microspores. Datura, Nicotiana and Atropa are among the genera in which a direct embryogenesis from the microspore is observed; it is interesting to note that all three genera belong to the family Solanaceae and are very rich in alkaloids. In almost all the other cases of in vitro induction of haploids, microspores produce calli from which plantlets can be differentiated, but this way of plant regeneration is less interesting because only few plantlets are obtained and it is not sure that each haploid comes from a single microspore. We examined the factors which could influence the transformation of microspores into embryoids in tobacco, namely: the developmental stage of microspore, the degeneration of tapaetal cells, the genotype of microspore, the composition of cultural media, the physiological conditions of the plant from which the anthers were taken. From a practical point of view, it would be desirable to have informations on methods giving a maximum number of haploid plants from one embryogenic anther and the greatest number of embryogenic anthers from the cultured anthers. Our recent experiments on anther culture in liquid shaken medium have yielded good results (about 7,000 embryoids from 25 embryogenic anthers). Further, we are conducting several experiments in order to synchronize the development of the microspores in the anthers; to this end, we analyse the effect of cold treatment, ionizing radiation and gravity force. Experiments are being performed with other cultivated species, beside tobacco, in order to solve some problems of plant breeding more easily and quickly through haploidy. With the aim of introducing, in cultivated tomato, some desirable characters from the wild species, Lycopersicum peruvianum, (self-incompatibility, disease resistance, simultaneous flowering), we have obtained the interspecific hybrid through in vitro culture of young embryos. Haploid production from this hybrid could allow to quickly obtain various genetic recombinations from these two species. For this purpose we are carrying out anther cultures as well as single microspore cultures. In rice, strawberry and L. peruvianum, several diploid and tetraploid plantlets were obtained from our anther cultures. Work is in progress to ascertain the mode of their origin.  相似文献   

19.
Anthers ofPsidium guajava cultivated on either Murashige-Skoog's or Nitsch's basal medium (BM) or the BM supplemented with 10−6 M benzylaminopurine (BAP) were observed to contain microspores undergoing androgenic segmentations as well as a few multicellular microscopic embryoids. However, final morphogenic response from such cultured anthers was the development of calli. These calli had restricted growth accompanied by their early browning. Suspecting the browning to be due to accumulation of polyphenols, the culture medium was fortified with polyvinylpyrrolidone (PVP). PVP, increased sucrose concentration in the medium and cold pretreatment of anthers decreased the proportion of anthers turning brown as well as delayed the browning of calli, but it was not possible to maintain the calli for differentiation. Cold pretreatment significantly increased the percentage of callusing anthers and also resulted in the early emergence of calli.  相似文献   

20.
Laticifers were identified in frozen sections of embryoids from callus and suspension cultures of Asclepias syriaca (common milkweed) by an indirect fluorescent antibody technique. Sections were treated with the IgG fraction of rabbit anti-latex antiserum, produced with field-collected A. syriaca latex as a source of antigens, and with fluorescein-conjugated IgG fraction goat anti-rabbit IgG. Laticifers were identified by their fluorescence in embryoids dissected from 3–4-month-old callus cultures and in embryoids from 2-month-old suspension cultures. Laticifers are not present in early globular embryoids of A. syriaca but embryoids similar in shape to late globular stage zygotic embryos possess branching laticifers typical of zygotic material. Sections on control slides, treated with whole serum or IgG fraction from whole serum, both from an uninjected rabbit, contained no fluorescent cells. No laticifers were detected with the fluorescent antibody assay in A. tuberosa embryoids.  相似文献   

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