Pollen Tube Culture on a Lactose Medium

A modified sugar-agar technic for growing pollen tubes is described in which a Van Tieghem cell is used as a moist chamber, and 12% lactose substituted for the normally used sucrose. The advantages offered over previous technics are: (1) direct observation of germination and growth at any stage and for any period of time, (2) easier prevention of bacterial contamination with the long growth periods required to obtain pollen tube divisions, (3) simple method for making permanent slides which are suitable for cytological study.     

Palm Chromosome Studies Facilitated by Pollen Culture on a Colchicine-Lactose Medium

This technique is very useful where pollen is readily available and when roots or microspore mother cells are difficult to obtain or to process. It provides a relatively uniform means of studying chromosomes in a great number of species. Pollen is collected from buds at anthesis or the day before and sown on a medium containing H₃BO₃, 100 ppm; colchicine, 0.04%; lactose, 12%; gelatin, 5%; egg albumen, 1 drop in 10 ml. The gametic chromosome complement is studied at mitosis of the generative cell in the pollen tube. In species which have sufficiently large chromosomes it is possible to construct idiograms for comparative studies. All palm species here reported have a haploid complement of n = 18, and the chromosomes range in length from 0.5-3.5 μ.     

Rice Immature Pollen 1 (RIP1) is a Regulator of Late Pollen Development

The above article was published     

Effect of Combined Changes in Culture Medium and Incubation Conditions on the Regeneration from Immature Embryos of Elite Varieties of Winter Wheat

In this study, tissue culture method for plant regeneration from immature embryos of elite Hungarian winter wheat varieties was established. The influence of the growth regulators and the concentration of macroelements in the regeneration medium and of the incubation temperature and light intensity on regeneration frequency were investigated. The most noticeable effect on regeneration frequency was achieved by simultaneously reducing both the incubation temperature to 23 °C and the concentration of macroelements in the regeneration medium to half-strength. This modification increased the average regeneration frequency from about 10–78%. Changes in the light intensity and temperature gave an average plant regeneration frequency of 83%.     

Reversible Changes in the Composition of the Population of Mtdnas during Dedifferentiation and Regeneration in Tobacco

Differences in the composition of the population of mtDNAs between green plants and calli of tobacco were detected by DNA filter hybridization analysis. The altered composition of the population of mtDNAs observed in calli returned to the composition typical of green plants during the process of regeneration. Quantitative assays revealed that the changes were associated with the differentiation and dedifferentiation of cells since the extent of the change in composition depended on the degree of differentiation of a population of cells. The sequence that accumulated in dedifferentiated cells was shown to be a product of recombination mediated by a 9-nucleotide repeated element, one of which is located at the 5' region of atp6. Although the recombinant sequence was not detected by a hybridization procedure in green plants, its presence was identified by a more sensitive polymerase chain reaction method. The recombination event was shown to result in a deletion that prevents reverse recombination. Therefore, the reversion from the altered composition to the normal state of the population of mtDNAs during regeneration is explained not by recombination but by the preferential amplification of subgenomic mtDNA molecules.     

六月鲜枣的幼胚培养

本实验以自然受粉的六月鲜枣为材料,培养枣的幼胚发育成为正常完整植株。通过正交实验筛选获得的优化组合为Nitsch基本培养基 3%蔗糖 1.0mg/L ZT 0.6mg/L IAA DJ PVP。     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期（即生殖细胞在形成后从靠壁部位移向中央部位的阶段）的水稻（Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程。在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成。微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状。尔后,中心微丝不断瞎长,最终在细胞中央的胞质内形成一个非常 类似多个纺锤体结合在一起的网络结构。这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质（或称周质）的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构。未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系。     

Eimeria tenella: Incomplete Excystation in the Presence of EDTA in a Taurodeoxycholate-Based Medium

ABSTRACT. Normally, sporozoites of Eimeria tenella are efficiently excysted in vitro with trypsin and bile salts. However, a one hour treatment at °40C with a chelator-supplemented excystation medium (purified trypsin and chymotrypsin, taurodeoxycholate and ethylenediaminetetraacetic acid in buffered saline) produced incomplete excystation. The treatment removed the sporocyst plug and left an opened sporocyst containing motile sporozoites, but the release of sporozoites was greatly reduced (<12% release). Some of the sporozoites extended a portion of their anterior end through the sporocyst opening then retracted it into the sporocyst. Sporozoites were released when magnesium was added to the chelator-supplemented medium. Manganese was less effective and calcium was ineffective in producing release. Also, sporozoites were released when the incompletely excysted sporocysts were transferred to buffered saline with albumin and this became the basis for a new assay. The assay demonstrated that ethylenediaminetetraacetic acid reduced release in the presence of taurodeoxycholate but not in its absence. Hydrophobic and hydrophilic chelators were tested in the assay. Ethylene-dioxy diethylene-dinitrilotetraacetic acid and 8-hydroxyquinoline were inactive. The chelator 1,10-phenanthroline did not require bile salt to reduce release. The inhibitory effects by phenanthroline were eliminated in the presence of magnesium or manganese, while calcium had no effect. Thus, although certain chelators can inhibit release, a consistent correlation between chelation and inhibition of release has not been established. The application of ethylenediaminetetraacetic acid with taurodeoxycholate as a reversible inhibitor of release is discussed.     

Extensin Secreted into the Culture Medium by Tobacco Cells I. Purification and Some Properties

The medium of 12-day-old culturs of tobacco cells (Nicotianatabacum L., var Xanthi; line XD-6S) contain c.a. 160mg/literof protein, of which 14% of the constituent ami no acids werefound to be hydroxyproline. By sequential column chromatographiesand CsCl density-gradient centrifugation, a basic hydroxyproline-richglycoprotein was purified from the medium and found to havean amino acid composition typical of extensin; with a high levelof hydroxyproline (33mole%), tyrosine (13%), and lysine (14%).The glycoprotein contained 42% (w/w) of sugars, among whicharabinose was the major component (85%). The proportion of thisextensin in the proteins in the culture medium was estimatedto be much higher than that of arabino-galactan protein (about5 times higher) on a protein basis, with extensin comprisingbetween 25% and 41%, and probably about 37% of the proteinsin the medium. (Received September 19, 1988; Accepted December 26, 1988)     

Pollen Dimorphism--Origin and Significance in Pollen Plant Formation by Anther Culture

Pollen dimorphism during the ripening of Nicotiana tabacum antherstakes the form of differentiation at the binucleate pollen stageinto normal (N) grains, characterized by their high frequency,larger size, densely–staining cytoplasm and high starchcontent and into smaller (S) grains characterized by their variableand low frequency and weakly–staining cytoplasm. Mostof the S grains show distinctive vegetative and generative nuclei(A grains); a small number have two vegetative–type nuclei(B grains). Evidence is presented that when excised anthersare cultured, pollen plants arise only from S grains. It issuggested that the differentiation into N and S grains arisesby an abnormal second meiotic division in the pollen mothercells. Nicotiana tabacum, tobacco, pollen dimorphism, anther culture     

培养基组分、激素及pH值对转基因白桦花粉萌发的影响

采用花粉离体培养法研究了培养基不同组分、激素及pH值对转基因白桦（Betula platyphylla Suk.）花粉萌发的影响。结果表明,培养基组分在一定范围内促进花粉萌发,转基因白桦花粉萌发对硼酸和Ca^2＋的反应敏感,培养基中缺乏该物质时花粉不萌发。激素类物质低浓度时促进萌发,高浓度时抑制萌发。最适合培养基组分为蔗糖18%,硼酸0.015%,Ca^2＋ 0.020%,pH6.0,NAA 10mg/ml,6-BA 1mg/ml。并对不同保存条件（4℃,-20℃）下转基因白桦花粉的萌发率进行了比较,4℃下干燥保存更有利于保持转基因白桦花粉的活力。     

Nucleic-Acid and Protein Contents of Embryogenic Tobacco Pollen

Anthers of Nicotiana tabacum cv. White Burley containing microsporesin mitosis were cultured for 12-14 d at 23° C in order toinduce mitosis of the vegetative cell (embryogenic grains).After this period the total DNA content of such grains estimatedby gallocyanin-chrome alum photometry was double that of themicrospores in mitosis, whereas the total RNA content was reducedby about one-third. Total protein estimated by naphthol yellowS photometry was approximately the same as at inoculation. Incontrast, total RNA and total protein contents of non-embryogenicgrains in the same anthers were at least four times greaterthan at inoculation. It is suggested that degradation of cytoplasmicinformation concerned in gametophytic differentiation takesplace prior to mitosis of the vegetative cell.     

The In Vitro Culture of Immature Barley Embryos on Different Culture Media

Immature barley embryos (Hordeum distichum var. Julia) of between0•20 and 0•80 mm in length, were isolated from thedeveloping grain and cultured in vitro on various culture media.The subsequent development of the embryos was followed overa period of weeks, and where germination ensued the growth rateof shoot and root meristems was compared with in vivo germinationrates. Various growth media were assessed for their abilityto support normal development of immature embryos. A numberof published media failed to support satisfactory developmentof young embryos. The addition of 1–15 per cent coconutmilk to Norstog's Medium I (mineral + vitamin solns) enhancedembryo development and lowered the threshold of viability fromembryos of 0•50 mm in length to 0•35 mm. Althoughin many cases germination ensued, embryo development was largelyabnormal. A slightly greater enhancement of growth was achievedwith 0•05–0•30 per cent casein hydrolysate asthe growth medium supplement, although abnormal developmentwas not eliminated. A further lowering of the viability thresholdto include embryos of 0•25 mm in length was obtained bycombining 2•7 mM glutamine with the casein hydrolysatesupplement. Normal development and germination of embryos assmall as O25 mm was however obtained on Norstog's Medium JJand the results were reproduced in four additional if . distichumvarieties. In each case the critical threshold of viabilitywas found to lie in embryos of 0•20–0•30 mmin length.     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

Disposition of Pollen in situ and its Relevance to Anther/Pollen Culture

Disposition of pollen in immature anthers of Hordeum vulgareis illustrated by scanning and transmission electron microscopy.Freeze-fracture confirms that the pollen is confined to a uniseriatecolumn aligned against the tapetum. There is no free pollenin the lumen of the anther loculi. In contrast, in Nicotianatabacum and Paeonia lactlflora the pollen is disposed at randomand occupies the whole of each loculus. Freezing preserves thefluid content of the loculi, appearing in fracture profilesas an amorphous matrix in which the pollen is embedded. Thematrix, which generally obscures the tapetum, is present throughoutthe microspore phase but diminishes as the spores enlarge. Itis still present in N. tabacum and H. vulgare at the first pollendivision, fragmentary at this stage in P. lactiflora, but isno longer discernible in any of the species at the onset ofpollen-grain maturation. Pretreatment of excised Hordeum spikes at 4 ?C during the microsporephase, a prerequisite for anther/pollen culture, disrupts thenormal developmental sequence but does not alter the uniseriatedisposition. Before the spores start to divide, however, thetapetum degenerates and the fluid phase is dispersed. The observations are discussed in relation to isolated pollenculture, float culture of anthers and the switch in programmefrom gametophytic to sporophytic development. Key words: Pollen, Tapetum, Freeze-fracture     

Extensin Secreted into the Culture Medium of Tobacco Cells II. Extensin Subfamilies of Similar Composition

Combining acetic acid extraction and high-performance gel chromatographyin guanidine HCl, extensin secreted into the medium by tobacco(Nicotiana tabacum L. var Xanthi) culture cells was separatedinto three component molecules, namely a major 74-kDa, and twominor 45-and 28-kDa components, in addition to larger oligomers.The sizes of these native extensin molecules were first reasonablyassessed using this gel-chromatography system. After deglycosylationwith hydrogen fluoride, the separation was improved and theestimated molecular sizes were reduced to 52 kDa, 34 kDa and18 kDa, respectively. The amino acid compositions of these componentswere similar, and N-terminal sequences of the 52- and 34-kDacomponents coincided. The relative abundance of the componentswas as follows: oligomers, 46%; 52-kDa, 44%; 34-kDa, 7.7%; 18-kDa,2.2%; respectively, on a protein basis (w/w). Fluorography ofthe acid extract of microsomes from cells labelled with ¹⁴C-prolinerevealed only one precursor band of 110-kDa or 42-kDa underthe glycosylating or non-glycosylating conditions, respectively.The smaller components in the medium may be derived, by proteolyticcleavage, from the major extensin molecule after secretion. (Received October 15, 1990; Accepted May 15, 1991)     

Culture of Pollen Tubes for Chromosomal Analysis at the Pollen Tube Division

It is possible to grow pollen tubes routinely for cytological analysis of nuclei at the pollen tube division. Pollen has been grown successfully after temperature, pressure, gas, moisture, and radiation treatments. The technic for growing Tradescantia pollen is described, but any method is satisfactory which ensures that: (a) the pollen is kept dry before sowing, (b) a minimum of time elapses between sowing pollen on culture medium and placing in a moist growing box, and (c) the growing pollen tubes are not allowed to dry out. Pollen of other species can be grown by the same methods.     

Effects of Nutritional Factors on the Formation of Ubiquinone by Tobacco Plant Cells in Suspension Culture

The ubiquinone (UQ) content of BY–2 cells on surface culture was examined to compare with that in suspension culture. The UQ content on surface culture was a little lower than that in suspension culture, but the pattern of the time-course of the UQ formation on surface culture was similar.

The changes of UQ content in BY–2 cells during autolysis were also examined. UQ in the cells subjected to autolysis was not rapidly metabolized nor excreted into the medium.

In order to obtain basic information for UQ formation by BY–2 cells in suspension culture, the cultural conditions, especially nutritional ones were investigated. Addition of 2,4-D was remarkably effective on UQ formation and a higher UQ content was observed with a higher 2,4-D concentration. Sucrose and glucose concentrations in the original medium were also influential factors. The UQ content tends to increase with the decrease of the sugar content. Precursors of UQ, amino acids, vitamins and organic acids were not effective on the UQ formation.