CHEMICAL ENGINEERING TRANSACTIONS VOL. 51, 2016 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Tichun Wang, Hongyang Zhang, Lei Tian Copyright © 2016, AIDIC Servizi S.r.l., ISBN 978-88-95608-43-3; ISSN 2283-9216 Effects of Various Explants and Hormone Combinations on in vitro Regeneration in Cucumber Yanhua Li, Yongdong Sun*, Weirong Luo, Lei Ni School of Horticulture and Landscape Architecture, Henan Institute of Science and Technology, Xinxiang, Henan, 45300, P. R. China, sunyd2001@163.com Effects of various explants and hormone combinations on in vitro regeneration in cucumber (Cucumis sativus) were studied using Jinyou No. 1. The results showed that explant types had a greater impact on the regeneration rate and number of regenerated buds in cucumber. When using 1–2-d-old sterilized cotyledons as explants, Murashige and Skoog (MS) supplemented with 6-benzylaminopurine (6-BA; 3.0 mg L-1), kinetin (KT; 1.0 mg L-1), abscisic acid (ABA; 1.0 mg L-1), and AgNO3 (2.0 mg L-1) was the best bud induction medium, with an adventitious bud regeneration rate of 73.3%, and 1.00 regenerated bud per explant. When using 5–6- d-old sterilized cotyledonary nodes as explants, MS supplemented with 6-BA (2.0 mg L-1) and AgNO3 (2.0 mg L-1) was the best, with an adventitious bud regeneration rate of 90.70% and 4.38 regenerated buds per explant. In this study, cotyledonary nodes were more suitable than cotyledons for the establishment of the in vitro regeneration system in cucumber, and MS containing 6-BA (2.0 mg L-1) was the best bud induction medium for cotyledonary nodes, which may have valuable applications for an efficient in vitro regeneration protocol. 1. Introduction Cucumber is an important member of the family Cucurbitaceae and is commonly used as a fruit and vegetable in regular diets. Cucumber plays an extremely important role in the vegetable supply and national economy (Wang et al., 2013). However, a low fruit set rate is a serious problem in agricultural production, especially under protected cultivation conditions because of low temperature and low light resulting from rain and snow or fog haze weather, which seriously affects the high yield and good quality of cucumber. Recent developments in plant in vitro culture techniques and genetic engineering have enabled the establishment of a highly efficient regeneration system and genetic engineering techniques for germplasm innovations, and these methods have improved the yield and quality of crops (Kielkiewicz et al., 2013). Therefore, it is imperative to use genetic transformation tools for the improvement of quality traits in cucumber. For the development of genetically transformed plants, an efficient in vitro regeneration protocol is required. In vitro regeneration of cucumber has been achieved by using different culture techniques (Malepszy 1988; Chee, 1990; Lou and Kako, 1994Mohiuddin et al., 1997; Selvaraj et al., 2007). Colijn-hooymans et al. (1994) found that seedling age has a high impact on cucumber regeneration, and 3–5-d-old cotyledon explants have the highest adventitious bud regeneration rate. Seo et al. (2000) established a shoot regeneration using leaf explants of cucumber as explants. Selvaraj et al. (2007) established a shoot regeneration from cotyledon explants of cucumber via organogenesis. Fan et al. (2008) used cotyledonary nodes as explants and optimized the seedling status, methods for explant dissection and inoculation, and genotypes to improve the cucumber regeneration system. Zhang et al. (2010) established a cucumber cotyledon regeneration system based on genotypes and hormones. Shukla et al. (2014) established in vitro shoot regeneration progress in Cucumis sativus by inhibition of endogenous auxin. There has been great progress in cucumber tissue culture techniques, however, issues such as low regeneration rate and low reproducibility still remain, seriously affecting the application of plant genetic engineering techniques to the molecular genetic improvement of cucumber. In the present study, effects of various explants and hormone combinations on cucumber in vitro regeneration were discussed, and the best bud induction medium for cotyledonary nodes were found, which DOI: 10.3303/CET1651006 Please cite this article as: Li Y.H., Sun Y.D., Luo W.R., Ni L., 2016, Effects of various explants and hormone combinations on in vitro regeneration in cucumber, Chemical Engineering Transactions, 51, 31-36 DOI:10.3303/CET1651006 31 may have valuable applications for an efficient in vitro regeneration protocol and genetic engineering studies in cucumber breeding. 2. Materials and methods 2.1 Plant material Cucumber seeds “Jinyou No. 1” were purchased from the Tianjin Kernel Cucumber Research Institute. 2.2 Preparation of sterile seedlings 2.2.1 Preparation of cotyledons from sterile seedlings Cucumber seeds of uniform size were selected and soaked in 55°C water for 5–20 min until the seed coat softened. The seed coat was removed from the wider side of the seed. The embryo without the seed coat was placed on a clean bench and sterilized with 70% alcohol for 1 min, followed by 0.1% mercuric chloride for 10 min, with gentle shaking. Embryos were rinsed with sterile water 3–5 times and inoculated onto the Murashige and Skoog (MS) medium. The MS medium (pH 5.8) consisted of 7 g/l agar and 30 g/l sucrose. The culture conditions were 25°C, with a light intensity of 2500 lx, and a photoperiod of 16 h/d. 2.2.2 Preparation of cotyledonary nodes from sterile seedlings Cucumber seeds of uniform size were selected and washed with running water for 4–5 h. Seeds were placed on a clean bench and sterilized with 70% alcohol for 1 min, followed by 0.1% mercuric chloride for 20 min with gentle shaking. Seeds were washed with sterile water 5–6 times, and inoculated onto the MS medium and cultured in the dark at 28°C 24 h, and then moved to the light conditions described above. 2.3 Induction of adventitious buds 2.3.1 Induction of adventitious buds in cotyledons Sterilized cucumber seedlings with cotyledons grown for 1–2 d but not yet fully expanded were selected. Both cotyledons were cut close to the growing point. The upper parts (1/2–1/3) of the cotyledons were removed and the central part was retained. Each cotyledon was cut into 4–6 pieces and placed upside down on the MS bud induction medium containing various concentrations of 6-benzylaminopurine (6-BA; 1.0, 2.0, and 3.0 mg L-1), kinetin (KT; 0, 0.5, 1.0, and 1.5 mg L-1), abscisic acid (ABA; 0, 0.5, and 1.0 mg L-1), and AgNO3 (2.0 mg L-1). The culture was grown in the dark at 28°C for 48 h and then switched to light conditions. The number of adventitious buds was measured after 20 d. 2.3.2 Induction of adventitious buds in cotyledonary nodes 5–6-d-old sterilized cucumber seedlings with cotyledons that were not fully expanded were selected. The growing point and the upper part (1/2–2/3) of the both cotyledons were removed. The hypocotyl was dissected longitudinally, and a 2-mm segment of hypocotyl was retained. The cotyledonary nodes were inoculated on the MS bud induction medium. 2.3.3 Calculation of the regeneration rate and the number of regenerated buds Regeneration rate (%) = (number of explants with regenerated adventitious buds / total number of explants) × 100. The number of regenerated buds per explant = the total number of adventitious buds / the total number of explants with adventitious buds. 2.4 Statistical analysis Statistical analysis was performed using SPSS 17.0 software. Analysis of variance (ANOVA) was followed by Tukey’s pair wise comparison tests, at a level of P<0.05, in order to determine the significant differences between means. 3. Results 3.1 Effects of various combinations and concentrations of hormones on in vitro regeneration from cucumber cotyledons Induction of adventitious buds was closely related to the combinations and concentrations of hormones (Table 1). For the same concentration of 6-BA, different concentrations of KT and ABA resulted in different trends in adventitious bud regeneration rate and the number of regenerated buds among explants. In C8 medium, the adventitious bud regeneration rate was 74.70%, significantly higher than that of other media, and the number of regenerated buds was 0.76. The adventitious bud regeneration rate using the C9 medium was 73.30%. However, it resulted in the highest number of buds, 1.00 per explant. Therefore, the C9 medium (MS + 3.0 mg L-1 6-BA + 1.0 mg L-1 KZ + 1.0 mg L-1ABA + 2.0 mg L-1AgNO3) was better to induce adventitious buds from cucumber cotyledons. 32 Table 1. Effects of various combinations and concentrations of hormones on in vitro regeneration from cucumber cotyledons and cotyledonary nodes Medium 6-BA (mg L- 1) KT (mg L- 1) ABA (mg L-1) Number of regenerated buds per explant Regeneration rate (%) cotyledons cotyledonary nodes cotyledons cotyledonary nodes A1 1.0 0 0 0.24 cde 1.40 abc 8.60 fg 65.40 abc A2 1.0 0 0.5 0.23 cde 1.11 bc 54.40 abcde 57.10 abc A3 1.0 0 1.0 0.24 cde 0.60 bc 15.10 fg 25.80 cd A4 1.0 0.5 0 0 e 1.37 abc 18.10 efg 81.90 ab A5 1.0 0.5 0.5 0.38 bcde 1.40 abc 46.40 abcdef 58.10 abc A6 1.0 0.5 1.0 0.41 bcde 1.24 bc 43.40 abcdef 50.90 abcd A7 1.0 1.0 0 0.22 cde 1.28 abc 45.50 abcdef 54.60 abcd A8 1.0 1.0 0.5 0.49 bcd 1.99 abc 45.80 abcdef 86.00 ab A9 1.0 1.0 1.0 0.48 bcd 0.60 bc 43.30 abcdefg 35.00 cd A10 1.0 1.5 0 0 e 0.91 bc 8.40 fg 38.40 bcd A11 1.0 1.5 0.5 0.46 bcd 1.20 bc 44.10 abcdef 55.00 abc A12 1.0 1.5 1.0 0.45 bcd 0.76 bc 55.20 abcde 50.90 abcd B1 2.0 0 0 0.31 cde 4.38 a 14.00 fg 90.70 ab B2 2.0 0 0.5 0.32 cde 0.82 bc 29.40 cdefg 44.10 bcd B 3 2.0 0 1.0 0.28 cde 0.54 bc 23.30 defg 43.00 abcd B 4 2.0 0.5 0 0 e 1.24 bc 0 g 63.00 abc B 5 2.0 0.5 0.5 0.61 abc 1.49 abc 56.80 cdefg 63.40 abc B 6 2.0 0.5 1.0 0.41 bcde 2.63 abc 38.30 abcdefg 52.40 abcd B 7 2.0 1.0 0 0.08 de 1.89 abc 8.90 fg 68.20 abc B 8 2.0 1.0 0.5 0.22 cde 2.21 abc 41.50 abcdefg 59.30 abc B 9 2.0 1.0 1.0 0.29cde 2.36 abc 29.70 cdefg 59.80 abc B 10 2.0 1.5 0 0 e 3.07 ab 11.70 fg 88.30 ab B11 2.0 1.5 0.5 0.24 cde 1.06 bc 35.60 abcdefg 40.60 bcd B 12 2.0 1.5 1.0 0.44 bcde 1.51 abc 34.40 abcdefg 45.60 abcd C1 3.0 0 0 0 e 2.44 abc 0 g 64.20 abc C 2 3.0 0 0.5 0.17 cde 1.86 abc 57.10 abcde 86.20 ab C3 3.0 0 1.0 0 e 1.46 abc 11.50 fg 66.50 abc C 4 3.0 0.5 0 0 e 3.30 ab 14.80 fg 96.20 a C 5 3.0 0.5 0.5 0.14 de 1.89 abc 62.80 abcd 75.80 abc C 6 3.0 0.5 1.0 0.33 cde 0.89 bc 34.80 bcdef 38.60 d C 7 3.0 1.0 0 0.36 bcde 3.28 ab 8.80 fg 77.20 abc C 8 3.0 1.0 0.5 0.76 e 1.72 abc 74.70 ab 71.80 abc C 9 3.0 1.0 1.0 1.00 a 2.10 abc 73.30 a 68.70 abc C 10 3.0 1.5 0 0.15 de 3.29 ab 0 g 90.20 ab C11 3.0 1.5 0.5 0.27 cde 2.89 abc 42.00 abcdef 68.50 abcd C 12 3.0 1.5 1.0 0.25 cde 2.50 abc 64.00 abc 51.90 abcd C13 0 0 0 0 e 0 c 0 g 0 d Note: Lower case letters indicate significant differences at p < 0.05. 33 3.2 Effects of various combinations and concentrations of hormones on the in vitro regeneration from cucumber cotyledonary nodes. As shown in Table 1, the regeneration rate and the number of regenerated buds varied markedly using cotyledonary nodes as explants, depending on the combinations and concentrations of hormones. In the control C13 medium, the growth point of the cotyledonary node did not bulge, and the number of adventitious roots grew substantially with increasing culture time. The adventitious bud regeneration rate in the B 1 medium was relatively high at 90.70%. The B1 medium also showed the highest number of regenerated buds, 4.38. The C4 medium had the highest regeneration rate of 96.20%, and the number of generated buds was 3.30. In the C10 medium, the regeneration rate was 90.20% and the number of regenerated buds was 3.29. Therefore, the B1 medium (MS + 2.0 mg L-1 6-BA + 2.0 mg L-1AgNO3) was the best bud induction medium for cotyledonary nodes in this study. 3.3 Effects of explant types on in vitro cucumber regeneration The explant types greatly affected cucumber regeneration (Table 1). In the C9 medium, the adventitious bud regeneration rate and the number of regenerated buds of cotyledon were 73.30% and 1.00, respectively, whereas those of the cotyledonary node were 68.70% and 2.10, respectively. In the B1 medium, the regeneration rate and the number of regenerated buds of the cotyledon were 14.00% and 0.31, respectively, whereas those of the cotyledonary node were 90.70% and 4.38, respectively. Thus, the number of regenerated buds for the cotyledonary nodes was much higher than that observed for the cotyledons under the same genotype and culture conditions, suggesting that cotyledonary nodes are more suitable for the establishment of the cucumber regeneration system. 3.4 Effects of 6-BA on in vitro regeneration using cotyledonary nodes In medium lacing KT and ABA and containing 2.0 mg L-1 AgNO3, different concentrations of 6-BA caused significant differences in the regeneration rate and the number of regenerated buds using cotyledonary nodes (Table 2). A 6-BA concentration of 2.0 mg L-1 resulted in the highest regeneration rate (90.70%) and the highest number of regenerated buds (4.38) for the cotyledonary nodes. Table 2: Effects of 6-BA on in vitro regeneration using cotyledonary nodes 6-BA (mg L-1) KT (mg L-1) ABA (mg L-1) Number of regenerated buds per explant Regeneration rate (%) 0 0 0 0 c 0 c 1.0 0 0 1.40 bc 65.40 ab 2.0 0 0 4.38 a 90.70 a 3.0 0 0 2.44 ab 64.20 ab Note: Lower case letters indicate significant differences at p < 0.05. 3.5 Effects of ABA on in vitro regeneration using cotyledonary nodes In medium lacking KT but containing 6-BA (2.0 mg L-1) and AgNO3 (2.0 mg L-1), the regeneration rate and number of regenerated buds of the cotyledonary nodes showed a decreasing trend with increasing concentrations of ABA (Table 3). For an ABA concentration of 0 mg L-1, the highest regeneration rate and the maximum number of regenerated buds were obtained, and these differed significantly from those of other treatment concentrations. Table 3: Effects of ABA on in vitro regeneration using cotyledonary nodes 6-BA (mg L-1) KT (mg L-1) ABA (mg L-1) Number of regenerated buds per explant Regeneration rate (%) 2.0 0 0 4.38 a 90.70 a 2.0 0 0.5 0.82 b 44.10 b 2.0 0 1.0 0.54 b 45.00 b Note: Lower case letters indicate significant differences at p < 0.05. 3.6 Effects of KT on in vitro regeneration using cotyledonary nodes As shown in Table 4, for medium lacking ABA but containing 6-BA (2.0 mg L-1) and AgNO3 (2.0 mg L-1), for increasing KT concentrations, both the regeneration rate and the number of regenerated buds using the cotyledonary nodes first declined and then rose. For a KT concentration of 0 mg L-1, both the regeneration rate 34 (90.70%) and the number of regenerated buds (4.38) were maximal. For a KT concentration of 0.5 mg L-1, both the regeneration rate (63.00%) and the number of regenerated buds (1.24) were the lowest. Table 4: Effects of KT on in vitro regeneration using cotyledonary nodes 6-BA (mg L-1) ABA (mg L-1) KT (mg L-1) Number of regenerated buds per explant Regeneration rate (%) 2.0 0 0 4.38 a 90.70 a 2.0 0 0.5 1.24 b 63.00 b 2.0 0 1.0 1.89 b 68.20 b 2.0 0 1.5 3.07 a 88.30 a Note: Lower case letters indicate significant differences at p < 0.05. 4. Discussion MS medium is often used as the basal medium for in vitro cucumber cultures. Supplementation with different types and concentrations of hormones results in regenerated plantlets. Hormone types, combinations, and concentrations used are generally selected based on genotype, regeneration method, and explant types (Kim et al. 1988). Optimal hormone types and concentrations are essential for a high frequency in vitro regeneration system. This study compared the effects of various combinations and concentrations of 6-BA, ABA, and KT on adventitious bud induction using cucumber cotyledons and cotyledonary nodes. We found that the number of regenerated buds for the cotyledonary nodes was much higher than that observed for the cotyledons under the same genotype and culture conditions, suggesting that cotyledonary nodes are more suitable for the establishment of the cucumber regeneration system. The addition of only 2.0 mg L-1 6-BA resulted in the growth and expansion of cucumber cotyledonary nodes, with the highest adventitious bud regeneration rate and the most regenerated buds. These results indicate that 6-BA is an essential hormone for in vitro regeneration, ABA and KT had little effect on the differentiation of adventitious buds using cucumber cotyledonary nodes. Wang et al. 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