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Objective To observe the somatic embryogenesis in in-vitro cultured Dendrobium candidum wall.Ex Lindl.(DCWL),and to supply evidence for its rapid propagation and germplasm preservation.Methods Protocorm-like bodies and callus of DCWL subcultured for 30 days was used as the explants,N6 was used as the basic culture with phytohormone added,and fungal extracts as the elicitor.Protocorm-like bodies and callus of DCWL were used for induction culture.Results Somatic embryogenesis in in-vitro cultured DCWL derived from the epithelial cells or inner cells of callus of DCWL.Conclusion A large amount of buds can be obtained by the induction and culture of protocorm-like bodies and callus of DCWL.
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[Objective] cDNA expression libraries from the leaves of Dendrobium candidum Wall. ex lindl. were constructed to supply evidence for the clones of the full-length genes involved in the biosynthesis of bioactive compounds. [Methods] Total RNA was isolated and purified from tender leaves of 4-year-growing Dendrobium candidum by using Trizol single-step method. cDNA was synthesized by long distance polymerase chain reaction (LD-PCR) and then was connected to ?TripIEX2. After that, the recombinant bacteriophages were packaged and cDNA library of Dendrobium candidum was constructed. The titer of cDNA library was expressed as the number of phage formation unit (pfu) per milliliter and the inserted fragment was identified by PCR amphfication. [Results] cDNA expression libraries from the leaves of Dendrobium candidum were constructed successfully. The titer of the original library was 4.3?105 pfu/mL and 3.1?105 clones in total, with a recombinant rate of 97.6%. The amplified titer was 6.8?109pfu/mL. PCR amplification suggested that the inserted cDNA fragments ranged from 0.5 to 2.0 kb, mostly from 1.2 to 1.7 kb. [Conclusion] The constructed Dendrobium candidum cDNA library has a higher titer, a higher recombinant percentage and larger inserted fragments.
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Objective To study the effects of two fungal elicitors on the growth of Dendrobium candidum protocorms cultured on the solid medium.Methods The medium for propagation of protocorms was selected and the growth curve on that medium was obtained.According to the curve,the two fungal elicitors were added at the different growth stages of protocorms.The fresh weight(FW) and dry weight(DW) of protocorms were measured.Results The medium for propagation of protocorms was 1/2MS+20% potato extract+3% sucrose+0.8% agar.Compared with the control MF23 elicitor added at weeks 11 and 13 could significantly increase the FW by 16.4%(P
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Objective To establish the fingerprint for the dry stem in Dendrobium candidum by RP-HPLC.Methods Kromasil○RKR100-5 C18 column(250 mm?4.6 mm,5 ?m) was used with a mixture of acetonitrile-0.4% phosphorus acid as mobile phase in a gradient mode and the data were analyzed with ″Computer Aided Similarity Evaluation″ software.Results The chemical constituents of D.candidum were optimally separated,among which 15 fingerprint peaks in common were confirmed.Conclusion This method is simple,accurate with good reproducibility,and can be used specifically for the quality control of D.candidum.
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Objective The high quality RNA from the plantlet of Dendrobium candidum was isolated efficiently.It laid a foundation to study the mechanism of molecular expression on its growth and metabolism under environmental stress.Methods The groups stimulated by sound wave and the control groups were established.Total RNA was prepared by a modified phenol-chloroform method.The content was determined by a spectrophotometer.The integrity of RNA was detected by electrophoresis with 1.0% denatured agarose gel.The total RNA obtained was amplified by RT-PCR.The differential cDNA bands were displayed on 6% denatured polyacrylamide gel by electrophoresis and silver staining.Results The ratios of total RNA A_(260)/A_(230) and A_(260)/A_(280) were 3.16 and 1.96,respectively.The yield was 0.27 ?g/mg fresh weight.The bands were clear on agarose gel and the integrity of RNA was good.The differential cDNA bands were screened with molecular weight of 240—600 bp.Conclusion The method of modified phenol-chloroform can be used to isolate RNA from D.candidium with high quality and high yield.
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Object To determine the diumal changes of photosynthesis, its relationships with light intensities and temperatures, and the contents of chloroophyll in leaves of Dendrobium candidum Wall. ex. Lindl., D. wilsonii Rolfe and D. hercoglossum Reichb. f. Methods The net photosynthetic rate (NPR) was measured at intervals of 1.5 h during daytimes 7:30—16:30, under the photosynthetic affective rates 0—140 mmol/(m 2?s), and under the temperatures 29.6—38.2 ℃; contents of chlorophyll a and b in leaves were measured. Results NPR, light saturation point and compensation point were low in each of the three Dendrobium L. species. The NPR peaked during 8:00 and 10:00 am, and thereafter decreased rapidly as the temperature and light intensity increased. Content of chlorophyll b was high in the leaves. Conclusion These results indicated that the Dendrobium L. species, which are sciophyte plants, have great adaptations to their growing habitats.
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Object To explore the protoplast fusion of Dendrobium candidum Wall ex. Lindl. and Gynostemma pentaphyllum. (Thunb) Makino. Methods The two kinds of protoplasts were fused by PEG method, and then cultured in the modified liquid medium containing 2 mg/L BA and 1 mg/L NAA. Results High yield, viability, and pure mesophyll protoplasts were isolated from D. candidum and G. pentaphyllum. Conclusion The first cell division occurres within three days after fusion. And some of the cells are divided three times.
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Object To optimize the fitting culture media for multiplication of adventitious bud and ra-dication of Dendrobium candidum Wall. ex Lindl. and the length of adventitious bud for succesive transfer multiplication. Methods In the same culture condition, the adventitious bud with different length developed in the various culture media for multiplication and radication. Facing the different growth status, the diversity test and general analysis were carried on for them. Results There were significant difference in different length of adventitious bud, difference between the multiplication and radication of culture media. Conclusion The adventitious buds in (1.2?0.1) cm length are superior for succesive transfer multiplication, MS adding BA 1.0 mg/L, NAA 0.2 mg/L is the best media for the adventitious buds multiplication, 1/2 MS adding NAA 0.2 mg/L is the test media for radication.
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Object To study the feasibility of suspension culture of protocorm in Dendrobium candidum Wall ex Lindl and effect of inoculum and medium volume on the growth of protocorm Methods Effect of four basic media MS, 1/2 MS, 67 V, and B 5, inoculum and medium volume on the growth of protocorm were studied by completely random experimental design and orthogonal test design Results The growth of D candidum protocorms in liquid medium was markedly better than that in solid medium (P0 05), B 5 was much better than 1/2 MS (P