Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.     

Higher flower bud formation in haploid tobacco is connected with higher peroxidase/IAA-oxidase activity,lower IAA content and ethylene production

Haploid tobacco plants (cv. Samsun) form inflorescences with a larger number of flowers than diploid plants. Leaves of haploid plants were shown to have lower free IAA level (by 40 %), higher peroxidase (by 160 %) and IAA-oxidase (by 70 %) activities and produce less ethylene (by 25 %) than leaves of corresponding diploid plants. The increase of peroxidase activity in haploids was due to the increase in the activity of the cathodic isozyme which is known to have high IAA-oxidase activity. It is proposed that higher peroxidase/IAA-oxidase activity in haploid plants may take part in IAA catabolism, at least duringin vitro culture of haploid explants. Lowered IAA level and ethylene production may then be directly correlated with a larger number of flower buds; as a higher IAA level is generally considered to act as a background inhibitor of flowering.     

Micropopulation differentiation in phenol content and susceptibility to herbivory in the Chilean kelp Lessonia nigrescenss (Phaeophyta,Laminariales)

Micropopulation differences in phenol content between intertidal and subtidal individuals of the kelp Lessonia nigrescens were found. Subtidal plants showed: (1) significantly higher phenol content than intertidal individuals, in vegetative and reproductive tissues, (2) intra-plant differences, with higher content in apical frond tissues, (3) higher resistance to consumption by herbivorous fishes. The microscopic progeny of subtidal plants showed the same trend as adult plants: (1) haploid spores from subtidal plants had higher phenol content than spores from intertidal individuals, and (2) the microscopic sporophytes derived from subtidal spores and gametophytes were less consumed by herbivorous snails (Tegula tridentata) than those derived from intertidal plant propagules. No increase in phenol content was detected after mechanical injury to experimental fronds, or after transplantation to the subtidal environment.In addition to the absence of inducible responses, the different phenol content between intertidal and subtidal individuals, in adult diploid plants and also in the haploid progeny, suggests that both environments differ someway enough to fix the mentioned features on the plants of Lessonia nigrescens. It is likely that the differences in herbivory between the two distributional extremes contributed to the observed pattern.     

Characterization of the antioxidant system during the vegetative development of pea plants

The antioxidative system was studied during the development of pea plants. The reduced glutathione (GSH) content was higher in shoots than in roots, but a greater redox state of glutathione existed in roots compared with shoots, at least after 7 d of growth. The 3-d-old seedlings showed the highest content of oxidised ascorbate (DHA), which correlated with the ascorbate oxidase (AAO) activity. Also, the roots exhibited higher DHA content than shoots, correlated with their higher AAO activity. The activities of antioxidant enzymes were much higher in shoots than in roots. Ascorbate peroxidase (APX) activity decreased during the progression of growth in both shoots and roots, whereas peroxidase (POX) activity strongly increased in roots, reflecting a correlation between POX activity and the enhancement of growth. Catalase activity from shoots reached values nearly 3 or 4-fold higher than in roots. The monodehydroascorbate reductase (MDHAR) activity was higher in young seedlings than in more mature tissues, and in roots a decrease in MDHAR was noticed at the 11^th day. No dehydroascorbate reductase (DHAR) was detected in roots from the pea plants and DHAR values detected in seedlings and in shoots were much lower than those of MDHAR. In shoots, GR decreased with the progression of growth, whereas in roots an increase was seen on the 9^th and 11^th days. Finally, superoxide dismutase (SOD) activity increased in shoots during the progression of growth, but specific SOD activity was higher in roots than in shoots.     

The biosynthesis of nicotinamide adenine dinucleotide during early stages of frog embryonic development of haploid and diploid embryos.

1. Concentration of NAD during embryonic development of haploid and diploid embryos of frog was followed. NAD content in haploid embryonic forms is twice that in diploid embryos. 2. The variation of the NMN adenylyltransferase activity in the oocytes and during the first states of embryonic development as surveyed in the nuclear soluble fraction and the nuclear insoluble fraction (chromatin). 3. The enzyme activity in the soluble fraction is low during embryonic development and shows higher values in haploid embryos. 4. In the nonfertilized mature oocytes, the NMN adenylyltransferase activity is sixfold higher in the insoluble chromatin fraction than in the soluble fraction. 5. The evolution of the NMN adenylyltransferase in the insoluble chromatin fraction also shows higher values in haploid embryos, as compared with diploid forms.     

Cytological Studies on Tissue Culture of Pinus cembra

Callus tissue of Swiss stone pine, Pinus cembra L. var. sibirica Loud., has been grown successfully for 9 months through 7 transfers. Excellent initial growth was obtained from hypocotyl segments of 7-day-old seedlings. A complex agar medium was used supplemented with 2 mg/l 2,4-D, 0.05 mg/l kinetin, 1 g/l Edamin and 10% v/v coconut milk. No deterioration of growth was observed on this medium after numerous transfers. The callus tissue did not produce shoots or roots, but showed an ability for cytodifferentiation. Tracheids or tracheid-like structures were formed in every passage, but the tracheids of the primary callus culture differed markedly from those of the subcultures. The tracheids of the primary culture probably already existed in the hypocotyl, whereas those of the subcultures were formed in vitro. The callus tissue was mainly diploid during the period studied, and more than 86% of the mitoses were diploid. A few mitoses with tetraploidy or a higher ploidy also occurred, but the tissue did not show any tendency to polyploidization.     

Callus growth and somatic embryogenesis in thalamus tissue ofRanunculus asiaticus L. CultivatedIn vitro: Cytokinin effect and phenol metabolism

Summary The effect of cytokinin on growth and plant regeneration of thalamus-derived calluses ofRanunculus asiaticus L. has been investigated with various concentrations of 6-benzyladenine and 6-furfurylaminopurine (kinetin), in a medium containing 2,4-dichlorophenoxyacetic acid levels, which was decreased to 0 over three subcultures. Cytokinins, although not essential, for initiating callus production, improved subsequent callus growth and plant regeneration. No somatic embryogenesis was observed on calluses grown on media lacking cytokinins or containing only kinetin. Calluses manifested embryogenesis on media containing 6-benzyladenie plus kinetin or only 6-benzyladenine. Nondifferentiating callus was characterized by a high content of phenolic polymers and an elevated peroxidase and polyphenol oxidase activity in comparison with differentiating callus. Differences in simple phenol concentrations were observed in the two kinds of callus.     

Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene

Ponkan (Citrus reticulata Blanco), one of the most important commercial cultivars of mandarin, is very seedy. In this study, the chimeric ribonuclease gene (barnase) driven by an anther tapetum-specific promoter (pTA29) was introduced into embryogenic callus of Ponkan by Agrobacterium-mediated transformation using the bar gene as a selectable marker. In contrast to previous reports, embryogenic calluses were used as the explant for Agrobacterium infection and transgenic plant regeneration. Selection of transformed callus was accomplished using basta. After 3 days of co-culture, calluses were transferred to MT medium with 50 mg/l basta and 400 mg/l cefotaxime. Resistant calluses were recovered and proliferated after three to four subcultures and then regenerated plantlets. A total of 52 resistant plants were recovered, of which 43 were verified to be transformants by polymerase chain reaction amplification of a fragment of the transgene. Southern hybridization of seven randomly selected transformed plants further confirmed their transgenic nature. The potential of this strategy for breeding citrus seedless types is discussed.     

The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

三七.人参和西洋参细胞悬浮培养的比较研究

用薄层层析对三七、人参和西洋参愈伤组织进行的初步鉴定表明,三种愈伤组织都含有皂甙和主要皂甙成分Rb_1、Rg_1,三七愈伤组织还含有一种抗癌皂甙Rh_1。对愈伤组织的生长,三七低于人参高于西洋参;对愈伤组织中总皂甙含量,三七均高于人参和西洋参。三种植物细胞悬浮培养结果类似于他们的愈伤组织培养,但生长又进一步提高。三七细胞悬浮培养中皂甙产生的时间进程几乎与生长平行,合适的收获期为培养30天。寡糖素不仅增强三七培养细胞的皂甙形成而且促进细胞生长,较合适的浓度为1.25 ppm。通过以上研究,使三七悬浮培养细胞的生长(干重增加178毫克)为最初培养愈伤组织的4倍以上,总皂甙产率高达20.6毫克,为最初培养愈伤组织的8.5倍。     

Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily ‘Connecticut King’

A system for producing haploid plants from anther cultures was developed for the Asiatic hybrid lily ‘Connecticut King’. Anthers containing microspores at the mid- to late-uninucleate stages were cultured on MS media supplemented with various plant growth regulators. Microspores containing 3 or 4 vegetative-like nuclei were observed 2 to 3 weeks later, and yellowish nodular calluses appeared within dehisced anthers 2 to 3 months after culture. Picloram was superior to 2,4-d for inducing nodular calluses. Anthers from greenhouse-grown plants required higher concentrations of both picloram and cytokinins than those from field-grown plants and most frequently produced nodular calluses (17.6%) on MS medium containing 2 mg 1⁻¹ picloram and 2 mg 1⁻¹ zeatin. The nodular calluses regenerated many bulblets following transfer to MS medium supplemented with 0.1 or 0.5 mg 1⁻¹ picloram and 0.01 mg 1⁻¹ BA, and the bulblets developed into plantlets (bulblets with scaly leaves and roots) after transfer to MS medium containing 0.1 mg 1⁻¹ NAA. Chromosome counts of root-tip cells of 11 plantlets revealed that five were haploids (2n = 12), two diploids (2n = 24), and four mixoploid. This result suggests that at least some plantlets were of gametophytic origin.     

Biochemical Properties of Haploid and Diploid Strains of Penicillium chrysogenum

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of β-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.     

黄芩过氧化物酶同工酶电泳和抗坏血酸过氧化物酶活性分析

对黄芩（Scutellaria baicalensis Georgi)二倍体和同源四倍体过氧化物酶进行了同工酶电泳分析及抗坏血酸过氧化物酶活性测定。结果表明,黄芩二倍体与同源四倍体过氧化物酶同工酶酶谱一致,但后者的着色程度大于前者,二年生黄芩叶子在快速区Rf为0．494和0．512处出现新的谱带,不同发育阶段和不同组织器官的谱带存在明显差异;根和叶中谱带数最多,花其次,种子最少;试管苗谱带数先减少后增加,并在整个培养过程中出现特征性谱带C。各组织器官抗坏血酸过氧化物酶总活力差异明显,顶芽最高,叶子次之,花最低。黄芩一年生和二年生各个多倍体株系叶子抗坏血酸过氧酶总活力均高于二倍体叶子抗坏血酸过氧化酶总活力。试管苗生长过程中抗坏血酶过氧化物酶活活力的变化与生长趋势一致,表明该酶与植株生长发育紧密相关。     

Effect of certain dicarboxylic acid monoesters on growth,chlorophyll content,chlorophyllase and peroxidase activities,and gas-exchange of young maize plants

The effect of some dicarboxylic acid monoesters on growth, chlorophyll content, chlorophyllase (EC 3.1.1.14), and total peroxidase (EC 1.11.1.7.) activities was examined in detached and intact leaves of maize (Zea mays) plants grown in a greenhouse. The -monomethyl ester of itaconic acid (MEIA) at 1250 ppm had no effect on growth. However, application of the monoethyl ester of succinic (MESA) and monoethyl ester of adipic (MEAdA) acids (1250 ppm) resulted in an increased leaf area, fresh and dry weight of leaves and stems. These compounds retarded chlorophyll degradation in both detached and intact leaves. Chlorophyllase activity of the control and treated leaves was measured and related to chlorophyll content. Delaying of senescence by treatment with monoesters resulted in greater chlorophyll and protein content, compared with the control. However, the chlorophyllase activity/chlorophylla ratio in the treated plants decreased. Total peroxidase activity was higher in senescent leaves, but all treatments inhibited the increase of this enzyme activity. Prolonged carbon assimilative activity and enhanced leaf water use efficiency in treated plants was noted.     

Evidence for a senescence-associated gene induced by darkness

Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H₂O₂ via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.     

A tissue culture method for the preservation of Soybean mosaic virus strains

The activity and longevity of Soybean mosaic virus (SMV) in soybean callus culture were investigated with 11 SMV strains which are distinguished by differential reactions on soybean cultivars [Glycine max (L.) Merr.]. Dual cultures (soybean callus and SMV) were initiated by direct culture of SMV-infected leaves from susceptible soybean plants on Msoy and MS agar medium. Established SMV-callus cultures were maintained at 25 °C under light, subcultured to fresh MS medium at 2-month intervals or as necessary, and assayed periodically for virus infectivity. The infected calluses on MS medium grew better and stayed active longer than those on Msoy medium. At 10–15 °C, calluses and SMV were viable and active for 13–15 weeks or longer without subculture. The infectivity of SMV from callus cultures was comparable with that of SMV from infected plants, and remained stable for more than a year through five successive subcultures. Callus tissues of dual cultures were uniformly infected by SMV, thus ensuring infectious subcultures by random transfers. Production of in vitro inoculum can be significantly increased by multiple subcultures. Biological integrity of the SMV cultures was maintained with no change of viral virulence and pathotype. The method is of value for preserving a collection of SMV strains in a highly infectious and readily available form and reduces the chance of contamination or loss in viability.     

Crystallization of Ribulose, 1,5-Bisphosphate Carboxylase of High Specific Activity from Anther-Derived Haploid Plants of Nicotiana tabacum

Haploid plants were generated from anther-culture of Nicotianatabacum cv. Burley Ky 14. Plants that developed from pollenwithout an intervening callus stage were grown to maturity.Ploidy of the anther-derived plants were verified by chromosomecounts in root tip squashes. Crystalline ribulose 1,5-bisphosphatecarboxylase was purified from leaves of mature haploid plantsgrown in a growth chamber. Crystals appeared to be similar tothose from the diploid parental line. The specific activityof the enzyme was higher than those reported earlier in theliterature using similar crystallization procedures in Nicotianatabacum Key words: Nicotiana, haploid, ribulose 1, 5-bisphosphate carboxylase     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).     

Comparative study of the effectiveness of selection in diploid and autotetraploid populations]

The mitotic activity and ploidy of cells of three strains of cellus tissue by the addition of the culture medium of 0,01; 0,1 and 1 mg/l of phytohemagglutinin was studied. These strains derived from the leaves of haploid, diploid, and tetraploid plants of Lycopersicon, esculentum. The number of dividing cells under the influence of phytohemagglutinin increased by 1;5 and 4% for haploid, diploid and tetraploid strains respectively. The shift of the peak of the mitotic activity at early stages of subculture under the effect of phytohemagglutinin was noticed. Phytohemagglutinin did not change the frequencies of cells of different ploidy in all the strains analysed. A weak mutagenic activity of phytohemagglutinin in the concentrations used was observed. Cells with single bridges were more frequently observed among the aberrant cells.     

Production,maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

A method is described for producing and maintaining Stevia rebaudiana suspensions and regeneration of plants from calli derived from cell suspensions. Suspension cultures composed of isolated cells (ca. 10%) and cellular aggregates (5–100 cells) were obtained in 20–30 days by using friable callus as the initial inoculum in liquid medi with BA (0.5 mg/l)+2,4-D (1.0 mg/l), and periodic filtering (100–500 m sieves) with 6–7 days interval between subcultures. Cultures derived from actively growing calli are mainly diploid (2n=22) whereas those derived from senescent calli showed a wide variation in chromosome number (55–200). Stock cell suspensions which had been maintained for 3 years were plated on basal LS agar medium with BA (0.5 mg/l)+2,4D (0.5 mg/l) to form callus. Calli originating from predominantly 2n cell suspensions when transferred to medium with K (2.0 mg/l)+NAA (0.02 mg/l) were able to form buds. Shoot elongation and further rooting of isolated shoots was better on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological characters and chromosome number was found amongst regenerated plants.Abbreviations BA Benzylaminopurine - 2,4-D 2,4 - Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - LS Linsmaier & Skoog