Physiological and Biochemical Analysis of Somaclones Derived from Leaf Explants of Lathyrus sativus

Low ODAP somaclones have been evaluated for physiological and biochemical parameters especially in relation to attributes that lead to increased biomass production. All the somaclones during development had substantially lower ODAP content in leaves as compared to parent P24. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. Somaclone Bio L12 had the highest whereas parent P24 and Bi0164 had the least leaf area. Harvest index was the highest and biomass production was the lowest in the Bio 164. Bio L08 gave the highest seed yield. Photosynthetic rates were also higher in Bio L12, although no significant positive correlation was observed in leaf photosynthesis and seed yield. The differences in physiolpgical and biochemical parameters indicate the possibility of development of high yielding genotypes. The results in present investigation show differences in photosynthetic rate, leaf characteristics, seed yield and ODAP content among somalones and parent. Somaclones with extremely low ODAP content with variability in leaf morphology and photosynthetic rate is indicative of variation induced during plant tissue culture.     

Gliadin and Glutenin Variation in Seeds of Somaclones of Triticum aestivum

Gliadin and glutenin of seeds of a pollen-derived pure line “H-33” and its 9 stable somaclones of wheat (Triticum aestivurn L.) from inflorescence callus were investigated using SDSPAGE. Among the 9 somaclones 6 were identical to the “H-33” in SDS-PAGE patterns of of gliadin and glutenin. The other 3 somaclones were identical with each other, but different from “H-33”. The varied electrophoretic bands of seed protein showed that somaclonal variation had occured at gene level during tissue culture in which the somaclones were produced.     

Isozyme Variability in Plants Regenerated from Calli of Cereus peruvianus (Cactaceae)

Morphological and isozyme variation was observed among plants regenerated from callus cultures of Cereus peruvianus. Different morphological types of shoots (68%) were observed in 4-year-old regenerated plants, while no distinct morphological variants were observed in plants grown from germinated seeds. Isozyme patterns of 633 plants regenerated from calli and of 261 plants grown from germinated seeds showed no variation in isocitrate dehydrogenase isozyme, and the differential sorbitol dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and peroxidase isozyme patterns observed in regenerated plants were attributed to nonallelic variation. Allelic variation was detected at three isoesterase loci. The proportion of polymorphic loci for both populations was 13.6% and the deviation from Hardy–Weinberg equilibrium for the Est-1 and Est-7 loci observed in somaclones was attributed to the manner in which the regenerant population was established. The high values for genetic identity among regenerant and seed-grown plant populations are in accordance with the low levels of interpopulation genetic divergence. In somaclones of C. peruvianus, morphological divergence was achieved within a short time but was not associated with any isozyme changes and also was not accompanied by biochemical genetic divergence.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.     

Molecular Characterisation of Low ODAP Somaclones of Lathyrus sativus

Somaclones of Lathyrus sativus having low ODAP contents were characterised at molecular level with respect to soluble protein pattern, esterase and ADH isozyme pattern, Southern hybridisation of genomic and mitochondrial DNA with specific probes. In 10-day-old seedling esterase isozyme pattern showed the presence of an unique band of Rm 0.73 in Bio L-12 and 15-8-1 and the absence of a band of Rm 0.80 in 15-8-1, as compared to parent and other somaclones. Southern hybridisation of genomic DNA with cDNA clone 29 showed an extra band of 13.5 kb in L-56 and 15-8-1 only. Hybridisation of mitochondrial DNA with mitochondrial specific ati ATPase probe showed differences with the pattern from Bio R-15 being unique. Some of these differences observed will be useful as marker for their identification.     

Superoxide Dismutases: II. Purification and Quantitative Relationship with Water-soluble Protein in Seedlings

Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Somaclonal Variation in a Food Legume—Lathyrus sativus

Somaclones of Lathyrus sativus cv P-24 were obtained from leaf, internode and root derived callus cultures. These showed significantly decreased neurotoxin (ODAP) content up to 0.03% compared to that of parent cultivar P-24 (0.3%). The somaclones also showed higher seed yield than parent P24. In addition. somaclone Bio 1–22 showed significantly decreased time for flowering. Other heritable morphological variant features were leaf length, leaf breadth, internode length, flower colour, seed colour, 100 seed weight, neurotoxin content of leaves and red markings on the pods.     

Variability for resistance to Fusarium solani culture filtrate and fusaric acid among somaclones in pea

Pea (Pisum sativum L.) somaclones of cultivars Adept, Komet and Bohatýr were obtained after selection in vitro with Fusarium solani filtrate and fusaric acid (FA). R2 regenerants were analysed by random amplification of polymorphic DNA (RAPD; OPAB4, P-14, UBC-556) and inter-retrotransposon amplification polymorphism (IRAP; Ogre) markers. Marker UBC-556 showed different banding patterns for each cultivar, but without specific bands for selected and control plants. Markers OPAB4, P14 and Ogre were useful for clear discrimination between selected and non-selected variants of all three cultivars. Flow cytometry analysis proved the same genome size of selected and non-selected pea lines. Therefore in vitro selection by pathogen derived agents could be the efficient method for obtaining of pea somaclones with increased resistance to F. solani.     

DNA Polymorphism among Rice Somaclones

Molecular markers were used to detect the influence of high concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D) in the callusing media on DNA variations in regenerated rice plants. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) based RFLP analysis were carried out on 12 somaclones of Oryza sativa L. cv. B-370. In vitro culture induced DNA variations were detected in the regenerated plants but the effect of high auxin concentration in the medium could not be revealed. In a second study, fingerprinting of 15 semi-dwarf, high yielding somaclones of B-370 was carried out using RAPD technique. Amplification using 20 random primers produced a total of 167 DNA bands out of which 97 bands were polymorphic. A total of 32 unique DNA bands were detected across all the somaclones and they could be grouped based on their similarity to B-370. RAPD analysis helped to reveal similarity or differences among the somaclones while fingerprinting using additional RAPD markers was not successful.     

Analysis of genetic diversity in cowpea (Vigna unguiculata L.Walp.) cultivars with random amplified polymorphic DNA markers

The genetic diversity among ten Indian cultivars of cowpea was analyzed using 18 sets of RAPD markers. A total of 181 bands with an average of 15 bands per primer were obtained. Out of 181 bands, 148 showed polymorphism (81.7%). The variation in genetic diversity among these cultivars ranged from 0.1742 to 0.4054. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA with high bootstrap values revealed two distinct clusters I and II comprised of two and seven cultivars, respectively. Cluster II was further differentiated into various subclusters. Cultivar IC-9883 was found to be unique based on its altogether distinct position in the dendrogram and two-dimensional space projections.     

Physiological and biochemical changes in different sugar beet genotypes infected with root-knot nematode

Ten genotypes of sugar beet plant either monogerm or multigerm seeds were screened under greenhouse conditions for both susceptibility and biochemical reaction to root-knot nematode (RKN) Meloidogyne incognita. All the tested genotypes were susceptible to nematode infection according to the number of root galls and gall indices. All infected genotypes exhibited significant reduction in chlorophyll a, b and carotenoids compared to non-infected ones. The total indole acetic acid and total phenolic compounds contents (mean of both shoot and root) increased significantly in most infected genotypes compared to non-infected genotypes except Disk-01-99 and Monte Rosa as well as LP16 and LP15 genotypes, respectively. Also, total polyamine contents (putrescine, spermidine and spermine) showed significant increases in response to infection with nematodes in all genotypes. The same trend was observed in lipid peroxidation expressed with malondialdehyde content in all tested genotypes. Activities of polyphenol oxidase, peroxidase, superoxide dismutase and catalase enzymes were also induced in most infected genotypes compared with non-infected genotypes. Generally, infection with RKNs induced the appearance of new protein bands at molecular masses 303, 288, 42 and 37?KDa in all infected genotypes. The differentiation in the appearance and/or disappearance of protein bands according to susceptibility to infection reflects the variation between genotypes in defense against infection.     

Isozyme Profile During Somatic Embryogenesis and In Vitro Flowering of Bambusa vulgaris

Peroxidase and esterase isozymes were investigated during plant regeneration via somatic embryogenesis in Bambusa vulgaris, The transition of non-embryogenic calli to embryogenic calli, somatic embryo development, germination and subsequent flowering of somatic embryo derived shoots were associated with selective expression or repression of isoforms of peroxidase and esterase. Non-embryogenic callus showed six peroxidase and four esterase bands. During somatic embryogenesis and germination of somatic embryos, some bands were suppressed and new isoforms of peroxidase and esterase appeared. During flowering, in addition to four peroxidase bands, a new unique esterase band ‘a’ appeared. Each developmental stage was thus associated with a definite isozyme profile.     

Somaclonal variant plants of wheat derived from mature embryo explants of three genotypes

Somaclones regenerated from three wheat (Triticum aestivum L.) cultivars, Glennson, Pavon and PAK-16171 were evaluated for variation in agronomic and morphological characters. Calli were initiated from germinating seeds on Linsmaier and Skoog (LS) medium plus 2 mg/l 2,4-dichlorophenoxyacetic acid, 2% sucrose and 1% agar. Calli were isolated and regenerated into whole plants on LS medium containing 0.1 mg/l indole - 3-acetic acid and 0.5 mg/l benzyladenine. Comparisons among the somaclones and their parents were made for plant height, spike length, number of grains per spike, and 100 grain weight. Significant variation was observed in these characters between the somaclones and parents. Genotypic differences were observed among the somaclones for many of these agronomic and morphological characters.     

Electrophoretic profiles of cyanobacterial membrane polypeptides showing heme-dependent peroxidase activity

The photosynthetic membranes of Anacystis nidulans R2 were examined electrophoretically following solubilization with lithium dodecyl sulfate. Electrophoresis yielded six prominent chlorophyll-containing bands. In addition, five polypeptides were observed which possessed heme-dependent peroxidase activity, monitored by incubating gels with 3,3′,5,5′-tetramethylbenzidine plus hydrogen peroxide. One such polypeptide, at 105 kdaltons, was removed by repeated washing of the membranes. Four remaining peroxidase-active polypeptides were observed at 7.2, 13.5, 18.5 and 33 kdaltons. Further examination of these four polypeptides yielded the following results. (1) The mobility of the 33 kdalton polypeptide was altered from 29 to 33 kdaltons upon heating (70°C) during membrane solubilization. (2) All four polypeptides showed stable heme-protein associations in the presence of 8 M urea; however, in the presence of urea, alterations in protein mobility were observed for each poly-peptide and only two (at 13.5 and 33 kdaltons) showed peroxidase activity following heating (70°C) during membrane solubilization. (3) The presence of thiols during membrane solubilization at 0°C was required to observe peroxidase activity at 7.2 kdaltons. These results, when compared to known properties of isolated cytochromes, suggest that the four polypeptides characterized here correspond to the subunits of photosynthetic cytochromes. Electrophoretic assessment of maize mutants lacking cytochrome f and b₆ activity supports this suggestion.     

Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

黑线姬鼠与大林姬鼠组织中超氧化物酶和过氧化物酶的分布与活性比较

以黑线姬鼠(Apodemus agrarius)和大林姬鼠(A. peninsulae)为研究对象,采用聚丙烯酰胺凝胶电泳(PAGE)不连续体系的方法,比较分析了心、肝、肾、肌肉、脑、肺6种器官和组织中超氧化物酶(SOD)和过氧化物酶(POD)活性,并建立了2种酶的电泳图谱。结果显示,上述2种酶在黑线姬鼠和大林姬鼠的6种器官和组织中均有表达并表现出明显的特异性,其中,2种鼠中超氧化物酶共分离出迁移率由0.15～0.66的9条电泳谱带,过氧化物酶共分离出迁移率由0.09～0.83的20条电泳谱带。在肝和肺中酶的活性最强,黑线姬鼠6种器官和组织中超氧化物酶活性均强于大林姬鼠,2种鼠组织中过氧化物酶的活性和分布相似,但在同一物种不同器官和组织间过氧化物酶的活性及分布存在明显差异。     

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B₅ medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R₁ generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R₂ generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.