Establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants

doi:10.6048/j.issn.1001-4330.2024.07.013

Abstract

Abstract:

【Objective】Study on establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants.【Methods】The cotyledons of four melon varieties, including Bawudun, Laohangua, Qiligan, and Xinmiza No. 11 (86-1).The sweet melon cotyledons were infected by Agrobacterium tumefaciens mediated method, and a genetic transformation and regeneration system of Laohan melon was established by tissue culture method and then genetically edited plants were obtained through PCR detection.【Results】The results showed that Laohan melon was selected as the dominant variety from four melon varieties as the explants. It was pre-cultured on MS+6-BA 1.0 mg/L+NAA 0.05 mg/L medium for 2 days and co-cultured for 3 days, then it was further cultured on MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+hygromycin 5 mg/L+cephalosporin 250 mg/L screened medium for 7 days, after that, small buds were seen growing on MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+hygromycin 5 mg/L+cephalosporin 250 mg/L adventitious bud induction medium for 1-2 weeks. The induced buds were placed on both non hygromycin and hygromycin added bud elongation medium for cultivation. It was found that the growth rate of the induced buds on the non hygromycin added bud elongation medium was faster than that on the hygromycin added medium, which shortened the growth of the buds to the two leaf seedling stage by 3 weeks. One bud DNA was cut off in a sterile environment and 21 transformed plants were examined by RT-PCR.【Conclusion】Gene edited plant buds were successfully selected on a shoot elongation medium with added hygromycin. The use of a shoot elongation medium without added hygromycin can shorten the time for obtaining gene edited plants by 3 weeks.

Key words: muskmelon; Agrobacterium mediated; genetic transformation; genome edited plant

摘要：

【目的】研究不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取。【方法】选用巴吾顿、老汉瓜、其里甘、新密杂11号(86-1)4个甜瓜品种的子叶为外植体,通过农杆菌介导法侵染甜瓜子叶,采用组织培养法建立老汉瓜的遗传转化再生体系,运用PCR法检测获取基因编辑植株。【结果】从4个甜瓜品种中筛选出老汉瓜为优势品种作为外植体,在MS+6-BA 1.0 mg/L+NAA 0.05 mg/L培养基预培养2 d和共培养3 d,在MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+潮霉素5 mg/L+头孢霉素250 mg/L筛选培养基上继续培养7 d,在MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+潮霉素5 mg/L+头孢霉素250 mg/L的不定芽诱导培养基上培养1~2周可见长出的小芽,将诱导芽分别放置于不加潮霉素和加潮霉素的芽伸长培养基上培养,诱导芽在不加潮霉素的芽伸长培养基上的生长速度比加潮霉素的培养基生长快,可使芽生长至2叶苗期缩短3周,在无菌环境下剪去一个芽提取DNA,并采用RT-PCR方法检测获得21株转化植株。【结论】筛选出基因编辑植株嫩芽,不加潮霉素的芽伸长培养基可将获得基因编辑植株周期缩短3周。

关键词: 甜瓜, 农杆菌介导, 遗传转化, 基因编辑植株

CLC Number:

S652

Kadierayi Maimaiti, ZHOU Tingting, HAN Sheng, Meilikehan Rexiti, Yushanjiang Maimaiti. Establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants[J]. Xinjiang Agricultural Sciences, 2024, 61(7): 1666-1672.

卡地尔阿依·买买提, 周婷婷, 韩盛, 梅丽克汗·热西提, 玉山江·麦麦提. 不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取[J]. 新疆农业科学, 2024, 61(7): 1666-1672.

Figures/Tables 9

Tab.1 Specific primers for disease resistance detection

引物名称 Primers	引物序列(5'-3') Sequence (5'-3')	扩增片段大小 Product size (bp)
HPT-1F	GAAAAGTTCGACAGCGTCTCC	407
HPT-1R	CGTCCATCACAGTTTGCCAGT	407
HPT-2F	TCGGACGATTGCGTCGCATC	720
HPT-2R	AGGCTATGGATGCGATCGCTG	720
HPT-3F	TGAACTCACCGCGACGTCTGT	980
HPT-3R	TGCGCCCAAGCTGCATCAT	980

Tab.2 Primer sequence

组分 Component	体积 Volume(μl)
Mix	10
正向引物 Forward primer	1
反向引物 Reverse primer	1
ddH₂O	7
模板 Template	1
总计Total	20

Tab.3 The difference of recovery rate and adventitious bud induction among infected varieties

品种名称 Variety name	总外植体数(块) Total number of explants (pieces)	出愈数(个) Number of healing points	出愈率 Healing rate (%)	不定芽诱导数(个) Adventitious bud inducing derivative (number)	不定芽诱导率 Adventitious bud induction rate (%)
巴吾顿 Bawudun	420	294	70	9	2.14
老汉瓜 Laohangua	420	362	86.19	36	8.57
其里甘 Qiligan	420	353	84.04	0	0
新密杂11号 Xinmiza 11	420	294	70	17	4.04

Tab.4 Differences in Browning rate and adventitive bud elongation among infected varieties

品种名称 Variety name	总外植体数(块) Total number of explants (pieces)	褐化数(个) Browning number (pieces)	褐化率 Browning rate (%)	芽伸长数(个) Number of bud elongation (pieces)	芽伸长率 Bud elongation rate (%)
巴吾顿 Bawudun	420	243	57.85	1	0.23
老汉瓜 Laohangua	420	200	47.61	8	1.90
其里甘Qiligan	420	260	61.90	0	0
新密杂11号 Xinmiza 11	420	220	52.38	1	0.23

Fig.1 Growth difference of gene-edited plants on bud elongation medium with and without hygromycin Note: A: Before culturing with hygromycin medium; B: After 2 weeks of cultivation on hygromycin medium; C: Before culturing in a culture medium without adding hygromycin; D: After 2 weeks of cultivation in medium without added hygromycin

Fig.2 Genome of bud extracted by gene editing Note: M: DNA marker of D2000; 1-21: DNA of leaves of genome editing plants

Fig.3 Partial gene-edited plants

Fig.4 PCR amplification results of gene-edited seedlings Note: M: DNA marker of D2000; 1-9 are gene edited seedlings obtained from SG10592; 10-21 are gene edited seedlings obtained from SG10610

Fig.5 Diagram of the entire process of obtaining gene edited plants Note: A: Agrobacterium activated by streak method; B: Aseptic seedlings obtained through inoculation of melon seeds; C: Cultivated sweet melon cotyledons; D: Buds screened for resistance to hygromycin; E: Genome editing buds elongate on bud elongation medium; F: Adventitious shoot roots in rooting culture medium for about 16 days; G: Genome editing seedlings of transplanted soil; H: Genome editing seedlings transplanted for 15 days; I: Genome editing plants were cultured in an artificial climate chamber

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