Albino Shoots Research Articles

CsCBF2 contributes to cold repression of chlorophyll and carotenoid biosynthesis in albino Camellia sinensis cv. Baiye 1.

C-repeat binding factors (CBFs) play a pivotal role in regulating cold response in higher plants. Camellia sinensis cv. Baiye 1, a representative albino tea cultivar, has been identified as temperature-sensitive based on long-term observations by tea farmers. However, it remains unclear whether CsCBFs are involved in temperature-mediated albinism and seasonal greening in 'Baiye 1', and the mechanisms by which CBFs regulate cold responses in albino leaves are unknown. In this study, we demonstrate that CsCBF2 suppresses the seasonal greening of albino leaves by inhibiting chlorophyll and carotenoid biosynthesis under cold stress. In tea plantations, the accumulation of chlorophylls and carotenoids in the albino shoots of 'Baiye 1' is closely correlated with the effective accumulated temperature during its seasonal greening process. Weighted Gene Co-expression Network Analysis revealed negative associations between CsCBF expression and chlorophylls, carotenoids, as well as their biosynthetic genes REVEILLE 1 (CsRVE1) and Zeaxanthin epoxidase 1 (CsZEP1) under temperature fluctuations during seasonal greening. Cold-induced upregulation of CsCBF2 expression and decreased chlorophylls and carotenoids under controlled climate conditions. Transient suppression of CsCBF2 by antisense oligodeoxynucleotide elevated expressions of target genes, and increased chlorophylls and carotenoids. CBF-binding cis-elements were identified in CsRVE1, Protochlorophyllide oxidoreductase A (CsPORA), and CsZEP1 promoters. Luciferase assays suggested CsCBF2 binding to the CRT/DRE cis-elements and repressing expression of CsRVE1, CsPORA, and CsZEP1. These findings highlight CsCBF2 as a key transcriptional repressor involved in the seasonal greening of albino 'Baiye 1' under cold stress, by modulating cold responses and inhibiting genes associated with chlorophyll and carotenoid biosynthesis.

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.

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis.

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

Widely targeted metabolomic analyses of albino tea germplasm ‘Huabai 1’ and ‘Baiye 1’

‘Huabai 1’ is a novel albino tea germplasm that has a stable albino phenotype and prolonged albino period. In this study, widely targeted metabolomics was employed to analyze the metabolic profile of ‘Huabai 1’, and compare metabolites between albino shoots of ‘Huabai 1’ (HBW) and albino shoots of the broadest cultivated cultivar ‘Baiye 1’ (BYW). A total of 359 differential metabolites were identified in ‘Huabai 1’. Comparative analysis of differential metabolites revealed that 83 and 161 differential metabolites were specific for HBW and BYW, respectively. The phenylpropanoid and flavonoids biosynthesis are the key metabolic pathways, contributing to the color change in ‘Huabai 1’, while the purine metabolism and lipid-related metabolites may play important roles in regulating albinism in ‘Baiye 1’. Comparative analysis of amino acids and catechins revealed that the umami taste of albino tea may be due to the internal balance of amino acid components and the decrease in phenol-ammonia ratio. In addition, lipid-related metabolites, the main contributors to the aroma of tea, were significantly enriched in BYW, while, HBW possess higher antioxidative components than that of BYW, such as apigenin and genistein etc., suggesting that the ‘Huabai 1’ may have high prospects in commercialization and future breeding programs.

Effects of Lignosulfonates on Callus Proliferation and Shoot Induction of Recalcitrant Indica Rice

In vitro culture of recalcitrant indica rice cultivar through intervening callus is difficult due to long regeneration period. Therefore, this study was undertaken to evaluate the growth promoting effects of lignosulfonate (LS) on callus proliferation and shoot induction of Malaysian recalcitrant indica rice cv. MR219. LS is a by-product of wood industry, commonly used as a plant growth enhancer. Seed derived calli were proliferated on Murashige and Skoog (MS) medium supplemented with different ion-chelated LS (calcium LS: CaLS and sodium LS: NaLS) at 50, 100, 150, and 200 mg/L. MS supplemented with 100 mg/L CaLS significantly increased the callus proliferation rate and adventitious root formation. In shoot induction study, both LSs did not enhance the shoot induction efficiency as compared to the control. However, the formation of albino shoot increased in MS fortified with 100 mg/L CaLS. Further chlorophyll and molecular analyses showed that, albino shoots induced from 100 mg/L CaLS had severe reduction in total chlorophyll content and expression of both chlorophyll-associated genes, chlorophyll a/b-binding protein 1 (OsCAB1R) and young seedling albino (OsYSA). Taken together, LS improves callus proliferation rate and modulate different physiological responses during plant growth of recalcitrant indica rice.

Obtention of new ornamental leaf variants of giant reed (Arundo donax L.) originated from somatic embryogenesis and their photosynthetic parameters

Giant reed (Arundo donax L.) is a perennial rhizomatous herbaceous plant, it has been widespread all over the world by human activities. It is a 2-8 meter high, polyploid, sterile species (not produce viable seeds), it can be propagated only by vegetative methods (rhizomes, stem cuttings, in vitro biotechnological methods). It has considered promising dedicated energy crop thanks to high biomass production (20-40 dry tons per hectare depending on microclimate), adaptability of different kind of soils and environment and low energy input required for its cultivation. It has been utilized for energetic purposes, biogas/bioethanol production, cellulose/paper production and ornamental purposes. The objectives of the present study were to determine morphological properties of new ornamental giant reed leaf variants originated from somatic embryogenesis and to evaluate their photosynthetic pigment content and photosynthetic activities. The most typical changes was the appearance of different colour (white, yellow, light green) longitudinal stripes on leaves (also on petiole and on leaf blade). It was significant differences (p<0.05) between green and leaf variants in case of photosynthetic pigments content and photosynthetic activity (Fv/Fm value). There was no detectable chlorophyll a or b content in the white bands of leaves and albino shoots. Total chlorophyll content of the white striped leaf variety was more than twice than the light green leaves. Photosynthetic activity and content of photosynthetic pigments also confirm and determine the morphological characteristics and growth habit of leaf variants.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process

Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2% in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Effects of sunlight on gene expression and chemical composition of light-sensitive albino tea plant

Light-sensitive albino tea plant grows yellow shoots under strong sunlight conditions. The yellow albino shoots contain high level of amino acids and medium level of polyphenols, which is beneficial for sensory quality of green tea. The effects of sunlight intensity on the transcriptions of genes involved in biosynthesis of chlorophylls and carotenoids, as well as the ultrastructure of chloroplasts, were investigated using a light-sensitive albino tea cultivar ‘Huangjinya’, and compared with a normal tea cultivar ‘Fudingdabai’. The results showed that strong sunlight induced hypoplasia of chloroplasts by suppressing the development of grana stacking and thylakoids, and down-regulated the expression levels of the 1-deoxy-d-xylulose-5-phosphate synthase gene family, involved in biosynthesis of precursors of carotenoids and chlorophylls, as well as the magnesium chelatase subunit D gene, involved in biosynthesis of chlorophylls, leading to the reduction of chlorophylls. Strong sunlight up-regulated the expression of genes involved in carotenoid pathway, resulting in the accumulation of carotenoids, including violaxanthin and lutein. The shortage of chlorophylls and the accumulation of carotenoids were considered to cause the yellow color of the albino shoots of the light-sensitive albino tea cultivar ‘Huangjinya’.

Studies to Investigate the Interactions of Genotypes, Culture Media and Culture Temperatures on Androgenesis in Recalcitrant Indica Rice (Oryza Sativa L.)

Production of doubled haploids through androgenesis is well established in japonica rice and successfully used to develop high yielding varieties. However, indica rice is remaining recalcitrant to this in vitro technique. To understand the interactions of genotypes, culture media and culture temperature, two indica varieties (Pokkali and Nona Bokra) were cultured on three culture media under normal (25°C) and alternate temperatures (30°C/ 20°C, 14 hours/10 hours). The responses of the recalcitrant varieties were examined at callus induction and regeneration stages. Pokkali was found responsive to culture media as well as culture temperatures. Although there was an increase in albino shoot production but green shoot productivity was recorded nine fold under alternate temperatures. Interactions of recalcitrant indica genotypes, culture media and culture temperatures were found significant at callus induction and regeneration stages. It is therefore reflected that to activate the recalcitrant genes for callus induction and regeneration of green shoots, interaction of genotypes, culture media and culture temperatures could play significant role. Pokkali responded well to SKI and SK II rather than others, indicated that alteration of culture media composition would be an important aspect in future investigation.

English

  White young shoots from albino tea cultivars have high level of amino acids and are rare and valuable materials for processing green tea. The effects of temperature on leaf colour, accumulation of chlorophylls and leaf ultrastructures of an albino tea cultivar ‘Xiaxueya’ were investigated. The study showed that the shoot albino phenomenon of ‘Xiaoxueya’ was controlled by temperature. The development of chloroplast from etioplast and the accumulation of chlorophylls a and b were blocked and albino shoots were observed at 15ËšC. However, the albino phenomenon was reversed when the plant was grown at 20ËšC or above. The authors recommended that the albino tea cultivars should be grown at high altitude or high latitude areas so as to prolong the production season of albino tea shoots.   Key words: Camellia sinensis, albino tea, chloroplast, etioplast, colour difference, amino acids.

Isolated microspore cultures of a Hungarian durum wheat ( Triticum turgidum L.) cultivar, Martondur 1

A number of sporophytically induced microspores and embryo-like structures (ELS) were obtained from isolated microspore cultures of durum wheat ( Triticum turgidum L. cv. Martondur 1). Various pre-treatments were screened, involving spike treatment at 4°C for 2, 7 or 14 days; anther treatment in 0.4 M mannitol containing macroelements at 33°C for 3 days, and various combinations of these. The frequency of embryogenic (star-like) microspores and the number of ELS showed a very high positive correlation in the cultures. Starvation at high temperature was necessary to achieve a reasonable frequency of microspore embryogenesis. The best results were achieved when starvation at high temperature was combined with no or short (2-day) cold treatment (212±77 and 203±34 ELS/100 anthers, respectively). However, the ELS failed to regenerate; only a few of them produced poorly-developed albino shoots. The present work could be a promising starting point for the production of doubled haploid durum wheat plants in Hungary via isolated microspore culture.

Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4-5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10-12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.

Selection of atrazine-resistant plants by in vitro mutagenesis in pepper (Capsicum annuum)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G4 were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l−1 atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l−1). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l−1atrazine, 1.0 mg l−1IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.

The effect of potato extract used as additive in anther culture medium on culture responses in Triticum aestivum

In the present study, potato extract was used as an additive supplemented to a synthetic induction medium in anther culture of wheat (Triticum aestivum), attempting to make reasonable utilization of potato extract and improve the efficiency of this induction medium. The concentration of the potato extract in the medium was 5% (water extract from 50g of potato tubers for a litre of medium), the basic synthetic medium used was W14 medium, but the inorganic salts (except iron) and the vitamins were reduced to three fourths of the original strength of W14 medium. The experimental results showed that the potato-extract-supplemented W14 medium could elicit more pollen-derived calli (or embryoids) and these calli possessed greater green shoot regeneration ability compared with both the original W14 medium and a modified W14 medium in which the five major inorganic salts were each reduced to three fourths strength of the original W14 medium, and in turn the calli produced on the original W14 medium possessed greater green shoot regeneration ability compared with the modified W14 medium. However there were no significant differences in albino shoot differentiation frequency of the calli between the above-mentioned three media, it varied only with different genotypes of the anther-donors. This indicates that the medium components affecting green shoot regeneration would not certainly affect albino shoot regeneration as well.

Investigation of the Callus Induction Medium and the Regeneration Medium in Rice Anther Culture.

We determined the callus induction medium and the regeneration medium to efficiently utilize the anther culture technique in rice breeding. The medium containing N6 basal medium supplemented with 4 mg L-1 of 2, 4-D effectively induced calli from microspores of some japonica varieties and two F1 hybrids with japonica varieties. The regenerated plants containing green and/or albino shoots were produced efficiently from microspore-derived calli on the regeneration medium containing LS basal medium supplemented with sorbitol (30000 mg L-1), CH (2000 mg L-1), MES (1000 mg L-1), NAA (2 mg L-1), and kinetin (0.5 mg L-1) solidified by gellun gum (4000 mg L-1).

Production of somatic hybrid plants between Iris ensata Thunb. and I. germanica L.

Wide hybridization that cannot be attained through conventional sexual crosses, can now be approached by somatic hybridization. Protoplasts of I. ensata and I. germanica were fused by electrofusion. For the selection of somatic hybrids, protoplasts of I. ensata which did not form colonies in protoplast culture and protoplasts of I. germanica which had regeneration ability for only albino shoot were used in symmetric fusion. On the other hand, the protoplasts of I. ensata and I. germanica protoplasts which were inactivated by iodoacetamide (IOA) treatment were used in asymmetric fusion. Five-six months after cell fusion, green plants were obtained in the symmetric and asymmetric fusion. In the random amplified polymorphic DNA (RAPD) analysis, the green plants had bands specific to both parental species. Therefore, these plants were somatic hybrids between I. ensata and I. germanica.

Induction of atrazine resistance and somatic embryogenesis in Solanum melongena

The effects of atrazine on cotyledon cultures of Solanum melongena were investigated with a view to establishing a system for in vitro selection of resistant mutants. At herbicide levels producing little growth inhibition some chlorophyll loss occurred associated with the production of albino shoots. At 15 mg/l bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenesis with EMS resulted in herbicide-resistant mutants based on the embryogenic ability of mutagenised explants placed on medium containing selective levels of sucrose (0.2%) and atrazine (15 mg/l). Different morphogenetic responses were observed when the levels of sucrose (0.2–5%) were altered. Somatic embryogenesis was observed at low sucrose concentrations (0.2–0.5%). Both embryogenesis and shoot regeneration occurred in 1% sucrose. Shoot regeneration was maximum in 2% sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3–5%). However, lowering of sucrose concentration from 2% to 0.2% caused complete bleaching, permitting the selection of herbicide-resistant mutants.

Plant Regeneration from Protoplast-derived Callus of Sugarcane.

In 5 sugarcane clones, cell suspension cultures were initiated and maintained in modified N6 Iiquid medium containing 2 mg/l 2, 4-D, 500 mg/l casein hydrolysate and 3% sucrose. These suspension cultures were able to regenerate plants for more than three months. Protoplasts isolated from these suspensions were embedded in 1.2% agarose (modified KM8P medium) and cultured in modified KM8P medium with the addition of nurse culture cells from the suspension culture. After several weeks of culttl[re, calluses were obtained in all the 5 clones. Calluses derived from protoplasts were cultured for 2 weeks on PR4 medium (pre-culture medium for regeneration), then transferred to R9 regeneration medium. In the subsequent 30-day period of culture, protoplast-derived calluses of US 76-9 and NiF 8 regenerated green shoots and albino shoots, respectively, while the other 3 clones did not form any organs. In the 6 experiments using suspension cultures of US 76-9 differing in age (8 to 31 weeks after initiation), regeneration of fertile green shoots from protoplast-derived calluses was observed. After the transfer to R1 medium for rooting, a few shoots developed to plants, while most of the shoots died without rooting.

Fertile plant regeneration from cell suspension and protoplast cultures of barley (t Hordeum vulgare cv. Schooner)

Fertile plants were regenerated from both cell suspension and protoplast-derived cultures of the two-row barley, Hordeum vulgare L. cv. Schooner. Embryogenic calluses, derived from immature embryos, were used to establish suspension cultures. More than 100 plants, with variable seed set, have been regenerated from six embryogenic cell suspension cultures. Protoplasts isolated from three suspension cultures divided and when the resultant embryogenic proto-calluses were transferred to regeneration medium both green and albino shoots were produced. The green shoots were transferred to growth regulator-free medium and plantlets that developed strong root systems were potted in soil and grown to maturity in the glasshouse. Root tip analysis of plants regenerated from cell suspension cultures revealed the expected 2N = 14 complement of chromosomes. However, chromosomal analysis of protoplast-derived plants showed numerical variation among a proportion of the regenerants.

Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL(-1) picloram + 0.01 mgL(-1) α-naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL(-1) BAP plus either 0.2 mgL(-1) picloram or 0.1 mgL(-1) indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.