卷丹新种质JD-h-15的形态特征与遗传变异分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0091

摘要/Abstract

摘要：

为培育优质的卷丹新种质，以秋水仙素诱变材料JD-h-15于田间种植3年分析鉴定染色体倍性。对JD-h-15和卷丹植株形态、柱头与花粉育性、叶表皮与淀粉粒特征以及鳞茎有效成分等进行比较，结合ISSR标记分析JD-h-15的遗传变异。结果表明，诱变株系JD-h-15已由非整倍的嵌合体恢复为3倍体植株。JD-h-15的株高、叶间距和叶面积与卷丹相比明显增加，茎粗和单株叶片数减少，单株花期提前约14 d。JD-h-15鳞茎独头率为93.75%，显著高于卷丹独头率70.00%。不同时期柱头可授性试验表明，JD-h-15在花苞生长至50-60 mm时表现出高于对照的活性。JD-h-15的花粉有13.71%可正常萌发，而卷丹花粉不萌发，与JD-h-15自交授粉后子房膨大相符。微形态分析表明，JD-h-15的花粉粒大于卷丹对照，气孔大小和密度与对照无差异，而珠芽和鳞茎淀粉粒小于对照。JD-h-15鳞茎中可溶性糖、总黄酮和总皂苷含量比对照分别增加42.37%、67.54%和45.65%，而抗坏血酸、总酚和总生物碱分别减少22.86%、33.02%和50.41%。ISSR标记分析发现3年生JD-h-15和卷丹植株遗传稳定，JD-h-15相比卷丹发生遗传变异，变异率为21.30%。研究结果为卷丹遗传性状改良和秋水仙素诱变百合新种质提供重要参考。

关键词: 卷丹, 遗传改良, 形态特征, 有效成分, ISSR标记

Abstract:

In order to cultivate a high-quality new germplasm of Lilium lancifolium，JD-h-15 induced by colchicine was used to analyze the chromosome ploidy after 3 years’ planting in the field. The plant morphology，stigma and pollen fertility，leaf epidermis and starch grain characteristics，and active components of bulb were compared in JD-h-15 and L. lancifolium control. Further their difference on molecular levels was analyzed by ISSR marker technology. The results showed that JD-h-15 has restored to be a triploid plant from an aneuploid chimera before. The plant height，leaf interval and leaf area of JD-h-15 significantly increased compared with that in the control，as well as the stem diameter and leaf number per plant decreased，in addition the flowering period per plant was advanced in JD-h-15 by 14 d. The single head rate of bulb in JD-h-15 was 93.75%，significantly higher than 70.00% of that in the control. Analysis of stigma acceptability in different flower periods revealed the stigma in 50-60 mm flower bud of JD-h-15 had a higher acceptability than that in the control. 13.71% of the pollen for JD-h-15 germinated normally，while the control pollen did not germinate，which was consistent with the expansion of ovary in JD-h-15 after self-pollination. Micromorphological analysis showed that the pollen grains of JD-h-15 were larger than those of the control，while the starch grains in bulbils and bulbs were much smaller than those of the control. In JD-h-15 bulbs，the contents of soluble sugar，total flavonoids and total saponins increased by 42.37%，67.54% and 45.65% respectively，while ascorbic acid，total phenolic acids and total alkaloids decreased by 22.86%，33.02% and 50.41% compared with that in the control. The 3 years’ old JD-h-15 and the control plants were genetically stable after ISSR marker analysis. Compared with the control，JD-h-15 plants experienced the genetic variation and the variation rate of JD-h-15 was 21.30%. These data provided a reference for the improvement of L. lancifolium genetic characters and the cultivation of new germplasm used by colchicine in lilies.

Key words: Lilium lancifolium, genetic improvement, morphological characteristics, active component, ISSR marker

符勇耀, 易德燕, 杨先茂, 蔡莉, 梁渝华, 雷美艳, 杨利平. 卷丹新种质JD-h-15的形态特征与遗传变异分析[J]. 生物技术通报, 2022, 38(11): 140-150.

FU Yong-yao, YI De-yan, YANG Xian-mao, CAI Li, LIANG Yu-hua, LEI Mei-yan, YANG Li-ping. Analysis of Morphological Characteristics and Genetic Variation in a New Germplasm Lilium lancifolium JD-h-15[J]. Biotechnology Bulletin, 2022, 38(11): 140-150.

图/表 12

图1 JD-h-15的染色体测试分析 A，B：流式细胞仪分析染色体倍性，A为卷丹，B为JD-h-15；C，D：根尖压片分析染色体数目，C为卷丹，D为JD-h-15

Fig. 1 Chromosome test analysis of JD-h-15 A，B：Analysis of the chromosome ploidy by flow cytometry，A is L. lancifolium，B is JD-h-15. C，D：Chromosome tableting using by root tips，C is L. lancifolium，and D is JD-h-15

图2 JD-h-15与卷丹植株的形态学比较 A：卷丹植株；B：JD-h-15植株；C：卷丹花部；D：JD-h-15花部；E-F：JD-h-15授粉后膨大果实；G-H：JD-h-15不同大小的无胚种子；标尺代表25 cm（A，B）、1.5 cm（C，D）和1 cm（E，H）

Fig. 2 Plant morphology comparing between JD-h-15 and L. lancifolium A：L. lancifolium plants；B：JD-h-15 plants；C：L. lancifolium flowers；D：JD-h-15 flowers；E-F：JD-h-15 fruit enlargement after pollination；G-H：different sizes of JD-h-15 embryoless seeds；scales represent 25 cm（A，B），1.5 cm（C，D）and 1 cm（E，H）

表1 JD-h-15与卷丹的主要形态特征

Table 1 Main morphological characteristics of JD-h-15 and L. lancifolium

性状 Traits	JD-h-15	卷丹L. lancifolium
株高Height/cm	123.89±2.56**	89.48±1.23
茎粗Stem diameter/cm	0.74±0.02	0.82±0.02**
单株叶片数 Leaf number per plant/cm	90.76±2.92	112.96±1.68**
叶间距Leaf interval/cm	1.39±0.03**	0.79±0.01
叶长Leaf length/cm	13.92±0.18**	10.83±0.09
叶宽Leaf width/cm	1.47±0.02**	1.16±0.01
叶长宽比Leaf length-width ratio	9.68±0.21	9.49±0.12
叶面积Leaf area/cm²	16.08±0.34**	9.80±0.12
单株叶片总面积Total leaf area per plant/cm²	1 465.80±68.09**	1 111.54±28.43
花径Flower diameter/mm	88.37±1.02	91.27±1.38
内轮花被片长Inner tepal length/cm	9.98±0.05	9.93±0.10
内轮花被片宽Inner tepal width/cm	2.63±0.03**	2.52±0.03
外轮花被片长Outer tepal length/cm	9.31±0.36	9.82±0.08
外轮花被片宽Outer tepal width/cm	1.82±0.03	1.71±0.05
花丝长Filament length/cm	7.31±0.05	7.18±0.05
花药长Anther length/cm	2.07±0.09	1.93±0.08
单个果实种子数 Number of seeds per individual fruit	236.64±5.34	0
单个果实正常大小种子数 Number of seeds in normal size per individual fruit	66.96±5.28	0
果实长Capsule length/cm	1.11±0.04	0
果实直径Capsule diameter/cm	2.83±0.06	0
鳞茎重量Bulb weight/g	54.99±5.98	61.17±6.71
鳞茎周径Bulb perimeter/cm	16.49±0.65	18.06±0.85
鳞茎最大直径Bulb max diameter/cm	5.50±0.21	6.23±0.29
珠芽重量Bulbil weight/g	0.54±0.02	0.55±0.02
珠芽最大直径Bulbil max diameter/cm	1.12±0.02	1.16±0.02

图3 JD-h-15与卷丹鳞茎特征比较 A：卷丹鳞茎；B：JD-h-15鳞茎；C：从上到下依次为卷丹内层、中层和外层鳞片；D：从上到下依次为JD-h-15内层、中层和外层鳞片；标尺代表6 cm（A，B）和2 cm（C，D）

Fig. 3 Comparison of the bulb traits between JD-h-15 and L. lancifolium A：L. lancifolium bulbs；B：JD-h-15 bulbs；C：L. lancifolium inner，middle and outer scales from up to down；and D：JD-h-15 inner，middle and outer scales from up to down. Scales represent 6 cm（A，B）and 2 cm（C，D）

图4 不同时期柱头的可授性分析 A，B：花苞长度40-50 mm的柱头；C，D：花苞长度50-60 mm的柱头；E，F：花苞长度60-70 mm的柱头；G，H：花苞长度70-80 mm的柱头；I，J：开花当天的柱头；A，C，E，G，I为卷丹，B，D，F，H，J为JD-h-15；标尺代表实际长度1 mm

Fig. 4 Analysis on the stigma acceptability of different periods A，B：Stigmas in 40-50 mm buds. C，D：Stigmas in 50-60 mm buds. E，F：Stigmas in 60-70 mm buds. G，H：Stigmas in 70-80 mm buds. I，J：Stigmas in the flowering days. A，C，E，G，I is L. lancifolium，and B，D，F，H，J is JD-h-15. Scales represent 1 mm

表2 不同时期柱头可授性分析

Table 2 Analysis on the stigma acceptability of different periods

材料 Materials	花苞40-50 mm Bud 40-50 mm	花苞50-60 mm Bud 50-60 mm	花苞60-70 mm Bud 60-70 mm	花苞70 mm以上 Bud > 70 mm	花开当天 Flowering day
JD-h-15	+	+	+ + +	+ + +	+ + +
卷丹L. lancifolium	+	+ +	+ + +	+ + +	+ + +

图5 花粉活性与花粉粒投影面积比较 A，B：卷丹和JD-h-15的花粉染色分析；C，D：卷丹和JD-h-15的花粉管萌发；E，F：卷丹和JD-h-15花粉粒投影面积分布情况；标尺代表实际长度100 μm（A，B）和200 μm（C，D）

Fig. 5 Comparison of the pollen activity and the projected area of pollen grain A，B：Staining of L. lancifolium and JD-h-15 pollens. C，D：Pollen tube germination in L. lancifolium and JD-h-15. E，F：Projection area of pollen grains in L. lancifolium and JD-h-15. Scales represent actual length 100 μm（A，B）and 200 μm（C，D）

表3 JD-h-15与卷丹的花粉、叶表皮和淀粉粒形态特征

Table 3 Morphological characteristics of pollen，leaf surface and starch grain in JD-h-15 and L. lancifolium

性状 Traits	JD-h-15	卷丹L. lancifolium
花粉粒径Pollen grain diameter/μm	66.23±0.39**	61.23±0.39
花粉粒投影面积Projected area of a pollen grain /μm²	2 023.35±33.48**	1 760.23±23.38
叶上表皮细胞长Length of leaf abaxial epidermis/μm	557.97±20.03	565.25±19.55
叶上表皮细胞宽Width of leaf abaxial epidermis/μm	73.56±1.27	70.92±1.35
叶上表皮细胞面积Area of leaf abaxial epidermis/μm²	40 646.72±1 382.35	39 513.50±1 274.04
保卫细胞长Guard cell length/μm	104.64±1.00	104.38±1.12
保卫细胞宽Guard cell width/μm	59.37±0.88	60.17±0.83
气孔长Stoma length/μm	72.16±1.28*	66.71±0.95
气孔宽 Stoma width/μm	15.91±0.34	18.48±0.95**
气孔密度Stoma density/（Stomas/mm²）	38.93±1.38	36.96±1.28
鳞茎淀粉粒径Bulb starch grain diameter/μm	23.42±0.50	26.98±0.48**
珠芽淀粉粒径Bulbil starch grain diameter/μm	17.61±0.54	23.00±0.52**
鳞茎淀粉粒投影面积Projected area of bulb starch grain/μm²	371.63±12.45	428.01±14.00**
珠芽淀粉粒投影面积Projected area of bulbil starch grain/μm²	167.46±5.48	289.54±14.02**

图6 卷丹和JD-h-15上表皮细胞和气孔观察 A，C：卷丹上表皮细胞；B，D：JD-h-15的上表皮细胞；E：卷丹叶片气孔；F：JD-h-15叶片气孔；标尺代表200 μm

Fig. 6 Observation on upper epidermal cells and stomas A，C：L. lancifolium upper epidermal cells. B，D：JD-h-15 upper epidermal cells. E：L. lancifolium leaf blade stomas. F：JD-h-15 leaf blade stomas. Scales represent 200 μm

图7 珠芽和鳞茎淀粉粒的形态比较 A：卷丹珠芽淀粉粒形态；B：JD-h-15珠芽淀粉粒形态；C：卷丹鳞茎淀粉粒形态；D：JD-h-15鳞茎淀粉粒形态；标尺代表100 μm

Fig. 7 Comparison on the morphology of starch grains in lily bulbils and bulbs A：Starch grain form in L. lancifolium bulbils. B：Starch grain form in JD-h-15 bulbils. C：Starch grain form in L. lancifolium bulbs. D：Starch grain form in JD-h-15 bulbs. Scales represent 100 μm

表4 JD-h-15与卷丹有效成分含量

Table 4 Content of active components in JD-h-15 and L. lancifolium

测试样品 Samples	淀粉 Starch/%	可溶性糖 Soluble sugar/%	抗坏血酸 Ascorbate/%	总黄酮 Total flavonoids/%	总皂苷 Total saponins/%	总酚 Total phenolic acids/%	总生物碱 Total alkaloids/%
卷丹L. lancifolium	28.805±0.007	1.310±0.001	0.070±0.001^**	0.114±0.007	0.455±0.005	0.106±0.007^**	0.613±0.004**
JD-h-15	28.452±0.518	1.865±0.022^**	0.054±0.003	0.191±0.005^**	0.668±0.012^**	0.071±0.002	0.304±0.017

图8 JD-h-15和卷丹的ISSR扩增 A：引物3A30；B：引物3A62；C：引物UBC814；D：引物UBC815；E：引物UBC835；F：引物UBC843；G：引物UBC844；H：引物UBC845；I：引物UBC857；J：引物UBC880；1-3为3个独立的卷丹样本，4-6为3个独立的JD-h-15样本，M为Marker 2000

Fig. 8 ISSR amplification of JD-h-15 and L. lancifolium A：Primer 3A30. B：Primer 3A62. C：Primer UBC814. D：Primer UBC815. E：Primer UBC835. F：Primer UBC843. G：Primer UBC844. H：Primer UBC845. I：Primer UBC857. J：Primer UBC880. 1-3 indicate three independent samples of L. lancifolium，4-6 indicate three independent samples of JD-h-15，M indicate Marker 2000

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