| Lujia 5 apple was used to compared the micro-structures of leaves and stems between the vitrified and the normal apple shoots in vitro by means of paraffin section and scanning electron microscopy (SEM), observed the physiological characteristics, agar, sucrose, macroelement and illumination were designed in orthogonal array to compare the influence on the vitrified of apple shoots in vitro, and the methylation-sensitive amplified polymorphism (MSAP) technique was employed to detect differences of methylation profiles between the vitrified and the normal apple shoots in vitro. The results showed that:1 There were significant differences in micro-structures of leaves and stems between the vitrified and the normal apple shoots in vitro. Compared with the normal shoots, the leaf thickness of the vitrified shoots was much thicker and the density of upper epidermis cells was remarkably lower. The size of upper epidermis cells became larger and vacuolated. The thickness of the palisade parenchyma(P) was reduced, while the thickness of the spongy parenchyma(S) was increased significantly. The major axis of stomatata apparatus did not change a lot, but the minor axis obviously became larger and the density of stomatata was significantly lower. The vascular tissue in the stem of the vitrified shoots appeared empty and collapsed, and there were many folds on the conduit wall. There was no accumulation of starch grains in the sieve tube.2 There were significant differences in physiological characteristics between the vitrified and normal apple shoots in vitro. Compared with the normal apple shoots, the total content of tissue water of the vitrified apple shoots was significantly increased, the content of the free water was increased by 43.26%, the content of bound water was decreased by 52.47%. The content of the chlorophyll of leaves was remarkably decreased by 45.60%, the membrane permeability was increased by 35.43%, the content of the soluble protein was remarkably increased by 38.48%. And the activity of SOD, POD and CAT was decreased in various degrees. The content of ZT and GA3 was almost not different, but the content of IAA was increased by 14.66% and. the content of the ABA was increased by 29.00%.3 The width of the stomata in three levels(from low to high) of these four factors was decreased at first and increased latter, and the most important factor among those was agar, then illumination while macroelement was the most unimportant factor. When the concentration of agar was 8 g·L-1 and source was 30 g·L-1, the percentage of macro element was three fourths and the illumination intensity was 2000LX, the apple shoots grew best and were difficult to vitrify. However, the apple shoots were easiest to vitrify when they were cultured on the medium with 6.5g·L-1 agar,25g·L-1source, half amout of macro elements and the 2000LX illumination intensity.4 The number of the total amplified bands of the vitrified and the normal apple shoots were 303 and 308, total methylated bands were 149 and 97, full methylation bands were 56 and 51, and hemi-methylated bands were 93 and 46. The frequency of total methylation of the vitrified and the normal apple shoots were 49.2% and 31.5%, the frequency of full-methylation were 37.6% and 52.6%, and the frequency of hemi-methylation were 62.4% and 47.4%. According to the different changes of the methylated bands from the normal to the vitrified apple shoots in vitro, the MSAP bands (195) were divided into 3 groups: the methylated bands were unchanged (type A), the methylated bands were decreased (type B) and the methylated bands were increased (type C) and the methylated bans were uncertained. Type A had 39 bands, the frequency in the MSAP bands was 20.00%. Type B had 43 bands, and the ratio of type B in the MSAP bands was 22.05%. Type C had 93 bands, and the frequency of type C in the MSAP bands was 47.70%. At last, the type D which has 20 bands, the ratio of the type D in the methylated bands was 10.25%. Therefore, it seems to be possible that the increased of gene methylation is closely related to the vitrified process of apple shoots in vitro, whether in the structures or the physiology. |
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