利用转基因毛状根高效培育大豆嵌合植株的方法

doi:10.11983/CBB23021

摘要/Abstract

摘要： 建立高效的大豆(Glycine max)转基因毛状根嵌合植株体系对于推动大豆功能基因组学研究具有重要意义。该研究利用3种大豆基因型材料比较了不同共培养条件下毛状根诱导率及成活率。结果显示, 用发根农杆菌(Agrobacterium rhizogenes)侵染外植体并在黑暗条件下共培养1天是诱导毛状根形成的有效策略。研究发现清除下胚轴处不定根可显著增加毛状根的数目并促进根系生长, 进而提高转基因毛状根的阳性率。毛状根诱导14天接种根瘤菌, 可增强生长初期转基因毛状根与根瘤菌的接触, 从而提高大豆的结瘤效率。该研究成功建立了一种高效培育大豆转基因毛状根嵌合植株的方法, 可广泛应用于大豆基因功能研究。

关键词: 大豆, 毛状根, 嵌合植株, 结瘤, 优化

Abstract: Chimeric soybean plants with transgenic hairy roots is very important for soybean functional genomics. In this study, we used three soybean genotypes to compare their hairy root induction rate and plant survival rate under different co-cultivation conditions. Our results showed that co-culturing the explants infected by Agrobacterium rhizogenes for 1 d under dark conditions was an effective strategy to induce hairy roots. We also found that removing the adventitious roots (AR) at hypocotyl significantly increased number of hairy roots, enhanced their growth and subsequently improved the positive rate of transgenic hairy roots. Furthermore, we found that the inoculation with rhizobium at 14 d of induction was able to enhance the contact between the bacteria and the transgenic hairy roots at early growth stages, and thus improved the soybean’s nodulation efficiency. Taken together, we successfully established a simple and efficient method to generate chimeric soybean plants with transgenic hairy roots. This method can be widely used in soybean gene functional studies.

Key words: soybean, hairy root, chimeric plant, nodulation, optimization

陈佳欣, 梅浩, 黄彩翔, 梁宗原, 全依桐, 李东鹏, 布威麦尔耶姆·赛麦提, 李欣欣, 廖红. 利用转基因毛状根高效培育大豆嵌合植株的方法. 植物学报, 2024, 59(1): 89-98.

Jiaxin Chen, Hao Mei, Caixiang Huang, Zongyuan Liang, Yitong Quan, Dongpeng Li, Buweimaieryemu·Saimaiti , Xinxin Li, Hong Liao. A Highly Efficient Method to Generate Chimeric Soybean Plant with Transgenic Hairy Roots. Chinese Bulletin of Botany, 2024, 59(1): 89-98.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB23021

https://www.chinbullbotany.com/CN/Y2024/V59/I1/89

图/表 6

图1 发根农杆菌诱导大豆下胚轴转基因毛状根产生嵌合植株流程 (A) 萌发4天后的大豆幼苗(bar=2 cm); (B) 在下胚轴处以45°角切开形成伤口(bar=1 cm); (C) 从培养皿中收集发根农杆菌(bar=1 cm); (D) 在伤口处涂菌(bar=1 cm); (E) 共培养(bar=1 cm); (F) 将外植体移入蛭石中(bar=5 cm); (G) 培养10天后诱导出愈伤组织(bar=1 cm); (H) 诱导14天后毛状根形成(bar=1 cm); (I) 毛状根生长20天后的状态(bar=2 cm); (J) I图的局部放大(bar=2 cm)

Figure 1 Procedure of generating chimeric soybean plants with Agrobacterium rhizogenes induced hypocotyl transgenic hairy roots (A) Soybean seedlings at 4 d post germination (bar=2 cm); (B) Hypocotyl was cut to form wound site with an angle of 45° (bar=1 cm); (C) A. rhizogenes were collected in petri dishes (bar=1 cm); (D) Apply bacteria to the wound (bar=1 cm); (E) Co-cultivation (bar=1 cm); (F) Inoculated explants were planted into vermiculite (bar=5 cm); (G) Callus came out after 10 d of cultivation (bar=1 cm); (H) Hairy roots emerged at 14 d post induction (bar=1 cm); (I) Growth performance of hairy roots after 20 d of growth (bar=2 cm); (J) Partial enlarged view of image I (bar=2 cm)

表1 不同基因型大豆在光/暗条件下毛状根(HR)形成的对比

Table 1 Comparison of hairy roots (HR) generated from different soybean genotypes under light/dark conditions

Treatments	Genotype	Co-cultivation time (d)	Efficiency of induction (%)	Number of chimeric plant with HR	Surviving rate (%)	Number of surviving plants	Total number of plants
Dark	Ws82	0	87.0	20	76.7	23	30
		1	96.2	25	86.7	26	30
		3	89.7	26	96.7	29	30
	HN66	0	87.5	21	80.0	24	30
		1	95.7	22	76.7	23	30
		3	83.3	15	60.0	18	30
	BX10	0	93.3	14	75.0	15	20
		1	94.7	18	95.0	19	20
		3	82.4	14	85.0	17	20
Light (100 µmol∙m^-2∙s^-1)	Ws82	0	82.4	21	76.7	23	30
Light (100 µmol∙m^-2∙s^-1)		1	91.3	21	76.7	23	30
		3	68.2	15	73.3	22	30
	HN66	0	90.0	18	66.7	20	30
		1	83.3	20	80.0	24	30
		3	90.5	19	70.0	21	30
	BX10	0	86.7	13	75.0	15	20
		1	83.3	15	90.0	18	20
		3	80.0	12	75.0	15	20

图2 清理下胚轴处的不定根对毛状根诱导的影响 (A) 不清理与清理不定根条件下毛状根生长对比(bars=1 cm); (B) 每条毛状根生长的侧根数目; (C) 根长; (D) 根表面积; (E) 根平均直径。数据为25个毛状根的平均值±标准差。***表示在0.001水平差异显著。AR: 不定根; HR: 毛状根

Figure 2 Effect of cleaning adventitious root at hypocotyl on induction of hairy roots (A) Comparison of hairy roots between non-clean and clean adventitious root condition (bars=1 cm); (B) Number of lateral roots per hairy root; (C) Root length; (D) Root surface area; (E) Average root diameter. Data are means±SD from 25 hairy roots. *** indicate significant differences at 0.001 level. AR: Adventitious root; HR: Hairy root

图3 清理下胚轴处的不定根对毛状根阳性率的影响 (A) 不清理与清理不定根条件下毛状根GUS染色, 椭圆形及箭头示非转基因毛状根(bars=1 cm); (B) 每个外植体产生的毛状根总数; (C) 每个外植体阳性毛状根数目; (D) 阳性率。数据为14个外植体的平均值±标准差。*和***分别表示在0.05和0.001水平差异显著。

Figure 3 Effect of cleaning adventitious root at hypocotyl on positive rate of transgenic hairy roots (A) GUS staining of hairy roots under non-clean and clean adventitious roots conditions, the oval and arrows indicate non-transgenic hairy roots (bars=1 cm); (B) Total number of hairy roots generated from each explant; (C) Number of transgenic hairy roots per explant; (D) Efficiency of transgenic hairy roots. Data are means±SD from 14 explants. * and *** indicate significant differences at 0.05 and 0.001 levels, respectively.

图4 清理不定根对毛状根结瘤的影响 (A), (D) 不清理(A)与清理(D)不定根的大豆嵌合植株(bars= 2 cm); (B), (E) 分别为图A和D方框中的局部放大(bars=1 cm); (C), (F) 毛状根GUS染色(bars=1 cm)。红色箭头指示毛状根上的根瘤。AR同图2。

Figure 4 Effect of cleaning adventitious root on nodulation of hairy roots (A), (D) Soybean chimeric plants without (A) or with (D) cleaning adventitious root (bars=2 cm); (B), (E) Partial enlarged view of image A and D in box (bars=1 cm); (C), (F) GUS staining of hairy roots (bars=1 cm). The red arrows indicate nodules on hairy roots. AR is the same as shown in Figure 2.

表2 不清理与清理不定根对转基因毛状根根瘤数目的影响

Table 2 Effect of non-clean (NCAR) and clean (CAR) adventitious root on nodule number in transgenic hairy roots

Treatments	Chimeric plants	Hairy roots	Nodule number
NCAR	1^st	1^st	3
		2^nd	4
		3^rd	2
	2^nd	1^st	2
		2^nd	1
		3^rd	3
	3^rd	1^st	4
		2^nd	2
		3^rd	1
Average			2.4
CAR	1^st	1^st	7
		2^nd	6
		3^rd	8
	2^nd	1^st	10
		2^nd	8
		3^rd	6
	3^rd	1^st	5
		2^nd	7
		3^rd	9
Average			7.3***

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