【作者】 任鹏；

【导师】 郭素娟；

【作者基本信息】 北京林业大学 ， 森林培育， 2005， 硕士

【摘要】 板栗(Castanea mollissima BL.)是我国的名优特干果,品种丰富,抗性强,兼有经济、生态和社会三大效益。它的栗仁不仅营养价值高,而且还具有较高的药用价值和保健功能,深受国内外消费者喜爱。然而,目前我国板栗仍处于实生苗嫁接良种的繁殖阶段,这大大限制了我国板栗良种的大面积推广。因此,开展我国板栗离体培养技术的研究,不仅对促进我国板栗事业的发展具有重要的理论与实践意义,而且也将为我国板栗品种的遗传改良奠定良好的技术基础。 本文以燕山板栗为研究材料,分别选择了成年树腋芽、3月生播种苗茎段、下胚轴以及子叶四种外植体进行培养,系统地对无菌培养过程的各个环节进行了研究。结果表明: 1、初代培养 采集3月生播种苗枝条中部茎段(顶芽下3～5节),用0.1%升汞表面灭菌16～18min后,以直插或斜插的方式接种在WPM+BA1.0mg/L培养基上,能够快速获得板栗无菌体系;以下胚轴为外植体,用0.1%升汞表面灭菌13～15min后,以平卧的方式接种在MS+BA2.0mg/L的培养基上的处理,同样能够快速获得无菌体系。而以腋芽、子叶作为外植体进行培养,没有获得成功。褐化、污染和不能进一步正常萌发生长是影响腋芽培养成功的关键限制因子。 2、继代培养通过二次旋转回转正交实验得出,以WPM为基本培养基,当培养基中BA的浓度控制在1.25～1.53mg/L的范围内,IBA的浓度范围在0.08～0.15mg/L时,增殖度达到6.5以上的概率为95%。 板栗继代培养主要采用切段或分株的增殖方式,其他培养条件为蔗糖30g/L、pH值为5.8,继代间隔周期为40天。 3、壮苗培养 在生根诱导前,对长势弱,长度较短的部分微枝进行壮苗培养,以培养基WPM+BA0.3mg/L获得的微枝质量最好。 4、生根培养 继代次数和生根诱导方式是影响板栗试管内生根的关键因子。对经过多次继代培养的微枝(7～9代),通过采用直接生根法和间接生根法进行生根诱导,获得成功。采用直接生根法诱导,微枝容易发生茎尖坏死现象,生根率很低。但切除坏死的茎尖后,微枝却又能正常生根。不过,常伴随有愈伤组织的产生。采用间接生根法,在黑暗条件下,先将微枝接种于含有高浓度IBA(3mg/L)的培养基,进行生根诱导,然后转入1/4MS空白培养基,生根效果最佳,不仅愈伤组织小,而且根粗壮,有须根。更多还原

【Abstract】 The objective of the studies is to achieve a reliable protocol of propagating Chinese chestnut (Castanea mollissima BL.) in vitro. At the beginning of this paper, the development of the researches on Plant Tissue Culture (PTC) in the past was reviewed on the whole, mainly focused on those of the Wood Plant (WP). Then the advances of micropropagation of chestnut (Castanea Miller) in vitro both home and abroad were summarized in terms of explants, multiplication, and rooting. Meanwhile, three obstacles including Low Survival Rate (LSR), Vitrification, and Shoot-tip Necrosis (STN), which have happened in many researches before, were also presented.On basis of the reviews, a research on Chinese chestnut’s PTC was conducted at the Beijing Forestry University (BFU) from the beginning of 2003. Some achievements have already been made. By comparisions of four explants types, like chestnut axillary bud from 3-month-old seedlings and adult plant, hypocotyls, and cotyledon, using chestnut axillary bud from 3-month-old seedlings and hypocotyls were successfully regenerated and proliferated in vitro. After the subculture for around 2 years, Chinese chestnut had been successfully rooted in vitro. However, the in vitro culture of axillary bud from adult material and cotyledon were not able to be established till present. The details in these regards are as follows:(i) Selection and Disposal of Explants. Axillary buds from 3-month-old-seedlings were sterilized by a quick dip in 75% alcohol followed by immersion for 16 to 18 minutes in 0.1 HgCl containing a few drops of Tween 80. The basal culture medium for initiation was Wood Plant Medium (WPM) with growth regulator benzyladenine (BAP) at the concentration of 1.0 mg/L. The dispodal of hypocotyls was as same as that of axillary buds but immersion for 13 to 15 minutes, Murashige and Skoog’s medium with BAP at the concentration of 2.0mg/L was appropriate.(ii) Shoots-multiplication. The basal medium for shoot-multiplication was WPM. By quadratic regressive orthogonal device for the choice of hormone, the best combination and concentration was BAP (1.25 to 1.53mg/L) with indolebutyic (IBA, 0.08 to 0.15mg/L). Other conditions should be sugar (30mg/L), pH (5.8). The microcuttings were subcultured on a 6-week-long cycle.(iii) Shoots-elongation. The WPM with BAP (0.3mg/L) was employed in the process of shoots-elongation. It has been proved to be efficient.(iv) Rooting. After the subculture for 7 to 9 generations, the Chinese chestnut was rooted in vitro. There were two ways to induce roots. One was that the microcuttings were cultured on the 1/4MS with IBA (1mg/L), 40% rooting rate was made. Callus and shoot-tip necrosis were happened in this way. The other was that the microcuttings were cultured on 1/4MS with IBA (3mg/L) at dark condition for 5 days, and then transfer to the 1/4MS with auxin-free, some roots were obtained with higher quality.更多还原