Considerations on overcoming intrasomatic selection during mutation breeding of vegetatively propagated plants

Summary When seeds and vegetative tissues are irradiated, a competition occurs in meristematic tissues between lethally and sublethally affected cells on one hand and unaffected cells on the other hand to the advantage of the latter. This phenomenon was first recognized by Freisleben and Lein (1943) and termed intrasomatic selection by Kaplan (1951) and diplontic selection by Gaul (1957). The work done on the nature of this phenomenon in seed-propagated plants is summarized along with suggested methods to reduce its intensity.The generally unencouraging results obtained so far in vegetatively propagated plants have been attributed to the more intensive operation of this phenomenon in this group of plants, consequent on the more complex nature of apical meristems and propagating materials. The work of Zwintzscher (1959) in apples and Bauer (1957) in black currants have shown that it is possible to overcome its effects with suitable handling of irradiated materials.The radiogenetic studies done by the author in the potato have been reviewed in this context. Low intensity irradiation, dose fractionation and irradiation of physiologically dormant tubers gave higher survival value. The method of isolating sprouts from irradiated tubers and growing them, and scoring for mutations in sproutling progenies was found to increase mutation frequency considerably. Other methods proposed for minimizing the effects of intrasomatic selection in vegetatively propagated plants are chronic irradiation, neutron irradiation, chemical mutagenesis and lastly, mutagenesis of isolated single cells and growing them into whole plants.

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Zusammenfassung Bei Bestrahlung von Samen und vegetativen Geweben konkurrieren in den meristematischen Geweben letal und subletal geschädigte Zellen einerseits und unbetroffene Zellen andererseits, wobei die letzteren überwiegen. Diese Feststellung wurde zuerst von Freisleben und Lein (1943) gemacht und durch Kaplan (1951) als intrasomatische Selektion und durch Gaul (1957) als diplontische Selektion bezeichnet. In der vorliegenden Arbeit sind die an samenvermehrten Pflanzen vorgenommenen Untersuchungen dieses Phänomens zusammengestellt mit den vorgeschlagenen Methoden zur Verminderung dieser Erscheinung.Die im allgemeinen wenig ermutigenden Ergebnisse, die bisher bei vegetativ vermehrten Pflanzen erzielt wurden, sind einem verstärkten Auftreten der intrasomatischen Selektion als Folge der komplexeren Art der apikalen Meristeme und der Fortpflanzungsorgane bei dieser Pflanzengruppe zugeschrieben worden. Zwintzscher (1959) und Bauer (1957) haben durch ihre Arbeiten an Äpfeln bzw. Schwarzen Johannisbeeren gezeigt, daß durch eine entsprechende Behandlung des bestrahlten Materials eine Ausschaltung dieses Effektes möglich ist.In diesem Zusammenhang wird über die Bestrahlungsuntersuchungen des Verfassers an Kartoffeln berichtet. Höhere Überlebenswerte wurden durch geringere Bestrahlungsintensität, durch Dosisfraktionierung und Bestrahlung physiologisch ruhender Knollen erreicht. Eine Isolierung und Aufzucht von Keimen bestrahlter Knollen ergab eine erhebliche Steigerung der Mutationshäufigkeit in den Nachkommenschaften. Andere zur Verminderung der Wirkungen der intrasomatischen Selektion vegetativ vermehrter Pflanzen vorgeschlagene Methoden sind Dauerbestrahlung, Bestrahlung mit Neutronen, chemische Mutagenese und schließlich Mutagenese isolierter einzelner Zellen und ihre Aufzucht als ganze Pflanze.

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Invitational Paper presented at the Seminar on Use of Radioisotopes and Radiations in Agriculture and Animal Husbandry, 28 March–1 April 1966, Indian Council of Agricultural Research, New Delhi, India.     

Naturally occurring anhydrovitamin A2: Transformation into retinene2

1. ;Naturally occurring anhydrovitamin A(2)' obtained from the liver oil of freshwater fish Bagarius bagarius yielded, after six-stage chromatography, a pure product showing characteristic bands at 350, 368 (E(1%) (1cm.) 1006) and 390mmu in ethanol, and producing a green colour with antimony trichloride (E(1%) (1cm.) 1884 at 693mmu). 2. On distribution of the material between light petroleum and 95% methanol, 70% of it is found in methanol, which points to its hydroxylic character. 3. It gives an acetyl derivative, from which the original hydroxy compound can be regenerated on hydrolysis. 4. The infrared spectrum shows, besides other bands, one at 3460cm.(-1) attributable to a hydroxy group. 5. On passing a light-petroleum solution of naturally occurring anhydrovitamin A(2) through manganese dioxide a 6% conversion into retinene(2) is observed. 6. A 3-hydroxyanhydroretinol structure is proposed for naturally occurring anhydrovitamin A(2) and a mechanism of its transformation into retinene(2) on this basis is suggested.     

Effect of soil solarization on nodulation,infection by mycorrhizal fungi and yield of cowpea

Solarization of soil was found beneficial for plant growth in cowpea under field conditions. Root nodulation, infection by mycorrhizal fungi and yield were higher in plants grown in solarized soil. These increases were to the extent of 104.7, 20.0 and 23.7 per cent respectively when compared to control treatment without solarization.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Effect of NMDA-receptor ligands on neocortical and hippocampal EEG activity of rat brain.

Neocortex and hippocampus play important role in motor activity, neuronal plasticity and learning and memory mechanisms. Electroencephalographic (EEG) activity of neocortex and hippocampus of rat following NMDA-receptor agonist, N-methyl-D-aspartate (NMDA), 0.25-2 nmol in 10 microliters, ICV and noncompetitive NMDA-receptor antagonists, MK 801 (0.025-0.1 mg/kg, ip) and ketamine (10-50 mg/kg, ip) at OH, 1/2H, 4H, 8H and 24H was recorded. The electrodes were implanted stereotaxically in hippocampus and neocortex respectively. NMDA (0.25 and 1 nmol) showed longer lasting decrease in amplitude in hippocampus and in frequency in cortical neurons while 2 nmol produced epileptogenic neurotoxicity. Opposite effect i.e. increase in amplitude in both, hippocampus and neocortex was observed with MK 801 and ketamine and these agents also showed longer lasting influence. Administration of MK 801 (0.05 mg/kg) and ketamine (50 mg/kg) prior to NMDA 2 nmol protected 40% animals from NMDA-induced neurotoxicity and blockade of NMDA-induced long term influence. The EEG effect of NMDA agonist and NMDA-induced neurotoxicity at higher dose and its modification by NMDA-antagonist, MK 801 and ketamine suggest that beside NMDA agonists (NMDA), its antagonists may, also affect long lasting changes in hippocampus and cortex. These antagonists reverse NMDA-mediated long term influence in these brain areas.