硬叶兰种子的迁地共生萌发及有效共生真菌的分离和鉴定

doi:10.3724/SP.J.1258.2012.00859

摘要/Abstract

摘要：

兰科植物的种子原地和迁地共生萌发技术是近年发展起来的开展兰科植物种子和共生真菌研究的有效方法。该研究对兰属(Cymbidium)附生植物硬叶兰(C. mannii)开展了种子的迁地共生萌发研究, 试图获得其种子萌发的有效真菌。利用硬叶兰成年植株根部周围的树皮、苔藓、枯枝落叶、腐殖质等作为培养基质, 进行种子的共生培养。在培养133天后, 成功地获得了处于不同阶段的已萌发种子、原球茎和幼苗, 并从原球茎中分离得到一种瘤菌根菌属(Epulorhiza)真菌。用所分离到的FCb4菌株和一种从兜唇石斛(Dendrobium aphyllum)分离到的胶膜菌属(Tulasnella) FDaI7菌株和硬叶兰种子在燕麦琼脂培养基上进行共生萌发, 设置不接菌作为对照处理, 以检验FCb4菌株对硬叶兰种子萌发的有效性。经过58天的培养, 不接菌的对照处理中种子没有萌发, 接种FCb4和FDaI7菌株的处理都有很高的种子萌发率, 两种接菌处理在不同光照条件下的种子萌发率均无显著性差异。但暗培养条件下, 种子萌发形成原球茎后, 表现出生长停滞的趋势, 仅有很少的原球茎继续生长达到幼苗阶段, 说明原球茎发育后期与幼苗发育阶段需要光照。在光照条件下, 接种FCb4菌株处理中达到幼苗阶段种子的比例为(25.67 ± 9.27)%, 显著高于接种FDaI7菌株处理的(3.04 ± 2.27)% (W = 56, p = 0.026, Mann-Whitney U-test), 表明此研究中分离到的瘤菌根菌属真菌能有效地促使硬叶兰种子萌发并生长发育到幼苗阶段。

关键词: 硬叶兰, 种子迁地共生萌发, 兰科植物保护, 原球茎, 共生真菌, 瘤菌根菌属

Abstract:

Aims The in situ and ex situ seed baiting techniques, which have been developed in recent years, are effective methods to study the compatible mycorrhizal fungi of orchids. Our aim was to obtain the compatible mycorrhizal fungi of epiphytic Cymbidium mannii using the ex situ seed baiting technique.
Methods Bark, moss, litter and humus around the roots of adult plants of C. mannii were collected as substrate to cultivate C. mannii seeds in the laboratory. The fungi were isolated from developed protocorms and identified by morphological and molecular characteristics. The effects of different mycorrhizal fungi and light on seed germination were examined by in vitro symbiotic germination.
Important findings The isolated fungus was identified as a species of the genus Epulorhiza and was named as FCb4. After 58 days cultivation, seeds inoculated with FCb4 strain and FDaI7 strain (Tulasnella, isolated from Dendrobium aphyllum) had a high germination ratio, whereas seeds without the fungus failed to germinate. There was no significant difference between the germination ratios of seeds inoculated with FCb4 and FDaI7 strains, but FCb4 was significantly superior for seedling formation and development compared with FDaI7 in light. This indicated a lower degree of symbiotic fungal specificity on seed germination than the protocorm development stage in C. mannii. The fungus we obtained was effective for seed germination, and protocorm and seedling development for C. mannii. Germination and protocorm production were higher in the dark (0/24 h light/dark) than the light condition (12/12 h light/dark), whereas the subsequent protocorm development was better with light. These findings will aid in seedling production and reintroduction of C. mannii.

Key words: Cymbidium manii, ex situ symbiotic seed germination, orchid conservation, protocorm, symbiotic fungi, Tulasnella

盛春玲, 李勇毅, 高江云. 硬叶兰种子的迁地共生萌发及有效共生真菌的分离和鉴定. 植物生态学报, 2012, 36(8): 859-869. DOI: 10.3724/SP.J.1258.2012.00859

SHENG Chun-Ling, LEE Yung-I, GAO Jiang-Yun. Ex situ symbiotic seed germination, isolation and identification of effective symbiotic fungus in Cymbidium mannii (Orchidaceae). Chinese Journal of Plant Ecology, 2012, 36(8): 859-869. DOI: 10.3724/SP.J.1258.2012.00859

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https://www.plant-ecology.com/CN/Y2012/V36/I8/859

图/表 5

表1 硬叶兰种子不同萌发阶段

Table 1 Different germination stages of Cymbidium mannii seeds

萌发阶段 Germination stage	描述 Description
0	未萌发的种子 Ungerminated seed
1	种胚膨大, 产生根状物(视为萌发) Enlarged embryo, production of rhizoid(s) (= germination)
2	种胚继续膨大, 突破种皮(形成原球茎) Continued embryo enlargement, rupture of testa ( = protocorm formation)
3	出现原生分生组织(原球茎发育阶段) Appearance of protomeristem (protocorm development)
4	长出第一片叶片及后续生长(幼苗发育初期) Emergence of first leaf and continue development (early stage of seedling development)

图1 硬叶兰种子迁地共生萌发133天后, 处于不同阶段的种子、原球茎和幼苗。A, 阶段0, 未萌发的种子。B, 阶段1, 种胚膨大, 产生根状物(视为萌发)。C, 阶段2, 种胚继续膨大, 突破种皮(形成原球茎)。D, 阶段3, 原球茎膨大, 出现原生分生组织。E, 阶段4, 幼苗。

Fig. 1 Seed, protocorm and seedling of Cymbidium mannii in different germination stages after 133 days of ex situ symbiotic seed germination. A, Stage 0, ungerminated seed. B, Stage 1, enlarged embryo, production of rhizoid(s) (= germination). C, Stage 2, continued embryo enlargement, rupture of testa (= protocorm formation). D, Stage 3, protocorm enlarges with appearance of protomeristem. E, Stage 4, seedling.

图2 硬叶兰种子及原球茎切片图。A, 未萌发的种子, 没有真菌侵染。B, 原球茎阶段, 菌丝团侵染原球茎基部。C, 原球茎形成第一片突起物(箭), 菌丝团侵染原球茎基部。D, 原球茎继续膨大, 形成顶端原生分生组织(箭), 此时菌丝团(箭头)较多。E, 图C的局部放大图, 箭所指为松散的菌丝团。F, 图D的局部放大图, 箭所指为被消解的菌丝团, 箭头所指为松散的菌丝团。

Fig. 2 Cross-section of seed and protocorm of Cymbidium mannii. A, Ungerminated seed without fungi infection. B, Protocorm with fungi infection on the base. C, Protocorm forms the first thrust (arrow), and fungi infect the base of protocorm. D, Protocorm enlarges with appearance of protomeristem (arrow) with lots of pelotons (arrowhead). E, Partial enlarged drawing of Fig. C, arrow indicates loosened pelotons. F, Partial enlarged drawing of Fig. D, arrow indicates digested pelotons, and arrowhead indicates loosened pelotons.

表2 在不同处理下培养58天后硬叶兰种子的萌发率、形成原球茎的比率, 以及处于不同阶段的已萌发种子、原球茎和幼苗的比率(平均值±标准误差)

Table 2 Seed germination percentage, protocorm ratio and the proportion of geminated seeds, protocorms and seedlings in different germination stages in different treatments after 58 days cultivation for Cymbidium mannii (mean ± SE)

处理 Treatment	光照 Photoperiod (h Light/Dark )	重复 Replication	萌发率 Germination (%)	原球茎比率 Protocorm ratio (%)	不同阶段萌发的种子、原球茎或幼苗的比率 Proportion of geminated seeds, protocorms and seedlings in different stages (%)
处理 Treatment	光照 Photoperiod (h Light/Dark )	重复 Replication	萌发率 Germination (%)	原球茎比率 Protocorm ratio (%)	阶段1 Stage 1	阶段2 Stage 2	阶段3 Stage 3	阶段4 Stage 4
FCb4菌株 FCb4 strain	12/12	7	73.64 ± 6.14	60.0 ± 12.08	13.63 ± 9.95	5.05 ± 4.05	29.30 ± 8.82	25.67 ± 9.27
FCb4菌株 FCb4 strain	0/24	7	85.04 ± 5.15	83.89 ± 5.42	2.48 ± 1.06	17.7 ± 12.85	66.6 ± 13.05	0.35 ± 0.35
FDaI7菌株 FDaI7 strain	12/12	10	69.82 ± 3.80	45.11 ± 8.74	24.47 ± 7.94	28.56 ± 7.35	13.51 ± 6.75	3.04 ± 2.27
FDaI7菌株 FDaI7 strain	0/24	10	75.20 ± 5.49	66.13 ± 8.56	9.48 ± 6.43	13.38 ± 4.72	50.56 ± 8.88	2.42 ± 1.00
对照 CK	12/12	10	0	0	0	0	0	0
对照 CK	0/24	10	0	0	0	0	0	0

图3 培养58天后硬叶兰种子处于不同萌发阶段的比率(平均值±标准误差)。A, 接种FCb4菌株处理在不同光照条件下, 各阶段萌发的种子、原球茎和幼苗的比率。B, 接种FCb4和FDaI7菌株处理在光照条件下(12/12 h 光/暗), 各阶段萌发的种子、原球茎和幼苗的比率。*表示同一阶段不同处理之间差异显著(α = 0.05)。

Fig. 3 Proportion of Cymbidium mannii seeds in different germination stages after 58 days cultivation (mean ± SE). A, Proportion of geminated seeds, protocorms and seedlings in different germination stages in the treatment inoculated with FCb4 strain under different light conditions. B, Proportion of geminated seeds, protocorms and seedlings in different germination stages in the treatments inoculated with FCb4 and FDaI7 strains under the light condition (12/12 h light/dark). * indicates the significant difference between different treatments within the same stage (α = 0.05).

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