Population Genetic Analysis of Paris polyphylla var. yunnanensis Based on cpDNA Fragments.

Paris polyphylla var. yunnanensis is a well-known medicinal plant that is mainly distributed in Southwest China; however, its genetic diversity and biodiversity processes are poorly understood. In this study, the sequences of cpDNA trnL-trnF fragments of 15 wild populations and 17 cultivated populations of P. polyphylla var. yunnanensis were amplified, sequenced, and aligned to study the population genetics of this species. Genetic diversity was analyzed based on nucleotide diversity, haplotype diversity, Watterson diversity, population-level diversity, and species-level genetic diversity. Genetic structure and genetic differentiation were explored using haplotype distribution maps and genetic distance matrices. A total of 15 haplotypes were identified in the 32 populations of P. polyphylla var. yunnanensis. Five unique haplotypes were identified from the fourteen haplotypes of the cultivated populations, while only one unique haplotype was identified from the ten haplotypes of the wild populations. The haplotype richness and genetic diversity of the cultivated populations were higher than those of the wild populations (HT = 0.900 vs. 0.861). In addition, there were no statistically significant correlations between geographic distance and genetic distance in the cultivated populations (r = 0.16, p > 0.05), whereas there was a significant correlation between geographical distance and genetic structure in the wild populations (r = 0.32, p > 0.05), indicating that there was a geographical and genetic connection between the wild populations. There was only 2.5% genetic variation between the wild populations and cultivated populations, indicating no obvious genetic differentiation between the wild and cultivated populations. Overall, the genetic background of the cultivated populations was complex, and it was hypothesized that the unique haplotypes and higher diversity of the cultivated populations were caused by the mixed provenance of the cultivated populations.

Dynamic variation of Paris polyphylla root-associated microbiome assembly with planting years.

MAIN CONCLUSION: P. polyphylla selectively enriches beneficial microorganisms to help their growth. Paris polyphylla (P. polyphylla) is an important perennial plant for Chinese traditional medicine. Uncovering the interaction between P. polyphylla and the related microorganisms would help to utilize and cultivate P. polyphylla. However, studies focusing on P. polyphylla and related microbes are scarce, especially on the assembly mechanisms and dynamics of the P. polyphylla microbiome. High-throughput sequencing of the 16S rRNA genes was implemented to investigate the diversity, community assembly process and molecular ecological network of the bacterial communities in three root compartments (bulk soil, rhizosphere, and root endosphere) across three years. Our results demonstrated that the composition and assembly process of the microbial community in different compartments varied greatly and were strongly affected by planting years. Bacterial diversity was reduced from bulk soils to rhizosphere soils to root endosphere and varied over time. Microorganisms benefit to plants was selectively enriched in P. polyphylla roots as was its core microbiome, including Pseudomonas, Rhizobium, Steroidobacter, Sphingobium and Agrobacterium. The network's complexity and the proportion of stochasticity in the community assembly process increased. Besides, nitrogen metabolism, carbon metabolism, phosphonate and phosphinate metabolism genes in bulk soils increased over time. These findings suggest that P. polyphylla exerts a selective effect to enrich the beneficial microorganisms and proves the sequential increasing selection pressure with P. polyphylla growth. Our work adds to the understanding of the dynamic processes of plant-associated microbial community assembly, guides the selection and application timing of P. polyphylla-associated microbial inoculants and is vital for sustainable agriculture.

[Phylogeography of Paris poliphylla var. yunnanensis based on chloroplast gene trnL-trnF sequences].

Phylogeography is a research hotspot in the field of the genetic diversity and core germplasm construction of endangered rare plants. Paris polyphylla var. yunnanensis is a rare plant species mainly distributed in China. Wild individuals have been overexploited for the last few decades because of increasing demand for such medicines. Therefore, it is great significance to study the phylogeography of P. poliphylla var. yunnanensis based on chloroplast gene trnL-trnF sequences. In this study, chloroplast genes trnL-trnF were used in the phylogeography analysis of 15 wild and 17 cultivated populations of P. polyphylla var. yunnanensis. This study revealed that based on the results of neutrality tests and mismatch analysis, the rapid expansion of wild population has not been detected in P. polyphylla var. yunnanensis. After aligning and sorting the obtained cpDNA sequences, a total of 15 haplotypes were detected in all 32 populations. One haplotype was unique to the wild population, and 5 haplotypes were unique to the cultivated population. It can be seen that the haplotype richness of cultivated population was higher than that of wild population. The wild populations of P. polyphylla var. yunnanensis were divided into two groups according to evolutionary relationship of haplotypes and distribution map of haplotypes. The haplotype of branch Ⅰ was mainly distributed in Guizhou, and the haplotype of branch Ⅱ was located in Yunnan and Huidong, Sichuan. Therefore, it's speculated that Guizhou and the west Yunnan region may be glacial refuge in the evolutionary history of wild populations of P. polyphylla var. yunnanensis, and in order to protect the wild resources more effectively, wild populations of P. polyphylla var. yunnanensis in these two areas should be included in the protection zone.

Transcriptome analyses of Paris polyphylla var. chinensis, Ypsilandra thibetica, and Polygonatum kingianum characterize their steroidal saponin biosynthesis pathway.

Steroidal saponins, one of the most diverse groups of plant-derived natural products, elicit biological and pharmacological activities; however, the genes involved in their biosynthesis and the corresponding biosynthetic pathway in monocotyledon plants remain unclear. This study aimed to identify genes involved in the biosynthesis of steroidal saponins by performing a comparative analysis among transcriptomes of Paris polyphylla var. chinensis (PPC), Ypsilandra thibetica (YT), and Polygonatum kingianum (PK). De novo transcriptome assemblies generated 57,537, 140,420, and 151,773 unigenes from PPC, YT, and PK, respectively, of which 56.54, 47.81, and 44.30% were successfully annotated, respectively. Among the transcriptomes for PPC, YT, and PK, we identified 194, 169, and 131; 17, 14, and 26; and, 80, 122, and 113 unigenes corresponding to terpenoid backbone biosynthesis; sesquiterpenoid and triterpenoid biosynthesis; and, steroid biosynthesis pathways, respectively. These genes are putatively involved in the biosynthesis of cholesterol that is the primary precursor of steroidal saponins. Phylogenetic analyses indicated that lanosterol synthase may be exclusive to dicotyledon plant species, and the cytochrome P450 unigenes were closely related to clusters CYP90B1 and CYP734A1, which are UDP-glycosyltransferases unigenes homologous with the UGT73 family. Thus, unigenes of ß-glucosidase may be candidate genes for catalysis of later period modifications of the steroidal saponin skeleton. Our data provide evidence to support the hypothesis that monocotyledons biosynthesize steroidal saponins from cholesterol via the cycloartenol pathway.

Comparative chloroplast genomes of Paris Sect. Marmorata: insights into repeat regions and evolutionary implications.

BACKGROUND: Species of Paris Sect. Marmorata are valuable medicinal plants to synthesize steroidal saponins with effective pharmacological therapy. However, the wild resources of the species are threatened by plundering exploitation before the molecular genetics studies uncover the genomes and evolutionary significance. Thus, the availability of complete chloroplast genome sequences of Sect. Marmorata is necessary and crucial to the understanding the plastome evolution of this section and facilitating future population genetics studies. Here, we determined chloroplast genomes of Sect. Marmorata, and conducted the whole chloroplast genome comparison. RESULTS: This study presented detailed sequences and structural variations of chloroplast genomes of Sect. Marmorata. Over 40 large repeats and approximately 130 simple sequence repeats as well as a group of genomic hotspots were detected. Inverted repeat contraction of this section was inferred via comparing the chloroplast genomes with the one of P. verticillata. Additionally, almost all the plastid protein coding genes were found to prefer ending with A/U. Mutation bias and selection pressure predominately shaped the codon bias of most genes. And most of the genes underwent purifying selection, whereas photosynthetic genes experienced a relatively relaxed purifying selection. CONCLUSIONS: Repeat sequences and hotspot regions can be scanned to detect the intraspecific and interspecific variability, and selected to infer the phylogenetic relationships of Sect. Marmorata and other species in subgenus Daiswa. Mutation and natural selection were the main forces to drive the codon bias pattern of most plastid protein coding genes. Therefore, this study enhances the understanding about evolution of Sect. Marmorata from the chloroplast genome, and provide genomic insights into genetic analyses of Sect. Marmorata.

Transcriptome-Wide Identification and Prediction of miRNAs and Their Targets in Paris polyphylla var. yunnanensis by High-Throughput Sequencing Analysis.

Long dormancy period of seeds limits the large-scale artificial cultivation of the scarce Paris polyphylla var. yunnanensis, an important traditional Chinese medicine. Characterizing miRNAs and their targets is crucial to understanding the role of miRNAs during seed dormancy in this species. Considering the limited genome information of this species, we first sequenced and assembled the transcriptome data of dormant seeds and their seed coats as the reference genome. A total of 146,671 unigenes with an average length of 923 bp were identified and showed functional diversity based on different annotation methods. Two small RNA libraries from respective seeds and seed coats were sequenced and the combining data indicates that 263 conserved miRNAs belonging to at least 83 families and 768 novel miRNAs in 1174 transcripts were found. The annotations of the predicted putative targets of miRNAs suggest that these miRNAs were mainly involved in the cell, metabolism and genetic information processing by direct and indirect regulation patterns in dormant seeds of P. polyphylla var. yunnanensis. Therefore, we provide the first known miRNA profiles and their targets, which will assist with further study of the molecular mechanism of seed dormancy in P. polyphylla var. yunnanensis.

Evidence of a Strong Domestication Bottleneck in the Recently Cultivated New Zealand Endemic Root Crop, Arthropodium cirratum (Asparagaceae).

We use chloroplast DNA sequencing to examine aspects of the pre-European Maori cultivation of an endemic New Zealand root crop, Arthropodium cirratum (rengarenga). Researching the early stages of domestication is not possible for the majority of crops, because their cultivation began many thousands of years ago and/or they have been substantially altered by modern breeding methods. We found high levels of genetic variation and structuring characterised the natural distribution of A. cirratum, while the translocated populations only retained low levels of this diversity, indicating a strong bottleneck even at the early stages of this species' cultivation. The high structuring detected at four chloroplast loci within the natural A. cirratum range enabled the putative source(s) of the translocated populations to be identified as most likely located in the eastern Bay of Plenty/East Cape region. The high structuring within A. cirratum also has implications for the conservation of genetic diversity within this species, which has undergone recent declines in both its natural and translocated ranges.

Diversity of Drimia indica (Roxb.) Jessop and its relationship to Drimia nagarjunae using phenotypic traits and molecular markers.

In the present study, we explored the diversity of different accessions of Drimia indica and its relation to D. nagarjunae using phenotypic traits and molecular markers. Twenty populations of D. indica, from different parts of India, were compared with D. nagarjunae, an endangered medicinal plant collected from Andhra Pradesh, India. Two species showed appreciable phenotypic diversity in number of leaves, leaf indices, bulb circumference, bulb length and length of roots. The principal component analysis (PCA) performed on above 5 quantitative characters to determine relationship among populations, has distinguished D. nagarjunae from D. indica phenotypically. Genetic diversity was analysed using RAPD and ISSR primers which produced reproducible bands in 8 RAPD and 3 ISSR primers. A total of 89 amplicons were observed, of which 69 (77.53%) were polymorphic. Cluster diagram and phylogenetic linkage showed that D. nagarjunae formed a separate cluster, showing no similarity with any of the populations of D. indica. The molecular marker data correlated with PCA of phenotypic traits. Current investigations have demonstrated that the statistical approach for phenotypic characters and molecular markers analysis can be applied to study diversity in Drimia species.

The complete chloroplast genome of colchicine plants (Colchicum autumnale L. and Gloriosa superba L.) and its application for identifying the genus.

MAIN CONCLUSION: The complete chloroplast genome of two colchicine medicinal plants is reported for the first time. Deletion of ycf 15 gene occurred only in Colchicum but not in Gloriosa and suggests this as a potential marker for delineating the two species. Colchicum autumnale L. and Gloriosa superba L. are well-known sources of colchicine, a type of alkaloid and an ancient anti-inflammatory drug used to prevent gout. Accordingly, this alkaloid has been used as a chemical marker for identifying the expanded Colchicaceae family. In the present study, we report the complete chloroplast genome (cpDNA) sequence of two colchicine medicinal plants (G. superba and C. autumnale) that belong to the tribe Colchiceae of the Colchicaceae family. In C. autumnale, the circular double-stranded cpDNA sequence of 156,462 bp consists of two inverted repeat (IR) regions of 27,741 bp each, a large single-copy region (LSC) of 84,246 bp, and a small single-copy region (SSC) of 16,734 bp. The cpDNA sequence of G. superba is longer than that of C. autumnale (157,924 bp), which consists of two IRs (28,063 bp), an SSC (16,786 bp), and an LSC (85,012 bp). Significant structural differences between them were observed in the ycf15 gene. ycf15 gene was absent from C. autumnale cpDNA and affected the length of the chloroplast genome between the species. Furthermore, this gene loss event was specific to the expanded genus of Colchicum sensu Vinnersten and Manning. Therefore, this gene may be an effective and powerful molecular marker for identifying the Colchicum genus within the family.

Microbial diversity in Paris polyphylla var. yunnanensis rhizomes of varying ages.

Endophyte microorganisms live inside plants without causing them any apparent damage. Recently, endophytic microorganisms have attracted attention because they can produce bioactive compounds of biotechnological interest. The endophytic microorganisms in Paris polyphylla var. yunnanensis (Liliaceae) - a species used since antiquity in traditional Chinese medicine - are under scrutiny because they may be responsible for producing the bioactive metabolites associated with the plant. The levels of bioactive metabolites in the rhizomes of P. polyphylla increase with rhizome age. To elucidate the roles played by endophytes in the accumulation of bioactive metabolites, we investigated the community structure and diversity of the endophytic microorganisms in P. polyphylla rhizomes of different ages (4, 6, and 8 years) using 16S rRNA and internal transcribed spacer (ITS) sequence analysis. 16S rDNA amplicon pyrosequencing revealed that the number of operational taxonomic units was lower in the 8-year-old samples than in the other samples. A total of 28 phyla were observed in the P. polyphylla samples and the predominant bacteria were of the Cyanobacteria and Proteobacteria phyla. Moreover, the percentage of Cyanobacteria increased with rhizome age. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes; some classes were more prevalent in the 8-year-old rhizomes than in younger rhizomes, indicating the importance in secondary metabolism in older rhizomes. Our study showed that endophyte microorganism diversity and prevalence depend on P. polyphylla rhizome age. There was also an indication that some endophyte microorganisms contribute to the higher saponin content in older P. polyphylla specimens.

[Phenotypic Trait Variation, Principal Component, Correlation and Path Analysis of Paris polyphylla var. yunnanensis].

OBJECTIVE: To investigate the phenotypic trait variation range of Paris polyphylla var. yunnanensis, and to look for phenotypic traits closely related with yield, in order to provide reference for its breeding in the future. METHODS: Wild plants of Paris polyphylla var. yunnanensis populations cultivated for three years in Xishuangbanna, Yunnan Province were chosen. The plants of Paris polyphylla var. yunnanensis were randomly labelled, its phenotypic traits were observed and analyzed by variation, principal component, correlation, variance and path analysis. RESULTS: Eleven phenotypic traits variation of the populations of Paris polyphylla var. yunnanensis were great, mainly distributed in yield, growth and genetic characteristics. Phenotypic trait of different growth years (4 - 10 years) including stem height, stem diameter, flower stem height, flower stem diameter, leaf number, leaf length and leaf width had no significant difference(P > 0. 05). The underground phenotypic traits including root weight, root length and root diameter increased significant (P <0. 05) with plant growth years. Effect of the other phenotypic traits on root weight was as follows: root diameter (0. 2550) > root growth years (0. 1183 ) > root length(0. 0202) > stem diameter(0. 0081) > stem height (0. 0044) > leaf number (0. 0025) > leaf length (0. 0018) > leaf width (0. 0004) > flower stem height (0. 0003) > flower stem diameter (0. 0001). CONCLUSION: The phenotypic traits on yield, growth and genetic characteristics have rich variations, and the phenotypic traits on the ground have no relationship with plant growth years, the plant growth years mainly affect the underground phenotypic traits. When choosing different root types, root diameter can be used as the preferred character, stem diameter as the second preferred traits when the root traits information not be easily obtained.

Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae).

Fritillaria (Liliaceae) is a genus of approximately 140 species of bulbous perennial plants that includes taxa of both horticultural and medicinal importance. As well as being commercially valuable, Fritillaria species have attracted attention because of their exceptionally large genome sizes, with all values recorded to date in excess of 30Gb. Despite such interest in the genus, phylogenetic relationships between the majority of species have remained untested. Here we present the first phylogenetic reconstruction of relationships to encompass most of the currently recognised species diversity in the genus. Three regions of the plastid genome were sequenced in 117 individuals of Fritillaria, representing 92 species (c. 66% of the genus) and in representatives of nine other genera of Liliaceae. Eleven low-copy nuclear gene regions were also screened in selected species for their potential utility. Phylogenetic analysis of a combined plastid dataset using maximum parsimony and Bayesian inference provided support for the monophyly of the majority of currently recognised subgenera. However, subgenus Fritillaria, which is by far the largest of the subgenera and includes the most important species used in traditional Chinese medicine, is found to be polyphyletic. Moreover, several taxa that were represented by multiple individuals show evidence of species non-monophyly. The Japanese endemic subgenus Japonica, which contains the species with the largest recorded genome size for any diploid plant, is resolved as sister to the predominantly Middle Eastern and Central Asian subgenus Rhinopetalum. Whilst relationships between most of the major Fritillaria lineages can now be resolved, our results also highlight the need for data from additional independently evolving loci; an endeavour that may be particularly challenging in light of the huge nuclear genomes found in these plants.

Somatic embryogenesis and synthetic seed production--a biotechnological approach for true-to-type propagation and in vitro conservation of an ornamental bulbaceous plant Drimiopsis kirkii Baker.

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4 ± 0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3 ± 0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24 ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.

De Novo transcriptome sequencing reveals important molecular networks and metabolic pathways of the plant, Chlorophytum borivilianum.

Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum.

Comparative genomics of four Liliales families inferred from the complete chloroplast genome sequence of Veratrum patulum O. Loes. (Melanthiaceae).

The sequence of the chloroplast genome, which is inherited maternally, contains useful information for many scientific fields such as plant systematics, biogeography and biotechnology because its characteristics are highly conserved among species. There is an increase in chloroplast genomes of angiosperms that have been sequenced in recent years. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Veratrum patulum Loes. (Melanthiaceae, Liliales) was analyzed completely. The circular double-stranded DNA of 153,699 bp consists of two inverted repeat (IR) regions of 26,360 bp each, a large single copy of 83,372 bp, and a small single copy of 17,607 bp. This plastome contains 81 protein-coding genes, 30 distinct tRNA and four genes of rRNA. In addition, there are six hypothetical coding regions (ycf1, ycf2, ycf3, ycf4, ycf15 and ycf68) and two open reading frames (ORF42 and ORF56), which are also found in the chloroplast genomes of the other species. The gene orders and gene contents of the V. patulum plastid genome are similar to that of Smilax china, Lilium longiflorum and Alstroemeria aurea, members of the Smilacaceae, Liliaceae and Alstroemeriaceae (Liliales), respectively. However, the loss rps16 exon 2 in V. patulum results in the difference in the large single copy regions in comparison with other species. The base substitution rate is quite similar among genes of these species. Additionally, the base substitution rate of inverted repeat region was smaller than that of single copy regions in all observed species of Liliales. The IR regions were expanded to trnH_GUG in V. patulum, a part of rps19 in L. longiflorum and A. aurea, and whole sequence of rps19 in S. china. Furthermore, the IGS lengths of rbcL-accD-psaI region were variable among Liliales species, suggesting that this region might be a hotspot of indel events and the informative site for phylogenetic studies in Liliales. In general, the whole chloroplast genome of V. patulum, a potential medicinal plant, will contribute to research on the genetic applications of this genus.

The chemistry and biological activity of the Hyacinthaceae.

The Hyacinthaceae (sensu APGII), with approximately 900 species in about 70 genera, can be divided into three main subfamilies, the Hyacinthoideae, the Urgineoideae and the Ornithogaloideae, with a small fourth subfamily the Oziroëoideae, restricted to South America. The plants included in this family have long been used in traditional medicine for a wide range of medicinal applications. This, together with some significant toxicity to livestock has led to the chemical composition of many of the species being investigated. The compounds found are, for the most part, subfamily-restricted, with homoisoflavanones and spirocyclic nortriterpenoids characterising the Hyacinthoideae, bufadienolides characterising the Urgineoideae, and cardenolides and steroidal glycosides characterising the Ornithogaloideae. The phytochemical profiles of 38 genera of the Hyacinthaceae will be discussed as well as any biological activity associated with both crude extracts and compounds isolated. The Hyacinthaceae of southern Africa were last reviewed in 2000 (T. S. Pohl, N. R. Crouch and D. A. Mulholland, Curr. Org. Chem., 2000, 4, 1287-1324; ref. 1); the current contribution considers the family at a global level.

Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers--a potential medicinal plant.

Malabar glory lily (Gloriosa superba L.) is a medicinally potent plant species used for the production of alkaloid colchicine. With ever increasing demand, there is a pressing need to conserve it through biotechnological approaches. A large number of complete plantlets were obtained by direct regeneration from the non-dormant tuber explants on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP)+0.5 mg/l α-naphthalene acetic acid (NAA). Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, the genetic stability of micropropagated clones was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. During the study a total of 80 (50 RAPD and 30 ISSR) primers were screened, out of which 10 RAPD and 7 ISSR primers produced a total of 98 (49 RAPD and 49 ISSR) clear, distinct and reproducible amplicons. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. This is the first report that evaluates the use of genetic markers to establish genetic fidelity of micropropagated G. superba using RAPD and ISSR, which can be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays for colchicine production to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses.

Characterization of Squalene synthase gene from Chlorophytum borivilianum (Sant. and Fernand.).

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.

[Breeding system of Paris polyphylla var. yunnanensis].

OBJECTIVE: To provide the basic guidance for seed breeding and cross-breeding of Paris polyphylla var. yunnanensis. METHOD: The floral behavior and pollinators were observed; 0.5% TTC solution was used for the pollen viability test and benzidine and -H2O2 was used for estimation of the stigma receptivity. The mating systems were tested by out crossing index (OC1), and pollination experiment was carried out by bagged and emasculated test in the field. RESULT: Commonly, stigma lobes spread slightly, and anthers started presenting the pollen from the outer ring while the flower was just beginning to open. Consequently, the distance between the stigma and its own pollen was relatively far, this "floral behavior" may be conducive to outcrossing. Then the flower entered the later period, while the stigma lobes spread widely, anthers all split, and this "floral behavior" shortened the stigma and its own pollen's distance, which may be conducive to selfing. P. polyphylla was partly protogynous. Stigma life-span was about 10-12 d. After anther dehiscence, the pollen viability maintained about 10% within 2 days, and 20% within 10 days. The value of out crossing index (OC1) was 4. By pollination experiment and pollinators observed, P. polyphylla was self-compatible, but no capacity for autonomous self-fertilization; In natural circumstances, outcrossing fructification rate was low, and mainly anemophilous. Assisted selfing-fertilization fructification rate was higher, spider was the main pollinators. CONCLUSION: P. polyphylla has a mixed mating system with self-pollination and cross-pollination characteristics. Floral behavior has important adaptive significance in avoiding female and male interference, outcrossing, and delayed selfing. P. polyphylla is ambophily (a combination of both wind and insect pollination), pollinators changes due to environment. Pollen limitation is the main cause of low fructification rate under natural conditions.

Differentially expressed transcripts from leaf and root tissue of Chlorophytum borivilianum: a plant with high medicinal value.

Chlorophytum borivilianum is one of the important medicinal plants used for treating different health problems such as diabetes, arthritis, physical weakness, etc. Saponins present in C. borivilianum are the primary source of its significant medicinal properties and are synthesized by mevalonate and non-mevalonate pathways in plants. However, the biosynthesis of these compounds at molecular level is not studied in C. borivilianum. Cloning and sequencing of genes involved in metabolic processes are prerequisite to study the gene expression, their regulation and genetic engineering experiments. Expressed sequence tags (ESTs) provide a quick insight into various genes and their tissue specific expression. Suppression subtractive hybridization (SSH) libraries were constructed using mRNA from leaf and root tissues of C. borivilianum. High quality non-redundant 506 and 303 ESTs were generated from leaf and root specific libraries respectively. These sequences were analyzed using bioinformatics tools and grouped into different categories based on their similarity and cellular functions such as photosynthesis, metabolism, transcription factors, cell signaling, defense, stress response etc. ESTs also showed similarity with genes involved in saponins biosynthesis such as squalene synthase, squalene epoxidase, cytochrome p450, glycosyltransferase, etc. Semi-quantitative analysis of some of the ESTs involved in saponins biosynthesis confirmed their differential regulation in leaves and roots. These ESTs will provide an efficient resource to accelerate gene discovery in C. borivilianum and will help in determining promising targets for genetic engineering of saponins pathway.