Transfer of germ plasm from the secondary to the primary gene pool in pennisetum

Summary Germ plasm from the A-genome of Pennisetum purpureum Schum. (AABB) of the secondary gene pool was transferred to cultivated pearl millet (AA) [P. glaucum (L.) R. Br.] by pollinating cytoplasmicnuclear male-sterile (cms) pearl millet with fertile allohexaploid pearl millet x P. purpureum hybrids (AAAABB). Certain allohexaploids used as pollinators on cms pearl millet resulted in 14-chromosome diploid pearl millet progenies. Three types of diploid pearl millet plants were produced in addition to the expected 28-chromosome AAAB-genome plants: (1) cms plants with only the A-genome, (2) cms plants with the A- and A-genomes, and (3) fertile plants with the A- and A-genomes. The latter group has allowed the utilization of genes for fertility restoration, stiff stalk, maturity, height, and morphological characteristics from the A-genome of P. purpureum in the pearl millet breeding program. Production of monoploid gametes by the allohexaploids appeared to be genetically controlled.     

Molecular analysis of mitochondrial DNA from rye (Secale cereale L.)

Summary Molecular characterization of mitochondrial (mt) DNA of rye (Secale cereale L.), free of significant amounts of contaminating chloroplast (cp) DNA, was initiated using the open-pollinated cultivar Halo as a source of mtDNA. Based on the compilation of data from restriction patterns, the molecular size of the mtDNA was estimated to be 410 Kb and its buoyant density was determined as 1.705 g/ml. Southern hybridization, using labelled cp genes (P700 and ribulosebiphosphate-carboxylase large subunit), indicated the presence of cpDNA-homologous regions on putative mtDNA fragments. Mt DNAs of inbred lines with fertile and cytoplasmic male sterile (CMS) Pampa cytoplasm were also analysed. Whereas the restriction patterns of mtDNAs of Halors and the fertile line turned out to be identical, Pampa mtDNA showed a unique restriction pattern, indicating (as in most other CMS systems) the involvement of mtDNA rearrangements in the expression of male sterility in rye. All 3 mtDNAs investigated contain regions homologous to the plasmid S1 of the CMS-S cytoplasm of Maize (Zea mays), as indicated by hybridization experiments. In Pampa cytoplasm the S-homologous sequence is located within a rearranged region of mtDNA.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.)

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m -naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.Abbreviations BA Benzyl adenine - IBA Indole-3-butyric acid - 2-ip N⁶-(-dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - NAA -Naphthaleneacetic acid     

A new type of cytoplasmic male sterility in rye (Secale cereale L.): analysis of mitochondrial DNA

Summary Mitochondrial (mt) DNA of a new type of rye cytoplasm (Gülzow, G) that induces cytoplasmic male sterility (CMS) was analyzed and compared with rye mtDNAs of different origins MtDNA of the G type was easily distinguishable from mtDNA of another CMS source, Pampa (P) type, and from mtDNA of fertile lines with respect to restriction fragment patterns and hybridization with mitochondrial genes. The results of the molecular analyses indicate a close, but not identical relationship between the mtDNA of the G type cytoplasm and that of cv Pluto.     

Asymmetric hybridization between cytoplasmic male-sterile (CMS) and fertile rice (Oryza safiva L.) protoplasts

Summary ⁶⁰Co-irradiated protoplasts of the cytoplasmic male-sterile line A-58 CMS (Oryza saliva L.) were electrofused with iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar Fujiminori. Seven of the colonies that formed were identified as cytoplasmic hybrids (cybrids): they all had the peroxidase isozymes of the fertile Fujiminori parent, but contained four plasmid-like DNAs (Bl, B2, B3 and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. In addition, digestion of cybrid mtDNA gave a set of restriction fragments that differed from those of the parents.     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Cytoplasmic male sterility in Petunia hybrida: factors affecting mitochondrial ATP export in normal and cytoplasmically male sterile plants

Summary In view of accumulating evidence that cytoplasmic male sterility (CMS) in some species results from an inability to generate the high ATP/ADP ratios required for specific stages of differentiation in the reproductive cycle, a number of aspects of ATP metabolism are being examined in CMS and male fertile plants.In experiments designed to test mitochondrial efficiency in ATP export, organelles from CMS plants performed very poorly when compared with normal lines. It is proposed that although most of the molecules involved in mitochondrial ATP production are nuclear encoded, the lesions in mitochondrial (mt)DNA known to accompany the CMS phenotype may be expressed as small modifications within the architecture of the mitochondrial membrane. To detect whether such changes could affect the ADP-ATP translocator in the membrane, two sets of experiments were carried out to determine a Km for the translocator. The two methods employed were based on different precepts, but nevertheless indicated a Km for the mitochondrial translocator in CMS lines which differed dramatically from that of male fertile plants. The view that CMS in Petunia hybrida thus might result from small differences in mtDNA encoded membrane proteins is considered in the light of the cytological changes seen to accompany CMS in these plants, as well as in the context of current theories advanced to explain CMS in other species.     

Novel cross-strand three-purine stack of the highly conserved 5′-GA/AAG-5′ internal loop at the 3′-end termini of Parvovirus Genomes

We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study a 2×3 asymmetric internal loop structure of the highly conserved `5-(GA)/(AAG)-5 bubble' present at the 3-end hairpin of the single-stranded DNA genome of parvoviruses. This motif contains an unpaired adenosine stacked between two bracketed sheared GA pairs. However, the phenomenal cross-strand G-G and A-A stacking in the tandem sheared GA pairs has undergone considerable change. A novel three-purine stacking pattern is observed instead; the inserted A18 base is completely un-stacked from its neighboring G17 and A19 bases, but well stacked with the cross-strand A4 and G3 bases to form a novel A4/A18/G3 stack that is different from the double G/G, A/A or quadruple G/G/G/G stack present in the 5-(GA)/(AG)-5 or 5-(GGA)/(AGG)-5 motifs. Unlike the bulged purine residue that usually causes about 20 degree kink in the helical axis of the parent helix when bracketed by canonical GC or AT base pairs, no significant kink is observed in the present helix containing a bulged-adenine that is bracketed by sheared G A pairs. The phosphodiesters connecting G3-A4 and G17-A18 residues adopt unusual torsional angles close to the trans domain, yet that connecting A18-A19 residues resumes the normal (g <sup>–</sup>) value. The well structured `5-(GAA)/(AG)-5' internal loop in the parvovirus genomes explains its resistance to single-strand specific endonuclease susceptibility.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The -isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and -ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate -ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the -ketoisocaproate-mediated inhibition is discussed.Abbreviations IPM -isopropylmalate - KIC -ketoisocaproate - KIV -ketoisovalerate - DTNB 5:5 Dithiobis-(2-nitrobenzoe acid)     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

Restoration of male fertileNicotiana by fusion of protoplasts derived from two different cytoplasmic male-sterile cybrids

Summary Using the donor-recipient protoplast-fusion technique, we have recently constructed several alloplasmic-like lines ofNicotiana in which the original cytoplasms (or part of them) of eitherN. tabacum orN. sylvestris were replaced respectively, either byN. undulata or byN. bigelovii cytoplasms. These cybridizations resulted in two kinds of cytoplasmic male-sterile (CMS) cybrid plants:N. tabacum withN. undulata-like cytoplasm andN. sylvestris withN. bigelovii-like cytoplasm. Fusion of protoplasts, derived from the above two CMS types, by the donor-recipient technique, lead to the recovery of 21 cybrid calli. One of these regenerated a cybrid with fertile pollen but having shortened filaments and slighly tappered anthers. Self pollination of the latter cybrid resulted in a second generation progeny having almost normal filaments and anthers. Further selfings produced a third generation in which numerous plants had normal stamens and fertile pollen. Mitochondrial DNA (mtDNA) analysis of second and third generation progenies revealed a novel pattern which differed from each of the parental CMS cybrids and also from the mtDNA of normal, male-fertileNicotiana species. The results suggest that mtDNA recombination between different types of CMS can lead to restoration of male-fertility.Paper presented at the 1st Plant Molecular Biology Congress, Savannah, Georgia, Oct./Nov. 1985.     

Glycogen phosphorylase ‘b’ in Dictyostelium: stability and endogenous phosphorylation

Summary The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase b form requires 5 AMP for activity and is present in early development. The active phosphorylase a form is 5 AMP independent and occurs during later development. We here show that the 92 kd b enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn²⁺ and ATP, the phosphorylase b shows apparent conversion into a 5 AMP independent form as measured by enzyme activity. In addition, Mn²⁺ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase b subunit. We also demonstrate phosphorylation of the b enzyme subunit in vivo by ³²-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase b subunit is characterized.     

The effect of leaf source and developmental stage on shoot organogenic potential of sweetgum (Liquidambar styraciflua L.) leaf explants

Leaf explants harvested from shoot proliferating cultures and intact plants of Liquidambar styraciflua Variegata were placed on solidified Woody Plant medium supplemented with 0.1 mgl^-1 (0.5 M) naphthaleneacetic acid and 2.5 mgl^-1 (11.1 M) benzyladenine to initiate shoot meristems directly. Leaves from intact plants produced over 4 times more adventitious shoots than leaves from in vitro shoots and had a greater tendency to form shoots on the lamina. The relative developmental age of leaf tissue used dramatically influenced the shoot organogenic response observed for leaf explants from intact plants of L. styraciflua Variegata and Moraine.-Leaves that were either 20% or 50% of full size and still actively expanding were superior to other developmental stages for shoot organogenesis. As developmental leaf age increased throughout the period of leaf expansion, the number of shoots forming on the petiole stub remained constant, whereas shoot formation on the lamina increased 8 fold. Shoots derived from Variegata leaves rooted well and grew normally as plants. Differences in rooting ability and plant size could be detected between groups that had been separated according to explant source (in vitro vs. intact plant) and the location of shoot formation (petiole vs. lamina).     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

High crossability of wild barley (Hordeum spontaneum C. Koch) with bread wheat and the differential elimination of barley chromosomes in the hybrids

Four bread wheat (Triticum aestivum L.) cultivars, Aobakomugi, Chinese Spring, Norin 61 and Shinchunaga, were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, Betzes, Kinai 5 and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, Betzes, and some Japanese wheat cultivars were better as the female parent than Chinese Spring. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from Betzes-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.     

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were Corbit (first subculture); GAF/Park and 88Ab3073 (second subculture); and GAF/Park and 87Ab5932 (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. GAF/Park, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes GAF/Park, 88Ab3073, and Tibor. The smaller clusters only regenerated plants for GAF/Park and 88Ab3073. From one gram of callus used to initiate suspensions of GAF/Park and 88Ab3073, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of GAF/Park, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

α-Subunit Composition of Nicotinic Cholinoreceptors of Neurons of the Guinea Pig Inferior Mesenteric Ganglion: an Immunohistochemical Study

Using immunoperoxidase labeling, we studied the subunit composition of nicotinic acetylcholine receptors, nAChR, in preparations of the inferior mesenteric ganglion, IMG, of the guinea pig. Antibodies against synthetic peptides corresponding to agonist-binding membrane components of the ₃, ₄, ₅, and ₇ nAChR subunits were used. The presence of ₃-specific antibodies was revealed on the membranes of about 58% of large neurons and of all small ganglionic cells (means of the greater and smaller diameters of the somata 53.8 ± 1.8 vs 33.6 ± 1.4 m, n = 20, and 14.1 ± 0.5 vs 7.5 ± 0.4 m, n = 50, respectively). Labeled cells of the rostral node were distributed evenly, while those of the caudal node were localized mostly within the regions of branching of the lumbar, colonic, and both hypogastric tracts. Immune labels to the ₄ subunit were observed only on the membranes of small ganglionic cells distributed mostly in the region of the internodal commissural tracts. ₅-Specific labeling was found on the membranes of about 63% large neurons, whose distribution was similar to that of the ₃-labeled units, and on all small cells. Immunoreactivity to the ₇ subunit was observed only on the membranes of small cells concentrated around unlabeled large neurons in the region of branching of the intermesenteric, colonic, and both hypogastric tracts. Thus, nAChR in the guinea pig IMG include ₃, ₄, ₅, and ₇ subunits. The nAChR with ₃ and ₅ subunits are localized on the membranes of large ganglionic neurons, whose number and topographical distribution are very close to each other. Our data agree with our results of earlier electrophysiological experiments and are indicative of the crucial role of the ₃- and ₅-containing nAChR in synaptic transmission via the ganglion under study. The presence of the ₄- and ₇-containing nAChR was found only on small ganglionic cells (which are, probably, not the relay units) and their processes.