A scale-up process of lettuce (Lactuca sativa L.) suspension culture in a 2-liter bioreactor was investigated. Factors that influenced cell growth and differentiation, including foaming, the wall effect (inoculum adhering onto the vessel wall above the medium level), aeration, and dissolved oxygen (DO), were tested. The wall effect resulted in severe inoculum loss (10%) in 24 hours. Inoculum loss significantly decreased shoot regeneration. The wall effect was caused by two factors: 1) foaming caused by the interaction between air bubbles and inoculum, and 2) the bubbles produced by aeration. Foaming could be prevented by sieving the inoculum through a 400-pm screen filter and then rinsing the inoculum thoroughly with distilled water to remove single cells, cell debris, and the contents of broken cells. The wall effect caused by air bubbles could be prevented by putting a 150-μm screen column in the center of the bioreactor to isolate the aeration area from the inoculum. After the wall effect was removed, shoot regeneration in the bioreactor increased significantly to a level similar to that in 125-ml flasks at an aeration rate of 1 to 2 vvm (liters air/liters medium per rein). DO for this shoot regeneration level was ≈ 70% to 80%of saturation at the end of bioreactor culture.
Whei-Lan Teng, Chiao-Po Lin and Yann-Jiun Liu
Whei-Lan Teng, Yann-Jiun Liu and Tai-Sen Soong
An efficient method for the regeneration of shoots directly from cell suspensions of three commercial cultivars of lettuce (Lactuca sativa L. cv. Great Lakes 659-700, Salad Bowl, and Prize Head) is described. Cell suspensions were prepared by osterizing cotyledon-derived callus for 60 seconds. The effects of callus quality, light intensity, carbohydrate type and concentration, auxins, and cytokinins on cell growth and differentiation in the suspension culture were examined. Among these factors, callus quality and carbohydrates were the most critical. The optimal medium for regeneration of shoots in suspension culture was SH (Schenk and Hilderbrandt) basal medium containing 1000 mg myo -inositol/liter, 1.5% glucose, 0.44 μm BA, and 0.54 μm NAA. The pH of the medium was adjusted to 5.8. Under such condition, hundreds of shoots could be produced from 50 to 55 mg (dry wt) of cell aggregates within 2 weeks. Chemical names used: α-] naphthaleneacetic acid (NAA); indole3-acetic acid (IAA); 6-benzylaminopurine (BA).
Whei-Lan Teng, Yann-Jiun Liu, Yu-Ching Tsai and Tai-Sen Soong
A bioreactor was used to establish a scale-up system for somatic embryogenesis in `Scarlet' carrot (Daucus carota L.). At a cell density of 1–2 × 106 cells/ml, mature and germinating embryos could be observed within 4 to 5 weeks. As cell density exceeded 2 × 106 cells/ml, the culture turned darker yellow, and embryo development was inhibited. Cell densities below 106 cells/ml resulted in abnormal embryos. Bioreactor design had a critical impact on somatic embryogenesis due to various types and the strength of shear forces generated. In this study, an air-lift bioreactor was selected from three different types (spinner flask, screen column bioreactor, and air lift) because it resulted in the highest biomass production and somatic embryogenesis. Foaming was eliminated by preculture of embryogenic cells in flasks; cells were then sieved on a 60-μm polyester screen and thoroughly rinsed with distilled water before being transferred to the bioreactor. Such preculture for at least 10 days significantly increased the regeneration of somatic embryos. During somatic embryogenesis, dissolved O2 concentrations decreased to 33% of saturation, and then increased up to 80% when embryo development approached maturity and mature embryos germinated. Bioreactor-cultured embryos germinated with relatively short cotyledons and long roots, whereas flask-cultured embryos germinated with relatively long cotyledons and short roots.
Jun Tang, Kang-Di Hu, Lan-Ying Hu, Yan-Hong Li, Yong-Sheng Liu and Hua Zhang
Hydrogen sulfide (H2S) has been shown to be a gaseous molecule in the regulation of many processes in plants such as abiotic stress tolerance, root organogenesis, stomatal movement, and postharvest fruit senescence. We studied the role of H2S in the regulation of senescence and fungal decay in fresh-cut sweetpotato (Ipomoea batatas L., cv. Xushu 18) roots. H2S donor sodium hydrosulfide (NaHS) alleviated senescence in fresh-cut sweetpotato root tissue in a dose-dependent manner with the optimal concentration of 2.0 mmol·L−1 NaHS solution. At the optimal concentration of 2.0 mmol·L−1 NaHS, H2S fumigation maintained higher levels of reducing sugar in sweetpotato fresh-cut root. H2S treatment also significantly increased the activities of guaiacol peroxidase (POD) and decreased those of polyphenol oxidase (PPO) in sweetpotato during storage. Further investigation showed that H2S treatment maintained a lower level of lipoxygenase (LOX) activity compared with water control. Consistently, the accumulation of malondialdehyde (MDA) was reduced in H2S-treated groups. Three fungal pathogens, Rhizopus nigricans, Mucor rouxianus, and Geotrichum candidum, were isolated from sweetpotato tissue infected with black rot or soft rot. H2S fumigation at 1 to 2.5 mmol·L−1 NaHS resulted in effective inhibition of the three fungi when grown on medium. When the three fungi were inoculated on the surface of sweetpotato slices, H2S fumigation greatly reduced the percentage of fungal infection. In conclusion, these data suggest that H2S effectively alleviated the senescence and decay in sweetpotato slices and might be developed into a novel fungicide for reduction of black rot or soft rot in sweetpotato.
Guangxin Liu, Yue Lan, Haoyang Xin, Fengrong Hu, Zhuhua Wu, Jisen Shi and Mengli Xi
Lily (Lilium L.) species produce among the most important cut flowers worldwide. China has ≈55 species of Lilium. Although many plants from this genus have been used in hybridization efforts, their cytology has remained unclear. The goal of the current study was to characterize the chromosomes of Lilium rosthornii Diels. Root tips were used to characterize Giemsa C-banding, propidium iodide (PI) banding, and 45S rDNA locations. The karyotype of L. rosthornii belongs to type 3B. C-banding revealed polymorphic banding patterns with the following formula: 2n = 24 = CI = 4C + 14CI+ + 2I+ +2I+ 2. Two of the four 45S rDNA hybridization sites were located at pericentromeric positions on the two short arms of the homologues of chromosome 1, and the other two were located on the long arms of one chromosome 6 homolog and one chromosome 11 homolog. Six of the eight PI bands were detected in the centromeres of the homologues of chromosomes 1, 5, and 8, and the other two PI bands were detected on the long arms of one chromosome 6 and one chromosome 11. Lilium rosthornii showed enriched banding in both Giemsa C-banding and PI painting. Interestingly, not all 45S rDNA was located in homologous chromosomal locations. These results may provide reference data for L. rosthornii for use in further Lilium breeding.
Guangxin Liu, Xiaoling Zhang, Yue Lan, Haoyang Xin, Fengrong Hu, Zhuhua Wu, Jisen Shi and Mengli Xi
Karyotype comparison and fluorescence in situ hybridization (FISH) were conducted to analyze the wild Lilium species distributed in China. The karyotype results revealed that all species except Lilium lancifolium (2n = 3X = 36) were diploid and had two pairs of metacentric or submetacentric chromosomes. The karyotypes of all species are similar. FISH analysis revealed that there are 5–12 45S rRNA gene loci dispersed on the chromosomes of the 14 diploid species, and 15 45S rRNA gene loci were detected in the triploid species L. lancifolium. Most of the FISH signals were detected on the long arms and the centromeric regions. Three samples of L. brownii [Hubei, China (lat. 31°28′N, long. 110°23′E); Liaoning, China (lat. 40°07′N, long. 124°19′E); and Guangxi, China (lat. 25°06′N, long. 107°27′E)] showed very similar chromosome patterns in both the karyotype and the FISH analyses, further demonstrating that these samples belonged to the same species. L. brownii is widely distributed in China from latitude 25°06′N to 40°07′N, indicating that it is highly adaptable to the environment.
Chengyan Yue, R. Karina Gallardo, Vicki A. McCracken, James Luby, James R. McFerson, Lan Liu and Amy Iezzoni
Rosaceous crops (e.g., almond, apple, apricot, caneberry, cherry, pear, peach, plum, rose, and strawberry) contribute to human health and well-being and collectively constitute the economic backbone of numerous North American rural communities. We conducted a survey of U.S. and Canadian rosaceous fruit crop breeders to assess priority setting in their programs, sources of information for setting priorities, and challenges in making technical and management decisions. Input from producers and consumers was most important in establishing breeding program targets, although respondents’ direct interaction with consumers was not frequent. Breeding targets and management decisions were mostly associated with the breeder’s type of organization, scope and range of crops, and intended use of the crop (fresh, processed, or both).
Quan Liu, Yan Lan, Feng Tan, Yunbiao Tu, Yingying Sun, Gajue Yougu, Zeshen Yang, Chunbang Ding and Tian Li
Water is essential for crops and plays a vital role in olive (Olea europaea) growth. Three irrigation treatments, rain-fed (CK), flood irrigation (FI), and drip irrigation (DI), were applied from late November to late May in a 2-year study (Nov. 2015 to Oct. 2017) on two olive cultivars, Coratina and Koroneiki. Shoot growth, flower and fruit characteristics, and olive and oil yields were measured. Compared with CK, FI had significantly higher values of vegetative growth, olive and oil yields, moisture content, and oil content. Although the fruit weight, pulp rate, and oil content with DI were the lowest, our results support that DI had the greatest positive effects on olive vegetative growth, flowers, fruit set, and olive and oil yields. It is suggested that DI in winter and spring is the best irrigation strategy for olive productivity in southwest China.
Shuai-Ping Gao, Kang-Di Hu, Lan-Ying Hu, Yan-Hong Li, Yi Han, Hui-Li Wang, Kai Lv, Yong-Sheng Liu and Hua Zhang
Hydrogen sulfide (H2S) was recently recognized as an endogenous gaseous molecule involved in seed germination, root organogenesis, abiotic stress tolerance, guard cell movement, and delay of senescence in plants. In the present study, we show that H2S participates in the regulation of postharvest ripening and senescence in fresh-cut kiwifruit, Actinidia deliciosa. Fumigation of fresh-cut kiwifruit with the H2S donor sodium hydrosulfide (NaHS) solution prolonged kiwifruit storage time and alleviated senescence and tissue softening in a dose-dependent manner at an optimal concentration of 1.0 mmol·L−1 NaHS. H2S treatment maintained higher levels of reducing sugars, soluble proteins, free amino acids, ascorbate, and chlorophyll and lowered carotenoid levels. H2S treatment also significantly decreased the contents of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide anion (•O2 −) during fruit storage compared with water controls. Furthermore, the activities of guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were increased by H2S treatment, whereas the activity of lipoxygenase (LOX) was decreased compared with untreated controls. Taken together, these results suggest that H2S is involved in prolonging postharvest shelf life and plays an antioxidative role in fresh-cut kiwifruit.
Chen Chen, Meng-Ke Zhang, Kang-Di Hu, Ke-Ke Sun, Yan-Hong Li, Lan-Ying Hu, Xiao-Yan Chen, Ying Yang, Feng Yang, Jun Tang, He-Ping Liu and Hua Zhang
Aspergillus niger is a common pathogenic fungus causing postharvest rot of fruit and vegetable, whereas the knowledge on virulence factors is very limited. Superoxide dismutase [SOD (EC 18.104.22.168)] is an important metal enzyme in fungal defense against oxidative damage. Thus, we try to study whether Cu/Zn-SOD is a virulence factor in A. niger. Cu/Zn-SOD encoding gene sodC was deleted in A. niger [MA70.15 (wild type)] by homologous recombination. The deletion of sodC led to decreased SOD activity in A. niger, suggesting that sodC did contribute to full enzyme activity. ΔsodC strain showed normal mycelia growth and sporulation compared with wild type. However, sodC deletion markedly increased the cell’s sensitivity to intracellular superoxide anion generator menadione. Besides, spore germination under menadione and H2O2 stresses were significantly retarded in ΔsodC mutant compared with wild type. Further results showed that sodC deletion induced higher superoxide anion production and higher content of H2O2 and malondialdehyde (MDA) compared with wild type, supporting the role of SOD in metabolism of reactive oxygen species (ROS). Furthermore, ΔsodC mutant had a reduced virulence on chinese white pear (Pyrus bretschneideri) as lesion development by ΔsodC was significantly less than wild type. The determination of superoxide anion, H2O2, and MDA in A. niger-infected pear showed that chinese white pear infected with ΔsodC accumulated less superoxide anion, H2O2, and MDA compared with that of wild type A. niger, implying that ΔsodC induced an attenuated response in chinese white pear during fruit–pathogen interaction. Our results indicate that sodC gene contributes to the full virulence of A. niger during infection on fruit. Aspergillus niger is one of the most common species found in fungal communities. It is an important fermentation industrial strain and is also known to cause the most severe symptoms in fruit during long-term storage (Pel et al., 2007). Meanwhile, plants activate their signaling pathways to trigger defense responses to limit pathogen expansion. One of the earliest host responses after pathogen attack is oxidative burst, during which large quantities of ROS are generated by different host enzyme systems, such as glucose oxidase (Govrin and Levine, 2000). ROS such as singlet oxygen, superoxide anion, hydroxyl (OH−), and H2O2 are released to hinder the advance of pathogens (Gara et al., 2003). ROS can react with and damage cellular molecules, such as DNA, protein, and lipids, which will limit fungal propagation in the host plant (Apel and Hirt, 2004).