Post-thaw survival and proliferation of friable and embryogenie callus in Hevea brasiliensis by liquid nitrogen vitrification tryopreservation method [Preprint]

Friable and embryogenic calli could be induced from the inner integument of the rubber tree clone Reyan 88-13. And they had been proliferated and maintained for more than 2 years. Somatic embryos and further regeneration plantlets had been obtained from these calli. In order to preserve these calli for a long time and maintain their genetic characters, a liquid nitrogen vitrification cryopreservation method had been studied with these calli as the material. These calli were inoculated on the pre-culture medium, and had been cultured for 2-3 days in the dark, the temperature was 25-28 oC. Then the calli were immersed for 10-30 min in the 60% PVS2 solution at room temperature. After dehydration treatment, these calli were immersed for 20-40 min in PVS2 solution (28%-32% glycerol + 12%-18% hexanediol + 12%-18% dimethyl sulphoxide + 136.9g1L sucrose) at O°C. Then the former PVS2 solution was removed, and new one was added. Afterward, these calli were thrown into liquid nitrogen (-196 OC) rapidly. The recovery of the calli was to take out of them from the liquid nitrogen quickly, immersed them in water for 2-3 min in which the temperature was 40 oC. After thaw and rinse, these calli were transferred on the subculture medium, and was cultured for 2-3 days in the dark, the temperature was 25-28 oC. Then they Were transferred on the fresh subculture medium and were cultured for 30 days. Most ofthe cryopreserved calli tumed brown and died. But sorne cell survived. So 30 days later, sorne tiny calli emerged, and grew. And they could be proliferated in the further subculture cycles. The post-thaw and survival calli could be used to induce somatic embryogenesis and further plant regeneration. The present results of the studies had the advantages that the experimental procedure was not complex, expensive apparatus could be avoided, repeatability and effect was good. So this method would be useful for long-term preservation of valuable germplasm materials, and would be beneficial for tissue culture and genetictransformation studies in Hevea brasiliensis. (Résumé d'auteur

In vitro culture method of powdery mildew (Oidium heveae Steinmann) of Hevea brasiliensis

A method for culturing powdery mildew (Oidium heveae) from isolated leaves of Hevea brasiliensis was evaluated, which included three steps: Leaves and fungi selection, nutrient solution and culture dish  preparation, fungi inoculation and culture. The culture time and produced conidia number were considered as decision index. We tested the influence of micro components of nutrient solution including 6-benzylaminopurine (6-BA), salicylic acid (SA) and vitamin C (VC) and evaluated the culture difference of various leaf phenological phases and rubber tree clones. The results show that the longest culture time of isolated leaves emerged on modified Murashige and Skoog (MS) macro elements with 4 mg/L 6-BA, 20 mg/L SA, 1 mg/L VC. The colour phase leaf was the preferable choice for culturing average 15 to 16 days and producing 3.2222 × 106 mL-1 conidia. The culture effects of using various rubber clones were different and higher resistance clones cultured less conidia. The method leading to mass production of powdery mildew was simple using a climate incubator to resolve problems linked to season and space limitation and preservation of powdery mildew. This method could improve rubber resistance breeding process.Key words: Hevea brasiliensis, Oidium heveae, in vitro culture, nutrient solution, phenological phase

Co-extraction of high-quality RNA and DNA from rubber tree (Hevea brasiliensis)

High-quality nucleic acids are the basic requirement for performing genomic research. A reliable and efficient method was developed for co-extracting high-quality DNA and RNA from rubber tree (Hevea brasiliensis) in this study. Polyethylene glycol (PEG) and cetyltrimethylammonium bromide (CTAB) extraction buffer with high concentrations of polyvinylpyrrolidone (PVP) and β-mercaptoethanol was used in this study. The results show that 3.2% polyethylene glycol 8000 is the optimal concentration for successful separation of DNA and RNA. Spectrophotometric determination (A260/A280 and A260/A230 ratios), agarose electrophoresis analysis and reverse transcription (RT-PCR) of isolated nucleic acids indicate that high-quality DNA and RNA were extracted by this method. The general applicability of this method was also evaluated, and the results show that it was suitable for a variety of plants.Key words: Hevea brasiliensis, polyethylene glycol (PEG), nucleic acid, co-extraction, higher plants

The rubber tree genome reveals new insights into rubber production and species adaptation

The Para rubber tree (Hevea brasiliensis) is an economically important tropical tree species that produces natural rubber, an essential industrial raw material. Here we present a high-quality genome assembly of this species (1.37 Gb, scaffold N50 = 1.28 Mb) that covers 93.8% of the genome (1.47 Gb) and harbours 43,792 predicted protein-coding genes. A striking expansion of the REF/SRPP (rubber elongation factor/small rubber particle protein) gene family and its divergence into several laticifer-specific isoforms seem crucial for rubber biosynthesis. The REF/SRPP family has isoforms with sizes similar to or larger than SRPP1 (204 amino acids) in 17 other plants examined, but no isoforms with similar sizes to REF1 (138 amino acids), the predominant molecular variant. A pivotal point in Hevea evolution was the emergence of REF1, which is located on the surface of large rubber particles that account for 93% of rubber in the latex (despite constituting only 6% of total rubber particles, large and small). The stringent control of ethylene synthesis under active ethylene signalling and response in laticifers resolves a longstanding mystery of ethylene stimulation in rubber production. Our study, which includes the re-sequencing of five other Hevea cultivars and extensive RNA-seq data, provides a valuable resource for functional genomics and tools for breeding elite Hevea cultivars. The rubber tree (Hevea brasiliensis, hereafter referred to as Hevea) is a member of the spurge family (Euphorbiaceae), along with several other economically important species such as cassava (Manihot esculenta) and the castor oil plant (Ricinus communis). Natural rubber (cis-1, 4-polyisoprene) makes up about one-third of the volume of latex that is essentially cytoplasm of the articulated laticifers in Hevea. The latex is extracted by tapping the bark, a non-destructive method of harvesting that facilitates continual production. As an industrial commodity, natural rubber is an elastomer with physical and chemical properties that cannot be fully matched by synthetic rubber1. In contrast to synthetics, the production of natural rubber is sustainable and environment friendly2. The commercial cultivation of Hevea, a native to the Amazon Basin, began in 1896 on a plantation scale in Malaya (now Malaysia) and expanded to other Southeast Asian countries that lead in world natural rubber production today3. Decades of selective breeding have resulted in a gradual improvement in rubber productivity, from 650 kg ha–1 derived from unselected seedlings during the 1920s to 2,500 kg ha–1 yielded by elite cultivars by the 1990s4. Nevertheless, the field production achieved so far is still well below the theoretical yield of 7,000–12,000 kg ha–1, as has been suggested for the rubber tree5. Meanwhile, conventional rubber breeding has been stagnating in the introduction of high-yield cultivars. The reasons include a narrow genetic basis for exploiting breeding potential and difficulty in introducing wild germplasms because of the genetic burden in removing unfavourable alleles6. The incorporation of marker-assisted selection and transgenic techniques offers promise to improve breeding efficiency for latex yield, and sequencing of the Hevea genome would uncover even more avenues leading to this end. The first draft Hevea genome was released by a Malaysian team7 that was participant to the recent boom in transcriptomic and proteomic studies of the species8,9,10,11. However, its low sequence coverage (∼13×) and a lack of large insert libraries (such as fosmid- or BAC-based clone libraries) have limited the success of genome assembly (a scaffold N50 size of 2,972 bp), precluding its application for furthering quality research in the field. Here, we report a high-quality genome assembly of Hevea Reyan7-33-97, an elite cultivar widely planted in China12,13 based on sequence data from both whole-genome shotgun (WGS) and pooled BAC clones. This assembly contains 7,453 scaffolds (N50 = 1.28 Mb), has a length of 1.37 Gb and covers ∼94% of the predicted genome size (1.46 Gb). Together with analysis of data from re-sequencing five other cultivars and comprehensive transcriptomic surveys, we aim to obtain new insights into the physiology of laticifers and molecular details of rubber biosynthesis, especially in relation to ethylene-stimulated rubber production. (Résumé d'auteur

Overexpression of a Hevea brasiliensis ErbB-3 binding protein 1 Gene Increases Drought Tolerance and Organ Size in Arabidopsis

Rubber trees are economically important tropical tree species and the major source of natural rubber, which is an essential industrial material. This tropical perennial tree is susceptible to cold stress and other abiotic stresses, especially in the marginal northern tropics. Recent years, the genome sequencing and RNA-seq projects produced huge amount of sequence data, which greatly facilitated the functional genomics study. However, the characterization of individual functional gene is in urgent demands, especially for those involved in stress resistance. Here we identified and characterized the rubber tree gene ErbB-3 binding protein 1, which undergoes changes in expression in response to cold, drought stress and ABA treatment. HbEBP1 overexpression in Arabidopsis increased organ size, facilitated root growth and increased adult leaf number by delaying the vegetative-to-reproductive transition. In addition, HbEBP1 overexpression enhanced the resistance of the Arabidopsis plants to freezing and drought stress, demonstrating that this gene participates in the regulation of abiotic stress resistance. RD29a, RD22 and CYCD3;1 expression was also greatly enhanced by HbEBP1 overexpression, which explains its regulatory roles in organ size and stress resistance. The regulation of drought stress resistance is a novel function identified in plant EBP1 genes, which expands our understanding of the roles of EBP1 gene in response to the environment. Our results provide information that may lead to the use of HbEBP1 in genetically engineered crops to increase both biomass and abiotic stress resistance

ASYMMETRIC PROTOPLAST FUSION BETWEEN PR107 AND REYAN8-79 BY USING PEG

The genetic improvement of rubber tree using traditional sexual hybridization is a time consuming and laborious task. Somatic hybridization through protoplast fusion is an alternative approach to improve rubber tree, for somatic hybrids had been obtained successfully in many other plant species, and protoplast regeneration systems for several rubber clones had been already established. An attempt was made to perform asymmetric protoplast fusion between two Hevea clones, PR 107 and Reyan 8-79,by using poly ethylene glycol (PEG). Protoplasts derived from embryogenics uspension cells of PR107 were used as donor protoplasts and treated with ultraviolet (UV) irradiation (380 μW/cm2) for 30, 60, 90, 120 and 180 s. Protoplasts derived from embryogenic suspension cells of Reyan 8-79 were used as recipient ones and treated withiodoacetamide (IOA) at concentrations of 1, 2, 3 and 4 mM for 15 min. PEG at 10, 20, 30 and 40% (w/v) were respectively tested to obtain a higher frequency of binary fusion. Results showed that the cell division of donor protoplasts was largely inhibited by UV irradiation for more than 90 s, and that the cell division frequency of recipientprotoplasts was decreased to less than 5% by 3 mM IOA. A maximal binary fusion frequency (about 20%) was obtained when the concentration of PEG was 30%. The donor protoplasts exposed to UVfor 90 s and 120 s were respectively fusedwith the 3 mM IOA-treated recipient protoplastsby using 30% PEG to produce somatic hybrid cells. The fusion products obtained were then transferred onto feeder layer culture.Visible cell colonies from these two fusion combinations were produced 28 and 35 d after feeder layer culture, and their formation frequencies were 146 ± 12 and 23 ± 5 per 105 protoplasts, respectively. They were transferred onto callus inducing medium for multiplication culture for one month, and then transferred onto differentiation medium for somatic embryogenesis. A total of 112 somatic embryos were produced from the cell colonies, but they failed to germinate as they turned brown or vitrified during their further culture. Keywords: Hevea brisiliensis, embryogenic cell suspensions, protoplast fusion, feeder layer culture, somatic embryogenesis, callus, rubbe

A rapid method for isolation of low-molecular-weight RNA from Arabidopsis using low salt concentration buffer

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<w:LsdException Locked="false" Priority="37" Name="Bibliography" /> <w:LsdException Locked="false" Priority="39" QFormat="true" Name="TOC Heading" /> </w:LatentStyles> </xml><![endif]--> <!--[if gte mso 10]> <mce:style><! /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.5pt; mso-bidi-font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:宋体; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:1.0pt;} --> <!--[endif]--><span lang="EN-US">We have developed a rapid extraction method using low salt concentration buffer for the isolation of low-molecular-weight RNA from <em>Arabidopsis</em> tissues. The method was quick and efficient, and the small scale extraction process took no more than 1 hour, while yield and RNA quality were comparable with those of previously reported. The LMW RNA isolated using this method was high quality, abundant in small RNA and free of high molecular weight RNA. This method can be used to extract low-molecular-weight RNA for the purpose of small RNA cloning and detection, and library construction.</span

COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALS EARLY GENE EXPRESSION PROFILE THAT CONTRIBUTES TO COLD RESISTANCE IN HEVEA BRASILIENSIS (PARA RUBBER TREE)

Rubber trees (Hevea brasiliensis) are tropical perennial woody plants that are susceptible to cold stress. In China, cold stress caused severe damages to rubber plantations in the past decades. Though several cold resistant Hevea clones have been bred, their cold hardiness mechanism is yet to be unraveled. In this study, the cold resistant clone CATAS93-114 and sensitive Reken501 were subjected to cold stress and their transcriptomes were analyzed at the time points of 0h, 2h, 8h and 24h. De novo transcriptome assembly and comparison were then carried out. The results showed that there were totally 7870 genes differentially expressed in these clones. The resistant clone CATAS93-114 had more genes being evoked from 2h to 8h during cold treatment, indicating more rapidly and intensively responsive profile than the sensitive clone Reken501. The DE genes can be further classified into seven major clusters, and GO terms in each cluster were enriched. The genes involved in ABA metabolism and signaling, ABA-independent pathway and early signal perception were noteworthy for their distinctive expression patterns toward cold stress between clones. Twenty-two genes playing a central role in these pathways were further investigated, and QPCR analysis of these genes confirmed the RNA-Seq results.Keywords:   cold resistance, Hevea brasiliensis, transcriptome, QPCR, RNA-seq, ABA, rubber, hevea, cold stress, gen

Identification, Functional Study, and Promoter Analysis of HbMFT1, a Homolog of MFT from Rubber Tree (Hevea brasiliensis)

A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical β-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species

Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis

The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 μM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.</jats:p