山核桃良种快繁关键技术及其产业化

本项目为森林培育学、经济林学领域的基础研究及应用基础研究。项目围绕浙江特色干果山核桃良种快繁，开展嫁接成活机理、体细胞胚胎发生等基础研究，形成了良种快繁关键技术体系，实现了山核桃良种的产业化。主要研究内容如下：1、山核桃良种快繁关键技术及其产业化研究。开展了山核桃采穗圃营建、砧木筛选、异砧嫁接、大苗培育等技术的研究，集成了嫁接苗产业化技术体系 山核桃嫁接产业化生产采用嫁接成活率高、愈合好的湖南山核桃砧木，嫁接成活率达到92.66%，接株当年生长量超过90 cm，造林保存率高达93.78%，缓苗期短、生长量大。系统研究了苗期营养动态变化，提出了营养快速诊断等大苗培育技术。3、山核桃不定芽再生和体胚发生技术研究。研究了胚龄、基本培养基、植物生长调节物质（BA、TDZ、2，4-D、picloram、NAA、IBA）种类和浓度对幼胚萌发、不定芽增殖、生根，胚性愈伤组织诱导、体胚发生、萌发等的影响，建立了不定芽组培再生体系和体胚发生体系，研发了不定芽的二步生根培养法，发现体胚发生经历了类似于合子胚的发育过程，即球形胚、心形胚、鱼雷胚以及子叶胚。3、山核桃嫁接成活机理研究。采用扫描电镜和免疫金定位技术研究了嫁接愈合过程，发现嫁接愈合过程中嫁接面愈伤组织细胞IAA的免疫反应特异性强，明确了砧穗质体中束缚态IAA的释放是启动砧穗愈合的主导因子IAA启动愈伤反应，形成愈伤组织，并进一步分化形成维管组织 用RACE和cDNA-AFLP技术得到山核桃嫁接过程中的20个差异TDFs，克隆到水通道蛋白基因CcPIP和生长素响应基因CcARF，分析了其在嫁接成活过程中的表达情况及其调控网络，从形态、生理生化和分子水平揭示了山核桃嫁接成活机理。

This project is a basic and applied basic research project in the disciplines of silviculture and economic forestry. Centering on the rapid propagation of improved varieties of Chinese hickory (Carya cathayensis), a specialty dry fruit in Zhejiang Province, this project carried out basic studies including the grafting survival mechanism and somatic embryogenesis, established a key technical system for rapid propagation of improved varieties, and realized the industrialization of Chinese hickory improved varieties. The main research contents are as follows:
1. Research on key technologies for rapid propagation of Chinese hickory improved varieties and their industrialization. We conducted studies on technologies including the establishment of Chinese hickory scion orchards, rootstock screening, heterologous rootstock grafting and large seedling cultivation, and integrated an industrial technical system for grafted seedlings. The industrialized grafting production of Chinese hickory uses the rootstock of Carya hunanensis, which features high grafting survival rate and good healing, achieving a grafting survival rate of 92.66%. The annual growth of grafted plants exceeds 90 cm, the afforestation survival rate is as high as 93.78%, with a short post-transplantation lag phase and robust growth. We systematically studied the dynamic changes of nutrient content during the seedling stage, and proposed large seedling cultivation technologies such as rapid nutrient diagnosis.
2. Research on adventitious bud regeneration and somatic embryogenesis technologies of Chinese hickory. We studied the effects of embryo age, basic medium, types and concentrations of plant growth regulators (PGRs including BA, TDZ, 2,4-D, picloram, NAA and IBA) on young embryo germination, adventitious bud proliferation, rooting, embryogenic callus induction, somatic embryogenesis and germination. We established an adventitious bud tissue culture regeneration system and a somatic embryogenesis system, developed a two-step rooting culture method for adventitious buds, and found that somatic embryogenesis undergoes a developmental process similar to zygotic embryogenesis, namely globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledonary embryo.
3. Research on the grafting survival mechanism of Chinese hickory. We used scanning electron microscopy (SEM) and immune-gold localization technology to study the graft healing process, and found that the immune reaction of IAA in callus cells at the graft interface is highly specific during the graft healing process. It was clarified that the release of bound IAA in the plastids of scion and rootstock is the dominant factor initiating the healing of scion and rootstock. IAA initiates the callus reaction, forms callus tissue, and further differentiates into vascular tissue. We obtained 20 transcript-derived fragments (TDFs) during Chinese hickory grafting using RACE and cDNA-AFLP technologies, cloned the aquaporin gene CcPIP and auxin response gene CcARF, analyzed their expression patterns and regulatory networks during grafting survival, and revealed the grafting survival mechanism of Chinese hickory from the morphological, physiological, biochemical and molecular levels.