一种有效地提高银杉人工菌根苗诱导率的方法

doi:10.17521/cjpe.2006.0043

摘要/Abstract

摘要：

银杉(Cathaya argyrophylla)是一种极其珍稀的濒危松科植物。该研究建立了一种有效的银杉人工菌根诱导技术。通过将银杉种子播种于琼脂培养基7 d后,切除1/5~1/3的种皮以及胚乳,从而使银杉种子的发芽率达到60%以上;同时筛选出既适合外生菌根菌土生空团菌(Cenococcum geophilum)平铺扩展生长的要求,又能满足银杉幼苗生长的培养基RM,使银杉的侧根能最大程度地与菌丝接触,确保了菌根的诱导率;通过紫外线照射加70%酒精或0.05%NaOCl的表面消毒的双重处理,使不能高压灭菌的容器达到表面消毒效果,同时验证了用该灭菌法既能达到完全的灭菌效果又不会对植物的生长产生不良影响,而且银杉小苗的菌根形成率达到87.5%。该方法对于银杉的菌根学研究以及其它基础研究具有实际应用价值。

关键词: 濒危植物, 种群复壮, 土生空团菌, 切割胚乳, 灭菌, 培养基

Abstract:

An effective in vitro ectomycorrhizal formation protocol of Cathaya argyrophylla, which is an endangered important tree belonging to the family Pinaceae, has been developed. Over 60% of the seeds germinated following 7 d on an agar medium that were treated by cutting and removing 1/5-1/3 of the seed coat and endosperms. A modified MS medium (MMS) containing 1/4 concentration of macro elements, 1/2 concentration of micro elements and full concentration of Fe salt and vitamins of the MS medium was used for seedling growth of C. argyophylla. An RM medium provided appropriate growth conditions for the hyphae of Cenococcum geophilum. The RM medium resulted not only in more rapid growth but also in progressively smoother fungal layers resulting in a thin homogeneous mycelia mat upon the medium surface.

The growth of C. argyophylla on the MMS medium was compared to growth on the RM medium. The results showed that seedlings of C. argyophylla also grew well on the RM medium. Therefore, the RM medium was selected for in vitro ectomycorrhizal formation between C. argyophylla and C. geophilum.

To solve the problem of sterilization of non_autoclavable containers, surface sterilization was achieved by 70% ethanol solution or 0.05% NaOCl solution followed by ultraviolet radiation. The results showed that all treated containers were sterilized and 87.5% of seedlings were mycorrhizal seedlings. This method will facilitate detailed studies on ectomycorrhizal development of C. argyrophylla and on ectomycorrhizal seedling applications.

Key words: Endangered species, Rejuvenate species, Cenococcum geophilum, Remove endosperm, Sterilization, Medium

邢树堂, 李玉花, 孙雪, 伦志明, 瓦里奥禄敏. 一种有效地提高银杉人工菌根苗诱导率的方法. 植物生态学报, 2006, 30(2): 323-329. DOI: 10.17521/cjpe.2006.0043

XING Shu_Tang, LI Yu_Hua, SUN Xue, LUN Zhi_Ming, VAARIO . AN EFFECTIVE IN VITRO PROTOCOL FOR INCREASING MYCORRHIZAL FORMATION RATE OF CATHAYA ARGROPHYLLA. Chinese Journal of Plant Ecology, 2006, 30(2): 323-329. DOI: 10.17521/cjpe.2006.0043

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链接本文: https://www.plant-ecology.com/CN/10.17521/cjpe.2006.0043

https://www.plant-ecology.com/CN/Y2006/V30/I2/323

图/表 7

图1 银杉种子的发芽率

Fig.1 The germination percentage of Cathaya argyrophylla

图2 银杉幼苗生长培养基筛选

Fig.2 Screening of basic medium for seedling growth of Cathaya argyrophylla

表1 WPM、SH、MMS培养基大量元素比较

Table 1 Comparing of macro nutritious elements of WPH、SH and MMS

大量元素 Macro elements	改良MS培养基 Modified MS medium MMS (mol·L^-1)	SH培养基 SH medium SH(mol·L^-1)	WPM培养基 WPM medium WPM(mol·L^-1)
N	1.49×10^-2	2.5×10^-1	1.48×10^-2
P	3.125×10^-4	2.61×10^-3	1.25×10^-3
K	4.7×10^-3	2.5×10^-1	1.265×10^-2
S	3.76×10^-4	1.6×10^-3	7.2×10^-3
Ca	5.6×10^-4	1.4×10^-3	3.05×10^-3
Mg	3.76×10^-4	1.6×10^-3	1.5×10^-3

图3 不同培养基上的土生空团菌菌落生长情况重复5次平均值±SD

Fig.3 Colony growth rate of Cenococcum geophilum on different media Mean values of 5 replicates±SD

图4 银杉幼苗在RM与MMS培养基上8周后生长情况的比较

Fig.4 Comparison of the growth status of Cathaya argyrophylla on RM and MMS after 8 weeks of incubation

表2 容器非高压灭菌法的灭菌效果以及其方法对于赤松种子发芽的影响

Table 2 The effects of non_autoclaved sterilization methods on surface sterilization on culture containers and germination rate of Pinus densiflore

处理 Treatments	容器污染状态 Contamination of containers		播种 Seeding		发芽率 Germination (%)
处理 Treatments	第一周 Week 1	第两周 Week 2	第三周 Week 3	第五周 Week 5	第五周 Week 5	第十周 Week 10
对照1 Control 1¹⁾	----+	_++++	+++++	+++++	NS	NS
对照2 Control 2²⁾	-----	-----	-----	-----	S	90.0
0.05% NaOCl	-----	-----	-----	-----	S	80.0
0.1% NaOCl	-----	-----	-----	-----	S	70.0
0.5% NaOCl	-----	-----	-----	-----	S	50.0
1.0% NaOCl	-----	-----	-----	-----	S	40.0
70%酒精 70% ethanol	-----	-----	-----	-----	S	90.0

图5 银杉-土生空团菌在非高压消毒容器中的菌根诱导状态

Fig.5 In vitro ectomycorrhization between Cathaya argyophylla and Cenococcum geophilum in non_autoclaving sterilized container

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