Establishment of an efficient in vitro regeneration protocol for rapid and mass propagation of Dendrobium chrysotoxum Lindl. using seed culture.
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ABSTRACT:
SUBMITTER: Nongdam P
PROVIDER: S-EPMC4221871 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:BackgroundSwitchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS).ResultsMicropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments.ConclusionsIn this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.
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Project description:Mycorrhizal fungi colonize orchid seed and induce the germination. This so-called symbiotic germination is a critical developmental process in the lifecycle of all orchids. However, the molecular changes taking place during the orchid seed symbiotic germination still remains largely unknown. To better understand the molecular mechanism of orchid seed germination, we performed comparative transcriptomic and proteomic analysis on Chinese traditional medicinal orchid plants, Dendrobium officinale to explore protein expression change at the different developmental stages between asymbiotic and symbiotic germination and identify the key proteins regulated symbiotic germination of orchid seeds. iTRAQ analysis from 8 samples identified 2256 plant proteins, of which, 308 proteins were differentially expressed across three developmental stages within asymbiotic or symbiotic accession and 229 proteins are differentially expressed in the symbiotic germination compared to asymbiotic germination. 32 proteins are co-upregulated in both proteomic and transcriptomic level for symbiotic germination compared to asymbiotic germination. Our results revealed that symbiotic germination of D. officinale seeds probably shares the common signal pathway with asymbiotic germination during the early germination stage.
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Project description:Kanamycin B as the secondary metabolite of wild-type Streptomyces kanamyceticus (S. kanamyceticus) ATCC12853 is often used for the synthesis of dibekacin and arbekacin. To construct the strain has the ability for kanamycin B production; the pSET152 derivatives from Escherichia coli ET12567 were introduced to S. kanamyceticus by intergeneric conjugal transfer. In this study, we established a reliable genetic manipulation system for S. kanamyceticus. The key factors of conjugal transfer were evaluated, including donor-to-recipient ratio, heat-shock, and the overlaying time of antibiotics. When spores were used as recipient, the optimal conjugation frequency was up to 6.7 × 10-6 . And mycelia were used as an alternative recipient for conjugation instead of spores; the most suitable donor-to-recipient ratio is 1:1 (107 :107 ). After incubated for only 10-12 hr and overlaid with antibiotics subsequently, the conjugation frequency can reach to 6.2 × 10-5 which is sufficient for gene knockout and other genetic operation. Based on the optimized conjugal transfer condition, kanJ was knocked out successfully. The kanamycin B yield of kanJ-disruption strain can reach to 543.18 ± 42 mg/L while the kanamycin B yield of wild-type strain was only 46.57 ± 12 mg/L. The current work helps improve the content of kanamycin B in the fermentation broth of S. kanamyceticus effectively to ensure the supply for the synthesis of several critical semisynthetic antibiotics.
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Project description:BackgroundIn plant breeding, one of the most cost-effective and efficient ways to increase genetic gain is to reduce the breeding cycle time. In general, modern breeding methods for self-pollinated crops should strive to develop fixed lines at the lowest possible cost and in the minimum possible amount of time. Previous studies on spring oat (Avena sativa L.) showed that combining high plant density with limited soil fertility and moisture levels in a growth media like sand effectively decreases the time and cost of generating fixed single-seed descent lines. More recently, 'speed breeding,' or the exposure to prolonged photoperiod regimes of 22 h, has been shown to decrease flowering time in oat significantly. The goal of this study was to combine 'speed breeding' with high-density planting in a limited soil fertility media to reduce further the costs and time required to develop oat single-seed-descent lines.ResultsWe grew oat plants at low density in potting-mix (control), high density in potting-mix (HD-soil), and high density in sand (HD-sand) under 16 and 22 h of day length. We observed that oat plants grown in HD-sand and exposed to 22 h day length reduced their flowering time by around 20 and 5 days on average compared to those grown in control conditions at 16 and 22 h, respectively. We also observed that 85% of plants grown at high density in sand produced a single seed when grown in bulk conditions. In contrast, only 40% of plants grown at high density in potting-mix produced a single seed.ConclusionsOur novel protocol showed that oat plants grown in high-density bulks, using sand media and 22-hour day length, reduced their flowering time by 20 days compared to control conditions and produced plants with single seeds, following closely single-seed descent assumptions while significantly reducing labor costs and greenhouse space. This methodology can be deployed in oat breeding programs to help them accelerate their rate of genetic grain for multiple traits.
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Project description:BACKGROUND:Rice genome sequencing projects have generated remarkable amount of information about genes and genome architecture having tremendous potential to be utilized in both basic and applied research. Success in transgenics is paving the way for preparing a road map of functional genomics which is expected to correlate action of a gene to a trait in cellular and organismal context. However, the lack of a simple and efficient method for transformation and regeneration is a major constraint for such studies in this important cereal crop. RESULTS:In the present study, we have developed an easy, rapid and highly efficient transformation and regeneration protocol using mature seeds as explants and found its successful applicability to a choice of elite indica rice genotypes. We have optimized various steps of transformation and standardized different components of the regeneration medium including growth hormones and the gelling agent. The modified regeneration medium triggers production of large number of shoots from smaller number of calli and promotes their faster growth, hence significantly advantageous over the existing protocols where the regeneration step requires maximum time. Using this protocol, significantly higher transformation efficiency (up to 46%) and regeneration frequency (up to 92% for the untransformed calli and 59% for the transformed calli) were achieved for the four tested cultivars. We have used this protocol to produce hundreds of independent transgenic lines of different indica rice genotypes. Upon maturity, these transgenic lines were fertile thereby indicating that faster regeneration during tissue culture did not affect their reproductive potential. CONCLUSIONS:This speedy, yet less labor-intensive, protocol overcomes major limitations associated with genetic manipulation in rice. Moreover, our protocol uses mature seeds as the explant, which can easily be obtained in quantity throughout the year and kept viable for a long time. Such an easy, efficient and generalized protocol has the potential to be a major tool for crop improvement and gene-function studies on the model monocot plant rice.
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