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  • Author or Editor: Lianghong Chen x
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Randomly amplified polymorphic DNA (RAPD) technique is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. It has been widely used for genetic mapping, plant and animal breeding programs, and DNA fingerprinting. However, there is no single set of RAPD-PCR conditions that can be applied to all situations. In order to adjust reaction component concentrations within suggested ranges for efficient amplification during the use of RAPD in detection of genetic variation of genus Camellia, crucial factors, such as concentrations of MgCl2 and DNA, annealing temperature (37 to 44 °C), and the use of an AmpliTaq® DNA polymerase and Stoffel fragment were examined. Five camellia cultivars, `Winter's Beauty', `Pink Icicle', `Polar Ice', `Winter's Hope', and `Snow Flurry', were under investigation. Clear and reproducible amplification products were produced with 3.0 μM MgCl2 and 30 ng template DNA/25 μL reaction mixer at annealing temperature 37 °C and 40 °C, compared with MgCl2 at 1.5, 2.0, and 2.5 μM. When annealing temperature increased, the RAPD-PCR stringency was increased, as expected. Stoffel fragment was found to provide highly reproducible results.

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To improve somatic embryogenesis of Daylilies (Hemerocallis hybrid); six types of explants namely immature seeds, immature embryos, mature embryos, young inflorescence, ovary-sections and filaments of daylily flower buds were investigated as source of explants. Explants from field grown plants were surface sterilized and followed by culturing on MS medium supplemented with 1.5 mg/L NAA and 0.5 mg/L BAP for four weeks. Explants were scored for development of embryogenic tissue and formation of somatic embryos with/without the formation of an intermediate callus stage. Both mature and immature embryo explants produced direct development of embryogenic tissue followed by somatic embryos. Young inflorescence explants developed compact calli and produced roots around cut ends and showed no somatic embryogenesis. Ovary explants exhibited swelling and not produced embryogenesis. Production of embryogenic callus and formation of somatic embryos in filament explants depended on the sizes of flower buds. Explants from 0.5 to 1.2 mm size flower buds produced calli and formed somatic embryos while explants from sizes over 1.2 mm flower buds only non-embryogenic calli. Immature seeds failed to grow. The results indicated that immature and mature embryos and filaments from young flower buds responded better than other explants for developing somatic embryos in daylily.

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During Summer 2004, a study was conducted to determine the effect of two types of fertilizers on the growth and development of tissue-cultured daylilies transferred to the greenhouse. Peters 20–20–20 water-soluble fertilizer and a slow-release fertilizer were the two fertilizers evaluated. Peters 20–20–20 fertilizer was used at 0 (control), 50, 100, and 200 mg·L–1 rates. The slow release fertilizer was used at 2.5 g per 10.2 cm pot. Each treatment was replicated four times in a randomized complete-block design. After 6 weeks of growth, the results showed that, when compared to the control, all treatments except for 200 mg·L–1 caused a significant increase in root growth. Shoot growth was significantly increased by the 100 mg·L–1 treatment, while the 200 mg·L–1 and slow-release treatments suppressed shoot growth. Similar to root and shoot growth, the 100 mg·L–1 treatment caused a significant increase in fresh weight, while the 200 mg·L–1 and slow-release treatments caused a reduction. These results imply that the 100 mg·L–1 Peters 20–20–20 fertilizer treatment is the best treatment for maximum growth and development of tissue-cultured daylilies transferred to the greenhouse.

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In this study research was conducted to evaluate the feasibility of characterizing genetic variation within camellia species using random amplified polymorphic DNAs (RAPD) markers. Eight varieties of species Camellia japonica and four varieties of species Camellia reticulata, provided by the America Camellia Society, Fort Valley, Ga., were investigated. RAPD profiles generated by five selected 10-based random primers (out of 20 primers) exhibited distinct patterns of amplified bands for all 12 tested varieties. A total of 344 bands were produced among the eight varieties of species C. japonica, with an average of 8.6 bands, ranging from 220 to 2072 bp in size, scored per primer. Among the 344 amplified bands, 74.4% of the bands presented polymorphic. The four varieties of species C. reticulata produced a total of 180 markers, with an average percentage of 57.8% polymorphisms. The amplified bands were in the range of 236–1760 bp. An average of nine amplified bands was generated per primer. The large percentages of polymorphisms displayed among 12 varieties within the two different species indicate that the expected genetic diversity among varieties within camellia species existed. It was concluded that the RAPD molecular markers are capable of revealing appreciable levels of genetic variation within camellia species.

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The reliability of the random amplified polymorphic DNA (RAPD) technique in amplifying polymorphisim among the hybrids and their parents' genomes of the genus Camellia was evaluated. Three hybrids (`Londontowne Blush', `Ashton's Snow', and `Ashton's Cameo') and one of the parents, C. oleifrea`Plain Jane', provided by the America Camellia Society, Fort Valley, Ga., were investigated. Twenty 10-based random primers were tested in this study. Five out of 20 primers were selected for RAPD analysis based on the ability to produce unambiguously scoreable RAPD bands for evaluation and comparison of the genotypes under investigation. The five primers were selected because they produced distinct patterns of amplified bands for each tested genotype. A total of 162 RAPD bands were produced. Among the 162 bands, 86 bands showed polymorphisms. The amplified band sizes ranged from 236 to 1656 bp. These data indicate that in the three hybrids and one of the parents exist unique genomic regions. Our investigation results showed that the RAPD molecular approach can be used to discriminate genetic variation among hybrids and their parents.

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During Summer 2005, a study similar to that of Summer 2004 was conducted to determine the effect of two type of fertilizers on the growth and development of tissue-cultured daylilies transferred to the greenhouse. Peters 20–20–20 water-soluble fertilizer and a slow-release fertilizer were the two fertilizers evaluated. Peters 20–20–20 fertilizer was used at 0 (control), 50, 100, 200 mg/L rates. The slow-release fertilizer was used at 2.5 g per 10.2-cm pot. Each treatment was replicated four times in randomized complete-block design. After 6 weeks of growth, the results showed that when compared to the control, all treatments except for 200 mg/L caused a significant increase in root growth. Shoot growth was significantly increased by the 100 mg/L treatment, while the 200 mg/L and the slow-release treatments suppressed shoot growth. Similar to the growth of roots and shoots, the 100 mg/L treatment caused significant increase in fresh weight, while both the 200 mg/L and slow-release treatments caused a reduction. Results obtained for Summer 2005 were similar to that of Summer 2004. These results imply that the 100 mg/L Peters 20–20–20 fertilizers treatment is the best treatment for maximum growth and development of tissue-cultured daylilies transferred to the greenhouse.

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This study was carried out to optimize conditions for plant regeneration of sweetpotato [Ipomoea batatas (L.) Lam] using shoot tips, petioles, and leaves of Selection 75-96-1 as explants in Murashige and Skoog (MS) with several growth regulators at different levels. Callus initiation and callus proliferation media were 9.0 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.0 μm 2,4-D + 1.1 μm N 6-benzyladenine (6-BA) in protocol I; 8.1 μm α-naphthaleneacetic acid (NAA) + 1.2 μm kinetin (KIN) and 5.4 μm NAA + 4.6 μm KIN in protocol II; 0.9 μm 2,4-D, and 0.9 μm 2,4-D + 1.2 μm N-isopenylamino purine (2iP) in protocol III; NAA (8.1 μm) + KIN (1.2 μm) and 2,4-D (0.9 μm) + 2ip (1.2 μm) in protocol IV, respectively. In protocol I and II, shoot tip, petiole, and leaf were used, but only petiole and leaf in protocol III and IV. In the protocol I and II, somatic embryos were obtained only from shoot tip explants; in protocol III and IV, only from petioles. The frequencies of somatic embryo development were 33.3% in protocol I, 42.1% in protocol II, 21.2% in protocol III, and 10.3% in protocol IV, respectively. The leaf explants failed to produce somatic embryos in all the experiments. In protocol I, somatic embryogenesis occurred through the well-known sequence of globular-, heart-shaped-, torpedo-, and cotyledon-type embryos. However, in protocol II, the structures resembling plumule and radicle were observed before the emergence of torpedo/cotyledon type embryo clusters. The somatic embryogenesis in protocol III and IV was similar to that in protocol I. Growth regulators influenced somatic embryo development. Further, this study showed that explant resource and growth regulators affected the frequency of plant regeneration in sweetpotato.

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