Periodic Reporting for period 1 - REDSTEM (Development of a new plant variety for the Asian market)
Période du rapport: 2015-03-01 au 2016-08-31
Résumé du contexte et des objectifs généraux du projet
Introduction
The kentia palm (Howea forsteriana) is endemic to a 12Km2 volcanic island in the Tasman Sea, Lord Howe Island (LHI). Despite its subtropical origin, it grows well in cool climates and under low light conditions, making a perfect indoor ornamental plant. It is one of the most traded houseplants in the world, and together with tourism, the pillar of the LHI economy.
The typical kentia palm presents a dark green-coloured stem; however, during an expedition on LHI, Professor Vincent Savolainen and his team discovered a new variety that presents a bright red stem. Red-stemmed palms are highly demanded as decorative plants, especially in Asia where red is thought to bring good luck. However, these red palm varieties require tropical or subtropical conditions to grow, hence commercialisation is limited. Thus, a red-stemmed variety of H. forsteriana, which is easily grown in a wide range of temperature, humidity, and light conditions, may have tremenmdous market potential. Nonetheless, palm trees grow slowly and reach maturity at 15-20 years old, making traditional breeding strategies insufficient to propagate the variety, difficulty that has to be addressed before accomplish commercialisation.
Objectives
We aimed to disentangle the underlying genetics of the red-stemmed phenotype in kentia palms, and to establish a protocol for the propagation of the variety. To this end, we proposed (i) to analyse the stability of such phenotype, (ii) to investigate the underlying genetics responsible for the coloration, (iii) and finally, to develop a protocol of somatic embryogenesis to propagate the variety.
Methods
Assessment of phenotype stability:
We planted 23 green-stemmed and 180 red-stemmed plantlets from LHI nursery in our greenhouse, using a mixture of soil that mimics calcarenite, the original soil on LHI. We visually checked for changes in coloration throughout 4 months.
High throughput RNA sequencing and gene expression analysis:
We sampled stems from 5 red and 5 green-stemmed adult individuals growing on calcarenite soil in LHI. After RNA extraction and library construction, samples were sequenced in an Illumina HiSeq genome analyser. After assessing sequence quality with FastQC program, reads were mapped against H. forsteriana transcriptome and expression values were calculated using RSEM software package. Finally, differential gene and isoforms expression was analysed with the R package EdgeR.
Anthocyanin content measurement:
We extracted anthocyanin from the stems of 78 green-stemmed and 13 red-stemmed young palms growing in our greenhouse using ethanol:HCl and chloroform. The absorbance of the extracts was measured at 535nm in a NanoDrop, and anthocyanin content was calculated using the following formula: C (μg/g) = (A/ε*L)*MW*1000; where C=concentration (µg/ml), A=absorbance (at 535nm), ε=Molecular extinction coefficient of cyanidin-3-glucoside (26900 M-1), L=Length in cm, MW = Molecular weight of cyanidin-3-glucoside (445).
In vitro propagation:
A total of 130 H.forsteriana embryos were extracted from their seeds and included in modified Murashige and Skoog (MSO) medium containing different concentrations of an artificial auxin: 0mg/l (55 embryos), 0.1mg/l (25 embryos), 0.3mg/l (26 embryos), and 0.5 mg/l (24 embryos). Germination incidence as well as callus formation was monitored. After six weeks, 15 plantlets were sectioned longitudinally and included in modified MSO medium supplemented with 3mg/l of artificial auxin to induce somatic embryogenesis.
Assessment of phenotype stability:
After 4 months, 85.6% of the red-stemmed plants had turned green-stemmed, while 100% of the green-stemmed ones did not show changes in coloration.
High throughput RNA sequencing and gene expression analysis:
We retrieved ca. 12M reads per sample, which we mapped against the previously published H. forsteriana transcriptome. We obtained a mean of 73.6±2.9% of reads mapped that corresponded to 13,868 genes. Differential gene expression analysis with EdgeR showed a total of 111 (P-value < 0.01) genes differentially expressed among both varieties. However, only four of those genes presented a False Discovery Rate (FDR) below the accepted threshold (0.1). Three of them were up regulated in red-stemmed samples and one was down regulated. BLAST analysis of the nucleotide sequences showed homology to oil palm genes: two of the up regulated genes are homologous to genes in the anthocyanin synthesis pathway (anthocyanidin 3-O-glucosyltransferase and chalcone synthase). The third up regulated gene is homologous to cytochrome b561, which is implicated in apoplastic (“front-line”) defence. The gene down regulated is homologous to a choloplastic gene involved in nitrogen assimilation (ferredoxin--nitrite reductase).
We also analysed the differential expression at the isoforms level. From the 31,693 isoforms mapped, 322 were differentially expressed (P-value < 0.01). Of those, 11 had a FDR<0.1; 6 isoforms were up regulated on red-stemmed samples, and 5 were down regulated. By homology analysis, we found that the up regulated isoforms are involved in anthocyanin synthesis, in light sensing, and in calcium signalling. Down regulated isoforms are involved in nitrogen metabolism and cell wall composition.
Anthocyanin content measurement:
Mean anthocyanin content in red stems was 2.8 fold more abundant than in green stems, being this difference statistically significant (p<0.001).
In vitro propagation:
After 2-3 weeks 93% of the embryos across the four treatments germinated (“germination” defined as “the appearance of a root and a shoot”). Hormone concentration did not affect germination rate (p=0.68). Approximately half of the embryos developed a callus (disorganised structure) independently of the hormone concentration in the medium.
Three out of fifteen plantlets monitored for somatic embryogenesis produced pre-embryonic tissue, and one produced a globular embryo surrounded by pre embryonic tissue.
The kentia palm (Howea forsteriana) is endemic to a 12Km2 volcanic island in the Tasman Sea, Lord Howe Island (LHI). Despite its subtropical origin, it grows well in cool climates and under low light conditions, making a perfect indoor ornamental plant. It is one of the most traded houseplants in the world, and together with tourism, the pillar of the LHI economy.
The typical kentia palm presents a dark green-coloured stem; however, during an expedition on LHI, Professor Vincent Savolainen and his team discovered a new variety that presents a bright red stem. Red-stemmed palms are highly demanded as decorative plants, especially in Asia where red is thought to bring good luck. However, these red palm varieties require tropical or subtropical conditions to grow, hence commercialisation is limited. Thus, a red-stemmed variety of H. forsteriana, which is easily grown in a wide range of temperature, humidity, and light conditions, may have tremenmdous market potential. Nonetheless, palm trees grow slowly and reach maturity at 15-20 years old, making traditional breeding strategies insufficient to propagate the variety, difficulty that has to be addressed before accomplish commercialisation.
Objectives
We aimed to disentangle the underlying genetics of the red-stemmed phenotype in kentia palms, and to establish a protocol for the propagation of the variety. To this end, we proposed (i) to analyse the stability of such phenotype, (ii) to investigate the underlying genetics responsible for the coloration, (iii) and finally, to develop a protocol of somatic embryogenesis to propagate the variety.
Methods
Assessment of phenotype stability:
We planted 23 green-stemmed and 180 red-stemmed plantlets from LHI nursery in our greenhouse, using a mixture of soil that mimics calcarenite, the original soil on LHI. We visually checked for changes in coloration throughout 4 months.
High throughput RNA sequencing and gene expression analysis:
We sampled stems from 5 red and 5 green-stemmed adult individuals growing on calcarenite soil in LHI. After RNA extraction and library construction, samples were sequenced in an Illumina HiSeq genome analyser. After assessing sequence quality with FastQC program, reads were mapped against H. forsteriana transcriptome and expression values were calculated using RSEM software package. Finally, differential gene and isoforms expression was analysed with the R package EdgeR.
Anthocyanin content measurement:
We extracted anthocyanin from the stems of 78 green-stemmed and 13 red-stemmed young palms growing in our greenhouse using ethanol:HCl and chloroform. The absorbance of the extracts was measured at 535nm in a NanoDrop, and anthocyanin content was calculated using the following formula: C (μg/g) = (A/ε*L)*MW*1000; where C=concentration (µg/ml), A=absorbance (at 535nm), ε=Molecular extinction coefficient of cyanidin-3-glucoside (26900 M-1), L=Length in cm, MW = Molecular weight of cyanidin-3-glucoside (445).
In vitro propagation:
A total of 130 H.forsteriana embryos were extracted from their seeds and included in modified Murashige and Skoog (MSO) medium containing different concentrations of an artificial auxin: 0mg/l (55 embryos), 0.1mg/l (25 embryos), 0.3mg/l (26 embryos), and 0.5 mg/l (24 embryos). Germination incidence as well as callus formation was monitored. After six weeks, 15 plantlets were sectioned longitudinally and included in modified MSO medium supplemented with 3mg/l of artificial auxin to induce somatic embryogenesis.
Assessment of phenotype stability:
After 4 months, 85.6% of the red-stemmed plants had turned green-stemmed, while 100% of the green-stemmed ones did not show changes in coloration.
High throughput RNA sequencing and gene expression analysis:
We retrieved ca. 12M reads per sample, which we mapped against the previously published H. forsteriana transcriptome. We obtained a mean of 73.6±2.9% of reads mapped that corresponded to 13,868 genes. Differential gene expression analysis with EdgeR showed a total of 111 (P-value < 0.01) genes differentially expressed among both varieties. However, only four of those genes presented a False Discovery Rate (FDR) below the accepted threshold (0.1). Three of them were up regulated in red-stemmed samples and one was down regulated. BLAST analysis of the nucleotide sequences showed homology to oil palm genes: two of the up regulated genes are homologous to genes in the anthocyanin synthesis pathway (anthocyanidin 3-O-glucosyltransferase and chalcone synthase). The third up regulated gene is homologous to cytochrome b561, which is implicated in apoplastic (“front-line”) defence. The gene down regulated is homologous to a choloplastic gene involved in nitrogen assimilation (ferredoxin--nitrite reductase).
We also analysed the differential expression at the isoforms level. From the 31,693 isoforms mapped, 322 were differentially expressed (P-value < 0.01). Of those, 11 had a FDR<0.1; 6 isoforms were up regulated on red-stemmed samples, and 5 were down regulated. By homology analysis, we found that the up regulated isoforms are involved in anthocyanin synthesis, in light sensing, and in calcium signalling. Down regulated isoforms are involved in nitrogen metabolism and cell wall composition.
Anthocyanin content measurement:
Mean anthocyanin content in red stems was 2.8 fold more abundant than in green stems, being this difference statistically significant (p<0.001).
In vitro propagation:
After 2-3 weeks 93% of the embryos across the four treatments germinated (“germination” defined as “the appearance of a root and a shoot”). Hormone concentration did not affect germination rate (p=0.68). Approximately half of the embryos developed a callus (disorganised structure) independently of the hormone concentration in the medium.
Three out of fifteen plantlets monitored for somatic embryogenesis produced pre-embryonic tissue, and one produced a globular embryo surrounded by pre embryonic tissue.