摘 要

为建立抗松材线虫病马尾松胚性细胞悬浮培养体系,本研究利用抗病马尾松未成熟胚诱导的胚性愈伤组织，通过定期对沉降细胞体积（SCV）、细胞密实体积（PCV）、细胞活力等进行测定，研究了初始接种量、继代周期、基本培养基种类、蔗糖浓度、植物激素2,4-D浓度对悬浮细胞体系的影响。主要结果如下：

（1）通过单因素试验筛选，研究4种不同初始接种量（0.5g/30mL、0.8g/30mL、1.0g/30mL、1.2g/30mL）对抗性马尾松胚性悬浮细胞生长的影响，确定了抗性马尾松胚性悬浮细胞初始接种量为0.8g/30mL-1.0g/30mL。

（2）通过测定不同继代周期对抗性马尾松胚性悬浮细胞生长的影响，经单因素试验筛选，确定了抗性马尾松悬浮培养最适宜的继代周期为7-9d。

（3）研究3种悬浮培养基本培养基LP、DCR、GD对抗性马尾松胚性悬浮细胞生长的影响，经单因素试验筛选，确定了抗性马尾松悬浮培养最适宜的基本培养基为LP和DCR。

（4）以蔗糖为碳源，通过研究碳源浓度对抗性马尾松胚性悬浮细胞生长的影响，确定了抗性马尾松悬浮培养最适宜的浓度为10g/L-20g/L。

（5）通过对2,4-D浓度对悬浮细胞生长的影响的研究，确定了抗性马尾松悬浮培养最适宜的2,4-D浓度为0.6mg/L。

研究结果为优化抗性马尾松胚性细胞稳定增殖体系及未来的规模化生产提供了技术支撑。

关键词：抗松材线虫病；马尾松；胚性细胞；悬浮培养

Establishment of cell suspension cultures in somatic embryogenesis of pine wilt disease-resistant Pinus massoniana Lamb.

Abstract

In order to establish the pines wilt disease-resistant Pinus massoniana Lamb. embryogenic cell suspension system, the study use the embryogenic tissue initiated from immature zygotic embryos in pine wilt disease-resistant Pinus massoniana Lamb. The effective of initial tissues weight, subculture cycle, basic medium, carbon source concentration, the type of carbon source, and hormone combination were investigated through measuring the sediment cell volume (SCV), packed cell volume (PCV) and cell viability during the establishment of the pines wilt disease-resistant Pinus massoniana Lamb. suspension culture. The main results were as follows:

1. The effects of 4 different initial inoculums concentration (0.5g/30mL,0.8g/30mL, 1.0g/30mL and 1.2g/30mL) on the growth of embryogenic suspension cells were studied by single factor experiment.And the experiment has determined the best initial tissues weight for the suspension culture was 0.8g/30mL-1.0g/30mL.
2. The effects of subculture cycle were studied by single factor experiment. And the experiment has determined the best subculture cycle for the suspension culture was 7d-9d.
3. With LP medium, DCR medium and GD medium as 3 different basic medium, the research was studied by single factor. And the experiment has determined the best basic medium for the suspension culture was LP medium and DCR medium.
4. Using sucrose as the carbon source, the effect of the concentration of carbon source on the growth of embryogenic suspension cells was studied, and the optimum concentration was 10g/L-20g/L.
5. The effect of 2,4-D concentration on the growth of suspension cells was studied. And the optimum concentration of 2,4-D in the suspension culture of resistant pine was 0.6mg/L.

The results provide technical support for the optimization of the control of the stability of the embryogenic cells of resistant pine embryos, and provide a feasible technology for the factory production of the resistant pine.

Key words: pines wilt disease-resistant Pinus massoniana Lamb. ; embryogenic cell ;suspension culture

目 录

1 前 言 - 1 -

2 文献综述 - 2 -

2.1 植物细胞悬浮培养的研究概况 - 2 -

2.2 植物细胞悬浮培养的影响因素 - 3 -

2.2.1 外植体的选择 - 3 -

2.2.2胚性愈伤组织的选择 - 3 -

2.2.3 适宜的培养条件 - 4 -

2.2.4 植物细胞悬浮培养存在的问题 - 4 -

3 材料与方法 - 6 -

3.1 试验材料 - 6 -

3.2 试验方法 - 6 -

3.2.1 抗性马尾松胚性悬浮培养初始接种量的测定 - 6 -

3.2.2 不同继代周期抗性马尾松胚性悬浮细胞生长状况测定 - 7 -

3.2.3 不同基本培养基对抗性马尾松悬浮细胞生长状况测定 - 7 -

3.2.4 不同浓度蔗糖抗性马尾松悬浮细胞生长状况测定 - 7 -

3.2.5 不同浓度的2,4-D对抗性马尾松悬浮细胞生长影响测定 - 7 -

4 结果与分析 - 9 -

4.1 抗性马尾松胚性细胞悬浮培养的初始接种量 - 9 -

4.2 不同继代周期对抗性马尾松悬浮细胞生长的影响 - 10 -

4.3 不同基本培养基对抗性马尾松悬浮细胞生长的影响 - 11 -

4.4 不同浓度蔗糖对抗性马尾松胚性悬浮细胞生长的影响 - 12 -

4.5 不同浓度的2,4-D对抗性马尾松胚性悬浮细胞生长的影响 - 13 -

5 结论与讨论 - 14 -

致 谢 - 16 -

参考文献 - 17 -

1 前 言

马尾松(Pinus massoniana Lamb.)属植物界，裸子植物门，松柏纲，松柏目，松科，松属。马尾松为常绿乔木，在我国分布十分广泛，从河南和山东以南到两广、台湾等都有分布，遍布于华中华南，一般在海拔600-700米以下的长江中下游分布。马尾松树干通直，是我国最主要的用材树种之一，也是荒山造林绿化的先锋树种。马尾松主要可以用作建筑材料，还含有丰富的树脂，应用十分广泛。

松材线虫病是由松材线虫（Bursaphelenchus xylophilus）引起的一种毁灭性的松树病害，松材线虫病极易传播，发病快，防治难。自1982年在南京中山陵发现以来，该病害就对我国的松林资源造成了极大的破坏和损失。虽然国内外的科学家采取了化学、物理、生物等多方面的防治方法，但总体成效并不是很明显。因此选育并推广抗松材线虫病的马尾松品种迫在眉睫。有研究表明，马尾松不同种源间和不同单株对松材线虫病的抗性差异显著，并且抗性稳定可靠。因此，培养和利用具有抗病性的马尾松是防治松材线虫病的有效途径之一。

目前在安徽已建立了马尾松抗松材线虫病候选个体保存园，并且现已收获了种子。但由于选育出的抗性马尾松初代结实率较低，种子少，自然繁殖较为困难，因此借助组织培养的方法，建立抗性优良的马尾松组织培养体系是批量培育抗性马尾松防治松材线虫病的可行途径。