TH19728Y - Methods of oil palm tissue culture. - Google Patents

Abstract

------06/11/2020------(OCR) This invention relates to a hybrid tissue culture method for oil palm using a modified N6 basic liquid medium formula combined with adenine sulfate, putrescine, and casein hydrolysate to induce callus formation. The callus obtained from this invention's method has significantly higher potential than that obtained by conventional methods of somatic embryo induction in solid medium. Furthermore, it enhances the development of seedlings by culturing in a modified MS basic solid medium containing putrescine and without hormones. This results in rapid seedling development in approximately 2-2.5 years, significantly different from the conventional method which takes at least 3-4 years. In addition, the liquid medium tissue culture method described in this invention results in significantly lower genetic variation in the resulting seedlings. ------------ Page 1 of 1 page. Summary of the Invention. This invention relates to a hybrid tissue culture method for oil palm using a modified N6 basic liquid medium combined with adenine sulfate, putrescine, and casein hydrolysate to induce callus formation. The callus obtained from this invented method has significantly higher potential than that obtained by conventional methods of somatic embryo induction in solid medium. Furthermore, the development of seedlings is improved by culturing in a modified MS basic solid medium containing putrescine and without hormones. As a result, oil palm tissue culture leads to rapid seedling development in approximately 2-2.5 years, significantly different from the conventional method which takes at least 3-4 years. In addition, this liquid medium tissue culture method for oil palm produces seedlings with significantly lower genetic variation.

Claims (5)

------06/11/2563------(OCR) 1. The method for tissue culture of oil palm involves the following steps: a) Culturing embryogenic callus in liquid medium using modified N6-1 medium under shaking conditions at 120-180 rpm, 25°C, in the dark for 2 months, with callus being transferred to fresh medium every 2 weeks. b) Transferring the newly developed embryogenic callus from the original callus obtained from step a) to solid medium using modified N6-2 medium until shoot development occurs, for 6-12 months. 2. The method for tissue culture of oil palm according to claim 1, which may be supplemented with the step of transferring somatic embryos beginning to develop shoots obtained from step b) to medium using modified MS-1 medium for 6-12 months. 3. The method for tissue culture of oil palm. Under Reputation 1, modified N6-1 liquid culture medium contains 213.5 mg/L ammonium sulfate ((NH4)2SO4), 3134 mg/L potassium nitrate (kN3), 540 mg/L potassium dihydrogen phosphate (Kh2PO4), 185 mg/L magnesium sulfate heptahydrate (MgSO4.7H2O), 166 mg/L calcium chloride dihydrate (CaCl2.2H2O), 0.8 mg/L potassium iodide (KI), 6.2 mg/L boric acid (H3BO3), 20 mg/L manganese sulfate tetrahydrate (MnSO4.4H2O), and 8.6 mg/L zinc sulfate heptahydrate (ZnSO4.7H2O). Cobalt chloride octahydrate (CoCl2.6H2O) 0.05 mg/L, copper sulfate heptahydrate (CuSO4.7H2O) 0.05 mg/L, sodium molybdate dihydrate (Na2MoO4.2H2O) 0.25 mg/L, ferrous sulfate heptahydrate (FeSO4.7H2O) 27.8 mg/L, disodium ethylenediamine tetraacetic acid (Na2EDTA) 37.2 mg/L, myo-enositol 100 mg/L, nicotinic acid 2.5 mg/L, pyridoxine hydrochloride 2.5 mg/L, thiamine hydrochloride 2.5 mg/L, sucrose 30 mg/L. Adenine sulfate 20-60 mg/L, putrescine 80-120 mg/L, casein hydrolysate 300-800 mg/L. 4. Method for oil palm tissue culture according to claim 1, where the modified N6-2 solid culture medium contains ammonium sulfate ((NH4)2SO4) 213.5 mg/L, potassium nitrate (KNO3) 3134 mg/L, potassium dihydrogen phosphate (KH2PO4) 540 mg/L, magnesium sulfate heptahydrate (MgSO4.7H2O) 185 mg/L, calcium chloride dihydrate (CaCl2.2H2O) 166 mg/L, potassium iodine (KI) 0.8 mg/L, and boric acid (H3BO3) 6.2 mg/L. Manganese sulfate tetrahydrate (MnSO4.4H2O) 20 mg/L, Zinc sulfate heptahydrate (ZnSO4.7H2O) 8.6 mg/L, Cobalt chloride octahydrate (CoCl2.6H2O) 0.05 mg/L, Copper sulfate heptahydrate (CuSO4.7H2O) 0.05 mg/L, Sodium molybdate dihydrate (Na2MoO4.2H2O) 0.25 mg/L, Ferrous sulfate heptahydrate (FeSO4.7H2O) 27.8 mg/L, Disodium ethylenediamine tetraacetic acid (Na2EDTA) 37.2 mg/L. Myo-inositol 100 mg/L, nicotinic acid 2.5 mg/L, pyridoxine hydrochloride 2.5 mg/L, thiamine hydrochloride 2.5 mg/L, sucrose 30 mg/L, adenine sulfate 20-60 mg/L, putrescine 120-160 mg/L, casein hydrolysate 300-800 mg/L, phytagel 2.5 mg/L. 5. Method for tissue culture of oil palm according to claim 1, where modified MS-1 culture medium contains ammonium sulfate ((NH4)2SO4) 1900 mg/L, potassium nitrate (kN3) 1650 mg/L, potassium dihydrogen phosphate (KH2PO4) 170 mg/L. Magnesium sulfate heptahydrate (MgSO4.7H2O) 370 mg/L, Calcium chloride dihydrate (CaCl2.2H2O) 440 mg/L, Potassium iodide (KI) 0.83 mg/L, Boric acid (H3BO3) 6.2 mg/L, Manganese sulfate tetrahydrate (MnSO4.4H2O) 22.3 mg/L, Zinc sulfate heptahydrate (ZnSO4.7H2O) 8.6 mg/L, Cobalt chloride octahydrate (CoCl2.6H2O) 0.025 mg/L, Copper sulfate heptahydrate (CuSO4.7H2O) 0.025 mg/L, Sodium molybdate dihydrate (Na2MoO4.2H2O) 0.25 mg/L. Ferrous sulfate heptahydrate (FeSO4.7H2O) 27.8 mg/L, Disodium ethylenediamine tetraacetic acid (Na2EDTA) 37.2 mg/L, Glycine 2.0 mg/L, Nicotinic acid 0.5 mg/L, Pyridoxine hydrochloride 0.5 mg/L, Thiamine hydrochloride 0.1 mg/L, Sucrose 30 mg/L, Putrescine 80-120 mg/L, Phytagel 2.5 mg/L. ------------ Edited 08/11/2018. No claims. ---------------------------------------------------------------------------------------------------- Page 1 of 3. Claims. 1. The oil palm tissue culture method involves the following steps: a) Culturing callus in liquid medium using modified N6-1 medium under shaking conditions at 120-180 rpm at 25°C in a cooled environment for 2 months. The callus is transferred to fresh medium every 2 weeks. b) Transferring the newly developed embryogenic callus from the original callus obtained in step a) to solid medium using modified N6-2 medium until shoot development occurs. This cultivation period is 6-12 months. 2. Cultivation method as per claim 1, which may be supplemented by a step of culturing somatic embryos beginning to develop a shoot, obtained from step b), in modified MS-1 culture medium for 6-12 months. 3. Culture Method According to Patent 1, modified N6-1 culture medium contains (NH4)2SO4 213.5 mg/L, KNO3 3134 mg/L, KH2PO4 540 mg/L, MgSO4.7H2O 185 mg/L, CaCl2.2H2O 166 mg/L, Kl 0.8 mg/L, H3BO3 6.2 mg/L, MnSO4.4H2O 20 mg/L, ZnSO4.7H2O 8.6 mg/L, CoCl2.6H2O 0.05 mg/L, CuSO4.7H2O 0.05 mg/L, Na2MoO4.2H2O 0.25 mg/L, FeSO4.7H2O 27.8 mg/L, and Na2EDTA 37.2 mg/L. Myorinositol 100 mg/L (Page 2 of 3), Nicotibic acid 2.5 mg/L, Pyridoxine hydrochloride 2.5 mg/L, Thiamine hydrochloride 2.5 mg/L, Sucrose 30 mg/L, Adenine sulfate 20-60 mg/L, Putrescine 80-120 mg/L, Casein hydrolysate 300-800 mg/L. 4. Culture Method. According to claim 1, the modified N6-2 culture medium contains (NH4)2SO4 213.5 mg/L, KNO3 3134 mg/L, KH2PO4 540 mg/L, MgSO4.7H2O 185 mg/L, CaCl2.2H2O 166 mg/L, Kl 0.8 mg/L, H3BO3 6.2 mg/L, MnSO4.4H2O 20 mg/L, ZnSO4.7H2O 8.6 mg/L, CoCl2.6H2O 0.05 mg/L, CuSO4.7H2O 0.05 mg/L, Na2MoO4.2H2O 0.25 mg/L, FeSO4.7H2O 27.8 mg/L, and Na2EDTA 37.2 mg/L. Myo-enositol 100 mg/L, Nicotinic acid 2.5 mg/L, Pyridoxine hydrochloride 2.5 mg/L, Thiamine hydrochloride 2.5 mg/L, Sucrose 30 mg/L (Page 3 of 3), Adenine sulfate 20-60 mg/L, Putrescine 120-160 mg/L, Casein hydrolysate 300-800 mg/L, Phytagel 2.5 mg/L. 5. Cultivation methods. According to claim 1, the modified MS-1 formula contains (NH4)2SO4 1900 mg/L, KNO3 1650 mg/L, KH2PO4 170 mg/L, MgSO4.7H2O 370 mg/L, CaCl2.2H2O 440 mg/L, Kl 0.83 mg/L, H3BO3 6.2 mg/L, MnSO4.4H2O 22.3 mg/L, ZnSO4.7H2O 8.6 mg/L, CoCl2.6H2O 0.025 mg/L, CuSO4.7H2O 0.025 mg/L, Na2MoO4.2H2O 0.25 mg/L, FeSO4.7H2O 27.8 mg/L, Na2EDTA 37.2 mg/L, Glycine 2.0 mg/L, and Nicotinic acid 0.5 mg/L. Pyridoxine hydrochloride 0.5 mg/L, Thiamine hydrochloride 0.1 mg/L, Sucrose 30 mg/L, Putrescine 80-120 mg/L, Phytagel 2.5 mg/L.