Studies On The Protoplast Separation, Purification And Culture Of Gladiolus

Gladiolus hybridus Hort, also called sword or gladiolus, is one of perennial monocotyledonous bulbous flowers of gladiolus Iridaceae, which is one of the four world-famous cut flowers. Cut flower production of gladiolus is the important parts of flower industry.Gladiolus is one of the most serious diseases and insect pests of all flowers. It brings the new hope for the disease-resistance breeding of the gladiolus to transform foreign genes by protoplast method. Now most of planting varieties and corms used in production come from foreign and lack of varieties breeding of the gladiolus in our country. The study on separation, purification and culture technique of the gladiolus will lay the foundation for the breeding of the gladiolus.The study took the offspring corms of the gladiolus as materials and esTab.lished the system of the separation, purification and culture technique of the gladiolus. The results were as follows:1. Culture medium of the induced callus system of the offspring corms of the gladiolus: 'Rose Supreme' MS+2.4-D 3.0 mg/L+6-BA 0.5 mg/L; 'Pink Supreme'MS+2,4-D4.0mg/L+ 6-BA0.5mg/L; and 'Blue Fairy' MS+2,4-D 4.0 mg/L+6-BA 0.5 mg/L.2. The study gained the callus of three types: type I Embryonic callus; type II Non-embryonic callus; type III middle types of callus. Thereinto callus culture of type I and type III may get the embryonic callus after 2-3 months. Culture medium with MS, 2,4-D 2.0 mg/L and 6-BA 0.5 mg/L will be favour for the vigour of the embryonic callus.3. Culture methods of suspending culture cells of the gladiolus were as following: First inoculation quantum was 2g/40ml, and first sub-culture cycle was 2-3d and 5~8d after a month. It was favour for the improving of protoplast vigour to the medium with 2mg/L Gly, 100mg/L Glu, 300mg/L LH, 100mg/L yeast extraction and 500mg/L BSA.4. Confirmed 0.6mol/L mannitol to be concentration of the osmotic pressure.5. Vigor and yields of lamina protoplast-pretreated 1h in 0.6mol/L mannitol were higher. Vigor and yields of protoplast, which was gained by the suspending culture system and sub-cultured for two month, were higher.6. Determined that the enzyme concentrations of lamina and suspending cells of gladiolus to separate protoplast respectively were Cellulase "Onzuka" RS 1.5%+Pectinase Y-23 0.5% and Cellulase "Onzuka" RS 2.0% + Pectinase Y-23 0.5%, PH5.6, the digestion time was 2h, and the digestion conditions were incubated at 40rpm/min, temperature 27℃ in the dark.7. Culture method of protoplast was first liquid and then embedded. Culture mediums were as follows: 'Rose Supreme' KM8P + 2,4-D1.0mg/L + NAA0.2mg/L + KT0.2mg/L; 'Pink Supreme' KM8P + 2,4-D 1.5mg/L + NAA0.2mg/L + ZT0.2mg/L; and 'Blue Fairy' KM8P +...