Cytological And Molecular Biology In Relation To The Growing Habit Of Columnar Apple

The tree form improvement of apple is one of important targets of molecular breeding. The growing habit can directly affect the tree form, flowering and fruit-setting ability. Columnar apple is believed to be an ideal variety resource for high density planting due to its particular growing habit such as short internodes, few branching and high bud burst rate. In this paper, the anatomical structure and chloroplast ultra structure of columnar and standard apples were studied using paraffin section method, scanning electronic microscope and transmission electronic microscope. The sensitivity to different hormones and physiological differences between columnar and standard apples were also evaluated. The DELLA protein genes related to gibberellins signaling transduction pathway were cloned using RACE and the expression patterns were showed by real time PCR. The results were as follows:1. The size of palisade tissue cells within the same layer of columnar apple was different and its array was irregular. The cells of different layers were intervened each other, and the border of different layers was not clear. The size of palisade tissue cells within the same layer of standard apple was equal and its array was quite regular and close together. The border of different layers was obvious to distinguish. The leaf total thickness, palisade tissue thickness and the ratio of palisade tissue thickness and spongy tissue thickness of columnar apple were obviously higher than those of standard apple. There were more wrinkles on the surface of the upper epidermal cells of columnar apple and the bulges of epidermal cells were higher compared with standard apple. There were no obvious difference for the length of stomata between columnar apple and standard apple, but the stomata density of columnar apple was obviously higher and its stomata width was shorter than those of standard apple. The grana or stroma lamellae of chloroplast of columnar apple were not parallel to the long axis of the chloroplast, grana lamellae were arrayed irregularly and the number of chloroplast lamellae was small. Whereas grana or stroma lamellae of standard apple were parallel to the long axis of the chloroplast, grana lamellae were arrayed regular and closely and the number of chloroplast lamellae were relatively more than those of columnar apple.2. According to the sensitivity to GA3, BR and IA A, the columnar apple was neither hormone defective nor hormone insensitive type. The new shoots were mostly sensitive to the spray of GA3 at 150 mg/L. Spray of BR at different concentrations could promote shoot growth of columnar apple, whereas IAA could inhabit the growth of the new shoots. During the period of same growing season, there were no obviously differences of the absolute contents of GA3, ZT, IAA and ABA and the ratios of GA3/ABA, ZT/ABA, IAA/ABA between columnar and standard apples.3. Exogenous GA3 could promote the activities of a amylase and POD, and inhibit the activity of SOD in the internodes of columnar apple. After spraying GA3 at the early stage the contents of GA3, ZT and IAA in the internodes of columnar apple showed a changing tendency as being decreased firstly, being increased then and being decreased finally. But the changing tendency of ABA contents was different, that is, being increased firstly, being decreased then and being increased finally. Higher concentration of GA3 (150mg/L or 200mg/L) could obviously promote the lengthening of the new shoots and was associated with the higher ratios of GA3/ABA, ZT/ABA and IAA/ABA.4. Full-length sequences of two genes encoding DELLA proteins named MdGAI1 and MdGAI2 were obtained from columnar apple "Lujia No.5" by means of RACE. The full length sequence of MdGAI1 was 2489bp, encoding 635 amino acids, and the full length sequence of MdGAI2 was 2274bp, encoding 639 amino acids. Their nucleic acid sequences and amino acid sequences were highly homologous with those of other plants. Sequence analysis of the products of the MdGAI1 and MdGAI2 indicated that they shared a highly conserved N terminus that contained two recognizable motifs DELLA and VHYNP, and a highly conserved C terminus that contained two recognizable motifs VHVID and SAW.5. Real-time quantitative PCR analysis showed that MdGAIl was moderately expressed in shoot apices of columnar and standard apples during the spring, summer and autumn growing season. In the same season, the MdGAI1 of three cultivars of columnar apple were expressed at higher level than that of three cultivars of standard apple. In the early spring, the MdGAI1 are expressed at higher levels in internodes of three cultivars of columnar apple compared with that of standard apple. MdGAI1 was moderately expressed in different tissues and organs at various developmental stages of columnar apple "Lujia No.5", with highest level in early stage of fruit development, the relative expression was 1.55, twice more than that of germinating seeds (0.61). After seeds germination and flower blooming, the relative levels of MdGAI1 gene expression were significantly decreased. During the whole growing season, the MdGAI1 gene of shoot apices were expressed stably.