Phenotypic and Marker-Assisted Genetic Enhancement of Parental Lines of Rajalaxmi, an Elite Rice Hybrid.

The cytoplasmic male sterile line system comprising CRMS 32A and its maintainer line CRMS 32B is a popular choice for the development of new hybrids in India as CRMS 32A, having Kalinga 1 cytoplasm (other than WA), is a viable alternative to WA cytoplasm. However, both lines are susceptible to bacterial blight (BB), a major disease on rice. As enhancement of host plant resistance is the most effective and economical strategy to control this disease, four resistance genes (Xa4, xa5, xa13, and Xa21) were transferred from a BB pyramid line of IR64, into the A and B lines using a marker-assisted backcrossing (MAB) breeding strategy. During the transfer of genes into CRMS 32B, foreground selection was applied using markers associated with the genes, and plants having resistance alleles of the donor, are selected. Selection for morphological and quality traits was practiced to select plants similar to the recurrent parent. The four gene and three gene pyramid lines exhibited high levels of resistance against the BB pathogen when challenged with eight virulent isolates. Using genome wide based SSR markers for background selection, pyramids having >95% of the recurrent parent genome were identified. With CRMS 32B gene pyramid as donor, the four resistance genes were transferred into the A line through repeated backcrosses and the A line pyramids also exhibited high level of resistance against BB. Through a combination of selection at phenotypic and molecular levels, four BB resistance genes were successfully introduced into two parental lines (CRMS 32 B and A) of Rajalaxmi, an elite popular hybrid. The pyramided B lines did exhibit high levels of resistance against BB. Selection for morphological and quality traits and background selection hastened the recovery of the recurrent parent genome in the recombinants. Through repeated backcrosses, all the four resistance genes were transferred to CRMS 32A and test crosses suggest that the maintenance ability of the improved CRMS 32B lines is intact. These improved maintainer and CMS lines can directly be used in hybrid rice breeding and the new hybrids can play an important role in sustainable rice production in India.

Molecular marker assisted gene stacking for biotic and abiotic stress resistance genes in an elite rice cultivar.

Severe yield loss due to various biotic stresses like bacterial blight (BB), gall midge (insect) and Blast (disease) and abiotic stresses like submergence and salinity are a serious constraint to the rice productivity throughout the world. The most effective and reliable method of management of the stresses is the enhancement of host resistance, through an economical and environmentally friendly approach. Through the application of marker assisted selection (MAS) technique, the present study reports a successful pyramidization of genes/QTLs to confer resistance/tolerance to blast (Pi2, Pi9), gall Midge (Gm1, Gm4), submergence (Sub1), and salinity (Saltol) in a released rice variety CRMAS2621-7-1 as Improved Lalat which had already incorporated with three BB resistance genes xa5, xa13, and Xa21 to supplement the Xa4 gene present in Improved Lalat. The molecular analysis revealed clear polymorphism between the donor and recipient parents for all the markers that are tagged to the target traits. The conventional backcross breeding approach was followed till BC3F1 generation and starting from BC1F1 onwards, marker assisted selection was employed at each step to monitor the transfer of the target alleles with molecular markers. The different BC3F1s having the target genes/QTLs were inter crossed to generate hybrids with all 10 stress resistance/tolerance genes/QTLs into a single plant/line. Homozygous plants for resistance/tolerance genes in different combinations were recovered. The BC3F3 lines were characterized for their agronomic and quality traits and promising progeny lines were selected. The SSR based background selection was done. Most of the gene pyramid lines showed a high degree of similarity to the recurrent parent for both morphological, grain quality traits and in SSR based background selection. Out of all the gene pyramids tested, two lines had all the 10 resistance/tolerance genes and showed adequate levels of resistance/tolerance against the five target stresses. The study demonstrates the potential of MAS for stacking of several genes into a single line with a high degree of parental genome recovery.

Genetic enhancement of host plant-resistance of the Lalat cultivar of rice against bacterial blight employing marker-assisted selection.

To incorporate durable resistance against bacterial blight, a major disease rice, three resistance genes, xa 5, xa13 and Xa21, from IRBB 60 were transferred through marker-assisted backcrossing using RG 556, RG 136 and pTA248 markers linked to the three genes to supplement the Xa4 gene present in Lalat, a popular rice cultivar. Effective selection enabled the transfer in three back-crosses and a generation of selfing and background selection employing morphological and grain quality traits and molecular markers, led to >90 % recovery of the recurrent parental genome. The gene pyramids exhibited high levels of resistance against the pathogen in multi-location evaluation trials conducted over several locations of bacterial blight in India. IL-2 (CRMAS2621-7-1), a gene pyramid, was identified as being promising for several endemic regions of bacterial blight and was released as Improved Lalat in one of the identified regions. The success of the study demonstrates the vast potential of marker-assisted selection for gene stacking and recovery of the parental genome with high precision.

Histamine induced decrease of lecithin levels in broncho-alveolar lavage fluid of rats is mediated by H2 receptor.

Lecithin, a major surface active substance of the surfactant system of the lung, was estimated in broncho-alveolar lavage (BAL) fluid in four groups of healthy adult male albino rats. Rats from group I were not administered any drug and acted as controls. Group II were administered histamine diphosphate. Group III were given H1 blocker (pyrilamine maleate) followed by histamine diphosphate. Group IV received H2 blocker (ranitidine hydrochloride) followed by histamine diphosphate. Lecithin content of BAL fluid in the control group was compared with that in the other three groups. A significant decrease in lecithin content was observed in the rats that received either histamine diphosphate or H1 blocker followed by histamine diphosphate. However, compared to control rats no significant difference in lecithin content was seen in rats that received H2 blocker followed by histamine diphosphate. The results clearly indicate that the decrease in surface active lecithin content in BAL fluid following administration of histamine diphosphate was unaffected by prior administration of H1 blocker, but was blocked by prior administration of H2 blocker. It was concluded that histamine induced decrease in lecithin content of BAL fluid is mediated through H2 receptors. Since the predominant source of intra-alveolar lecithin are Type II cells of the alveolar epithelium, It is possible that Type II cells have H2 receptors, stimulation of which resulted in decreased intraalveolar lecithin.

Isolation of an unknown compound, from both blood of Bhopal aerosol disaster victims and residue of tank E-610 of Union Carbide India Limited--chemical characterization of the structure.

A total of more than 28 chemical entities/reaction products in the form of gases, vapour and particulate matter were reported from the tank E-610 of methyl isocyanate (MIC) storage tank of Union Carbide India Limited on the night of 2/3 December 1984 in Bhopal. In earlier studies, methyl isocyanate and its trimer, with a few other compounds, were reported in the human victims preserved in deep freeze. Randomly selected samples were analysed by gas chromatograph coupled with mass spectrometer (ITD-800, Finnigan MAT, UK). Four of the cases showed the peaks and fragmentation pattern identified with one of the unidentified compound of molecular weight 269 amu in the Tank Residue, which constituted about 0.2 area per cent on GC-ITD. After isolation by column chromatography and being exposed to characterization, it was identified as a Spiro compound. It was possibly formed by the polymerization of five molecules of methyl isocyanate.

Distribution of malathion in body tissues and fluids.

Six cases of suspected poisoning were studied. The various body tissues and fluids of all the cases were analysed and malathion was found positive. The quantitative analysis was performed using high performance liquid chromatography. The distribution of malathion was studied in lungs, liver, kidneys, spleen, brain, heart, blood, muscles, urine and gastric contents.

GC-NPD and GC-MS analysis of preserved tissue of Bhopal gas disaster: evidence of methyl carbamylation in post-mortem blood.

Twenty-five preserved autopsy blood samples of Bhopal toxic gas exposed victims were analysed by gas chromatography (GC) coupled with either Nitrogen-Phosphorous detector (NPD) or mass spectrometer (MS) for the presence of methyl carbamyl valine in terms of valine methyl hydantoin (VMH). 84% of these samples showed a positive test for VMH on GC-NPD and the identity of the peaks were further confirmed on GC-MS. The concentration of VMH in the gas-affected positive blood samples ranged from 2.56 to 51.28 nanomoles. These results indicate entry of methyl isocyanate (MIC), one of the constituents of the toxic cloud caused by the disaster, into the blood stream of victims who had inhaled gas.

GC-MS identification of MIC trimer: a constituent of tank residue in preserved autopsy blood of Bhopal gas victims.

Based on the external and internal findings of Bhopal gas disaster victims, it was apparent that the gases and particulate matter came out as an aerosol. This was possibly the pyrolysed, reformulated, reconjugated suspension of constituents of the tank E-610 of Union Carbide India Limited, Bhopal, while it was claimed to be methyl isocyanate (MIC) only. It was postulated by the manufacturer of MIC, that the material inhaled by the victims of the Bhopal gas disaster does not cross the lung barrier (UCC press conference on 14th December 1984). It was observed that the more the victims ran, the more aerosol they inhaled and the fatalities were observed in such victims. The tissues, which were preserved in the deep freeze, were randomly selected and analysed by GC coupled with MS (ITD) Finnigan MAT, UK. 14 out of 34 autopsy cases showed MIC trimer peak in extracts of blood. This was one of the constituents of the aerosol and was also located in the tank residue, thereby proving that the trimer had passed the lung barrier.

High performance liquid chromatographic determination of alcohols with reference to body distribution of methanol.

A method has been developed on reverse phase high performance liquid chromatography for simultaneous determination of methyl, ethyl and isopropyl alcohols under refractive index detection using pure water as the mobile phase. A good separation has been achieved between these alcohols. Detector response was linear with a detection limit of 5 mg/100 ml. Recovery studies were performed by adding known amounts of methyl and ethyl alcohols to blood, lung and liver within the range 80-90%. The reproducibility of the results was always greater than 90%. The quantitative distribution of methyl alcohol in postmortem body tissues and fluids has been reported in three cases of poisoning.

Effect of drugs used in pulmonary edema (frusemide and combination of etofylline and theophylline) on surfactant system of lung.

Pulmonary surfactant activity of healthy male albino rats was estimated in terms of the maximum and minimum surface tension values of alveolar washings and the phospholipid content of the extract. The results obtained in these (control) animals were compared with those in two groups of animals treated with therapeutic doses of frusemide and a combination of etofylline and theophylline. A significant increase in surfactant activity in terms of surface tension values and phospholipid content was observed with frusemide, whereas a significant increase in phospholipid content without a change in surface tension values was observed in the case of combination of etofylline and theophylline. These findings suggest that frusemide in addition to its diuretic action, increases the surfactant activity of lung. This might be another mechanism by which it provides relief in pulmonary edema patients. The study also indicates that phospholipid concentration need not always reflect surfactant activity of lung.

High performance liquid chromatographic method for the analysis of organophosphorus and carbamate pesticides.

A method has been developed for the rapid quantitative analysis of organophosphorous and carbamate pesticides using HPLC. Good separation was obtained among the four major groups of organophosphorus pesticides [i.e. aryl phosphorothionate (methyl parathion), alkyl phosphorothionate (malathion), enolphosphate (phosphomidon, monocrotophos, dichlorvos), heterocyclic phosphorothionates (quinalphos)] and carbamates [viz. Carbaryl (Sevin) and Baygon (Dalf)] with a detection limit of 100 ng for all the pesticides. Separation was measured in terms of capacity factor (k') resolution (R) and selectivity factor (alpha ii). The method described can be used for the analysis of biological samples for the presence of organophosphorus and carbamate pesticides in the cases of poisoning. Recovery studies were made in the blood, lung and liver and found to be 85-97% with reproducibility at greater than 95%.

Effect of bronchodilators on surfactant system of lung.

Pulmonary surfactant activity of healthy male albino rats was estimated in terms of the maximum and minimum surface tension values of alveolar washings and the phospholipid content of the extract. The results obtained in these (control) animals were compared with those in three groups of animals treated with therapeutic doses of terbutaline, adrenaline and aminophylline. A significant decrease in the surface tension values without a significant increase in the phospholipid content was observed with aminophylline, whereas a significant increase in phospholipid concentration without a significant decrease in surface tension values was observed in case of terbutaline and adrenaline. These findings suggest that aminophylline, in addition to a bronchodilator action, lowers the elastic resistance of lung. The study also indicates caution in interpreting phospholipid concentration as surfactant activity.

Inhibition of rabbit liver arylsulfatase B by phosphate esters.

Arylsulfatase B (aryl-sulfate sulfohydrolase, EC 3.1.6.1) purified from rabbit liver is competitively inhibited at modest concentrations by a variety of phosphate esters derived from amino acids, amines and simple sugars. Phospho-L-serine coupled to Sepharose 4B could be used as an affinity column to enhance the purity of a crude preparation of the enzyme. It is suggested that phosphate esters containing functional groups can be used to obtain affinity reagents to purify arylsulfatases and also to probe their active sites.

Telo aneuploids in pearl millet.

Nomenclature and chromosome constitution for twenty different aneuploids with telocentrics in Pennisetum typhoides are presented.

Reaction of 4-methylumbelliferylguanidinobenzoate with proteases in human amniotic fluid.

An arginine esterase activity similar to that observed in plasma has been demonstrated in second trimester and term human amniotic fluid. Like plasma, the protease(s) hydrolyzed esters of arginine, were reactive towards 4-methylumbelliferylguanidinobenzoate (MUGB), a sensitive active site titrant of trypsin-like enzymes, and had a pI of 5.1--5.4. The pH optimum for proteolytic activity was 8.0. This protease activity was inhibited by soybean trypsin inhibitor (STI), benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate (NPGB), and was insensitive to 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) and p-hydroxymercuribenzoic acid (HMB). Upon gel filtration, two MUGB-reactive fractions were observed, one with an apparent molecular weight of 200,000 and the other, 100,000. Both fractions had arginine esterase activity and appeared to be sensitive to inhibition by STI and benzamidine. The mean MUGB titre value (nmoles of 4-methylumbelliferone released per ml amniotic fluid) for 300 mid-trimester amniotic fluids was 11.40 +/- 2.40 nmoles MU/ml. The mean specific activity was 2.36 +/- 0.41 nmoles MU/mg protein. Two amniotic fluids from pregnancies which delivered children with cystic fibrosis (CF) were analyzed in blind samples sent from other laboratories. The MU titre values obtained were 4.73 and 4.32 with specific activities of 1.24 and 1.30 respectively. A third was identified in our screening program of amniotic fluids obtained from amniocenteses done for the intrauterine detection of genetic abnormalities. The MU titre value was 5.52 nmoles/ml with a specific activity of 1.34. The specific activities of these fluids when compared to the controls were significantly different (p less than 0.001). The mean titre value for 23 term amniotic fluids samples was 8.14 +/- 1.69 nmoles MU/ml. The mean specific activity was 3.37 +/- 0.76 nmoles MU/mg protein. A term amniotic fluid obtained from a woman who delivered a baby with CF showed a markedly reduced level of MUGB reactivity (3.01 nmole/ml). The specific activity was 1.06 which was significantly different from the control term fluids. The MU titre values and specific activities of amniotic fluids obtained from abnormal pregnancies (such as those with neural tube defects, chromosomal abnormalities and polymorphisms, abortions and stillbirths) and fluids with elevated alphafetoprotein and maternal blood contaminants did not significantly vary from the mean control values (Table 3).

Protease deficiency in plasma of patients with cystic fibrosis. Reduced reaction of 4-methylumbelliferylguanidinobenzoate with plasma of patients with cystic fibrosis.

Protease activity in plasma is assayed using 4-methylumbelliferylguanidinobenzoate. The assay is modified by carrying out the reaction in the presence and absence of benzamidine, a competitive inhibitor of trypsin-like proteases. The parameters of the assay are described in detail. Using this assay, our earlier demonstration of a deficiency of protease activity in plasma of patients with cystic fibrosis is confirmed. The activity, corrected for the nonspecific hydrolysis of 4-methylumbelliferylguanidinobenzoate by benzamidine, is expressed as nanomoles of 4-methylumbelliferone released per milliliter plasma. Under standard conditions, the activity in plasma activated with chloroform-ellagic acid was 127.2 +/- 23.1 in 7 controls, 70.4 +/- 11.7 in 11 obligate heterozygotes, and 48.7 +/- 16.6 in 12 patients with cystic fibrosis. Identical results were obtained when unactivated plasma was used. These data demonstrate that the judicious use of specific inhibitors such as benzamidine might be useful in assaying low levels of protease activity in crude systems.

Reaction of 4-methylumbelliferylguanidinobenzoate with cultivated human skin fibroblasts derived from patients with cystic fibrosis.

Protease activity in cultivated human skin fibroblasts has been quantitated using 4-methylumbelliferylguanidinobenzoate (MUGB), an active site titrant of trypsin-like proteases (7). The reaction of the proteases with MUGB was complete in 1 hr, inhibited both by benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate, but not by p-hydroxymercuribenzoate. The extent of reaction was proportional to protein concentration and independent of MUGB concentration. This activity was present in the particulate fraction of the cell. The mean "titre" values (nanomoles of 4-methylumbelliferone released per mg protein) of the proteases in fibroblasts from eight controls (N), 8 obligate heterozygotes (H), and 14 patients with cystic fibrosis (CF) were: N, 1.27 +/- 0.11; H, 0.82 +/- 0.12; CF, 0.66 +/- 0.10. The differences in the "titre" values for N:CF and N:H were significant (p less than 0.001) as were those for H:CF (p less than 0.01). The mean "titre" value obtained for cultivated control amniotic fluid cells was 1.29 +/- 0.17. These data indicate a reduction in the MUGB-reactive proteases in skin fibroblasts derived from patients with CF when compared either to control or to obligate heterozygotes. These data are consistent with our earlier suggestion (11, 15) that decreased proteolytic levels in the tissues and fluids of patients with CF may be a generalized phenomenon.