Effect of certain inorganic chemicals on growth and spore germination of Alternaria tenuis auct., the fungus causing core rot of mandarin oranges in India

Summary Laboratory studies to determine the tolerance of an isolate ofAlternaria tenuis causing a black core rot of mandarin oranges (Citrus reticulata) have shown that presence of low amounts of manganese, zinc, iron, copper, boron, and molybdenum in a synthetic medium stimulated growth and sporulation of the fungus. Concentrations of 20 ppm or more of copper sulphate, boric acid, and ammonium molybdate, however, caused significant reduction of growth. Conidia of the fungus were very sensitive to even low amounts of copper, zinc, and iron. Copper sulphate was most toxic to spore germination followed, in order of inhibitory effect, by zinc sulphate, ferrous sulphate, and boric acid. Ammonium molybdate and manganese sulphate were stimulatory to spore germination at 5 ppm. There was a positive correlation between percentage of spore germination and average length of germ tubes in different concentrations of various chemicals. The possibility of using copper sulphate, zinc sulphate, and ferrous sulphate as spray materials for trees and as dip for ripe fruits to prevent black core rot are discussed.Assoc. Professor and Research Assistant, respectively. Publication under journal series No. 27 from Experiment Station, U.P. Agricultural University.     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.     

Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris

Summary In Brewbaker and Kwack's medium (BK) only 16% of the pollen grains germinated, and these produced pollen tubes having a maximum length of 25 m. With a solution based on Monnier's medium 47% germination and 160-mlong pollen tubes were observed. Calcium was shown to be essential for germination; the optimal concentration was 880 mg/l calcium chloride. The optimal concentrations of magnesium sulphate and boric acid were 360 and 50 mg/l, respectively. Germination at pH 4.0 but also pH 8.0 and the presence of vitamins B1 and B6 (1 mg/l each) were stimulatory. Polyethylene glycol (PEG) was superior to sucrose as an osmoticum and germination and tube length were significantly improved using PEG 4000 at a concentration of 120 g/l (0.03 M). Equimolar concentrations of PEG 400 and PEG 600 gave inferior results. Combining PEG with sucrose in the medium did not improve germination or increase tube length.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

The reversible inhibition of pollen germination of Nicotiana tabacum L.: an entry into a transformation system

Summary The hydrodynamics of mature pollen rehydration in Nicotiana tabacum was used to study reversible inhibition of pollen germination in vitro. Tobacco pollen was incubated for various times in media containing calcium, potassium and magnesium salts, boric acid, and exhibiting different osmotic pressures as a function of sucrose concentration. Total inhibition of germination with complete viability preservation was achieved for 56 h by keeping the grains in medium with 80% sucrose, since typical percentages of germination and pollen tube lengths were recovered after this treatment. These effects were considered as consequences of natural osmoregulation of rehydration/germination in mature pollen. The possibility of applying these findings to the incubation of pollen with Agrobacterium tumefaciens to develop a pollen transformation method is discussed.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

紫荆花粉离体萌发条件的研究

以紫荆(Cercis chinensis Bunge)花粉为材料,采用人工培养法研究了紫荆花粉离体萌发的最适条件.结果表明:蔗糖、硼酸在一定质量浓度范围内能促进花粉的萌发,最适花粉萌发的质量浓度分别为蔗糖50 mg/L以及硼酸20 mg/L,最佳萌发温度20℃.正交试验设计结果表明:花粉萌发的最佳质量浓度分剐为70 mg/L蔗糖以及18mg/L硼酸,最佳萌发温度为22℃,此时的萌发率可达60.51％.     

温度、蔗糖和硼酸对含笑花粉离体萌发的影响

以含笑(Michelia figo)花粉为试验材料,采用花粉人工培养法研究了含笑花粉离体萌发的最适条件。结果表明:蔗糖、硼酸在一定浓度范围内能促进花粉萌发,含笑花粉萌发的最适蔗糖和硼酸分别为50 mg.L-1和5mg.L-1,萌发率分别为21.51%和44.14%;花粉最佳萌发温度为25℃,萌发率达53.33%。正交试验设计结果表明,花粉萌发的最佳组合为70 mg.L-1蔗糖+5 mg.L-1硼酸+28℃,萌发率可达65.33%。     

STUDIES ON POLLEN GERMINATION OF CERTAIN CUCURBITACEAE

Vasil , I. K. (Delhi U., Delhi, India.) Studies on pollen germination of certain Cucurbitaceae. Amer. Jour. Bot. 47(4) : 239—247. Illus. 1960.—Pollen grains of 8 members of the family Cucurbitaceae were cultured in artificial nutrient media. Among the sugars, sucrose proved to be the best for germination of pollen. Although growth regulators, vitamins, antibiotics and some other chemical substances also improved germination of pollen and length of pollen tubes, the effect of boric acid (0.005—0.02%) and borax (0.01%) was most outstanding. The role of sugars and boron in the germination of pollen is discussed.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

硼酸和蔗糖对‘柔毛齿叶’睡莲花粉萌发的影响

以‘柔毛齿叶’睡莲（Nymphaea tetragona）花粉为试验材料,采用花粉人工培养法研究花粉离体萌发的最适条件。结果表明：蔗糖、硼酸在一定浓度范围内能促进花粉萌发,睡莲花粉萌发的最适蔗糖和硼酸浓度分别为60mg／L和15mg／L,萌发率分别达30．83％和23．73％。     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

东方百合花粉萌发培养基组分的优化

以东方百合6个品种的花粉作实验材料,选用22个培养基配方,在研究硼酸和蔗糖浓度对花粉萌发和花粉管伸长影响的基础上,对东方百合花粉培养基配方进行了优化。研究结果表明东方百合离体花粉萌发培养基的最佳组成是:蔗糖13%,硼酸143mg·kg^-1,琼脂1%。     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.     

In vitro pollen functionality of attacin-transgenic “Royal Gala” apple plants and apples transformed with 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-antisense vector

Abstract

To assess pollen functionality of transgenic apple trees, in vitro pollen germination and tube growth were evaluated. Flowers of transgenic “Royal Gala” apple lines containing attacin E gene to confer resistance to fire blight (Erwinia amylovora), or antisense 1-aminocyclopropane-1-carboxylic acid synthase (ACS) construct to improve fruit storage life, were collected, and pollen was harvested. Amongst the 19 transgenic lines, pollen from three lines transformed with an ACS-antisense vector consistently had significantly lower germination rate compared to “Royal Gala”; however, no correlation between ACS level in fruit and pollen germination rate was observed. Western blots showed that the amounts of the lytic protein, attacin, varied in pollen of the four attacin-transgenic lines sampled. There was no significant correlation between attacin level in the pollen and pollen germination rate or pollen tube growth. The addition of boric acid to the germination buffer enhanced germination in attacin-transgenic lines, as well as in lines down-regulated in ethylene synthesis and control “Royal Gala”. This initial study suggests that the majority of transgenic lines tested do not differ from the control “Royal Gala” in pollen germination, and that attacin or down-regulation of ethylene does not influence in vitro pollen functionality.     

小果短柱茶花粉离体培养系统的研究

山茶的短柱茶组是优良种质资源,有必要对小果短柱茶(Camellia confusa Chang 1941)的花粉萌发和花粉管生长的生理特性进行研究.本文研究了花粉生活力、培养温度及pH对小果短柱茶花粉萌发和花粉管生长的影响.结果表明:最适离体萌发培养基为5%蔗糖、0.003%的硼酸,0.005%的氯化钙和12%的PEG...     

ULTRASTRUCTURAL STUDY OF CAMELLIA JAPONICA POLLEN TREATED WITH MYRMICACIN,AN ANT-ORIGIN INHIBITOR

The germination and tube growth of Camellia japonica pollen were stopped on a myrmicacincontaining (50–200 ppm) culture medium, but were restored again when the pollen grains were transferred to a myrmicacin-free medium. Myrmicacin inhibits the movement of Golgi vesicles utilized for the formation of the callose layer in the pollen grain before the germination, and the growing tube tip wall.     

Development of an improved medium for germination of Cajanus cajan (L.) Millsp. pollen in vitro.

A simple, reliable medium for pollen germination of Cajanus cajan was developed by modifying Brewbaker and Kwack (BK) medium. Past attempts of C. cajan pollen germination in artificial media were not successful. A medium containing polyethylene glycol 4000 (PEG) showed more than 90% germination for C. cajan var. Pusa 33 only when the young buds (36 h before anthesis) were kept in pollen germination medium (PGM) for 36 h before pollen extraction. Supplementation of PGM with epsilon-amino caproic acid (EACA), an amino acid, showed improved pollen germination in Pusa 33 and also helped to avoid preconditioning of young buds before pollen extraction. It was also observed that there is a genotypic difference in the level of EACA required for in vitro pollen germination. Thus a complete medium for C. cajan genotypes consists of 37.5% sucrose+ 15% PEG 4000+250 mg l(-1) boric acid+300 mg l(-1) calcium nitrate+100 mg l(-1) potassium nitrate+ 200 mg l(-1) magnesium sulphate+1% agar+EACA (0, 100, 250, 500, 750 or 1000 mg l(-1)).     

梓树属花粉生活力的研究

以梓树属楸树组3种楸树花粉为试材,采用固体培养基法进行离体培养,研究不同浓度蔗糖、硼酸对花粉萌发的影响,以确定最适宜培养基条件下最佳的培养时间.结果表明,3个种花粉的最佳离体萌发条件一致.培养基中含有20%的蔗糖、0.005%的硼酸时较适合3种楸树花粉萌发,楸树、灰楸和滇楸萌发率分别为28.34%、12.24%和60.22%;3个种的花粉最佳培养时间均为6 h;不同重复数之间萌发率差异不显著,以3个重复为宜.花粉种内单株之间萌发率差异显著,且滇楸花粉萌发率明显高于楸树和灰楸.