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兰花诱变育种研究进展

花匠小妙招
2024-11-18 00:12

兰花是兰科（Orchidaceae）的总称，具有很高的观赏价值、药用价值、食用价值和/或文化价值，深受世界各国人们的喜爱。迄今，已鉴定出兰科植物27 801种，899属[1]，在英国皇家学会登录的集体杂种超过15万个。在我国，传统意义上的兰花是指兰科兰属（Cymbidium）植物，特别是其中的地生种类，也就是今天的国兰。兰花是中国传统十大名花之一，是世界著名的观赏植物，也是当今生物学领域研究生命和进化的理想模式植物。

新品种选育是兰花产业高质量发展的基础。兰花新品种选育的主要方法有引种驯化、选择育种、杂交育种、诱变育种和倍性育种等。诱变育种是指通过人为控制化学、物理因素诱导植物，使其发生遗传变异，从可遗传变异性状中挑选出有利目标性状，最终培育出新品种或种质资源的方法[2]，具有育种年限短、突变方向不确定、突变范围广、可以产生特殊变异等特点。自1979年KOZLOWSKA-KALISZ[3]使用γ射线辐照兰花以来，兰花诱变育种取得了巨大进步，获得了一批叶色叶型、花色花型改变以及抗性提升等特性的突变体。本文对兰花诱变育种研究进行综述，旨在明确兰花诱变育种现状和影响兰花诱变育种效果的因素，总结兰花诱变机理，找出进一步提高兰花诱变育种效率和效果的方法，为更好地利用诱变育种技术培育兰花新品种，深入阐明兰花诱变和进化机理提供参考。

1 兰花诱变方法

兰花诱变方法主要包括物理诱变、化学诱变、空间诱变，这3类诱变方法在兰花育种中均有研究报道，其中物理诱变的研究报道最多，成效最大。

1.1 物理诱变

物理诱变育种是指利用物理因素诱导动植物的遗传特性发生变异，再从变异群体中选择符合人们某种要求的单株或个体，进而培育成新的品种或种质的育种方法[4]。迄今，至少已在兰属、蝴蝶兰属、石斛兰属等13个属44种兰花中开展了物理诱变育种研究，获得突变体716个，培育出小兰屿蝴蝶兰飞兰等兰花新品种12个（表1），其中5个来自突变体库（The Mutant Varieties Database of the International Atomic Energy Agency, IAEA）。

表1 物理诱变在兰花育种中的应用

Tab. 1 Application of physical mutagenesis in orchid breeding

辐照源
Radiation source 种类
Species 辐射材料
Irradiated
material 辐射结果
Radiation result 参考文献
Reference γ射线春剑 Cym. longibracteatum 根状茎 RD50为20 Gy；再生植株主要表现为叶色失绿变淡，获得1个叶艺突变体 [5-6] γ射线寒兰 Cym. kanran 根状茎 LD50为80.30 Gy；再生植株叶片数减少、植株瘦弱低矮黄化、叶片黄化等 [7] γ射线墨兰 Cym. sinense 根状茎 RD50为10 Gy；低剂量（5 Gy）增加可溶性蛋白质含量和POD活性、促进根状茎的生长，高剂量（10 Gy以上）则抑制其生长甚至导致死亡 [8] γ射线春兰 Cym. goeringii 根状茎 LD50为20 Gy；高剂量（30 Gy以上）抑制增殖与分化，获得4株叶片边缘白化苗 [9] γ射线建兰 Cym. ensifolium
墨兰 Cym. sinense 幼芽 LD50为16~18 Gy；再生植株矮化变粗，叶片短小、扭曲等，获得矮小和叶片嵌有金色线条的变异单株 [10] γ射线春兰 Cym. goeringii 成熟植株产生斑缟艺、片缟艺、中斑艺、粉斑艺等叶艺突变体 [11] γ射线建兰 Cym. ensifolium
墨兰 Cym. sinense
蕙兰 Cym. faberi 成熟植株新叶生长趋缓，出现扭曲、旋转，叶色变浓；花葶缩短或加长、颜色变深；花朵出现并蒂花、多花等性状 [12] γ射线墨兰 Cym. sinense
建兰 Cym. ensifolium
杂交兰 Cym. hybird 成熟植株 LD50为10~20 Gy；出现叶片短小、叶片旋转、线艺、并蒂花、多花、葡匐茎等不同的变异类型 [13] γ射线春兰 Cym. goeringii
蕙兰 Cym. faberi
建兰 Cym. ensifolium
寒兰 Cym. kanran 成熟植株 LD50为5~20 Gy；辐照后导致叶片枯黄、死亡和抑制花芽分化 [14] γ射线冬凤兰 Cym. dayanum
竹叶兰 Arundina graminifolia
碧玉兰 Cym. lowianum
西藏虎头兰 Cym. tracyanum 组培苗冬凤兰、竹叶兰、碧玉兰和西藏虎头兰的LD50分别为20.72、26.31、29.88、41.04 Gy；出现植株矮化、变粗，叶变宽、叶尖分叉、叶上有淡绿斑、叶扭曲等变化 [15] γ射线兰属 Cymbidium －得到1个叶艺矮化新品种Dong-i [16] γ射线兰属 Cymbidium 类原球茎/
芽/植株类原球茎、芽和植株的LD50分别为35.0、41.0、83.1 Gy；出现叶艺、矮化、叶片变大等变异 [17] γ射线玉女兰 Cym. Yunv 类原球茎 LD50为60 Gy [18] γ射线兰属 Cymbidium 分生组织 8 Gy促进原球茎形成和生长，200 Gy完全抑制生长，700 Gy致死 [3] γ射线杂交兰 Cym. tracyanum × Cym. iridioides 组培苗得到2个叶艺突变体，其植株矮化，但根数不减，叶片数增多，气孔特征和染色体结构发生了变化 [19-20] γ射线杂交兰(Cym. sinense × Cym. goeringii) × Cym. spp. 类原球茎 RD50为40 Gy [21] γ射线杂交兰Cym. sinense × Cym. goeringii 根状茎基于植株高度的RD50为51.2 Gy，基于植株鲜重的RD50为48.0 Gy [22] γ射线杂交兰 Cym. sinense × Cym. goeringii 根状茎再生植株出现多种黄色线艺、黄色斑艺、叶色变淡、矮化、叶卷曲等变异 [23-24] γ射线杂交兰(Cym. sinense × Cym. goeringii) × Cym. spp. 类原球茎 LD50分别为1 h辐照16.1 Gy，4 h辐照23.6 Gy，8 h辐照37.9 Gy，16 h辐照37.9 Gy，24 h辐照40.0 Gy [25] γ射线蝴蝶兰 Phalaenopsis aphrodite 盆栽组培苗出现花瓣缺失、重瓣花等花型变异 [26] γ射线蝴蝶兰 Phal. aphrodite －辐照处理后植株叶长、叶宽改变，有2株提前开花 [27] γ射线蝴蝶兰 Phal. aphrodite 原球茎 LD50为50~68 Gy；低剂量辐射对原球茎生长影响不明显，高剂量辐照后其存活率、增殖系数和分化率均明显下降 [28,34] γ射线蝴蝶兰 Phal. amabilis 盆栽苗出现花型变异 [29] γ射线蝴蝶兰 Phal. amabilis 盆栽组培苗最佳辐射诱变剂量为15 Gy；植株和花梗明显矮化，叶片增厚，花期推迟且花量减少 [30] γ射线蝴蝶兰Phal. equestris 盆栽实生苗产生花瓣表型突变体，得到蝴蝶兰迅兰、飞兰、繁兰3个新品种 [31] γ射线蝴蝶兰 Phal. violacea 植株出现叶片变异，叶片颜色较深且狭长，厚度增加，茎基部出现侧枝 [32] γ射线蝴蝶兰属 Phalaenopsis 花粉 LD50为60~80 Gy；选育出1个株形、叶形、花形和花色变异优良株系 [33] γ射线蝴蝶兰属 Phalaenopsis 原球茎出现生长量减小、叶片增厚、叶近圆形的变异苗和金色叶缘的变异苗 [35] γ射线蝴蝶兰属 Phalaenopsis 原球茎/小苗出现较多的株型变异 [36] γ射线蝴蝶兰属 Phalaenopsis 盆栽组培苗辐照植株花期提前或延迟、花梗分枝增多、矮化、花型变异等 [37] γ射线石斛 Dendrobium Sonia 离体再生芽 LD50为15~30 Gy；单色光显著影响辐照后苗的成活率和生长；黄光和红光处理显著增加辐照后芽的鲜重、芽长度和叶绿素含量 [38] γ射线石斛 Den. Sonia 类原球茎出现花型、花色突变体，得到6个石斛新品种KeenaOval、KeenaRadiant、KeenaHiengDing、KeenaAhmadSobri、KeenaPearl、KeenaPastel [39⇓⇓⇓-43] γ射线石斛 Den. Sonia 类原球茎 LD50为43 Gy；低剂量辐照促进再生植株根、茎、叶的发育；高剂量辐照后，再生植株气孔显著变小 [44] γ射线石斛 Den. Sonia 幼苗 LD50为30~60 Gy；不同品种间LD50存在差异；辐照剂量对叶片宽度影响不明显，对株高、假鳞茎长、叶片长均有抑制作用，品种间有一定的差异 [45] γ射线石斛 Den. Sonia Kai 类原球茎再生植株花变大或变小，花色出现纯白色，出现畸形花等 [46] γ射线石斛 Den. candidum 种子种子LD50为62 Gy，与不同胶膜菌属菌株共生萌发的LD50为69 Gy和63 Gy；低剂量促进萌发、高剂量抑制萌发 [47] γ射线石斛 Den. Emma White 类原球茎 GR50为25.52 Gy；突变体植株出现花期提前、叶色变黄、叶片形态不对称和卵圆形到心形的变异 [48] γ射线石斛 Den. lasianthera 类原球茎 LD30为19.7697 Gy，LD50为67.3504 Gy；再生植株出现叶宽增加、叶片卷曲等变异 [49] γ射线石斛 Den. bigibbum 芽出现紫色叶片突变 [50] γ射线石斛 Den. lodoardi －得到1个叶艺新品种Hwancho [51] γ射线石斛 Den. lodoardi 植株突变植株株高增加，叶增宽，根数和根长减少，叶形改变，叶色改变等 [52] γ射线石斛 Den. officinale 原球茎 LD50为86. 4 Gy [53] γ射线石斛 Den. officinale 原球茎 LD50为67.23 Gy；变异苗出现茎分叉、叶片缺绿或白绿相间等现象，其倍性发生改变，出现非整倍体 [54] γ射线杂交石斛 Den. hybrid －得到1个叶艺新品种Seolhwa [51] γ射线兜兰 Paphiopedilum delenatii
兜兰 Paph. callosum 类原球茎/
芽/植株 Paph. delenatii类原球茎、芽、植株的LD50分别为20.0、23.7、38.0 Gy；Paph. callosum类原球茎、芽、植株的LD50分别为23.0、27.1、40.4 Gy [55] γ射线兜兰属 Paphiopedilum 种子/不定芽
/幼苗/小苗种子LD50为6.92 Gy；种子萌发、芽苗阶段的最佳辐照剂量分别为5 Gy和20 Gy；再生苗矮化、花叶、卷曲或裂叶等 [56] γ射线树兰 Epidendrum secundum 蒴果种子LD50为78.08 Gy；20 Gy促进种子萌发；增加辐照剂量对株高、叶长有显著抑制作用，叶片数次之 [57] γ射线蕾丽兰 Laelia autumnalis 原球茎 LD50为53 Gy，RD50为28 Gy；5 Gy促进幼苗生长，20~30 Gy促进叶绿素形成，高剂量抑制原球茎存活 [58] γ射线杂交卡特兰 Cattleya hybrid 类原球茎 LD50为20~60 Gy [59] γ射线紫花苞舌兰Spathoglottis
plicata
苞舌兰 Spath. kimballiana
苞舌兰 Spath. tomentosa 原球茎突变植株白化、叶色加深、叶分叉、分枝增多等 [60] γ射线紫花苞舌兰 Spath. plicata 幼苗 LD50为14.3 Gy；突变植株矮化、分蘖少、花序变短、花形改变、花香变浓等 [61] γ射线短足兰Brachypeza indusiata 类原球茎 20 Gy可提高移栽成活率，降低株高；剂量越高，植株越小 [62] γ射线文心兰 Oncidium lanceanum 类原球茎再生植株花出现突变 [63] γ射线指甲兰 Aerides crispa 原球茎 LD50为2 Gy；再生植株出现叶斑、叶尖变圆、株高增加、根变粗变长等变异 [64] γ射线白及 Bletilla striata 种子 LD50为150 Gy；叶绿素含量降低，植株矮化，出现叶艺 [65] γ射线香荚兰 Vanilla planifolia 离体再生芽 LD50为60 Gy；低剂量（20 Gy）促进新芽形成，高剂量（60~100 Gy）造成死亡；用ISSR评估γ射线辐照剂量方面未观察到聚类趋势 [66] 重离子束春兰 Cym. goeringii
寒兰 Cym. kanran 根状茎根状茎白化，再生植株出现叶艺 [67] 重离子束文心兰 Oncidium lanceanum 类原球茎再生植株出现叶片变窄、叶片分布不均匀等变异 [68] 重离子束君豪兰 Cym. Junhao 根状茎植株粗壮，矮小瘦弱或徒长，颜色变浅或加深，叶片挺立、叶数增多、叶宽增加、叶线艺、畸形等 [69] 重离子束小凤兰 Cym. Xiaofeng 根状茎 LD50为60 Gy；当辐照剂量超过60 Gy时，根状茎失去芽分化能力 [70] 重离子束玉女兰 Cym. Yunv 类原球茎 LD50为49 Gy；获得抗茎腐病突变体3个抗性系 [71] 重离子束石斛 Den. mirbellianum
石斛 Den. crumenatum 类原球茎 Den. mirbelianum的LD50为2.55 Gy；再生植株出现叶形变异、抗螨突变体；Den. crumenatum的再生植株中出现叶艺、花朵宽度增加、花梗变长等变异 [72⇓-74] 重离子束兜兰 Paph. delenatii
兜兰 Paph. callosum 类原球茎再生植株出现叶艺、叶片变大、叶片变窄、芽变大等变异 [55] 质子束杂交兰 Cym. hybrid 根状茎基于植株鲜重的RD50为35 Gy [22] 快中子蝴蝶兰火鸟 Doritaenopsis
Taisuco Firebird 原球茎 LD50为2.5×1011~3.5×1011·cm-2；过高注量（>3.5×1012·cm-2）的辐照使原球茎生长完全受到抑制甚至大量死亡 [75] 快中子蝴蝶兰火鸟 Dorit. Taisuco Firebird
蝴蝶兰内山姑娘 Dorit. Neyshanguniang 原球茎／
幼苗茎段火鸟品种原球茎和茎段的LD50分别为3.3×1011·cm-2和2.2×1011·cm-2；内山姑娘品种原球茎和茎段的LD50分别为4.1×1011·cm-2 和2.3×1011·cm-2 [76] 紫外线春兰 Cym. goeringii 原球茎再生植株出现部分植株变矮，叶片增宽增厚，少数叶片出现艺兰的形状 [77]注：-表示不详。Note：- indicates unknown.

用于兰花辐照的辐照源有γ射线[3,5⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓-66]、重离子束[55,67⇓⇓⇓⇓⇓⇓-74]、快中子[75-76]和紫外线[77]等。60Co-γ射线是最常用的辐照源，近年来，采用重离子束辐照逐渐增多，与γ射线辐射相比，碳重离子辐照的植株突变频率更高，突变谱更宽[78]。LUAN等[55]分别使用60Co-γ射线和12C6+重离子辐照2种蝴蝶兰，在60Co-γ射线辐照的2种蝴蝶兰中均未发现变异系，而在12C6+重离子（3 Gy）辐照的2种样本中筛选出24个变异系。

除了辐照源外，辐照材料、剂量和剂量率也是影响兰花辐照效果的重要因素。在兰花诱变育种中，多数研究人员将半致死剂量（LD50）作为最佳诱变剂量，采用的诱变材料多为原球茎、类原球茎和根状茎。不同种、品种、辐照材料的LD50不同，因此在兰花诱变育种中，确定辐照材料的LD50值十分重要。兰花根状茎辐照后，有时不同剂量处理后的材料均能存活，但生长停滞。因此，当无法用半致死量对辐射诱变效果进行评估时，有使用半减少剂量（the 50% reduction dose, RD50）作为最佳诱变剂量的报道[21,23]。

物理诱变引起兰花株型、株高、分蘖性，叶数、叶形、叶色、花序、花数、花型、花色、花期，抗病性、抗虫性、组培快繁特性等性状的变化（表1）。其中叶色突变出现的频率最高，表明通过物理诱变选育兰花叶色变异新品种的成功率高。

一般认为诱变育种对改变植物个别性状是有效的，但越来越多的研究结果表明，辐照能改变兰花的多个性状。主要原因是辐照能够引起染色体数目和结构变异，导致多个基因的改变，此外一因多效等也会导致多个性状发生改变。耿庆芝[69]采用重离子辐照君豪兰根状茎，获得了株型、叶数、叶色变异等多个突变体，其中叶艺突变体植株生长缓慢、植株矮小、适应性差（图1）。

图1 君豪兰及其重离子辐照处理叶艺突变体

A：君豪兰（左）及其重离子辐照处理叶艺突变体（右）叶片；B：君豪兰（左）及其重离子辐照处理叶艺突变体（右）植株。标尺=10 cm。

Fig. 1 Cym. Junhao and it’s mutant with leaf color and pattern from heavy ion irradiation

A: Cym. Junhao (left) and it’s mutant with leaf color and pattern from heavy ion irradiation (right) leaves; B: Cym. Junhao (left) and it’s mutant with leaf color and pattern from heavy ion irradiation (right) plants. Scale bar=10 cm.

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1.2 化学诱变

化学诱变是指利用化学药剂与遗传物质发生生物化学反应，引起基因发生点突变[2]，具有容易操作、剂量易控制、对基因组损伤小、突变率高等特点[79]。迄今，至少已在兰属、石斛兰属、蝴蝶兰属等6个属13个兰花种中开展了化学诱变育种研究。已有结果表明，化学诱变可导致兰花株高、叶数、叶色、叶宽、抗病性、抗逆性等性状的改变，至少已获得叶色改变、矮化、抗病、抗寒等突变体206个，其中叶色改变和矮化突变体是常见的变异类型（表2）。

表2 化学诱变在兰花育种中的应用

Tab. 2 Application of chemical mutagenesis in orchid breeding

诱变剂
Mutagen 种类
Species 诱变材料
Mutagenic-
material 诱变结果
Mutagenic result 参考文献
Reference EMS 春兰 Cym. goeringii 根状茎再生植株出现矮化、白化、网纹斑艺和线艺突变体 [82] EMS 寒兰 Cym. kanran 根状茎再生植株产生黄色线艺和白化植株等变异 [83] EMS 寒兰 Cym. kanran 根状茎 LD50为0.607% EMS处理1 d，0.237% EMS处理2 d，0.145% EMS处理3 d，0.119% EMS处理4 d [84] EMS 寒兰 Cym. kanran 原球茎 LD50为0.038% EMS处理6 d，0.018% EMS处理11 d，0.012% EMS处理16 d；再生植株叶色变淡，瘦弱低矮，白化 [80] EMS 君豪兰Cym. Junhao 根状茎 LD50为0.2% EMS处理4 d；再生植株出现颜色变浅、瘦弱、矮壮，叶宽增加、畸形、叶色加深等 [69] EMS 玉女兰 Cym. Yunv 类原球茎 LD50为0.3% EMS处理4 d [18] EMS 蝴蝶兰属 Phalaenopsis 花梗再生苗叶尖部出现锯齿，对持续低温抵抗能力高 [85] EMS 蝴蝶兰属 Phalaenopsis 类原球茎 LD50为0.4% EMS处理6~8 h；细胞体积增大，形状改变；较高浓度EMS处理后的细胞中出现微核等异常现象 [81] EMS 石斛 Den. friedericksianum 类原球茎 LD50为0.8% EMS处理90 min；再生植株白化，具白绿或黄绿嵌合叶片 [86] EMS 石斛 Den. officinale 原球茎 LD50为1.0% EMS处理6 h；获得抗炭疽病植株 [87] EMS 石斛 Den. Earsakul 类原球茎 LD50为1.8% EMS处理4 h；获得抗炭疽病植株 [88] EMS 石斛 Den. Sonia 类原球茎再生植株生长快，叶片增大，根系增粗，气孔密度降低 [89] EMS 文心兰金西 Onc. Kinsei 类原球茎 LD50为0.8% EMS处理4 d；类原球茎生长受到抑制，再生苗数量减少，出现多叶丛生变异植株 [90] EMS 白及 Ble. striata 愈伤组织 LD50为0.6% EMS处理6 h [91] EMS 指甲兰 Aerides crispa 原球茎 LD50为0.05% EMS处理5 d [64] NaN3 蝴蝶兰霍娅淑女
Phal. Tsuei Foa Lady 类原球茎 LD50为4~8 mmol/L NaN3处理6 h；出现白化苗 [92] NaN3 蝴蝶兰属 Phalaenopsis 类原球茎 LD50大约为5 mmol/L NaN3处理8 h。NaN3处理可使细胞体积增大、形状改变。高浓度NaN3处理细胞中出现双核等现象 [81] NaN3 石斛 Den. Earsakul 类原球茎 LD30和LD50分别为0.1、0.5 mmol/L NaN3处理1 h；得到抗黑腐病的再生植株，均为混倍体 [93⇓-95] NaN3 文心兰金西 Onc. Kinsei 类原球茎 LD50为3 mmol/L NaN3处理4 d，6 mmol/L NaN3处理2 d，12 mmol/L NaN3处理1 d；再生试管苗普遍矮小 [96] NaN3 白及 Ble. striata 愈伤组织 LD50为6 mmoL/L NaN3处理6 h [91] DES 寒兰 Cym. kanran 原球茎 LD50为0.040% DES处理6 d，0.026% DES处理11 d，0.014% DES处理16 d；再生植株叶片黄化或细长 [80] DES 铁皮石斛 Den. officinale 原球茎 LD50为0.3%~0.4% DES处理2 h；得到抗寒突变体 [97] 咖啡碱寒兰 Cym. kanran 原球茎 LD50为0.135%咖啡碱处理6 d，0.094%咖啡碱处理11 d，0.078%咖啡碱处理16 d [80] 5-BU 寒兰 Cym. kanran 原球茎 LD50为0.083% 5-BU处理6 d，0.068% 5-BU处理11 d，0.037% 5-BU处理16 d [80] MH 寒兰 Cym. kanran 原球茎 LD50为0.075% MH处理6 d，0.048% MH处理11 d，0.036% MH处理16 d [80] Na2SO3 寒兰 Cym. kanran 原球茎 LD50为10.46% Na2SO3处理6 d，9.41% Na2SO3处理11 d，7.58% Na2SO3处理16 d [80]

在兰花上使用的化学诱变剂有烷化剂[甲基磺酸乙酯（EMS），硫酸二乙脂（DES）]、呼吸抑制剂[叠氮化钠（NaN3）]、碱基类似物[马来酰肼（MH）]以及其他诱变剂如亚硫酸钠（Na2SO3）和咖啡碱等。其中烷化剂是最常用的化学诱变剂，占65.4%；其次是叠氮化钠，占19.2%（表2）。不同诱变剂对兰花的诱变效率不同。李夏[80]使用多种化学诱变剂对寒兰原球茎进行诱变处理，结果表明，诱变效率从高到低的排序为EMS>DES>MH>Na2SO3>咖啡碱>5-BU。陈超等[81]用EMS和NaN3对蝴蝶兰类原球茎（PLB）进行处理，结果发现EMS的诱变效果比NaN3好。

诱变剂浓度、诱变方法和材料选择是影响兰花化学诱变效率的主要因素。根状茎、原球茎和类原球茎等中间繁殖体是常用的诱变材料，浸泡法是最常用的处理方法。不同材料和不同诱变剂，其诱变剂量和处理时间不同，诱变效率也不同。对兰花中间繁殖体，EMS的常用浓度为0.05%~1.00%，NaN3浓度为2.0~8.0 mmol/L。EMS能在溶液中和细胞内部水解产生强酸，不仅使溶液中的EMS浓度降低，而且酸性水解产物对细胞有毒害作用，对处理材料造成生理损伤，降低存活率，从而降低了诱变效率[98]，因此EMS溶液一般用浓度不超过0.1 mol/L的磷酸缓冲液（pH 7.0）配制，但也有将EMS直接加入液体培养基进行诱变处理，并得到较多突变体的报道[82-83]。

1.3 空间诱变

空间诱变育种是指利用返回式卫星、宇宙飞船或高空气球将农作物种子带到太空，利用太空特殊的环境（空间宇宙射线、微重力、高真空、弱磁场等因素）诱发植物产生变异，再返回地面选育新品种的技术[99]，该技术具有突变谱广、诱变频率高和对植株损伤小等特点[100]。空间诱变至少已应用于石斛兰属和蝴蝶兰属2个属4个种中，已获得8个突变体、选育出4个新品种。空间诱变导致金钗石斛矮化、茎变粗、分蘖多；能促进铁皮石斛生长、提高产量；使蝴蝶兰花变大、花朵数变多、观赏期延长、抗病性抗逆性增强（表3）。石斛种子经过空间诱变选出了矮化新品种Uju[51]。陈肖英等[101]利用空间诱变技术，培育出花大色艳、抗性强和适应性好的优良蝴蝶兰新品种航蝴1号。

表3 空间诱变在兰花育种中的应用

Tab. 3 Application of space mutagenesis in orchid breeding

种类
Species 诱变材料
Mutagenic material 诱变结果
Mutagenic result 参考文献
Reference 蝴蝶兰 Phal. amabilis 丛生芽块组织获得花变大，花朵数量增加，花期延长，耐热性和耐寒性增强以及抗病虫能力提升的蝴蝶兰新品种航蝴1号 [101]
蝴蝶兰 Phal. amabilis - 获得花变大，花朵数增加，花色加深，花期延长，抗病虫性和抗逆性提升的蝴蝶兰新品种航蝴2号 [102]
石斛 Den. moniliforme - 获得矮化叶艺新品种Uju [51] 石斛 Den. officinale 种子获得生长加快，产量提高的铁皮石斛新品种仙斛3号 [103] 石斛 Den. nobile 种子大部分太空诱变的金钗石斛幼苗呈现矮化，茎加粗，分蘖多，叶片变短的趋势 [104]注：-表示不详。Note：- indicates unknown.

2 兰花突变体的筛选鉴定

表型鉴定是兰科植株突变体鉴定中简单、经济、实用的方法。在组培过程中或田间观察到有植株与对照植株在株型、叶色、叶型、花色、花形、花期、抗性等方面存在明显差异时，该植株会被单独选出，通过分株繁殖、扦插繁殖或组培快繁生产株系，如果变异性状在株系内一致且稳定，一般就认为该变异植株是突变体。采用分子标记对变异植株和对照植株进行分析，如果变异植株和对照植株在DNA水平上存在差异，则进一步证明变异植株是突变体[105]，但是分子鉴定结果与植株表型性状的变化之间可能并不存在一一对应关系[6]。

兰花的营养生长期一般为3~5 a。为了提早进行突变体筛选和鉴定，对于肉眼不可见、不稳定或不容易鉴定的性状，开发出新的育种目标性状突变体筛选和鉴定技术是十分重要的。在一些兰花上已建立分子标记辅助选择技术[106]、试管花诱导技术体系[107]、茎腐病抗性品种筛选和鉴定技术[70]等，有的已应用于突变体筛选。

3 兰花诱变机理

诱变可以引起兰花染色体数目和结构、基因结构和表达的变异，从而导致性状改变。γ射线辐照能引起染色体数目改变，形成非整倍体[54]，也能引起核型改变[19]。对叶艺隆昌素叶艺形成的分子机理研究结果表明，编码尿卟啉原脱羧酶基因HEME在叶艺隆昌素中表达下调，而叶绿素生物合成中编码镁原卟啉Ⅸ甲基转移酶基因CHLM、谷氨酸-tRNA还原酶基因HEMA和叶绿素降解相关基因表达上调，说明色素合成、叶绿体发育相关基因的差异表达可能是导致隆昌素叶艺形成的原因[5,108-109]。KIM等[24]对兰花黄叶突变体进行RNA测序分析，发现叶绿素代谢或离子运输的改变有可能导致兰花叶色变黄。LIM等[50]对石斛紫叶突变体进行转录组分析，发现突变体叶片变紫色与花青素生物合成途径中生物合成酶基因和MYB2等几个转录因子表达增加有关。卓志勇[110]用Illumina HiseqX技术对君豪兰和重离子辐照诱变形成的线艺君豪兰叶片、根和茎混样进行转录组测序和基因表达分析，筛选出2个与兰花线艺性状突变有关的候选基因，分别为CYP76B10和PSBO。

4 展望

兰花是中国的传统十大名花之一，中国人赏兰不仅赏花，而且赏叶。诱变处理可以诱导兰花产生丰富的遗传变异，特别是诱导兰花产生丰富的株型和叶部性状变异，因此诱变育种非常适合我国兰花育种工作。随着我国经济发展和科技进步，可以进一步提高诱变当代突变效率和突变谱的诱变新技术（重离子辐照、空间诱变等）的不断出现，兰花诱变育种技术有望成为进一步加快我国兰花育种进程，尽快缩小与国外在兰花育种上差距的关键技术之一。

进一步提高诱变育种效率和效果是今后兰花诱变育种研究的主要任务，可以主要从以下几个方面深入系统地开展研究工作。首先是进一步提高诱变效率。筛选高效诱变剂、适宜的诱变材料和方法依然是建立高效诱变育种技术体系的基础性工作，在此基础上，可通过多种诱变技术的结合进一步提高诱变效率[64]。其次是建立高效突变体筛选和鉴定技术体系。随着基因组测序和功能基因组学研究的不断深入，兰花高密度分子遗传图谱的建立和主要育种目标性状关键基因的挖掘不断取得新进展[111]，在此基础上，建立高效分子标记辅助选择体系和定向诱导基因组局部突变（Targeting Induced Local Lesions IN Genomes, TILLING）等技术成为可能，这些技术在兰花诱变育种上的应用将进一步提高突变体筛选和鉴定效率。再次是建立兰花快速高效育种技术。将杂交、诱变和细胞工程技术相结合，建立多种育种技术相融合的兰花快速高效育种技术不仅可以提高兰花育种效率，而且可以缩短兰花育种周期，快速培育优良兰花新品种。最后是进一步明确兰花诱变育种的倍性效应。多数兰花商业化品种是多倍体[112-113]，不同倍性兰花品种诱变效果可能有差异，因此系统开展倍性对兰花诱变育种效果的影响是非常有必要的。

诱变机理研究对进一步提高兰花诱变育种效率具有重要的促进作用。大量研究表明，诱变通过引起染色体数目改变[54]、染色体结构变异[19]、基因结构变异和表达水平变化[108-109]等改变植物性状。不同诱变剂诱导遗传物质改变的类型、频率和诱发植物变异的性状不完全相同，不同类型遗传物质的改变导致兰花性状改变的机理也不同。诱变剂是如何引起遗传物质发生改变，从而进一步导致性状改变的，目前这些机理尚不十分清楚，今后应进一步加强这方面的研究。

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摘要

以兰花春剑隆昌素品种为材料,用不同剂量60Co γ射线辐照处理其根状茎,研究辐照诱变技术对兰花根状茎组织培养成活率及分化率的影响,并用ISSR分子标记技术检测春剑隆昌素组培苗辐射诱变前后引起的DNA变异情况。结果表明:10 Gy辐射处理后的根状茎分化苗率为93.2%,20Gy辐射处理后根状茎分化苗率为54.4%,而40Gy和60Gy剂量处理后的根状茎几乎全部死亡。主要变异性状是叶色失绿变淡。通过对春剑隆昌素辐射后代形态变异株的ISSR基因检测,发现经过30多年继代培养的春剑隆昌素组培苗的变异率为6%,而辐射诱变后代的变异率为18%。说明经过60Co γ辐射诱变的根状茎所获得的变异比组织培养所获得变异高。可疑突变株和对照组在15个ISSR引物扩增结果中并未出现明显的差异,ISSR 分子标记结果与植株表现出的形态变化之间可能并不存在一一对应关系。

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Inter-Simple Sequence Repeat(ISSR) molecular markers were used to analyze the Cymbidium ‘longchangsu’ seedlings in vitro, mutagenic progenies and the leaf shape mutants by 60Co γ rays. Results showed that the differentiation rate of the radiated rhizomes by 10Gy and 20Gy were 93.2% and 54.4%,respectively. The rhizomes radiated by 40Gy and 60Gy were all died. The mutation rates of seedlings by repeatedly subcultured in vitro were 6% and the mutagenic progenies by radiation were 18%.It is indicated that the mutagenic rates caused by radiation is higher than that by tissue culture. No significant differences were observed between suspicious mutants and the control after the gene detection of the leaf shape mutants. It suggests that,maybe,there were not significant correlation between ISSR results and the morphological changes.

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摘要

以不同剂量的60Co γ射线对‘铁骨素’建兰、‘十八学士’建兰、‘长汀’墨兰3种国兰成熟植株进行辐照，对各处理植株的新长幼芽、假鳞茎、花期、植株形态等进行观测。结果显示：在10～50 Gy辐照剂量范围内，受照植株成活率随剂量的加大下降，而辐照效应却增强。不同品种的国兰耐辐射能力有所差异，‘长汀’墨兰＞‘铁骨素’建兰＞‘十八学士’建兰；同一植株不同器官组织，不同发育阶段的耐辐射能力也有所不同，成熟假鳞茎＞幼芽＞花芽；植株形态变异一般只在VM1代的幼芽上有所发生。3种国兰成熟植株辐射诱变适宜的处理剂量为16～18Gy。

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摘要

为了解60Co-γ射线对铁皮石斛种子萌发和幼苗形成产生的辐射效应，以及胶膜菌属（Tulasnella）菌根真菌对辐射后铁皮石斛种子萌发的影响，本研究采用不同梯度剂量（20~120 Gy）60Co-γ射线辐射处理铁皮石斛种子，对辐射种子分别进行非共生萌发［1/2MS、燕麦培养基（OMA）］、菌根真菌共生萌发（OMA接种菌株JL4、JL2）培养，比较分析非共生和共生培养方法对辐射种子萌发率和成苗率的影响，观察幼苗表型变化。结果表明，在非共生萌发（1/2MS）条件下，铁皮石斛种子半致死剂量为62 Gy，在与不同胶膜菌属菌株共生萌发后，半致死剂量为69 Gy（JL2）和63 Gy（JL4）。种子萌发率随辐射剂量的增高而降低，低剂量（20 Gy）处理加速了幼苗形成，高剂量处理（90、120 Gy）抑制了幼苗形成；培养115 d后，20 Gy处理成苗率较对照显著提高，分别达到18.26%（1/2MS）、15.00%（JL2）和17.86%（JL4）；高剂量（90、120 Gy）处理种子在共生萌发条件下可获得表型变化更明显的幼苗，具体表现为幼苗高长、假鳞茎粗壮。本研究将种子辐射与兰科菌根真菌共生萌发相结合，为促进铁皮石斛辐射种子恢复、提高辐射诱变效率、高效创造铁皮石斛新种质提供了技术参考和科学依据。

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DINARTI D

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The objective of this research was to determine the effects of gamma irradiation on protocorm like bodies (PLBs) Dendrobium lasianthera and Lethal dose (LD) 30 and 50 of gamma irradiation. The irradiation was conducted at the Center of Technology Application of Isotops and Radiation, Nuclear Energy Agency (PATIR-BATAN) and culture at Tissue Culture Laboratory of IPB from February 2014 to July 2014. The treatments were arranged in a completely randomized design (CRD) with a single factor of gamma irradiation doses, include i,g. 0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, and 100 Gy. Each dose of gamma irradiation treatment was repeated 5 times. There were 30 experimental units. Each experimental unit consisted of five culture bottles containing 4 individually planted PLBs Dendrobium lasianthera. The results of this research showed that the increasing doses of gamma irradiation significantly decreased the percentage of alive PLBs, PLBs germination percentage, number of leaves, number of roots, the percentage of rooted PLBs. Morphological changes among other wider and spiral leaves were observed in the treated plantlets. Lethal dose 30% (LD30) was at 19.7697 Gy and LD50 was at 67.3504 Gy.Keywords: Dendrobium lasianthera, gamma irradiation, in vitro, Lethal dose (LD), mutation ABSTRAKPenelitian ini bertujuan untuk mengetahui pengaruh iradiasi gamma terhadap pertumbuhan protocorm like bodies (PLBs) anggrek Dendrobium lasianthera serta menentukan Lethal dose (LD) 30 dan 50 dari iradiasi sinar gamma. Proses iradiasi dilakukan di Pusat Aplikasi Teknologi Isotop dan Radiasi, Badan Tenaga Nuklir Nasional (PATIR-BATAN) Proses kultur dilakukan di Laboratorium Kultur Jaringan IPB. Penelitian dilakukan dari bulan Februari 2014 hingga Juli 2014. Penelitian disusun menggunakan rancangan acak lengkap (RAL) dengan faktor tunggal yaitu dosis iradiasi gamma meliputi 0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, dan 100 Gy. Setiap dosis perlakuan iradiasi gamma diulang 5 kali, seluruhnya terdapat 30 satuan percobaan. Setiap satuan percobaan terdiri atas lima botol kultur yang masing-masing ditanam 4 PLBs anggrek Dendrobium lasianthera. Hasil penelitian ini menunjukkan bahwa peningkatan dosis iradiasi gamma nyata menurunkan persentase hidup PLBs, persentase PLBs berkecambah, jumlah daun, jumlah akar, dan persentase PLBs berakar. Perubahan planlet in vitro yang teramati antara lain daun melebar dan daun spiral. LD30 berada pada 19.7697 Gy dan LD50 pada 67.3504 Gy.Kata kunci: Dendrobium lasianthera, in vitro, iradiasi gamma, Lethal dose (LD), mutasi

KIM S W

RYU J

KANG S Y

KIM J B

KIM S H

. Upregulation of the MYB2 transcription factor is associated with increased accumulation of anthocyanin in the leaves of Dendrobium bigibbum[J]. International Journal of Molecular Sciences, 2020, 21(16): 5653.

Orchids with colorful leaves and flowers have significant ornamental value. Here, we used γ-irradiation-based mutagenesis to produce a Dendrobium bigibbum mutant that developed purple instead of the normal green leaves. RNA sequencing of the mutant plant identified 2513 differentially expressed genes, including 1870 up- and 706 downregulated genes. The purple leaf color of mutant leaves was associated with increased expression of genes that encoded key biosynthetic enzymes in the anthocyanin biosynthetic pathway. In addition, the mutant leaves also showed increased expression of several families of transcription factors including the MYB2 gene. Transient overexpression of D. biggibumMYB2 in Nicotiana benthamiana was associated with increased expression of endogenous anthocyanin biosynthesis genes. Interestingly, transient overexpression of orthologous MYB2 genes from other orchids did not upregulate expression of endogenous anthocyanin biosynthesis genes. Together, these results suggest that the purple coloration of D. biggibum leaves is at least associated with increased expression of the MYB2 gene, and the MYB2 orthologs from orchids likely function differently, regardless of their high level of similarity.

KIM W J

IM J

KANG K W

KIM S H

JO Y D

KANG S Y

LEE J H

HA B K

. Single nucleotide polymorphism (SNP) discovery through genotyping-by-sequencing (GBS) and genetic characterization of Dendrobium mutants and cultivars[J]. Scientia Horticulturae, 2019, 244: 225-233.

HARTATI S

YUNUS A

. Diversity induction with gamma ray irradiation on Dendrobium odoardi orchid[C]// The 7th International Conference on Sustainable Agriculture and Environment, Surakarta, 2021.

谢小波, 孙崇波, 宋仙水. 铁皮石斛γ射线诱变及后代变异研究[J]. 浙江农业科学, 2016, 57(5): 687-690.

XIE X B

SUN C B

SONG X S

. Study on γ-ray mutagenesis and progeny variation of Dendrobium officinale[J]. Journal of Zhejiang Agricultural Sciences, 2016, 57(5): 687-690. (in Chinese)

詹忠根, 徐程, 席玙芳. 137Cs γ射线辐照对铁皮石斛种胚原球茎的诱变研究[J]. 核农学报, 2009, 23(5): 816-819.

摘要

研究了不同剂量的137Csγ射线辐照对铁皮石斛种胚原球茎生长和分化效应的影响。结果表明：辐照处理对种胚原球茎的生长和分化有抑制作用，尤其是对根的抑制更为明显；由于种胚原球茎的完整性好，耐受性强，其辐照半致死剂量(LD50)为67.23Gy；流式细胞分析表明，大多数外部形态发生改变的植株其细胞内DNA的倍性发生了改变，出现相当多的非整倍体；此外，辐照产生的变异苗出现茎分叉、叶片缺绿或白绿相间等现象，可能是由于某些基因的表达与关闭引起的。

ZHAN Z G

XU C

XI Y F

. Study mutagesis of Dendrobium officinale protocorm from embryo irradiated by 137Cs γ-rays[J]. Journal of Nuclear Agricultural Sciences, 2009, 23(5): 816-819. (in Chinese)

UYEN N H P

HA V T T

. In vitro mutation breeding of Paphiopedilum by ionization radiation[J]. Scientia Horticulturae, 2012, 144: 1-9.

孙音, 郝军, 房义福, 张谦, 姜楠南. 60Co-γ辐射对兜兰组培苗的诱变效应[J]. 中国农学通报, 2022, 38(15): 45-52.

摘要

为了选育兜兰新品种,明确60Co-γ对兜兰不同发育时期的诱变效应,筛选适宜辐射剂量。以种子、瓶苗（不定芽增殖、不定根分化阶段）及小苗为材料,采用0、5、10、20、30、40 Gy的60Co-γ进行辐射,观测生长发育和变异情况,测定生理生化指标。结果表明,种子萌发阶段和不定根分化阶段为5 Gy,不定芽增殖阶段和小苗阶段为20 Gy。6.92 Gy是兜兰种子的半致死剂量。随着辐射剂量增大,种子的发芽率、不定芽殖增率、不定根分化率、小苗生长势下降,变异增多。同时,电导率、丙二醛含量、可溶性糖含量、可溶性蛋白含量、脯氨酸含量以及过氧化物酶、超氧化物歧化酶活性呈现先升高后降低的趋势。兜兰种子在萌发阶段和不定根分化阶段需要适宜的低辐射剂量,不定芽增殖阶段和小苗阶段较高。在合适的辐射剂量下,诱导的变异性状明显,效果稳定。

SUN Y

HAO J

FANG Y F

ZHANG Q

JIANG N N

. Mutagenic effect of 60Co-γ on tissue culture seedlings of Paphiopedilum[J]. Chinese Agricultural Science Bulletin, 2022, 38(15): 45-52. (in Chinese)

周亚倩, 姚娜, 魏莉, 李潞滨, 刘蕾. 60Co-γ射线对树兰蒴果辐照生物学效应研究[J]. 核农学报, 2017, 31(9): 1693-1699.

摘要

为探讨辐照处理对树兰种子的生物学效应,以树兰蒴果为研究对象,60Co-γ射线为辐照源,设置不同剂量(0~200 Gy)进行辐照处理, 在组织培养条件下研究其生物学效应。结果表明,当辐照剂量为20 Gy时可提高种子萌发率,并缩短种子萌发时间,而当辐照剂量为200 Gy时,种子不再萌发;树兰种子的半致死剂量为78.08 Gy。辐照处理对树兰再生植株的影响明显,与辐照剂量呈正相关,对株高、叶长影响最大,叶片数量次之,叶宽最小。本研究结果为树兰属植物离体诱变育种提供了一定的科学依据。

ZHOU Y Q

YAO N

WEI L

LI L B

LIU L

. Effect of 60Co-γ irradiation on capsule of Epidendium secundum[J]. Journal of Nuclear Agricultural Sciences, 2017, 31(9): 1693-1699. (in Chinese)

PEDRAZA-SANTOS M E

LOPEZ P A

CRUZ-TORRES E D L

FERNANDEZ-PAVIA S P

MARTINEZ-PALACIOS A

MARTINEZ-TRUJILLO1 M

. DL50 and GR50 determination with gamma rays (60Co) on in vitro Laelia autumnalis protocorms[J]. Agrociencia, 2017, 51(5): 507-524.

. Effects of gamma irradiation on protocorm-like bodies of Cattleya alliances[C]// The Sixth Conference on Nuclear Science and Technology, Bangkok: Office of Atomic Energy for Peace, 1996: 18-30.

. Radiosensitivity of three species of ground orchids (Spathoglottis plicata, S. kimballiana var. angustifolia and S. tomentosa) to acute gamma radiation[D]. Nueva Ecija: Central Luzon State University, 2007.

. Acute effect of gamma radiation on stable characteristics of Spathoglottis plicata Blume[J]. Acta Horticulturae, 2012, 953: 173-180.

PUSPITANINGTYAS D M

DINARTI D D

. Pengaruh iradiasi sinar gamma terhadap variasi pertumbuhan anggrek Brachypeza indusiata (Reichb.f) garay secara in vitro[J]. Buletin Kebun Raya indonesia, 2007, 10(2): 53-59.

BASIRAN M N

. Mutagenesis of Oncidium lanceanum[C]// Research and Development Seminar, Bangi: Malaysian Institute for Nuclear Technology Research, 2004: 57-62.

GAYATRI M C

SARANGI S K

. In vitro mutagenesis and characterization of mutants through morphological and genetic analysis in orchid Aerides crispa Lindl.[J]. Indian Journal of Experimental Biology, 2018, 56(6): 385-394.

. γ-ray radiation on Bletilla striata R. seeds and occurrence of variation in the seedlings[D]. Korea: Chonbuk National University, 2005.

GOMEZ-MERINO F C

CRUZ-IZQUIERDO S

SPINOSO-CASTILLO J L

BELLO-BELLO J J

. Gamma radiation (60Co) induces mutation during in vitro multiplication of Vanilla (Vanilla planifolia Jacks. ex Andrews)[J]. Horticulturae, 2022, 8(6): 503.

In vitro mutagenesis is an alternative to induce genetic variation in vanilla (Vanilla planifolia Jacks. ex Andrews), which is characterized by low genetic diversity. The objective of this study was to induce somaclonal variation in V. planifolia by gamma radiation and detect it using inter-simple sequence repeat (ISSR) molecular markers. Shoots previously established in vitro were multiplied in Murashige and Skoog culture medium supplemented with 2 mg·L−1 BAP (6-benzylaminopurine). Explants were irradiated with different doses (0, 20, 40, 60, 80 and 100 Gy) of 60Co gamma rays. Survival percentage, number of shoots per explant, shoot length, number of leaves per shoot, and lethal dose (LD50) were recorded after 60 d of culture. For molecular analysis, ten shoots were used for each dose and the donor plant as a control. Eight ISSR primers were selected, and 43 fragments were obtained. The percentage of polymorphism (% P) was estimated. A dendrogram based on Jaccard’s coefficient and the neighbor joining clustering method was obtained. Results showed a hormetic effect on the explants, promoting development at low dose (20 Gy) and inhibition and death at high doses (60–100 Gy). The LD50 was observed at the 60 Gy. Primers UBC-808, UBC-836 and UBC-840 showed the highest % P, with 42.6%, 34.7% and 28.7%, respectively. Genetic distance analysis showed that treatments without irradiation and with irradiation presented somaclonal variation. The use of gamma rays during in vitro culture is an alternative to broaden genetic diversity for vanilla breeding.

LYU J I

ABE T

YANO Y

YOSHIDA S

LEE Y I

LEE H Y

. Effect of heavy-ion beam irradiation on in vitro cultured plant organisms[C]// Proceedings of APAC, Gyeongju, Korea, 2004: 444-445.

HASSAN A A

IDRIS N A

BASIRAN M N

TANAKA A

SHIKAZONO N

OONO Y

HASE N

. Effects of ion beam irradiation on Oncidium lancianum orchids[J]. Journal of Nuclear and Related Technologies, 2015, 3: 1-8.

耿庆芝. 君豪兰诱变技术体系的建立和突变体筛选[D]. 广州: 华南农业大学, 2015.

GENG Q Z

. Establishment of technique system of mutation breeding and mutant creation of Cymbidium ‘Junhao’[D]. Guangzhou: South China Agricultural University, 2015. (in Chinese)

袁红丽. 兰花茎腐病抗性鉴评和资源创新[D]. 广州: 华南农业大学, 2017.

YUAN H L

. Evaluation on resistance to stem rot and creation of germplasm resource in Cymbidium[D]. Guangzhou: South China Agricultural University, 2017. (in Chinese)

郭和蓉, 张腾, 袁红丽, 曾瑞珍, 张志胜, 谢利. 利用12C6+重离子辐射和尖孢镰刀菌毒素筛选杂交兰抗茎腐病突变体[J]. 核农学报, 2021, 35(12): 2688-2695.

摘要

为创建抗茎腐病杂交兰资源,本研究以玉女兰类原球茎为材料,采用12C6+重离子辐射技术结合尖孢镰刀菌(Fusarium oxysporum)粗毒素筛选抗茎腐病突变体。结果表明,经不同剂量12C6+重离子辐射后,玉女兰类原球茎的死亡率差异显著,辐射剂量为50 Gy时,死亡率为54.66%;尖孢镰刀菌粗毒素对玉女兰类原球茎存活率影响显著,当粗毒素滤液浓度为80%时,玉女兰类原球茎存活率为41.98%。采用逐步筛选法对经50 Gy12C6+辐射后的玉女兰类原球茎进行抗茎腐病筛选,获得14个抗性系类原球茎,筛选率为4.67%;对抗性系类原球茎进行分化、生根壮苗和移栽,获得3个抗性系Z50Gt1、Z50Gt2和Z50Gt3。在含80%粗毒素滤液的培养基上抗性系类原球茎的超氧化物歧化酶(SOD)和过氧化物酶(POD)的活性均高于对照,POD活性峰值比对照提高了867.67 U·g-1·h-1;丙二醛(MDA)含量呈现先高于对照,随着毒素胁迫时间的延长,转变为低于对照的趋势。对3个抗性系试管苗和小苗进行人工接种鉴定,结果表明,抗性系Z50Gt2和Z50Gt3的再生植株具有稳定的茎腐病抗性,2个抗病株系7个月大的盆栽植株病情指数分别为20.00%和19.33%,均为抗性级别。本研究结果为采用重离子辐射技术选育抗茎腐病杂交兰新品种奠定了基础。

GUO H R

ZHANG T

YUAN H L

ZENG R Z

ZHANG Z S

XIE L

. Screening mutants resistant to stem rot using 12C6+ heavy ion beam irradiation and crude toxin of Fusarium oxysporum in hybrid Cymbidium[J]. Journal of Nuclear Agricultural Sciences, 2021, 35(12): 2688-2695. (in Chinese)

ARIFFIN S

AHMAD Z

BASIRAN M N

OONO Y

HASE Y

SHIKAZONO N

NARUMI I

TANAKA A

. Mutation induction of orchid plants by ion beams[C]// Japan Atomic Energy Agency Review, Ibarakiken, 2015: 3-18.

SAKINAH A

ZAITON A

MOHD NAZIR B

TANAKA A

NARUMI I

OONO Y

HASE Y

. Mutation induction in orchids using ion beams[C]// Japan Atomic Energy Agency Takasaki Annual Report, Ibarakiken, 2008: 61.

ARIFFIN S

HASSAN A A

AHMAD RAMLI R A

BASIRAN M N

IBRAHIM R

. Induction of insect resistance in Dendrobium mirbelianum using ion beam irradiation[J]. Acta Horticulturae, 2015(1087): 499-505.

张银洁, 李杰, 郑春. 快中子辐射对蝴蝶兰的诱变效应[J]. 江苏农业科学, 2014, 42(5): 158-159.

ZHANG Y J

LI J

ZHENG C

. Mutagenesis effect of fast neutron radiation on Phalaenopsis[J]. Jiangsu Agricultural Sciences, 2014, 42(5): 158-159. (in Chinese)

张银洁, 李杰, 王芝娜, 郑春. 快中子辐射对蝴蝶兰原球茎和茎段增殖分化的影响[J]. 核农学报, 2014, 28(3): 440-445.

摘要

为了获得蝴蝶兰新种质以及开辟新的蝴蝶兰育种方法，利用快中子脉冲堆对蝴蝶兰不同品种原球茎和幼苗茎段进行辐照处理，结果表明：通过对处理材料的回归分析得到，火鸟原球茎的半致死注量为3279×108·cm-2，内山姑娘原球茎的半致死注量为4063×108·cm-2，火鸟茎段的半致死注量为2239×108·cm-2，内山姑娘茎段的半致死注量为2298×108·cm-2。辐射敏感性表现为火鸟>内山姑娘，幼苗茎段>原球茎。对于原球茎，辐照注量小于600×108·cm-2，对其增殖和分化有一定的促进作用；对于茎段，辐照注量小于600×108·cm-2，对其所形成的幼苗的生根没有影响，但小于300×108·cm-2，对所形成幼苗的增殖有促进作用。高注量（>2500×108·cm-2）辐照对原球茎的增殖和分化以及对茎段所形成的幼苗的增殖和生根均有显著的抑制作用。

ZHANG Y J

LI J

WANG Z N

ZHENG C

. Effect of fast neutron radiation on the proliferation and differentiation of protocorm-like bodies and seedlings stem segments of Phalaenopsis[J]. Journal of Nuclear Agricultural Sciences, 2014, 28(3): 440-445. (in Chinese)

林芬, 邓国础. 春兰人工诱变的研究[J]. 湖南农业大学学报(自然科学版), 1997, 23(4): 337-340.

LIN F

DENG G C

. Studies on the inductive mutation of spring orchid[J]. Journal of Hunan Agricultural University (Natural Sciences Edition), 1997, 23(4): 337-340. (in Chinese)

YASUNO N

OHTSUKA M

TANAKA A

SHIKAZONO N

HASE Y

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刘子瑜, 黄先群, 唐章林, 黄团, 李丽. 马铃薯诱变育种研究进展[J]. 西南农业学报, 2010, 23(6): 2124-2128.

LIU Z Y

HUANG X Q

TANG Z L

HUANG T

LI L

. Progress in mutation breeding of potato[J]. Southwest China Journal of Agricultural Sciences, 2010, 23(6): 2124-2128. (in Chinese)

李夏. 寒兰离体化学诱变研究[D]. 南昌: 南昌大学, 2007.

LI X

. The research on chemical mutation in vitro of Cymbidium kanran Makino[D]. Nanchang: Nanchang University, 2007. (in Chinese)

陈超, 王桂兰, 乔永旭, 张莉敏. 蝴蝶兰类圆球茎的化学诱变试验[J]. 核农学报, 2006, 20(2): 99-102.

CHEN C

WANG G L

QIAO Y X

ZHANG L M

. The PLB chemical induced mutagenesis and regeneration of Phalaenopsis[J]. Journal of Nuclear Agricultural Sciences, 2006, 20(2): 99-102. (in Chinese)

KANG S Y

LEE H Y

. Development of leaf mutant cultivars of Cymbidium goeringii by ethyl-methane- sulfonate (EMS) treatment[J]. Korean Journal of Plant Resources, 2011, 24(1): 17-22.

JUNG J S

LEE J S

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王朝雯. EMS和秋水仙素对寒兰根状茎化学诱变的研究[D]. 南昌: 南昌大学, 2012.

WANG C W

. Research on the mutation effects ofthe rhizomes of Cymbidium kanran Makino in vitro after EMS and colchicine treatment[D]. Nanchang: Nanchang University, 2012. (in Chinese)

龚妮. 蝴蝶兰的组织培养和EMS诱变的初步研究[D]. 武汉: 华中农业大学, 2007.

GONG N

. Preliminary studies on tissue culture and EMS induction mutation in Phalaenopsis[D]. Wuhan: Huazhong Agricultural University, 2007. (in Chinese)

SOMPONG T C

. Use of EMS to induce mutation in Dendrobium friedericksianum Rchb.f.[J]. Journal of Agriculture, 2008, 24(2): 153-164.

闫小巧. 铁皮石斛抗病突变体筛选及基于RNA-seq的抗病机理初步研究[D]. 广州: 广州中医药大学, 2018.

YAN X Q

. Primary screening of disease-resistance mutants of Dendrobium officinale and preliminary study on resistance mechanism based on RNA-seq[D]. Guangzhou: Guangzhou University of Chinese Medicine, 2018. (in Chinese)

HUALSAWAT S

CHUEAKHUNTHOD W

THARAPREUKSAPONG A

TOMSANTIA B

YENCHON S

PAPAN P

TANTASAWAT P A

. Selection and characterization of in vitro-induced mutants of Dendrobium ‘Earsakul’ resistant to black rot[J]. In Vitro Cellular & Developmental Biology-Plant, 2022, 58(4): 577-592.

SOMPONG T C

SUREERAT Y

. Effect of ethyl methanesulphonate (EMS) on Dendrobium Sonia[J]. Khon Kaen Agriculture Journal, 2014, 42(3): 506-511.

崔广荣, 张子学, 张从宇, 胡能兵, 隋益虎, 李杰勤. 文心兰EMS离体诱变及再生苗RAPD检测[J]. 热带作物学报, 2011, 32(2): 261-268.

CUI G R

ZHANG Z X

ZHANG C Y

HU N B

SUI Y H

LI J Q

. Mutation induction of EMS in vitro cultured Oncidium and RAPD analyzing of regenerated plantlets[J]. Chinese Journal of Tropical Crops, 2011, 32(2): 261-268. (in Chinese)

钟程, 田鑫, 王天钊, 陈约约. 白及愈伤组织诱导及EMS、NaN3诱变对其干旱胁迫响应的影响[J]. 黑龙江农业科学, 2022(2): 63-69.

ZHONG C

TIAN X

WANG T Z

CHEN Y Y

. Induction of the callus of Bletilla and the effect of EMS and NaN3mutagenesis on its response to drought stress[J]. Heilongjiang Agricultural Sciences, 2022(2): 63-69. (in Chinese)

马玉涵, 赵岩, 张强, 孙玉军, 周正义. 叠氮化钠诱变对离体蝴蝶兰类原球茎生理的影响[J]. 核农学报, 2010, 24(2): 411-414.

摘要

以2、4、6和8mmol/L的叠氮化钠浸泡6h处理蝴蝶兰类原球茎后进行组织培养，测定了处理组和对照的蝴蝶兰类原球茎中脯氨酸、丙二醛和可溶性糖含量以及超氧化物歧化酶和过氧化氢酶的活力。结果表明，经叠氮化钠处理的类原球茎中的超氧化物歧化酶活力、脯氨酸和丙二醛含量均有上升，而过氧化氢酶活力与可溶性糖含量均有下降，并呈现浓度梯度效应。叠氮化钠诱变的半致死剂量介于4~8mmol/L之间，其中4mmol/L的叠氮化钠对蝴蝶兰类原球茎生理损伤较小，因此认为4mmol/L的叠氮化钠是诱变蝴蝶兰类原球茎较为合理的浓度。

MA Y H

ZHAO Y

ZHANG Q

SUN Y J

ZHOU Z Y

. Effect of azide sodium in mutagenesis on physiological traits of Phalaenosis protocorm-like body in vitro[J]. Journal of Nuclear Agricultural Sciences, 2010, 24(2): 411-414. (in Chinese)

KHAIRUM A

CHUEAKHUNTHOD W

THARAPREUKSAPONG A

TANTASAWAT P A

. Profiling of black rot resistant Dendrobium ‘Earsakul’ induced by in vitro sodium azide mutagenesis[J]. European Journal of Horticultural Science, 2022, 87(2): 1-13.

KATIVAT C

TANTASAWAT P A

. Mutation induction of Dendrobium ‘Earsakul’ using sodium azide[J]. Hortscience, 2016, 51(11): 1363-1370.

Dendrobium ‘Earsakul’ is an important commercial orchid in Thailand. Breeding new Dendrobium varieties for improved quality and yield is crucial. The objectives of this research were to perform in vitro mutagenesis of Dendrobium ‘Earsakul’ protocorm-like bodies (PLBs) using sodium azide (NaN3) and to select and evaluate the putative mutants using morphological characters, molecular markers, and the cytological method. The percentages of mortality of PLBs increased as concentrations of NaN3 increased. At 2 weeks, the lethal dose 30 (LD30) and LD50 were obtained with 0.1 and 0.5 mm NaN3, respectively. These two NaN3 concentrations were used for in vitro mutagenesis with reverse osmosis water (ROW; control 1) and 0 mm NaN3 (control 2) as controls. After the plants were cultured for 6 months, morphological differentiation was observed in some putative mutants: reduced height, higher numbers of nodes, reduced node length, shorter and thicker leaves, and shorter and fewer roots, compared with controls. When genetic profiles of 24 putative mutants were compared with controls, altered DNA profiles were found in 20 of 24 putative mutants (83.33%). Sixty-three polymorphic bands were produced from a total of 181 bands (34.81%) amplified by 10 polymorphic intersimple sequence repeat (ISSR) primers. When genetic diversity and relatedness, which were evaluated by ISSR analysis, and morphological characters were compared, the two markers were found to be uncorrelated. ISSR had a higher mutant differentiation capability than the morphological characters, indicating its higher efficiency. The chromosome numbers were similar in putative mutants and controls (2n = 2x = 24), suggesting that neither of the concentrations of NaN3 had any effect on the chromosome numbers in this experiment. These results indicate that NaN3 can be used effectively to mutagenize Dendrobium ‘Earsakul’ PLBs, and ISSR is a powerful tool for the identification of mutants. Chemical name: sodium azide (NaN3); reverse osmosis water (ROW).

POOLSAWAT O

CHAOWISET W

TANTASAWAT P A

. Evaluation of genetic variability in in vitro sodium azide-induced Dendrobium ‘Earsakul’ mutants[J]. Genetics and Molecular Research, 2014, 13(3): 5333-5342.

崔广荣, 张子学, 张从宇, 胡能兵, 隋益虎, 李杰勤. 文心兰NaN3离体化学诱变及RAPD检测[J]. 广西植物, 2011, 31(6): 836-843.

CUI G R

ZHANG Z X

ZHANG C Y

HU N B

SUI Y H

LI J Q

. Study on NaN3 chemical induction for Oncidium in in vitro culture and RAPD screening[J]. Guihaia, 2011, 31(6): 836-843. (in Chinese)

胡松梅, 周永胜, 康和英. 硫酸二乙酯诱变筛选铁皮石斛耐寒突变体的研究[J]. 江西农业, 2019(18): 114-115.

HU S M

ZHOU Y S

KANG H Y

. The research of Dendrobium officinale cold-tolerant mutants induced by diethyl sulfate[J]. Jiangxi Agriculture, 2019(18): 114-115. (in Chinese)

夏英武. 作物诱变育种[M]. 北京: 中国农业出版社, 1997: 56.

XIA Y W

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温贤芳, 张龙, 戴维序, 李春华. 天地结合开展我国空间诱变育种研究[J]. 核农学报, 2004(4): 241-246.

WEN X F

ZHANG L

DAI W X

LI C H

. Study of space mutation breeding in China[J]. Journal of Nuclear Agricultural Sciences, 2004(4): 241-246. (in Chinese)

王艳芳, 王世恒, 祝水金. 航天诱变育种研究进展[J]. 西北农林科技大学学报(自然科学版), 2006, 34(1): 9-12.

WANG Y F

WANG S H

ZHU S J

. Advance in the space flight mutation breeding[J]. Journal of Northwest A & F University (Natural Science Edition), 2006, 34(1): 9-12. (in Chinese)

陈肖英, 郑平, 徐明全, 赵贵林, 刘敏. 航蝴1号蝴蝶兰选育研究初报[J]. 热带农业科学, 2011, 31(11): 23-27.

CHEN X Y

ZHENG P

XU M Q

ZHAO G L

LIU M

. Space breeding research on Hanghu No. 1 of Phalaenopsis amabilis[J]. Chinese Journal of Tropical Agriculture, 2011, 31(11): 23-27. (in Chinese)

陈肖英, 彭宝富, 徐明全, 赵贵林, 郑平, 刘敏. 蝴蝶兰新品种航蝴2号选育结果与分析[J]. 卫星应用, 2013(1): 61-65.

CHEN X Y

PENG B F

XU M Q

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ZHENG P

LIU M

. Breeding results and analysis of a new variety of Phalaenopsis ‘Hanghu No.2’[J]. Satellite Application, 2013(1): 61-65. (in Chinese)

徐靖, 王晓彤, 胡凌娟, 李明焱, 邵清松, 贾宸. 铁皮石斛新品种“仙斛3号”的选育及特征特性研究[J]. 中国现代中药, 2017, 19(3): 337-341.

XU J

WANG X T

HU L J

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SHAO Q S

JIA C

. Breeding of Dendrobium officinale var. “Xianhu 3” and its characteristics[J]. Modern Chinese Medicine, 2017, 19(3): 337-341. (in Chinese)

彭曦, 叶庆生. 太空诱变对金钗石斛光合特性和生长的影响[J]. 热带亚热带植物学报, 2017, 25(5): 480-488.

PENG X

YE Q S

. Effects of space mutation on photosynthetic characteristics and growth of Dendrobium nobile[J]. Journal of Tropical and Subtropical Botany, 2017, 25(5): 480-488. (in Chinese)

高祥云. 重离子辐照对杂交兰的组培和诱变效应[D]. 广州: 华南农业大学, 2018.

GAO X Y

. Effects of heavy ion irradiation on tissue culture and mutagenesis in hybrid Cymbidium[D]. Guangzhou: South China Agricultural University, 2018. (in Chinese)

肖文芳, 李佐, 陈和明, 吕复兵. 基于SLAF-BSA技术的蝴蝶兰花底色关联SNP分子标记开发与验证[J]. 中国农业大学学报, 2021, 26(9): 92-100.

XIAO W F

LI Z

CHEN H M

LYU F B

. ldentification and validation of single nucleotide polymorphism markers linked to flower ground color in Phalaenopsis by using combined specific-locus amplified fragment sequencing and bulked segregant analysis[J]. Journal of China Agricultural University, 2021, 26(9): 92-100. (in Chinese)

陈如梦, 彭梦萱, 魏倩, 曾瑞珍, 宿庆连, 张志胜. 影响兰花试管花诱导和发育的因素[J/OL]. 分子植物育种, 2022. [2023-07-23]. https://kns.cnki.net/kcms/detail/46.1068.S.20220829.1026.002.html.

CHEN R M

PENG M X

WEI Q

ZENG R Z

SU Q L

ZHANG Z S

. Factors influencing on the induction and development of test-tube flower in Cymbidium[J/OL]. Molecular Plant Breeding, 2022. [2023-07-23]. https://kns.cnki.net/kcms/detail/46.1068.S.20220829.1026.002.html. in Chinese)

蒋彧, 涂勋良, 何俊蓉. 国兰叶色突变体叶片差异表达基因分析[J]. 园艺学报, 2023, 50(2): 371-381.

摘要

中国兰春剑‘隆昌素’（Cymbidium longibracteaturn）及其叶色突变体‘叶艺隆昌素’是研究兰花叶色突变形成机制的良好材料。采用Illumina Hiseq2500对两种材料的叶片进行de novo转录组测序，以此鉴定与叶色突变性状形成相关的差异表达基因。结果表明，色素合成、叶绿体发育相关基因可能是导致‘隆昌素’与‘叶艺隆昌素’叶片性状差异的原因。

JIANG Y

TU X L

HE J R

. Analysis of differential expression genes in leaves of leaf color mutant of Chinese orchid[J]. Acta Horticulturae Sinica, 2023, 50(2): 371-381. (in Chinese)

The Chinses orchid‘Longchangsu’and its leaf color mutant‘Yeyi Longchangsu’are excellent materials to study the formation mechanism of the leaf color of orchid. Differentially expressed genes（DEGs）in the leaves of‘Longchangsu’and‘Yeyi Longchangsu’were identifed by next-generation sequencing using Illumina Hiseq2500. Through the analysis of different expression genes，it was found that the genes related to pigment synthesis and chloroplast development might be the cause of the differences between‘Longchangsu’and‘Yeyi Longchangsu’.

蒋彧, 何俊蓉. 国兰叶色突变体根状茎差异表达基因分析[J]. 核农学报, 2022, 36(3): 497-508.

摘要

兰花叶色对其观赏及商业价值具有非常重要的意义。国兰春剑隆昌素(Cymbidium longibracteaturn Longchangsu)及其叶色突变体叶艺隆昌素是研究兰花叶色形成机制的重要材料。本研究采用Illumina Hiseq2500对这2种材料的根状茎进行denovo转录组测序,以鉴定其与叶艺形成相关的差异表达基因(DEGs)。差异表达基因分析结果发现色素合成、叶绿体发育等相关的基因在隆昌素与叶艺隆昌素根状茎中的表达存在显著差异。本研究获得的序列数据极大地丰富了国兰可利用的基因资源,为进一步明确国兰叶色形成的分子机制提供了科学参考。

JIANG Y

HE J R

. Analysis of differential expression genes in rhizome of leaf color mutant of Chinese orchid[J]. Journal of Nuclear Agricultural Sciences, 2022, 36(3): 497-508. (in Chinese)

The color of orchid leaf is very important for its ornamental and commercial value. The Chinese orchid Longchangsu and its leaf color mutant Yeyi Longchangsu are important materials to study the formation mechanism of the leaf color of orchid. Next-generation sequencing on Illumina Hiseq2500 platform was used to identify differentially expressed genes (DEGs) in the rhizomes of Longchangsu and Yeyi Longchangsu. The expressions of genes related to pigment synthesis and chloroplast development were significantly different between Longchangsu and Yeyi Longchangsu rhizomes. The sequence data obtained in this study greatly enriched the available gene resources of Chinese orchid and provided scientific reference for further clarifying the molecular mechanism of leaf color formation of Chinese orchid.

卓智勇. 兰花重离子辐照效应与叶片线艺突变机理研究[D]. 广州: 华南农业大学, 2020.

ZHUO Z Y

. Study on effect of heavy ion irradiation and mechanism of thread art mutation on leaf in Cymbidium[D]. Guangzhou: South China Agricultural University, 2020. (in Chinese)

DONG N

ZHAO Y M

WU S S

LIU Z J

ZHAI J W

. A review for the breeding of orchids: current achievements and prospects[J]. Horticultural Plant Journal, 2021, 7(5): 380-392.

吴婷, 贾瑞冬, 杨树华, 赵鑫, 于晓南, 国圆, 葛红. 蝴蝶兰多倍体育种研究进展与展望[J]. 园艺学报, 2022, 49(2): 448-462.

摘要

对蝴蝶兰离体加倍诱导、2n配子诱导、有性杂交和分离内多倍体等诱导途径,以及形态学、细胞学和染色体计数等多倍体鉴定方法的研究进展进行综述,总结了近年来蝴蝶兰多倍体育种的成果。从筛选不同化学试剂和植物材料,加强对2n配子的研究和利用,加强有性杂交、细胞融合与多倍体诱导技术相结合,加强诱导后植株的研究和应用等方面对蝴蝶兰多倍体育种进行展望。

WU T

JIA R D

YANG S H

ZHAO X

YU X N

GUO Y

GE H

. Research advances and prospects on Phalaenopsis polyploid breeding[J]. Acta Horticulturae Sinica, 2022, 49(2): 448-462. (in Chinese)

罗远华, 方能炎, 樊荣辉, 黄敏玲. 兰科植物多倍体诱导研究进展[J]. 江苏农业科学, 2022, 50(1): 6-13.

LUO Y H

FANG N Y

FAN R H

HUANG M L

. Research progress of polyploid induction in orchids[J]. Jiangsu Agricultural Sciences, 2022, 50(1): 6-13. (in Chinese)

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