Normal Green Plants Research Articles

Haem Oxygenase 1 is a potential target for creating etiolated/albino tea plants (Camellia sinensis) with high theanine accumulation.

Theanine content is highly correlated with sensory quality and health benefits of tea infusion. The tender shoots of etiolated and albino tea plants contain higher theanine than the normal green tea plants and are valuable materials for high quality green tea processing. However, why these etiolated or albino tea plants can highly accumulate theanine is largely unknown. In this study, we observed an Arabidopsis etiolated mutant hy1-100 (mutation in Haem Oxygenase 1, HO1) that accumulated higher levels of glutamine (an analog of theanine). We therefore identified CsHO1 in tea plants and found CsHO1 is conserved in amino acid sequences and subcellular localization with its homologs in other plants. Importantly, CsHO1 expression in the new shoots was much lower in an etiolated tea plants 'Huangkui' and an albino tea plant 'Huangshan Baicha' than that in normal green tea plants. The expression levels of CsHO1 were negatively correlated with theanine contents in these green, etiolated and albino shoots. Moreover, CsHO1 expression levels in various organs and different time points were also negatively correlated with theanine accumulation. The hy1-100 was hypersensitive to high levels of theanine and accumulated more theanine under theanine feeding, and these phenotypes were rescued by the expression of CsHO1 in this mutant. Transient knockdown CsHO1 expression in the new shoots of tea plant using antisense oligonucleotides (asODN) increased theanine accumulation. Collectively, these results demonstrated CsHO1 negatively regulates theanine accumulation in tea plants, and that low expression CsHO1 likely contributes to the theanine accumulation in etiolated/albino tea plants.

Registration of Albino‐Virescent Leaf peanut genetic stock

AbstractRecently, an Albino‐Virescent Leaf peanut plant (Reg. no. GS‐10, PI 693218) (Arachis hypogaea L.) was identified in an F10 advanced breeding line, GA 082524, at the University of Georgia, Gibbs Research Farm in Tifton, GA. GA 082524 was the result of a three‐way cross between ‘Georgia‐02C’ × (‘Georgia‐01R’ × ‘COAN’). Similar to GA 082524, the Albino‐Virescent Leaf peanut genetic stock has high‐oleic acid concentration, as well as root‐knot nematode resistance. The Albino‐Virescent Leaf mutant is essentially albino upon emergence; then following a short lag period, chlorophyll gradually accumulates in older leaves, with new leaves emerging deficient of chlorophyll. Eventually, as the plant matures and new leaves are no longer added, the plant appears entirely green and is indistinguishable from a normal green plant. The mutation is controlled by a recessive mutation at a single locus, designated avl, with phenotypic segregation following a 3 normal:1 Albino‐Virescent Leaf pattern (p = .95) when crossed to a normal green genotype. The Albino‐Virescent Leaf mutant described here represents a presumably unique spontaneous mutation that has not been released previously to the public.

Molecular mapping of a new red mutant gene (Rs) in upland cotton

In recent years, there has been slow progress in improving cotton yield. It is known that the F1 generation from the cross of the new red mutant and the normal green leaf plant has high photosynthetic efficiency. Therefore, cloning the new red mutant gene and further introducing it into other crops through transgenic techniques is a promising approach for achieving high photosynthetic efficiency through breeding. To map this new mutant gene, tentatively named Rs, the authors constructed an F2 generation containing 1214 individual plants from mutant EH083 (Gossypium hirsutum) and Hai 7124 (Gossypium barbadense). Fifty-five pairs of simple sequence repeats and sequence-related amplified polymorphism (SRAP) primers on chromosome 7 were selected to screen the two parents. Finally, the Rs gene was mapped at the 0.3 cM interval flanked by markers NAU3735 and NAU1048.

Genetic analysis of a novel aurea mutant in wheat

A novel spontaneous chlorina mutation of wheat (Triticum aestivum L.) was found in 2000. Three types of phenotypes, i.e., green plant, intermediate yellow-green plants (or chlorina), and yellow plant (aurea), were observed in the selfed progenies of the mutant. The aurea plants usually died before heading stage, and the intermediate plants were viable but less vigorous and developed later than the green plant. The mutant wheat was analyzed genetically after self-pollination and crossing with normal green plants. Selfing the M1 plant (chlorina) results in 1:2:1 segregation ratio of aurea:chlorina:green plants. The progenies of M1 green plants were all green. The test cross between a chlorina plant and a normal plant (as male or female) gave a 1 normal green: l yellow-green segregation ratio. The result can be explained on the assumption that the aurea is homozygous for au and the chlorina heterozygous for Au. Based on these findings, this mutant is controlled by a partial dominant nucleus gene. The genotypes of aurea, chlorina, and normal plants are au/au, Au/au, and Au/Au, respectively.

Differential translocation of phosphorus in chlorotic and normal green plants of sugarcane ratoon

A study was carried out on the first ratoon of sugarcane Saccharum spp. hybrids, (cv.CoLk 8102) exhibiting chlorosis under subtropical conditions to know the accumulation and translocation of phosphorus in normal green and chlorotic green plants. The phosphorus content in the root of normal green plant was twice that of chlorotic plants suggesting relatively lesser accumulation in the roots of chlorotic than normal green plants. The stalks of chlorotic plants recorded lower phosphorus content than that of normal green plants, while the leaf sheaths of normal green registered lower phosphorus content than chlorotic ones. But leaf laminae of normal green plant accumulated more than two times of phosphorus than chlorotic ones. Thus, there was a progressively higher accumulation of phosphorus in the above ground parts of normal green plants as compared to the chlorotic ones. From this study it may be concluded that some restriction mechanism is working for translocation of phosphorus to the leaf laminae of chlorotic plants as compared with normal green plants.

Preferência de Bemisia tabaci biótipo B em linhagens mutantes de algodoeiro

Os efeitos de caracteres mutantes morfológicos do algodoeiro (Gossypium hirsutum L. r. latifolium Hutch.): folha okra, bráctea frego e planta vermelha, em relação à resistência à mosca-branca (Bemisia tabaci biótipo B Hemiptera: Aleyrodidae), foram avaliados em experimentos com ou sem chance de escolha. Os experimentos foram conduzidos em casa-de-vegetação, no delineamento de blocos ao acaso, em fatorial 23 + 1, com quatro repetições. O mutante com a característica planta vermelha foi menos atrativo e menos preferido para oviposição, em relação à planta verde, em ambos os ensaios, com ou sem escolha. Não houve preferência quanto à forma da folha e ao tipo de bráctea.

Inheritance of a Recessive Transgene‐Associated Character Controlling Albinism in Transgenic Bean (Phaseolus vulgaris L.)

We identified a transgenic line exhibiting albinism during our work to introduce genes through genetic engineering in dry bean (Phaseolus vulgaris). The transgenic mother plant (R0) presented a normal phenotype and generated albino and normal green plants in the first generation (R1). The segregation ratio of the albino character in the R1 and R2 generations fitted the expected ratio for a character controlled by a single recessive gene linked to a foreign gus gene, suggesting that albinism could be a consequence of insertional mutation caused by introduction of the exogenous gene. Analysis by electron microscope revealed that the albino cells possessed no chloroplasts and a greater number of mitochondria when compared to normal green plants. This transgenic bean line may be used in understanding the genetic control of chloroplast genesis, for acquiring additional knowledge of genomic structure or in physiological studies. This is the first described transgene-associated mutant bean plant.

A Light-Intensity Sensitive Chlorophyll Mutant in Sorghum

A light-intensity sensitive chlorophyll mutant, gs3, was discovered in a local sorghum [Sorghum bicolor (L.) Moench] population at Hays, Kansas, and analyzed genetically after crosses with normal green inbreds. The mutation causes the loss of chlorophyll of varying degrees and formation of lengthwise white stripes on leaves, leaf sheaths, glumes, and white kernels at soft dough stage. In field tests, the mutant behaved like a simply inherited recessive trait, producing a ratio of 3 normal green:1 chlorophyll deficiency in the F2 generation and a ratio of 1 normal green:1 chlorophyll deficiency in the backcross populations. The selfed progeny from the mutant were virtually all mutant type, and the F2 green plants segregated into 2 segregating:1 true breeding F3 families, confirming the F2 data and showing that the mutant is under the control of a single recessive allele. However, the chlorophyll mutant often failed to show under low light intensity (585 μmol/m2/s) when grown in a growth chamber or in a greenhouse during the winter months, and no definitive segregation ratio could be determined even if both green and striped trait did appear, suggesting that the mutant allele is light intensity regulated. Instability or occurrence of variegated phenotypes, specific chlorophyll deficient patterns, could not breed true, and the rare reversion to normal green plants suggested that the expression of chlorophyll deficient trait may be regulated by a transposable genetic system.

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions ofyoung-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% ofthe same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity ofless than 10%) irradiated with UV at an intensity of 300 muW/cm(2) for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts ofthe recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.

Maternal inheritance of plant variegation in cowpea, Vigna unguiculata (L.) Walp.

A chimeric plant was observed in the F2 generation of a cross between a mutant cultivar, Ife BPC, and a germplasm line, TVu 2, in cowpea, Vigna unguiculata (L.) Walp. The chimeric plant had four lateral branches, one of which was intensely variegated, while the others were mostly green with few white sectors. F3 progeny from the intensely variegated branch of this plant were all variegated, while seed derived from the mostly green branches produced only green progeny. In subsequent generations, the descendants of the variegated branch bred true for the variegated trait, while those of the mostly green branches were also true-breeding for green colour. No pure-green or pure-white shoots were observed in any of the variegated plants examined in this study. Consequently, no pure-green or pure-white seedlings were produced from seeds harvested from the variegated plants. The results of reciprocal crosses between variegated and normal green plants indicate that variegation is inherited in a strictly uniparental maternal fashion. This is the first report of a cytoplasmically inherited mutation affecting foliage colour in cowpea.

Marking cell layers with spectinomycin provides a new tool for monitoring cell fate during leaf development.

Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow-green (YG) plants resulting from spectinomycin-treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG-plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10-20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000-4000 anticlinal divisions for the adaxial epidermis, was 2-3-fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell-autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.

A chlorophyll‐reduced seedling mutant in oilseed rape, Brassica napus, for utilization in F1 hybrid production

AbstractA mutant chlorophyll‐reduced (Cr) seedling with yellow‐green cotyledons and leaves was obtained from the Brassica napus inbred line 3529 induced by fast neutron and diethyl sulphate (DES). Genetic analysis revealed that the Cr seedling marker trait was controlled by a pair of recessive genes. A randomized complete block design was used to evaluate its agronomic performance. Results from 2 years of tests indicated that the seed yield of Cr lines was significantly lower than that of normal green plants; however, when the Cr gene was in the heterozygous condition, no deleterious effects on yield characteristics and disease resistance were observed. The Cr seedling marker trait was introduced into male‐sterile lines, and Cr male‐sterile lines revealed the same superior combining ability as normal chlorophyll (Nc) lines. The Cr trait can therefore be used as a marker to produce hybrid seed.

Analysis of randomly selected cDNAs reveals the expression of stress- and defence-related genes in the barley mutant albostrians

Randomly selected cDNAs obtained from white leaf material of the albostrians mutant of Hordeum vulgare were analysed. About one third of the cDNAs identified due to their homology to known plant genes are coding for defence- or stress-related proteins. These include several different lipid transfer proteins, an ascorbate peroxidase, a cytochrome P450, a 14-3-3 protein, a thionin, a phenylalanine ammonia lyase, proteases, as well as protein kinases and phosphatases and a heat shock cognate protein. Northern analysis with selected clones showed that the mRNA accumulation in white albostrians leaves differs fundamentally from that in normal green plants. Consequently, the albostrians material provides a unique source of cDNAs only rarely expressed during normal leaf development, and might be of potential value in the identification of novel pathogenesis- and stress-related proteins.

Correction of chlorophyll deficiency in alloplasmic male sterileBrassica junceathrough recombination between chloroplast genomes

SummaryBrassica junceacv. Pusa Bold carryingB. oxyrrhinacytoplasm (oxycytoplasm) was male sterile and chlorotic under field conditions at low temperature (Prakash & Chopra, 1990). Leaf protoplasts of the chlorotic male sterile alloplasmic line (2n = 36) were fused with hypocotyl protoplasts of green male fertile,B. junceacv. RLM-198 (2n = 36) using polyethylene glycol. Of the 1043 plants regenerated from 10 fusion experiments, 123 had ‘gigas’ features and were identified as presumptive fusion products. Among field-grown population, one plant was dark green even at low temperatures and male sterile. It possessed 72 chromosomes which formed 36 bivalents at late diakinesis of meiosis-I. This plant was back-crossed toB. junceacv. Pusa Bold (the maintainer line) for three successive generations. One male sterile, normal green BC3 progeny plant with 2n = 36 was analyzed for organelle constitution. Probing its total DNA with the mitochondrial gene for cytochrome oxidase subunit I revealed that it possessed mitochondria ofB. oxyrrhina. Southern hybridization pattern with the gene for ribulose bisphosphate carboxylase oxygenase-large subunit (rbcL) revealed that the chloroplast genome of the chlorophyll deficiencycorrected plant had characteristics of bothB. junceaandB. oxyrrhina. The deficiency correction has been attributed to recombination between chloroplast genomes of the two species.

Plant regeneration from protoplasts isolated from embryogenic suspension cultures of creeping bentgrass (Agrostis palustris Huds.)

A rapidly growing embryogenic suspension culture cell line of creeping bentgrass cv Penncross (Agrostis palustris Huds.) was established from callus derived from the culture of mature seeds. High concentrations of 2,4-D were required for the induction of callus (3 mg/1) as well as for the maintenance of the cell Une (2 mg/1) on modified B5 medium of Gamborg. Protoplasts isolated from the suspension cultures were successfully cultured in Murashige and Skoog's basal medium supplemented with only 0.1 mg/1 2,4-D. Although protoplast plating efficiency was rather low (0.36%), 30% of the protocalli formed normal green plants that were successfully established in soil.

Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Hartog)

Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength of the macroelements, 5 μm 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25%. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome complement for wheat (2n = 42).

D-rice seedling development in periodically stirred water in sealed environments

Summary Large numbers of germinating seedlings of two rice cultivars exhibited d- type development consisting solely of completely white coleoptile growth after 15 days in the light in both static and periodically stirred water environments in completely sealed jars. Thus, whether germinated in static or agitated water in the sealed environments, coleoptile greening and subsequent normal seedling development were inhibited by hypoxic stress caused by the large numbers of germinating rice seedlings competing for the limited environmental oxygen supply. Consequently, the evidence presented points away from the formation of unfavourable oxygen diffusion gradients in static water environments in the sealed jars as having been responsible for d type seedling development observed in previous investigations. Continuous aeration of the 15-day-old static and periodically stirred aquatic environments, respectively, resulted in complete reversal of all d seedlings into normal green plants.

Hypoxic inhibition of rice seedling development in the presence of exogenous nitrate

Summary Hypoxic stress resulting from large numbers of germinating rice seedlings competing for the limited oxygen supply in initially air-saturated unagitated water in air-tight containers with and without exogenous nitrate supplementation inhibited normal seedling development. Subsequent aeration of the hypoxic aquatic environments with and without exogenous nitrate supplementation resulted in complete reversal of all non-normal seedlings into normal green plants. Reversal of hypoxic stress inhibition of normal rice seedling development during exposure to aeration confirms previous findings that exogenous nitrate supplementation was unable to serve as an alternative electron acceptor in place of molecular oxygen needed for normal seedling development. These observations are consistent with reports at a recent symposium on nitrate assimilation in that dissimilatory nitrate reduction has not been observed in higher plants.

The Ascorbic Acid System in Cuscuta reflexa Roxb.

Summary Seedlings of the parasitic plant Cuscuta reflexa Roxb. synthesize ascorbic acid (AA); however, the conversion of galactono-γ-lactone (GL) to AA is low in respect to other angiosperms. The AA content of the seedlings, as well as the AA/dehydroascorbic acid (DHA) ratio, is much lower than in any other higher plant. Cuscuta has no AA oxidase, but it does have AA peroxidase, its activity being similar to that found in normal green plants. Both the reducing enzymes-ascorbic free radical (AFR) reductase and DHA reductase- are present in this parasitic plant. DHA reductase activity is fairly high; while, that of AFR reductase is ten times lower than in etiolated and green plants. These results suggest that AFR reductase is a key enzyme for maintaining the AA system in the reduced state and explain why the AA/DHA ratio is low in this parasitic plant.

High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis

A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F1 progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment.