星蕨体外快繁技术

doi:10.11983/CBB24190

摘要/Abstract

摘要： 为加强野生蕨类植物的保护与开发, 优化星蕨(Microsorum punctatum)孢子萌发方法和条件, 比较分析了不同因素对原叶体增殖、孢子体诱导、绿色球状小体(GGBs)诱导及其发育为幼孢子体的影响, 建立人工高效快繁技术体系。以成熟孢子为材料, 分别以MS、1/2MS、1/3MS和1/4MS为基本培养基在无菌条件下萌发; 通过L₉(3⁴)正交试验, 研究无机盐浓度、植物生长调节剂及其质量浓度对原叶体发生和增殖的影响。当原叶体增殖到一定数量时, 再以MS、1/2MS、1/3MS和1/4MS为基本培养基筛选适宜诱导孢子体的培养基; 随后, 以幼孢子体为材料诱导GGBs发育为新的幼孢子体并炼苗移栽。适宜孢子萌发的培养基为1/2MS, 原叶体在MS+0.3 mg·L^-16-BA+1.5 mg·L^-1 NAA培养基中大量增殖, 60天后增殖系数约为9.6; 将原叶体切割后接入1/4MS培养基, 加无菌水培养90天后, 幼孢子体发生系数约为10.0; 幼孢子体在1/2MS+1.5 mg·L^-16-BA+0.1 mg·L^-1 NAA培养基中可诱导出GGBs, 诱导率达93.3%, GGBs在此培养基中的增殖系数达32.0; 在1/2MS培养基中GGBs的分化成苗率较高, 最高约为92%; 试管苗经炼苗移栽成活率在90%以上。该研究建立了原叶体-受精-孢子体和幼孢子体-GGBs-幼孢子体2个技术体系, 尤其是GGBs的产生, 极大缩短了植株的再生周期。研究结果可为优质种苗及其它蕨类植物的人工繁育提供技术支撑。

关键词: 星蕨, 孢子萌发, 原叶体, 幼孢子体, 绿色球状小体

Abstract: INTRODUCTION: The wild populations of Microsorum punctatum face endangerment due to habitat degradation and low spore reproductive efficiency. Fern life cycles involve alternating gametophyte and sporophyte generations, where gametophyte development and sporophyte transition represent critical bottlenecks in in vitro propagation, heavily influenced by environmental factors and culture conditions. Although asexual propagation techniques such as green globular bodies (GGBs) have been successfully applied in some fern species, low sporophyte induction efficiency and proliferation challenges persist, hindering large-scale production. This study employed M. punctatum spores to systematically investigate sterile germination mechanisms, gametophyte proliferation, and sporophyte regeneration. A dual-pathway rapid propagation system was established, integrating high-efficiency prothallus proliferation with GGBs induction, aiming to provide both theoretical insights and practical solutions for conserving endangered fern resources and advancing industrial-scale cultivation.
RATIONALE: The unique alternation of generations life cycle in ferns, characterized by independent gametophyte survival, provides a theoretical framework for in vitro propagation. Studies have demonstrated that gametophyte homogenization culture and GGBs induction can overcome sporophyte regeneration barriers, while medium composition and phytohormone ratios critically regulate developmental phase transitions. To address the challenges of low spore propagation efficiency and habitat sensitivity in M. punctatum, this study leverages its gametophyte proliferation potential and rhizome meristematic activity in sporophytes. By optimizing aseptic systems and induction conditions, as well as mimicking the natural fertilization microenvironment, a dual-path regeneration system integrating prothallus proliferation and GGB-based propagation was established, laying a theoretical foundation for efficient conservation of endangered ferns.
RESULTS: Spore germination was optimally achieved in 1/2MS medium. Prothalli exhibited vigorous proliferation in MS medium supplemented with 0.3 mg·L^-1 6-BA and 1.5 mg·L^-1 NAA, reaching a proliferation coefficient of 9.6 after 60 days of culture. Fragmented prothalli transferred to 1/4MS medium with sterile water supplementation achieved a young sporophyte induction coefficient of 10.0 following 90 day cultivation. GGBs were successfully induced from young sporophytes in 1/2MS medium containing 1.5 mg·L^-1 6-BA and 0.1 mg·L^-1 NAA, showing 93.3% induction efficiency and a remarkable proliferation coefficient of 32.0. The GGB differentiation into plantlets was most efficient in 1/2MS medium, yielding a conversion rate of 92%. Acclimatized plantlets demonstrated over 90% survival rate post-transplantation.
CONCLUSION: This study successfully established an efficient in vitro rapid propagation system for M. punctatum spores. Optimization of sterilization duration and culture medium types significantly enhanced spore germination rates. A prothallus culture protocol with a high proliferation coefficient was developed, overcoming bottlenecks in gametophyte mass propagation. Liquid immersion-assisted fertilization technology enabled efficient induction of young sporophytes, while the GGBs induction system markedly shortened the regeneration cycle. For the first time, a dual-pathway rapid propagation strategy—“prothallus proliferation-sporophyte induction” combined with “GGBs cyclic regeneration” was proposed. The study demonstrated that the meristematic properties of M. punctatum GGBs are distinct from callus tissue, providing a robust technical framework for the conservation of endangered ferns and industrial-scale seedling production.

Formation of antheridia, archegonia, and sporophyte production in Microsorum punctatum

Key words: Microsorum punctatum, spore germination, prothallus, young sporophyte, green globular bodies

葛晓青, 李梦瑶, 黄衡宇, 张爱丽. 星蕨体外快繁技术. 植物学报, 2025, 60(6): 944-956.

Xiaoqing Ge, Mengyao Li, Hengyu Huang, Aili Zhang. Rapid Propagation Technology of Microsorum punctatum in Vitro. Chinese Bulletin of Botany, 2025, 60(6): 944-956.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24190

https://www.chinbullbotany.com/CN/Y2025/V60/I6/944

图/表 10

表1 原叶体增殖L9(34)正交试验

Table 1 The orthogonal design L9(34) of prothallus proliferation

Levels	Factors
Levels	A (basic medium)	B (mg·L^-16-BA)	C (mg·L^-1 NAA)
1	MS	0.3	0.5
2	1/2MS	0.6	1.0
3	1/4MS	1.2	1.5

表2 孢子萌发及其生长状况

Table 2 Spore germination and growth status

Basic medium	Disinfection time (min)	Contamination rate (%)	Growth situation
MS	5	100.00±0.00 a	The spore germination time was second only to 1/2MS, and the green flaky bodies were partially aggregated on the surface of the medium, and some were sparsely distributed or even blank
	8	74.67±0.04 b
	10	55.33±0.02 c
	12	32.00±0.02 d
1/2MS	5	100.00±0.00 a	The flaky bodies appeared first and were widely distributed on the surface of the medium. Some of them gathered into pieces, which were green in color and stretched out in shape
	8	74.67±0.04 b
	10	50.67±0.02 c
	12	22.00±0.03 d
1/3MS	5	100.00±0.00 a	The flaky bodies were sparsely distributed on the surface of the medium
	8	80.67±0.04 b
	10	50.67±0.04 c
	12	18.67±0.02 d
1/4MS	5	100.00±0.00 a	Green spots appeared at the latest, and the flakes were scattered on the surface of the me- dium
	8	72.00±0.11 b
	10	53.33±0.05 b
	12	21.33±0.05 c

图1 孢子萌发过程 (A) 20天后无菌体系建立(bar=2 cm); (B) 30天后孢子萌发(bar=2 cm); (C) 45天后小团状片状体形成(bar=2 cm); (D) 70天后原叶体形成(bar=2 cm); (E) 孢子萌发(bar=50 μm); (F) 丝状体时期(bar=100 μm); (G) 早期片状体时期(bar=100 μm); (H) 心形原叶体时期(bar=50 μm)

Figure 1 Spore germination process (A) Establishment of aseptic system (20 days) (bar=2 cm); (B) Germination of fertile spores (30 days) (bar=2 cm); (C) Prothallus (45 days) (bar=2 cm); (D) Prothallus (70 days) (bar=2 cm); (E) Spore germination (bar=50 μm); (F) Filamentous period (bar=100 μm); (G) Early lamellar period (bar=100 μm); (H) Cardioprothallus stage (bar=50 μm)

表3 原叶体增殖L9(34)正交试验结果

Table 3 The orthogonal design L9(34) result of prothallus proliferation

Number		Factors				Multiplication coefficient
Number		A (basic medium)	B (6-BA)	C (NAA)	D (error)	Multiplication coefficient
1		1	1	1	1	8.73±0.07
2		1	2	2	2	8.33±0.07
3		1	3	3	3	9.60±0.12
4		2	1	2	3	7.53±0.18
5		2	2	3	1	6.47±0.07
6		2	3	1	2	6.80±0.23
7		3	1	3	2	6.60±0.12
8		3	2	1	3	7.00±0.12
9		3	3	2	1	6.00±0.12
Multiplication coefficient	K₁	8.89	7.62	7.51	7.07
	K₂	6.93	7.29	7.29	7.24
	K₃	6.53	7.56	7.56	7.04
	R	2.36	0.33	0.27	0.20

图2 原叶体增殖和幼孢子体诱导 (A) 转接15天后的原叶体团(bar=2 cm); (B) 转接30天后的原叶体团(bar=2 cm); (C) 转接45天后的原叶体团(bar=2 cm); (D) 转接60天后的原叶体团(bar=2 cm); (E) 原叶体(bar=500 μm); (F) 精子器(bar=40 μm); (G) 局部颈卵器(bar=200 μm); (H) 颈卵器(bar=40 μm); (I) 加水培养20天后的原叶体和幼孢子体(bar=2 cm); (J) 加水培养60天后的原叶体和幼孢子体(bar=2 cm); (K) 加水培养90天后的原叶体和幼孢子体(bar=2 cm); (L) 孢子体生根培养(bar=2 cm)

Figure 2 Proliferation of prothallus and induction of young sporophytes (A) Prolobular mass was transferred after 15 days (bar=2 cm); (B) Prolobular mass was transferred after 30 days (bar=2 cm); (C) Prolobular mass was transferred after 45 days (bar=2 cm); (D) Prolobular mass was transferred after 60 days (bar=2 cm); (E) Gametophyte (bar=500 μm); (F) Antheridium (bar=40 μm); (G) Local archegonium (bar=200 μm); (H) Archegonium (bar=40 μm); (I) Prothallus and young sporophyte cultured with water after 20 days (bar=2 cm); (J) Prothallus and young sporophyte cultured with water after 60 days (bar=2 cm); (K) Prothallus and young sporophyte cultured with water after 90 days (bar=2 cm); (L) Sporophyte rooting culture (bar=2 cm)

表4 不同培养基类型对原叶体重量增加的影响

Table 4 Effect of different culture media on weight increase of prothallus

Basic medium	Fresh weight before multiplication (g)	Fresh weight after multiplication (g)	Multiplication weight (g)	Multiplication coefficient
MS	3.92±0.54 a	37.69±0.77 a	33.77±0.47 a	9.81±0.62 a
1/2MS	4.17±0.61 a	30.72±0.98 b	26.55±0.83 b	6.36±0.90 b
1/4MS	4.03±0.77 a	28.01±0.82 c	23.98±0.37 c	5.95±0.56 c

图3 不同无机盐浓度对孢子体诱导及绿色球状小体(GGBs)发育成苗率的影响不同小写字母表示差异显著(P<0.05)。

Figure 3 Effects of different inorganic salt concentrations on sporophyte induction and plantlet rate of green globular bodies (GGBs) development Different lowercase letters indicate significant differences at the 0.05 level.

表5 不同植物生长调节剂浓度对绿色球状小体(GGBs)诱导和增殖的影响

Table 5 Effects of different plant growth regulators concentrations on green globular bodies (GGBs) induction and proliferation

No.	Plant growth regulators		Induction rate (%)	Multiplication coefficient
No.	6-BA (mg·L^-1)	NAA (mg·L^-1)	Induction rate (%)	Multiplication coefficient
CK	0	0	21.33±0.03 g	2.63±1.43 d
R1	0.5	0.1	76.67±0.03 cd	18.73±3.40 c
R2	0.5	0.6	64.67±0.08 de	20.60±3.64 bc
R3	0.5	1.2	49.33±0.01 f	19.40±2.50 bc
R4	1.0	0.1	80.00±0.04 bc	19.87±1.65 bc
R5	1.0	0.6	70.67±0.05 cde	13.73±2.32 c
R6	1.0	1.2	63.33±0.05 e	19.13±1.92 bc
R7	1.5	0.1	93.33±0.03 a	32.00±3.30 a
R8	1.5	0.6	89.33±0.03 ab	28.20±2.66 ab
R9	1.5	1.2	65.33±0.04 de	16.60±3.17 c

图4 对星蕨孢子体绿色球状小体的诱导及绿色球状小体(GGB)植株的再生与驯化 (A)以孢子体为材料诱导GGBs (bar=1 cm); (B) 孢子体长出绿色球状小体(bar=1 mm); (C) 根状茎分离出绿色球状小体(bar=1 mm); (D), (E) 绿色球体培育成的GGBs聚合体((D) bar=1 mm; (E) (bar=1 cm); (F) GGBs聚合体中分离出单个GGB (bar=500 μm); (G), (H) GGBs分化长出幼孢子体((G) bar=5 mm; (H) bar=1 mm); (I) 孢子体根状茎长出GGB (bar=1 cm); (J) GGB增殖(bar=1 cm); (K) GGB分化成苗(bar=1 cm); (L) 培养60天后孢子体生根(bar=1 cm); (M)-(P) 孢子体驯化((M) bar=5 cm; (N)-(P) bars=3 cm)。因孢子体驯化生长空间有限(M), 后续将其转入面积较大的隔间驯化(N)-(P)。

Figure 4 Green globular induction of sporophytes and regeneration and domestication of green globular body (GGB) plants of Microsorum punctatum (A) GGBs induced by sporophyte (bar=1 cm); (B) Spore grows green globular bodies (bar=1 mm); (C) Rhizomes isolated green globules (bar=1 mm); (D), (E) GGBs polymer cultivated from e-green spheroids ((D) bar=1 mm; (E) bar=1 cm); (F) A single GGB was isolated from GGBs polymer (bar=500 μm); (G), (H) GGBs differentiated into young sporophytes ((G) bar=5 mm; (H) bar=1 mm); (I) Sporophyte rhizomes grow GGB (bar=1 cm); (J) GGB multiplication (bar=1 cm); (K) GGB differentiate into plantlets (bar=1 cm); (L) The sporophyte took root after 60 days of culture (bar=1 cm); (M)-(P) Sporophytic domestication ((M) bar=5 cm; (N)-(P) bars=3 cm). Due to limited space for the domestication and growth of spores (M), they were later transferred to larger compartments for domestication (N)-(P).

图5 星蕨体外再生过程 GGBs: 绿色球状小体

Figure 5 Regeneration process of Microsorum punctatum in vitro GGBs: Green globular bodies

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