A study on multiplication of oncidium-sweet sugar by using cell tissue culture

Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 67 A STUDY ON MULTIPLICATION OF ONCIDIUM-SWEET SUGAR BY USING CELL TISSUE CULTURE Le Hoai Thanh, Le Huu Can1 Received: 10 November 2014 / Accepted: 18 April 2016 / Published: May 2016 ©Hong Duc University (HDU) and Journal of Science, Hong Duc University Abstract: Orchid is a specious kind of flower that brings about high economic value to flower production and flower business. Production of Orchids in Vietnam has been limited to its development, especially production of Oncidium-Sweet Sugar Orchids. Research on a process of rapid multiplication of Oncidium-Sweet Sugar Orchids by using cell tissue culture in order to create high quality varieties that help serve flower production industry. This is also hoped to contribute to the Orchids production in Thanh Hoa province. - The most appropriate mode of disinfection for bud samples is 7 minutes + 1 minute, and for blooming samples is 7 minutes. - Both BA and Kinetine have effects on the forming generation of the blooming Oncidium Orchids. The best treatment for BA is MS+2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar + 2 pmm BA, and for Kinetine is MS + 2% sugar + 0.1g/liter of Inositol + 6.5g agar + 2 ppm Ki. - The best medium that contains the growth control substances for forming generation of slicing is MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar + 1 pmm BA. - The best medium for the Oncidium Orchids’ bud multiplication when added with growth control substances is: MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar + 0.5 ppm Ki. - During the process of rapid multiplication of the Oncidium Orchids’ buds, it is possible to add organic extracts to the medium (like bananas and potatoes) following the treatment: MS + 2% of sugar + 0.1 g/liter of Inositol + 6.5g of agar + 50g Potato + 50g Banana. Keywords: Sweet Sugar, disinfection, organic substances, bud multiplication process 1. Introduction Orchid is a specious kind of flower that brings about high economic value to flower production and flower business. However, the production of Orchids in Vietnam has been Le Hoai Thanh Department of postgraduate management and education, Hong Duc University Email:Lehoaithanh@hdu.edu.vn () Le Huu Can Faculty of Agricultural, Forestry and Fishery, Hong Duc University Email: Lehuucan@hdu.edu.vn () Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 68 limited to its development, especially production of Oncidium-Sweet Sugar Orchids. Our attempt in this study is to research on a process of rapid multiplication of Oncidium-Sweet Sugar Orchids by using cell tissue culture in order to create high quality varieties that help serve flower production industry. This is also hoped to contribute the Orchids production in Thanh Hoa province. 2. Subjects and research methods 2.1. Subjects for research Fresh, yellow Oncidium Sweet Sugar Orchids, small flowers, and many flowers on a branch. 2.2. Research methods Experiments implemented in the artificial conditions, which enable adjustments in lights and temperature, in the plant cell tissue culture labs of Hong Duc University. The experiments were arranged as follows: - Randomly, repeated every three times, each treatment using 10 samples. - Regularly observed, data collected and calculated after every 10 to 15 days. - Data analyzed using IRRISTAT. 3. Results and discussion 3.1. Effects of disinfection time on clean scale and forming generation scale of samples after 8 weeks cultured Table 3.1. Effects of disinfection time on clean scale and forming generation scale of samples after 8 weeks cultured Observation Index Treatment (T) Auxiliary bud samples Blooming samples Infected samples % Clean samples Infected samples % Clean samples Dead scale % Forming generatio n scale % Dead scale % Forming generatio n scale % T1: 7 mins 95.1 0.0 4.9 40.0 13.3 46.7 T2: 10 mins 95.0 5.0 0.0 26.7 60.0 13.3 T3: 7 mins + 1 min 20.0 20.0 60.0 20.0 73.3 6.7 T4: 10 mins + 1 min 15.0 65.0 20.0 0.0 100.0 0.0 Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 69 The Table 3.1 shows that: The single disinfection for 7 minutes is best for blooming. Dual disinfection for 7 minutes + 1 minute is best for the samples as axillary buds. 3.2. Effects of Benzamin Adenin (BA) on forming generation scale of Oncidium blooming samples Table 3.2. Effects of the BA on forming generation scale of Oncidium blooming samples after 8 weeks cultured Observation Index Treatment (T) Forming generation scale (%) Protocorm scale (%) Bud scale (%) T1: MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar 5.3 9.4 90.6 T2: 0.5 ppm BA 40.0 66.2 34.8 T3: 1 ppm BA 75.2 88.3 11.7 T4: 2 ppm BA 84.7 96.6 3.4 T5: 3 ppm BA 45.3 60.7 39.3 The data of the Table 3.2 show that: The best treatment for forming generation and Protocorm generation of blooming Oncidium Orchids is T4 (2 ppm). 3.3. Effects of Kinetine on form generation of oncidium blooming orchids after 8 weeks in culture medium Table 3.3. Effect of Kinetine on form generation scale and protocorm scale after 8 weeks Observation Index Treatment (T) Form generation scale (%) Protocorm scale (%) Bud scale (%) T1: MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar 13.3 9.4 90.6 T2: 1 ppm BA 65.7 76.9 23.1 T3: 2 ppm BA 100.0 89.2 10.8 T4: 3 ppm BA 83.3 71.5 28.5 T5: 4 ppm BA 50.0 50.2 49.8 The data show that: The concentration of Ki that is best for blooming Oncidium Orchids’ forming generation and protocorm is 2ppm. Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 70 3.4. Effects of growth control substance on bud multiplication process 3.4.1. Effects of the BA on bud multiplication process Table 3.4.1. Effect of the BA on multiplying coefficient after 8 weeks of culture Observation Index Treatment (T) Bud multiplying coefficient (times/sample/ total time) Bud creation scale (%) Protocorm creation scale (%) T1: MS + 20 g/ liter of saccarose + 0.1g/ litter of Inositol + 6.5g of agar 0.38 6.01 93.09 T2: T1+0.5 ppm BA 1.63 53.53 46.47 T3: T1+1 ppm BA 1.63 71.24 28.76 T4: T1+1.5 ppm BA 2.12 76.15 23.85 T5: T1+2 ppm BA 1.43 68.47 31.53 LSD (5%) 0.66 CV% 2.20 The data show that: Concentration of the BA that is best for bud multiplication process is 1.5 ppm BA. 3.4.2. Effects of Kinetine on the process of bud multiplication Table 3.4.2. Effects of Kinetine on bud multiplying coefficient after 8 weeksof culture Observation Index Treatment (T) Bud multiplying coefficient (times/ sample/ time) Bud creation scale (%) Protocorm creation scale (%) T1: MS + 20g/ liter of saccarose + 0.1g/ litter of Inositol + 6.5g of agar 0.38 8.56 91.44 T2: T1+0.5 ppm Ki 2.47 93.30 6.70 T3: T1+1 ppm Ki 2.02 86.71 13.29 T4: T1+1.5 ppm Ki 2.10 70.27 29.73 T5: T1 + 2 ppm Ki 2.20 56.50 43.50 LSD (5%) 0.10 CV% 2.80 The data from Table 3.4.2 show that: The adding of Ki to the culture medium would increase in the bud multiplying coefficient (T1-T4). The concentration of Kinetine which is best for bud multiplication of Oncidium Orchids is 0.5 ppm of Kinetine. Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 71 3.5. Effects of organic substances on bud multiplication process Table 3.5. Effects of organic extract on bud multiplying coefficient after 8 weeks of culture Observation Index Treatment (T) Bud multiplying coefficient (times/ sample/ time) Bud height (cm) Number of leaves/buds (leaves) T1: MS + 20g/ liter of saccarose + 0.1g/ litter of Inositol + 6.5g of agar 0.06 0.17 0.57 T2: T1+ 30g Potato 1.85 1.00 2.86 T3: T1+ 50g Potato 2,54 1.10 3.45 T4: T1+ 70g Potato 1.74 1.10 2.10 T5: T1+ 100g Potato 1.53 1.19 2.25 T6: T1+ 30g Banana 1.80 1.01 2.91 T7: T1+ 50g Banana 2.00 1.06 3.17 T8: T1+ 70g Banana 1.76 1.06 2.15 T9: T1+ 100g Banana 1.50 1.15 2.96 T10: T1+ 30g Banana + 30g Potato 1.30 1.39 2.86 T11: T1+ 50g Banana + 50g Potato 1.49 1.11 2.99 T12: T1+ 70g Banana + 70g Potato 1.49 1.03 2.47 T13: T1+100g Banana + 100g Potato 1.30 1.31 2.73 LSD (5%) 0.10 CV% 3.70 The data of the Table 3.5 show that: The treatments that are added with organic extract produce higher quality of bud multiplying coefficient and bud shoot than Treatment 1 that is not added with organic extract. The different organic extracts (potatoes and bananas) in an appropriate concentration produce good quality of buds and bud multiplying coefficient. The combination of these two kinds also show good results, and the best treatment is 50g Potato + 50g Banana. Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 72 3.6. Effects of the BA and Kinetine on developmental formation of the thin slicing samples after 8 weeks observed 3.6.1. Research on the effects of the BA on developmental formation of the thin slicing samples Table 3.6.1. Effects of the BA and Kinetine on developmental formation of the thin slicing samples after 8 weeks observed Observation Index Treatment (T) Formation generation scale (%) Protocorm scale (%) Number of Protocorm/LC T1: MS + 20g/ liter of saccarose + 0.1g/ liter of Inositol + 6.5g of agar 6.7 100 2.4 T2: T1+ 0.3 ppm BA 100 100 2.6 T3: T1+ 0.5 ppm BA 100 100 4.4 T4: T1+ 1 ppm BA 100 100 5.2 T5: T1+ 2 ppm BA 90.1 100 4.6 The Table 3.6.1 shows that the best culture medium for slicing is MS+ 2% sugar + 6.5g agar + 1 ppm BA. 3.6.2. Research on effects of Kenetine on developmental formation of the thin slicing samples Table 3.6.2. Effects of Kenetine on developmental formation of the thin slicing samples Observation Index Treatment (T) Formation generation scale (%) Protocorm scale (%) Number of Protocorm/LC T1: MS + 20g /liter of saccarose + 0.1g/liter of Inositol + 6.5g of agar 6.67 100 2.43 T2: T1+ 0.3 ppm Ki 87.50 100 4.15 T3: T1+ 0.5 ppm Ki 100.00 100 6.75 T4: T1+ 1 ppm Ki 75.12 100 5.38 T5: T1+ 2 ppm Ki 75.05 100 5.35 The Table 3.6.2 shows that the BA has more significant effects on the development of slicing than Ki, especially when BA added, the scale of forming generation and number of Protocorm/LC are higher than when Ki added. Journal of Science Hong Duc University, E.2, Vol.7, P (67 - 73), 2016 73 4. Conclusion and recommendations 4.1. Conclusion The most appropriate mode of disinfection for bud samples is 7 minutes + 1 minute, and for blooming samples is 7 minutes. Both BA and Kinetine have effects on the forming generation of the blooming Oncidium Orchids. The best treatment for BA is MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar + 2 pmm BA, and for Kinetine is MS + 2% sugar + 0.1g/liter of Inositol + 6.5g agar + 2 ppm Ki. The best medium that contains the growth control substances for forming generation of slicing is MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar + 1 pmm BA. The best medium for the Oncidium Orchids’ bud multiplication when added with growth control substances is: MS + 2% of sugar + 0.1g/ liter of Inositol + 6.5g of agar + 0.5 ppm Ki. During the process of rapid multiplication of the Oncidium Orchids’ buds, it is possible to add organic extracts to the medium (like bananas and potatoes) following the treatment: MS + 2% of sugar + 0.1 g/liter of Inositol + 6.5g of agar + 50g Potato + 50g Banana. 4.2. Recommendations It is possible to apply researched techniques to the multiplication of the Oncidium Orchids. 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