RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.     

小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

Cracks in the palisade cuticle of soybean seed coats correlate with their permeability to water

BACKGROUND AND AIMS: Soybean (Glycine max) is among the many legumes that are well known for 'hardseededness'. This feature can be beneficial for long-term seed survival, but is undesirable for the food processing industry. There is substantial disagreement concerning the mechanisms and related structures that control the permeability properties of soybean seed coats. In this work, the structural component that controls water entry into the seed is identified. METHODS: Six soybean cultivars were tested for their seed coat permeabilities to water. To identify the structural feature(s) that may contribute to the determination of these permeabilities, fluorescent tracer dyes, and light and electron microscopic techniques were used. KEY RESULTS: The cultivar 'Tachanagaha' has the most permeable seed coat, 'OX 951' the least permeable seed coat, and the permeabilities of the rest ('Harovinton', 'Williams', 'Clark L 67-3469', and 'Harosoy 63') are intermediate. All seeds have surface deposits, depressions, a light line, and a cuticle about 0.2 microm thick overlaying the palisade layer. In permeable cultivars the cuticle tends to break, whereas in impermeable seeds of 'OX 951' it remains intact. In the case of permeable seed coats, the majority of the cracks are from 1 to 5 micro m wide and from 20 to 200 micro m long, and occur more frequently on the dorsal side than in other regions of the seed coat, a position that correlates with the site of initial water uptake. CONCLUSIONS: The cuticle of the palisade layer is the key factor that determines the permeability property of a soybean seed coat. The cuticle of a permeable seed coat is mechanically weak and develops small cracks through which water can pass. The cuticle of an impermeable seed coat is mechanically strong and does not crack under normal circumstances.     

DNA strand breakage by wheat germ type 1 topoisomerase

Properties of strand breakage in duplex and single-stranded DNA by the wheat germ type 1 DNA topoisomerase were investigated. Strand breakage in duplex DNA is dependent upon the use of denaturing conditions to inactivate the enzyme and terminate the reaction, whereas breakage of single-stranded DNA occurs under the normal reaction conditions and is not dependent upon denaturation. Breakage generates a free 5' hydroxyl group and enzyme bound to the 3' side of the break, presumably via the 3' phosphate group. The location of sites of breakage with both duplex and single-stranded DNA is not random. In all these respects the wheat germ enzyme closely resembles the rat liver type 1 topoisomerase. A comparison of the locations of the sites of breakage in duplex DNA generated by the topoisomerases from wheat germ and rat liver indicates a number of common sites, although the patterns of breakage are not identical.     

Differences in protodermal cell wall structure in zygotic and somatic embryos of <Emphasis Type="Italic">Daucus carota</Emphasis> (L.) cultured on solid and in liquid media

The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.     

Within-tree distribution of Ecdytolopha torticornis (Lepidoptera: Tortricidae) oviposition on macadamia nuts

Vertical distribution of eggs of the macadamia nutborer Ecdytolopha torticornis Meyrick (Lepidoptera: Tortricidae) and its preference of oviposition sites within and between macadamia cultivars were studied in Turrialba, Cartago, Costa Rica, in 1992 (N = 6,939). E. torticornis eggs were found throughout the foliar parts of the tree, but fewer eggs were laid in the crown top than in the mid or lower crown. Differences in the horizontal distribution of the eggs were not significant, albeit more eggs were found in the outer positions. The numbers of eggs found within the crowns of different clones were similar, implying that the nutborer has no preference for a particular cultivar.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

The fate of surface cell layers of Daucus carota (L.) embryos raised in suspension culture

The ultrastructure and fate of surface cells covering mature somatic embryos of Daucus carota grown in suspension culture were analyzed and new information obtained concerning somatic embryogenesis in these conditions. Our studies showed that during some developmental stages, these embryos were covered irregularly and discontinuously by cells with a typical protodermal phenotype characterized by a cuticle on the outer cell wall. We observed that cells with cuticles were peeled off from the surface of mature embryos. Before peeling off, these cells underwent programmed cell death, which was confirmed by the TdT-mediated dUTP nick end labeling method. Transmission electron microscopy revealed advanced processes of autophagy in these cells.     

Apple Fruit Cuticles and the Occurrence of Pores and Transcuticular Canals

Developmental studies were made on the dewaxed thin-sectionedapple fruit cuticles of 10 Malus sylvestris Mill, cultivarsfrom 4 weeks before anthesis through fruit maturation and harvest.Cuticular development appears to correlate well with the generalgrowth of the fruit. However, no correlation exists betweenfruit size and cuticle thickness. Cuticular pores were evidentas early as 1 week before anthesis and transcuticular canalsbecame evident by 1 week following anthesis. Dewaxed thin-sectioned,as well as isolated, mature fruit cuticles of 16 cultivars andfour crab-apples consistently revealed the distinct presenceof ubiquitous pores and canals. Evidence is provided by lightmicroscopy and scanning electron microscopy (SEM). Measurementswere taken of cuticle thicknesses during development and ofcuticular pore dimensions, and calculations were made of poreand canal numbers. Fruit size alone is not directly indicativeof total pore numbers per surface area. Canal lengths are directlyrelated to the developmental thickness of the cuticle. No correlationwas found between the thickness of the mature cuticle and eitherthe number of pores present or the pore diameters. Malus sylvestris Mill, apple fruit, morphology, cuticle, cuticular flanges, cuticular pores, transcuticular canals, ultrastructure     

Late Embryogeny of Pinus bungeana

Except for Pseudolarix, which is endemic to China, the late embryogeny of ten genera of Pinaceae has been reported before in the diffferent degree. Among them the mature embryos of Keteleeria evelyniana are different from those of the others in having well-developed cotyledons and a very short hypocotyl. As far as information we have is concerned there are three types in structure of the mature embryos of Pinaceae. The first type occurs in Keteleeria and Cedrus, which have very well-developed cotyledons; the second one appears in Picea and Larix, in which the hypocotyl and the root cap are equal in length; the third one, to which Pinus bungeana belongs, includes all the other genera of Pinaceae. The last type is of a prominent hypocotyl in the mature embryos. It is interesting to note that the mature embryos of Torreya grandis and Keteleeria evelyniana are very similar in having specially developed cotyledons, while the proportions of the various tissues in the mature embryo in Fokienia of Cupressaceae as well as Taxus and Amentotaxus of Taxaceae are similar to those of Pinus bungeana. The pith and secretory cells are usually present in the mature embryos of Pinaceae. Although no pith is present in those of Metasequoia and Taiwania of Taxodiaceae, the secretory cells generally occur in their embryos in Fokienia of Cupressaceae and Taxus and Pseudotaxus of Taxaceae, neither pith nor secretory cells are present in their hypocotyl. From above, the structures of mature embryos among Pinaceae, Taxodiaceae and Cupressaceae are different from one another to some degree. The most outstanding feature of the matur embryos in Pinus bungeana is that the shoot apex is very well developed, with a high H/D ratio, about 0.83 on an average, even up to 0.96 in some case. Above-mentioned H/D ratio of Pinus bungeana is rare in the mature embryos of conifers. Gifford (1943) reports that the average H/D ratio of shoot apex of Ephedra altissima is from 0.44 to 0.68, while in 5-year-old branch apex of Pinus ponderosa, the average ratio is about 0.25, and that in l5-year-old branch apex is about 0.35. For the apex of the dormant short shoots of Pinus densiflora the average ratio is about 0.35, but that of shoot apex is about 0.52 when new buds have just formed. According to the present data about shoot apices of both branches and mature embryos, the average ratio of shoot apex of mature embryos in Pinus bungeana is the largest one. From the present investigation the shoot apex of mature embryos of Pinus bungeana exhibits four distinct tissue zones, i.e. the apical initials, the central mother cell zone, the peripheral tissue zone and the rib meristem. It is worthy of note that the shoot apex of Pinus strobus may be divided into five zones, including transition zone between central mother cells and rib meristem (Owston, 1968). Four zones are recognized in the shoot apex of Pinus lambertiana and P. ponderosa, without transition one (Sacher, 1954). From cytological zonation, the shoot apex of mature embryos in Pinus bungeana is rather similar to that of Pinuslambertiana and P. ponderosa.     

In vitro plant regeneration from immature cotyledon explants of macadamia (Macadamia tetraphylla L. Johnson)

The macadamia tree, an Australian native, is highly valued for its nuts. Macadamia improvement programs so far have relied on conventional breeding and selection. The production of improved cultivars required to meet future demands could be accelerated by the application of modern biotechnological techniques, but this requires an efficient and reproducible regeneration system that has not yet been established for macadamia. We report here shoot regeneration from immature cotyledon explants of macadamia. Adventitious buds were induced on the cotyledon explants from fruits collected at 140 and 190 days after full bloom (DAFB) on MS medium supplemented with either 10 or 15 μM TDZ. The addition of 2% coconut milk (CM) to 10 μM TDZ containing media resulted in enhanced adventitious bud induction from 190 DAFB explants. Further shoot development from the induced buds was depressed in media containing TDZ + CM; the addition of 0.001 μM IAA to this combination doubled shoot development, from 1.9–3.9 shoots per explant. The transfer of bud clumps to media supplemented with 8.8 μM BA alone or in combination with either 0.14 μM GA₃ or 0.001 μM IAA significantly increased shoot production from the previously induced explants by 1.5–2 times of that observed in TDZ + CM medium. Histological examinations revealed that shoot regeneration was primarily by organogenesis originating from cells on or just below the cut surfaces of explants.     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

Gravitropic response of primary maize rootlets as influenced by light and temperature

Abstract. The gravitropic curvature of primary maize rootlets was measured as a function of temperature, both in the presence and absence of light. In two different cultivars, light strongly increased the downward curvature of roots developing from horizontally-oriented embryos. At 15–20°C, the bending angle was in the range of 70–80° in the light, and 25–50° in the dark, depending on the cultivar. When the temperature was increased above the 15–20°C range, marked differences were found between the two cultivars in their response to light. In one variety tested, JX180, the effect of light was relatively small at 30–35°C. Gravitropic curvature in another variety, Halamish, depended strongly on light throughout the temperature range tested. In both cultivars, gravitropic curvature was only slightly temperature dependent when germination and growth were in total darkness. In the dark, the extent of gravitropic curvature also depended on whether the kernels were oriented with their embryos facing upwards or downwards. Under continuous light, the gravitropic bending of roots of cultivar Halamish did not show a marked temperature dependence. When the seedlings were subjected to only a 15 min illumination, their gravitropic response was partial, and the dependence on temperature somewhat increased. In cultivar JX180, a combination of temperature and light modulates gravitropism. The gravitropic response of different maize cultivars thus differs considerably in its combined dependence on light and temperature.     

Embryo formation and regeneration in Triticum turgidum ssp. durum anther culture

Field grown Moroccan genotypes of durum wheat were tested for their capacity for androgenesis. The cultivar '1726' gave the best results with 25.37% embryos (embryos per 100 anthers) whereas '1715' was the least favorable with 3.17%. Spikes excised in early spring gave the best percentages of embryos. A chemical hybridizing agent (CHA) treatment of donor plants doubled the yield of embryos for almost all genotypes. A thermal pretreatment of spikes at 3 °C for 8 days improved androgenetic capacity. The C17 medium for embryo induction was consistently the best. Two media containing potato extract (BPTG and P2) gave intermediate results and N6 gave the lowest response. The highest percentage regeneration was obtained on C17 medium containing regulatory and amino substances, using embryos about 1 mm in diameter transferred to regeneration medium 21 days after their appearance. Regenerated plants were albino except for two green plants from cultivars 'Marzak' and '1658'. This revised version was published online in June 2006 with corrections to the Cover Date.     

棉花纤维发育过程中内源激素含量和纤维品质变化及其关系研究

以新疆棉区优质棉品种‘新陆早16号’、品质中等品种‘新陆早10号’和‘新陆早13号’以及品质较差品种‘02-DB’为材料,测定了棉纤维发育过程中内源生长素(IAA)、赤霉素(GA4)、玉米素(ZR)和脱落酸(ABA)含量和主要纤维品质指标的变化,分析内源激素含量变化与纤维品质形成的关系。结果表明:不同品种棉花纤维发育中纤维内源激素变化趋势基本相似,其差异主要表现在IAA、GA4、ZR和ABA的含量大小及峰值出现的时间方面。‘新陆早16号’在纤维发育前期有较高IAA、GA4、ZR含量和较低的ABA含量,表现出纤维伸长速率较高、快速伸长时期较长等特征;而且在次生壁加厚期ZR峰值出现较早,有利于棉纤维成熟,从而表现出较优的纤维品质。‘02-DB’在纤维发育前期由于ABA含量较高影响了纤维伸长速率和快速伸长期的时间,同时后期ZR峰值出现晚,使纤维发育受到影响,而最终品质较差。可见,在棉花纤维伸长期IAA、GA4、ZR含量高而ABA含量低、次生壁加厚期ZR峰值出现早则有利于优质棉纤维形成。     

Aflatoxin formation and varietal difference of cow pea (Vigna unguiculata (L.) Walp.) and garden pea (Pisum sativum L.) cultivars

Different cultivars of cow pea and garden pea seeds were surveyed for susceptibility or resistance towards the toxigenic and aflatoxin-producing mould (Aspergillus flavus IMI 102135). The results show that aflatoxin production varied among the different cultivars of both cow pea and garden pea. Morphological and histological characters of the different cultivars tested did not show any relation between colour, shape and size of seeds and the amount of aflatoxin produced. The chemical analysis of the different constituents obtained from both seed coats and seed kernels with susceptible, partially resistant and resistant cow pea and garden pea cultivars revealed that the resistant cultivars of cow pea (namely: Balady cultivar) and garden pea (namely: Melting Sugar cultivar) contained lower levels of sodium and higher levels of phosphate and potassium.     

Improved Regeneration Efficiency from Mature Embryos of Barley Cultivars

A reliable protocol for plant regeneration from mature embryo derived calli of nine barley (Hordeum vulgare) cultivars has been developed. The auxins 2,4-dichlorophenoxyacetic acid, picloram and dicamba proved effective in inducing callus from mature embryos of most of the barley cultivars. The induced primary callus was loose, friable and translucent. It ultimately yielded creamy white and compact callus after 2 - 3 transfers on fresh medium of the same composition. Callus induction and regeneration capacity were highly cultivar dependent. Addition of a high concentration of picloram (4 mg dm^-3) promoted regeneration in 3 cultivars (Tallon, Grimmett and Sloop). In cv. Arapiles, abscisic acid and betaine were crucial in generating morphogenic callus from the mature embryos. Plants regenerated from these calli were hardy and developed roots readily when transferred to hormone free medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

黄瓜对霜霉病抗性的显微和超微结构的研究

利用显微及透射和扫描电镜方法,研究了黄瓜对霜霉菌[Pseudoperonosporacubensis(Berk.et Curt.)Rostow.]的抗性机理,结果如下:抗病与感病品种叶表面的气孔密度和大小无明显区别,而病菌分生孢子在叶表面萌发情况有差异。抗病品种被病菌侵染后,细胞迅速颗粒化,与病菌一起死亡,两个侵染点之间的叶肉细胞大量繁殖,细胞中叶绿体减少,膜系统受到破坏,壁增厚,叶表面出现少量很小的病斑;中抗品种表现为少量菌丝蔓延,并产生分生孢子囊梗和孢子,受侵细胞的线粒体和叶绿体膜系统被破坏,随后内细胞和菌丝死亡,但比抗病品种慢,在叶表面形成很多病斑;感病品种表现为叶肉细胞内有大量菌丝蔓延,并产生分生孢子囊梗和孢子,受侵细胞被破坏、解体成碎片,最后与菌丝一起死亡,在叶表面联成大量的病斑。     

Genotype-specific normalization of soybean somatic embryogenesis through the use of an ethylene inhibitor

The effect of ethylene on somatic embryogenesis from cotyledons of soybean (Glycine max) cultivars `Bragg', `IAS-5', and `RS-7' was studied through the application of silver nitrate or aminoethoxyvinylglycine. The addition of these chemicals to the induction medium had no effect on embryo induction, in spite of aminoethoxyvinylglycine having decreased ethylene production and silver nitrate enhancing it. However, subsequent histodif-ferentiation and conversion capacity of somatic embryos was affected by treatments applied to the induction medium. The effects of ethylene on embryo histodifferentiation and conversion were genotype-specific. Cultivars `IAS-5' and `RS-7' produced high frequencies of dicotyledonous embryos and had high conversion rates. These were also the least affected by alterations in ethylene production. For `Bragg', which has a low regeneration capacity, the use of aminoethoxyvinylglycine led to a significant improvement in the frequency of normal embryo formation as well as in the frequency of conversion into plants. The results suggest that the use of ethylene inhibitors during the induction process may facilitate plant recovery from soybean genotypes, such as `Bragg', which have a low regeneration capacity. Received: 8 October 1996 / Revision received: 6 May 1997 / Accepted: 3 June 1997     

Regeneration of cassava plants via shoot organogenesis

A novel regeneration system based on direct shoot organogenesis is described for cassava. Plants could be regenerated at high frequency by inducing shoot primordia on explants derived from cotyledons of cassava somatic embryos. After a passage on elongation medium, the regenerated shoots were easily rooted in hormone-free medium and could be successfully transplanted to soil. Using the shoot-organogenesis-based regeneration method, up to eight transplantable plantlets per explant could be regenerated. The system was optimised first for one cassava cultivar, and then its transferability to three other cultivars was demonstrated. This method widens the scope of in vitro regeneration modes of cassava, and is also compatible with Agrobacterium-mediated transformation. To develop an efficient system for production of somatic embryos for regeneration experiments, conditions for inducing primary and cycling somatic embryos were also studied, and highly efficient plant regeneration via germination of somatic embryos was achieved using maltose instead of sucrose in the culture medium, and combining paclobutrazol with 2,4-dichlorophenoxyacetic acid in the embryo induction medium. Received: 25 January 1997 / Revision received: 10 February 1997 / Accepted: 20 February 1997