什么是PTC?
Plant Tissue Culture is
a collection of techniques used
to maintain or grow
plant cells, tissues or organs
under sterile conditions
on a nutrient culture medium
-Wikipedia
这是维基百科上关于植物组织培养(Plant Tissue Culture, PTC)的定义,其中值得注意的重点在于维持植物组织的生长发育,以及无菌条件的培养。除此以外,定义里面还强调了PTC是个很广的概念,其中包含了一系列培养方法和技术,如下图所示:1)Enbryo Culture,胚细胞培养;2)Organ Culture,植物器官培养;3)Callus Culture,愈伤组织培养;4)Cell Culture,体细胞培养。
作者眼中的PTC
就像我们可以把核酸提取这样一类分子生物学实验粗暴地划分成细胞裂解和核酸纯化两个步骤一样,作者认为植物组织培养也可以分为两个核心步骤,野生材料驯化和组织形态建成。这两个核心步骤看起来复杂,其实只要注意每一步操作的细节,是可以保证成功率的。下面我们就把每个步骤拆解开,简单分析一下。
野生材料的驯化
很多时候,进行植物组织培养的目的往往是出于对遗传转化受体的需要,从而进行不同条件的培养以提取代谢物进行差异分析,出于这样的前提,实验者往往只需借鉴文献里给出的培养条件或者做出特定改动,缺乏的只是植物材料而已。在大量培养植物材料的过程中,无菌培养是十分重要的,是这一类组织培养实验能否成功的关键,集中体现在无菌操作、外植体消毒等一系列与污染作斗争的操作上。
野生材料驯化的几点细节
1. 灭菌
通常,我们需要对使用的培养基、培养皿、镊子、解剖刀、去离子水和滤纸进行彻底的灭菌。其中,培养基、镊子、解剖刀、去离子水等液体和金属器皿通常采用121℃ 20-30min的湿热灭菌法;玻璃器皿常用180℃ 2h的干热灭菌法;而抗生素、激素等不耐高温的试剂常用过滤灭菌法。
2. 消毒
消毒是指外植体和操作台面的消毒,目的是在不伤害植物的前提下,抑制和杀死大部分微生物。其中,外植体消毒的方式主要分为表面消毒和侵入消毒两种,对于操作台面的消毒通常是指超净工作台使用前进行的的30min紫外灯消毒和操作过程中的空气滤过消毒,值得一提的是,在使用超净台之前用70 % 酒精擦拭台面会有一定效果。
对于大部分种子、陆生植物的茎叶等,表面消毒足以杀死可能会在组织培养过程中导致污染的微生物,比如使用70 % 乙醇、5 % 过氧化氢,均可以在短时间内有效杀死表面的微生物;但对于一些生长在潮湿阴暗环境中的植物来说,通常有着大量的组织内生菌,而这些内生菌往往不是表面消毒可以杀死的,一些侵入式消毒剂会比较有效,如使用升汞、次氯酸钠等。
3. 无菌操作
无菌操作即杜绝一切来自操作者的污染或因操作者失误导致的其他污染,一些常见的操作手法如下:
-设立单独的组培间,并设立与外界污染区的缓冲间,在缓冲间内更衣进入组培间;
-进入组培间前要在缓冲间内进行全身的酒精喷雾消毒,同时换上组培室专用拖鞋;
-操作过程中,佩戴手套或肢体不从一切开口器皿的上方经过;
-器皿不要遮盖超净台出风口,超净台不要开口太大;
-在超净台中点燃酒精灯,操作尽量在酒精灯火焰周围进行;
-使用的金属工具和玻璃器皿每进行一阶段的操作后及时进行灼烧灭菌;
-镊子、枪头等接触培养基的工具在接触到其他固态表面后立即灼烧灭菌或更换;
-做其他分子实验的超净台应与进行组织培养接种的超净台分开。
4. 富内生菌类型的野生材料的驯化
部分野生材料难以实现有效的消毒,即便是两种消毒方法复合使用也收效甚微,此时应使用试管苗培养的方法,大批量接种培养,经过两周后筛选未污染的植株再进行扩大培养,此方法可有效驯化野生植株获得无菌系,是一些苔藓植物常用这样的方法。注意,在培养过程中一旦发生污染,整个培养基的植株都应该丢弃。
如果与内生菌的斗争过于艰难,可以尝试使用组织培养抗菌剂PPM,这是一种广谱的抗菌剂,但是也会对某些植物有着抑制效果,建议在驯化获得无菌系之后停止使用。
组织形态建成
在有些时候,我们需要对一些从未进行过组织培养的植物进行人工的繁育,以适应实验需求,那么我们往往需要自己进行组织形态建成的探究。
正确地改变培养条件,是建成想要的组织形态的一种策略。
首先要说明的是,在更换培养基的时候,需彻底清除残余的培养基并将植株剪切或者其他方式进行处理后移到新的培养基上,否则残余的培养基将影响植株的状态。
那么有哪些培养条件可以进行人为的改变呢?
1. 光照的强度、光质与光照时间;
2. 培养基的无机成分、有机成分,尤其是生长调节剂;
3.培养基的PH值;
4. 培养基的一些添加剂;
5. 培养的温度、湿度、培养瓶通气性等。
1. 光照对植物组织的影响
当发生以下问题时,需要考虑光照因素:
诱导营养生长与生殖生长——光质、光周期,远红光通常可诱导生殖生长;
组织褐变率高——光强过强、光照时间过长;
叶片黄化或生长速度慢——光照不足。
2. 培养基成分的影响
植物组织培养的培养基通常需要考虑添加以下成分:
无机盐成分——其中,氨态氮、硝态氮的比例和组织的脱分化与再分化有关,Fe、Mg与叶绿素生成有关、Ca与愈伤组织生长有关,还有很多针对不同植物特殊需求而添加的元素,等等;
琼脂等固相支持物——与培养基的凝固能力有关,通常添加0.8 % – 1.2 %的琼脂即可满足实验需要;
蔗糖、葡萄糖等碳源——与组织的生长速度有关,通常不会影响组织的形态,一般添加0.5 % – 4 %,通常2 % 的蔗糖可供组织吸收一个月;
维生素、烟酸、肌醇等其他有机成分——可促进胚状体的形成,协助组织进行各项代谢。
3. 培养基PH的影响
培养基PH不适宜可能导致组织褐变和玻璃化,但不同植物对PH的敏感性不同,如苔藓植物对培养基PH的敏感性较低,而一些单子叶植物则对培养基PH十分敏感。
4. 活性炭、椰奶等添加剂
活性炭可吸收受伤的植物组织产生的酚类,同时保护易降解的植物激素,在容易发生褐变与玻璃化的组织培养驯化过程中,可以尝试在培养基中加入活性炭,一般1 g/L。
椰奶、洋葱、土豆泥、香蕉泥、胡萝卜汁等添加成分,其化学成分不完全清楚,但往往具有着一定的调节作用,诸如香蕉泥可以稳定PH有利于壮苗、土豆泥可以补充维生素有利于生根等。
5. 培养室的一些环境参数
培养室的环境尤其温度会对植物培养造成一定的影响,一般植物组织培养可使用23 – 27 ℃的温度区间。在一定范围内,湿度、通气性等条件通常不会对组织造成关键性的影响。
6. 特别地,植物生长调节剂在组织培养中的关键作用
植物生长调节剂可分为生长素类、细胞分裂素类、赤霉素、特殊调节剂类四种。
生长素类通常可以诱导植物组织生根、生芽,与细胞分裂素类搭配可以使高等植物向生芽、生根和保持愈伤组织状态生长,而低等的苔藓植物没有根茎叶的明显分化,其形态建成较为复杂。通常,IBA与NAA、IAA造成的效果相似但相对更强,2,4-D造成的效果与另外三种生长素有着显著不同。细胞分裂素可显著提高愈伤组织的生长速度,但对低等植物效果不明显。
特殊调节剂,如酚噻隆、PSK等,具有着强烈的调节效果但机理尚不明确,可在培养基中尝试添加。
References
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