The tissue culture experiments that have been done now mainly include shoot tip culture and immature embryo culture.
During shoot tip culture, long branches should be collected from the middle and upper parts of ginkgo plants to be propagated, leaves and petioles should be cut off, wrapped with moist gauze and brought back indoors, and washed repeatedly with distilled water, bleaching powder solution, distilled water, mercuric chloride water and distilled water in turn. Then, under aseptic conditions, cut off the stem tip about 0.5 cm long, and finally inoculate the stem tip on the prepared culture medium, and cover the nozzle to prevent pollution. The bud tip used for tissue culture can be terminal bud or lateral bud. Considering that the tissue of ginkgo tree has obvious site effect, it is generally not suitable to collect lateral buds extending outward as tissue culture materials.
Generally, the medium used for shoot tip culture is improved white medium to induce callus and embryonic stem, and N6-based rooting medium is used to promote rooting.
The composition of the improved white medium is: white medium+Ba0.5-1.0+NAA0.5+NH4Cl 5.34+sugar 4%.
The composition of rooting medium is N6+2,4-D3+BA1+NAA1+3% sugar +0.5g agar.
(The above concentration units, except sugar agar, are milligrams per liter of solution. )
The culture conditions are: temperature 25-29℃. Firstly, a week of dark culture was carried out to form callus. From the eighth day, it was transferred to natural scattered light and cultured for another week to promote the differentiation of embryonic stem. Two kinds of leaf buds can be seen at this time. One lives in groups and the other lives alone. Stem segments formed by independent buds were selected and transferred to rooting medium for subculture. After three weeks, the root system can gradually grow and form a complete ginkgo plant.
The results showed that the bud induction rate of shoot tip tissue of Ginkgo biloba L. was 83.65438 0% when adding NH4Cl instead of 2,4-D, and sometimes it could even induce rooting.
Young embryo culture is mainly used to save some hybrid embryos or abnormal embryos that are not fully mature or can not grow into new plants for other reasons, which is conducive to the preservation of new genotype offspring.
The specific method is as follows: firstly, remove the seed coat of Ginkgo biloba from the bone, sterilize the seed kernel (actually the endosperm here) with 70% alcohol for 2-3 minutes, then put it in 0. 1% mercuric chloride aqueous solution for 20 minutes, and then rinse it with sterile water for 3 times. Seed embryos are picked out under aseptic conditions, then the embryos are cut into whole or several pieces, and the water is absorbed by sterilized filter paper, and then inoculated on the surface of agar medium in test tubes respectively.
As the medium for immature embryo culture, N6 and MS are the basic medium for callus formation. After 10- 15 days, buds will form at the hypocotyl incision. Three commonly used budding media are composed of:
N6+KT 1+NAA 1+ 3% sugar +0.5% agar.
MS+BA3+NAA0.5+ sugar 3%+ agar 0.5%
Ms+2,4-D2+IH 300+3% sugar +0.5% agar.
Transfer the embryo culture block with bud to the medium of white+2,4-D2+NH4Cl 5.34+IH 300+sugar 4%+ activated carbon 0.05%+ agar 0.5%, and after 6- 10 days, the root can grow from the bud and form a complete plantlet.
The specific culture conditions are: the temperature is kept at about 25℃, and the upper and lower temperature is not more than 2℃. In the stage of differentiation and culture, 40W fluorescent lamp was used for irradiation 10 hour during the day and kept dark at night. When the seedlings grow 4-7 leaves, they are cut into 2-3 sections, cultured in chronological order, and then transferred to rooting medium after budding.
Institute of Botany, Chinese Academy of Sciences, etc. Using bee milk as culture medium to cultivate young embryos of Ginkgo biloba has also achieved success. It can be seen that the normal development of Ginkgo biloba embryos can be guaranteed as long as there are suitable hormones and nutrients necessary for cell division and proliferation.