Studies on Conditions for Cell Division and Embryogenesis in Isolated Pollen Culture of Nicotiana rustica

A method for the induction of a high rate of cell division and embryogenesis of Nicotiana rustica pollen was developed. Binucleate pollen grains were fractionated by Percoll density gradient (35/45%) centrifugation and cultured in 0.4 molar mannitol at 30°C (the first culture). After 3 days in culture pollen was recollected by a second Percoll fractionation (0/30%) and transferred to and cultured in a medium containing the Murashige-Skoog macro-elements, 0.4 molar mannitol, 40 millimolar galactose, 3 millimolar glutamine, and 5 micromolar ABA for 10 days (the second culture). The cell population consisting of about 80% dividing pollen was transferred to a Murashige-Skoog medium containing 0.4 molar mannitol, 3 millimolar glutamine, and no phytohormone (the third culture), where about 40% of dividing pollen developed into embryos or embryogenic calli.     

Embryoid Formation in Pollen Grains of Nicotiana tabacum

Anthers of Nicotiana tabacum (n = 24) were cultured on nutrientagar and examined at intervals for pollen embryoids. Embryoidswere formed in anthers of varying developmental stage, the youngestof which coincided with the liberation of free microspores fromtetrads, and the oldest with the formation of bicellular grains.This period in the development of the anther occupied 4–5days. Older anthers within this range were more successful thanyounger anthers. The first mitosis of the pollen was typicallyasymmetric and resulted in the formation of unequal generativeand vegetative cells. Some of the grains then went into a lagphase for at least 5–6 days, after which the mitotic conditionwas restored. Embryoids were formed by repeated division ofthe vegetative cell. If the generative cell divided, it didso only once or twice. Occasionally the first mitosis was symmetricand gave rise to equal cells, and in these instances both cellsprobably participated in embryoid formation. The youngest anthersexamined were probably less successful because fewer grainssurvived to enter mitosis. The number of embryoids produced varied considerably from oneanther to another both within the same bud and between differentbuds: values ranging from less than 400 to 10 000 per antherwere encountered. Most of these degenerated after the firstfew divisions, partly because they burst prematurely from thepollen grain wall. Embryoids which continued to develop formedplantlets and/or callus. The largest number of plantlets obtainedfrom one anther was 32. Haploid plantlets were also regeneratedfrom callus by transferring it to a low-sugar medium withoutauxin. The behaviour of grains not forming embryoids was also noted.     

Problems Encountered in the Culture of Isolated Pollen of a Burley Cultivar of Nicotiana tabacum

Pollen isolated from anthers of a Burley cultivar of N. tabacumby an anther homogenization procedure failed in culture to retainviability and produce plantlets even when isolation was precededby 14 d of anther preculture. By surgical manipulation of anthersprior to their culture it was shown that pollen viability wasvery sensitive to injury caused to the anther wall. Plantletscould be obtained however by culture of anthers in which oneor both pollen sacs had been carefully opened along the dehisenceline (DL). The somewhat lower yield of plantlets from such antherscompared to that from intact anthers is considered to resultnot from any change in the atmospheric environment of the pollenas a result of opening the pollen sac but from the inabilityin every case to keep the incisions strictly along the dehiscenceline. Pollen isolated from 14 d precultured anthers by this DL techniquewas able to yield plantlets when cultured in a simple definedmedium. However DL-isolated grains from 4 d precultured anthersfailed to retain viability in culture. It is concluded that pollen of this cultivar suffers severeinjury when isolated by the homogenization method and that thiscan be overcome by the DL technique. However conditions of culturehave not been established which reproduce for this cultivarthe nutritional conditions operating within the anther duringits early period in culture and which are essential to plantletformation.     

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 – 16 kDa (group 1), 27 – 28 kDa (group 3: chitinases) and 36 – 40 kDa (group 2a: -1,3-glucanases).     

Osmotic Adjustment of Cultured Tobacco Cells (Nicotiana tabacum var. Samsum) Grown on Sodium Chloride

Tobacco cell cultures (var. Samsum) were grown on increasing levels of NaCl to select variants for increased salt tolerance. The osmotic adjustment of NaCl-adapted and nonadapted cell lines was studied. Both cell lines were grown on modified Linsmaier and Skoog medium with or without NaCl. Few differences were found in the response of adapted and nonadapted lines to NaCl.     

Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.     

Embryogenesis and Plantlet Formation in Tissue Cultures of Celery

Callus cultures were initiated from sections of petioles ofcelery (Apium graveolens) var. Lathom Blanching on a modificationof the Murashige and Skoog medium. The callus, when isolatedfrom the parent explant, could be sub-cultured at regular intervalsforming both new callus and embryoids during each subculture.The embryoids appeared to be initiated on the callus surfacewhere early embryonic forms were found, but their continueddevelopment into plants only occurred when embryoids were transferredto a hormonefree medium. Embryoids were also formed in suspensionculture following inoculation of the liquid medium by differentiatingcallus, but again development of these structures was limitedto the early embryonic forms. When transferred to a solid hormone-freemedium, the embryoids, from either callus or suspension culture,developed into plantlets which could be transferred to soiland grown to maturity with only a few losses. The relevanceof this system as an aid in the breeding programme of self-blanchingcelery is discussed.     

Anther Culture in Nicotiana tabacum: The Role of the Culture Vessel Atmosphere in Pollen Embryo Induction and Growth

A comparison of four volumes of culture vessel atmosphere revealedthat this variable greatly influenced the induction and growthof pollen embryos from anthers of Nicotiana tabacum. The optimumfrequency of anthers producing embryos and plantlets was foundwith a culture atmosphere of 15 ml per anther, whereas the optimumnumber of plantlets was found with 5.5 ml per anther. A smallvolume (0.5 ml per anther) almost completely suppressed embryoinduction. Removal of specific components of the culture atmosphere(ethylene, carbon dioxide, oxygen) influenced the response ofthe anthers but did not produce a satisfactory explanation ofthe inhibition of pollen embryogenesis by the small cultureatmosphere volume. In particular, the influence of ethyleneabsorption on embryo induction and growth depended both on theculture atmosphere volume and on the stage of development ofthe pollen at the start of culture. Using anthers containingpollen at a stage after the first pollen grain mitosis. ethyleneabsorption was found to increase the survival of induced embryos.Treatment of anthers for 3 d with silver nitrate, a known antagonistof ethylene action, was not an efficient means of increasingthe yield of pollen plantlets.     

烟草花发育过程中花药和花粉IAA分布规律的研究

以烟草（Nicotiana tabacumL.）花药为材料,通过4’,6-二脒基-2-苯基吲哚（DAPI）染色详细观察花粉发育过程,获得了花药发育时期与花蕾大小的对应关系;通过吲哚乙酸（IAA）单克隆抗体、免疫组织化学技术以及DR5∶∶GUS转基因植株的GUS活性对花药和花粉发育过程中生长素的分布规律进行了研究。免疫酶标记结果表明,在不同的花药发育时期IAA水平呈现出明显的差别。小孢子母细胞时期,IAA在整个花药中均有分布,并且在小孢子母细胞发育晚期,IAA信号集中在小孢子母细胞的细胞核中;随着小孢子母细胞减数分裂后形成四分体,IAA信号逐渐减弱,四分体中几乎没有信号;单核花粉期的花药中IAA信号进一步减弱,仅存在于花药壁中;待小孢子继续发育为成熟二核期时,花粉和整个花药组织中均出现较强的IAA信号。GUS活性检测结果表明,烟草DR5∶∶GUS转基因植株中花药和花粉粒的GUS信号与IAA免疫酶定位结果基本一致。总的来说,IAA在烟草花药和花粉中的积累呈现出由强到弱、再由弱到强的分布规律,暗示IAA在被子植物花药和花粉发育过程中可能起着较为重要的作用。     

Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

The Presence of the N' Gene in Nicotiana tabacum L. cv. Samsun NN

The N′ gene was shown to be present in the ‘Samsun NN’ line of Nicotiana tabacum by its differential response to tobacco mosaic (TMV) and tomato mosaic (ToMV) viruses in the segregating and test-cross generations of the cross ‘Samsun NN’בSamsun nn’. The appearance of phenotypes reacting with local necrosis to ToMV and with systemic mosaic to TMV confirmed that the genes N and N′ are on distinct loci. An isogenic N′;N′ Samsun line was selected.     

低温胁迫下甘肃黄花烟草愈伤组织的抗氰呼吸

继代培养的ｉｃｏｔｉｏｎｏ ｒｕｓｉｔｏａ Ｌ．ｃｖ．Ｇａｎｓｕ Ｙｅｌｌｏｗ Ｆｌｏｗｅｒ）愈伤组织经低温（４℃）处理后,交替途径容量９Ｖａｌｔ）和实际运行量（ρＶａｈ）均被显著地诱导,同时其电子传递途径也在发生交替变化。低温胁迫开始时以细胞色素途径为主,至第２天时则以交替途径为主,并一直持续到第５天,以后则又转为经细胞色素途径为主,而对照却始终以细胞色素途径为主。经放红菌酮或氯霉素处理后,受低     

The Cyanide-resistant Respiration in Callus of Nicotiana rustica cv. Gansu Yellow Flower Under Low Temperature

The increasement of the operation (ρValt) and the capacity of the alternative pathway (Valt) in tobacco (Nicotiana rustica L. cv. Gansu Yellow Flower) callus that treated by low temperature (4 ℃) were observed. The two electron transport pathways were changed alternatively, the cyanide-resistant respiration was the main respiratory pathway from the 2nd day to the 5th day, while the cytochrome pathway was the main pathway consistently in control callus. Cycloheximide and chloramphenicol treatment which inhibited protein synthesis resulted in dramatic and partly inhibition of Valt induced by low temperature respectively. This further proved above result that low temperature induced Valt. The same result was found when two inhibitors were used together. The analysis of heat production of callus indicated that the operation of the alternative pathway could be regulated to improve the micro-environmental temperature of tobacco callus to adapt to low temperature. The possible participation of the alternative pathway in the response to low temperature and its physiological roles were discussed.     

Analysis of fertility in somatic hybrids of Nicotiana rustica and N. tabacum and progeny over two sexual generations

Summary Somatic hybrid plants, produced between Nicotiana rustica and N. tabacum by heterokaryon isolation and culture and also by mutant complementation, were examined regarding their ability to set seed. From a total of seventeen independent somatic hybrids, three were found to be partially self-fertile while the others did not set seed. Differences regarding the methods of hybrid selection, parental varieties and chloroplast composition of hybrids did not appear to be significant regarding the ability of plants to set seed. Much variation in fertility was observed in subsequent generations and by recurrent selection of the most fertile, over two generations, it was possible to increase the level of self-fertility in some of the progeny. One R2 derivative possessed approximately a tenfold higher level of self-fertility than it's somatic hybrid parent. The presence of genetic markers from both parents were observed in all progeny indicating their hybrid nature.     

皱叶山苏的组织培养和植株再生

1植物名称皱叶山苏(Asplenium antiquum Makinocv‘.Victoria’)。2材料类别孢子。3培养条件孢子萌发及原叶体形成的培养基:(1)1/2MS。丛芽分化及增殖培养基:(2)1/2MS;(3)1/2MS 6-BA0.5mg·L-1(单位下同) NAA0.1;(4)1/2MS 6-BA1.0 NAA0.1     

Effect of X-radiation on pollen tube growth and seed setting in crosses between Nicotiana tabacum and N. rustica

Summary In vitro andin vivo studies of the effect of irradiating pollen with different doses of X-rays were carried out inNicotiana rustica andN. tabacum. Dosages upto 9600 r were found to enhance the rate of growth of pollen tubers per unit time. Advantage was taken of this observation to overcome the incompatibility normally found in the crossN. tabacum x N. rustica as a result of the inadequate growth ofrustica pollen tubes intabacum style. Thus, whenrustica pollen exposed to 4800 r and 9600 r of X-rays were used to pollinatetabacum, fertilization resulting in the formation of viable hybrid seeds occurred. In the crossN. rustica×N. tabacum there is partial seed failure as a result of the hyperplastic development of the nucellar and endothelial cells. However, when X-rayedtabacum pollen were used in the cross, seed development registered a marked improvement. Since there is now evidence for suggesting that the contents of even those pollen tubes which do not participate in fertilization have some role to play in the development of the seed, it seems likely that radiation-induced biochemical changes in pollen may prove advantageous when conditions for seed development are abnormal as in instances of somatoplastic sterility. Besides such indirect biochemical effects, irradiation of pollen may also lead to genetic changes resulting in the inactivation of zygotic lethals. Thus, there is much scope for investigating the use of radiation to overcome inter-specific and inter-generic cross-incompatibility barriers.With 2 Figures in the Text