Effects of different hormone combinations on tomato in vitro regeneration and related gene expression

doi:10.3969/j.issn.1004-1524.2022.09.08

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (9): 1889-1900.DOI: 10.3969/j.issn.1004-1524.2022.09.08

• Horticultural Science • Previous Articles Next Articles

Effects of different hormone combinations on tomato in vitro regeneration and related gene expression

JIN Baoxia(), WANG Weijie, ZHU Xiaolin, WANG Xian, WEI Xiaohong()

Gansu Key Lab of Crop Genetic & Germplasm Enhancement, Gansu Provincial Key Lab of Aridland Crop Science, College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China

Received:2022-01-07 Online:2022-09-25 Published:2022-09-30
Contact: WEI Xiaohong

Abstract

Abstract:

Cotyledons and hypocotyls of cherry tomato material J6 were cultured on MS medium supplemented with hormone combinations A(2 mg·L^-1 6-BA+0/0.1/0.2/0.3 mg·L^-1 IAA, numbered A1, A2, A3, A4) and B (2 mg·L^-1 ZT+0/0.1/0.2/0.3 mg·L^-1 IAA, numbered B1, B2, B3, B4), respectively, so as to study the effects of different hormone combinations on the dedifferentiation and redifferentiation of cotyledons and hypocotyls at different stages, as well as the expression of dedifferentiation genes and redifferentiation genes. The results showed that the dedifferentiation rate of cotyledons was 100% in all treatments at the 10th day, but expression level of dedifferentiation genes differed greatly. Compared with combination B, the dedifferentiation rate of hypocotyls under combination A was higher and expression level of all dedifferentiation genes were also significantly (P<0.05) up-regulated. On the 20th day, callus morphology of cotyledon was better under combination B, and expression level of dedifferentiation genes Solyc01g091420.2.1, Solyc04g072900.1.1 and Solyc04g054910.3.1 were significantly up-regulated under treatment B1 and B2. The callus morphology of hypocotyl was loosed and bright under all treatments, and the expression level of all dedifferentiated genes were significantly up-regulated only under treatment B2.On the 60th day, the redifferentiation rate of cotyledons under treatment B1 and B2 reached 67% and 58%, and the redifferentiation genes Solyc05g013540.1.1, Solyc02g022850.1.1 and Solyc10g052510.2.1 were highly expressed. The redifferentiation rate and gene expression level in hypocotyls were lower in all treatments. In conclusion, cotyledons of J6 had the best regeneration effect in vitro under the treatment of 2 mg·L^-1 ZT+0/0.1 mg·L^-1 IAA. The dedifferentiation genes Solyc01g091420.2.1, Solyc04g072900.1.1 and Solyc04g054910.3.1, and the redifferentiation genes Solyc05g013540.1.1, Solyc02g022850.1.1 and Solyc10g052510.2.1 were closely related to the regeneration process. It was also confirmed that different hormone combinations had important effects on regulating the dedifferentiation and redifferentiation process of different types of tomato explants.

Key words: cherry tomato, dedifferentiation, redifferentiation, hormone combination, qRT-PCR, molecular mechanism

CLC Number:

S641.2

JIN Baoxia, WANG Weijie, ZHU Xiaolin, WANG Xian, WEI Xiaohong. Effects of different hormone combinations on tomato in vitro regeneration and related gene expression[J]. Acta Agriculturae Zhejiangensis, 2022, 34(9): 1889-1900.

Figures/Tables 11

Table 1 Information of primers for dedifferentiated and redifferentiated genes of tomato

基因类型 Genetic types	基因注册号 Gene registration number	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')
脱分化基因	Solyc09g066260.3.1	GTCCTGCTAGATTTGCAGCCA	ACATCGATCGGGGACAGGAA
Dedifferentiated genes	Solyc06g083930.2.1	TCCTGTCTATGGTTGTGTTGCC	AAGCTTTGTGCTAACTGCCC
	Solyc01g091420.2.1	AGTGTGTTGCCGGTTGCATA	GGGTTGTTAGGTGGGCTTGT
	Solyc04g072900.1.1	CTAAGAACCGAACCCGCCTT	CCTTGTCGTAAGCCAGAGCA
	Solyc12g056980.1.1	CAATTGGGCTGAACCAAGCC	CGTTATGACGGAGATGGGGG
	Solyc04g054910.3.1	CCCAATGAAGCAAACGGGTG	AGCCAAGCTTTGGCAAATGG
再分化基因	Solyc05g013540.1.1	TGTACGAACAGCTGCCGAAT	TCATTTGCGGTAGCACCACT
Redifferentiated genes	Solyc03g082980.3.1	TCACAGACGTCCTGTTTCCG	GTACATTCACCCATGCCCCA
	Solyc12g042210.2.1	CTGCACGAGTATGGCTAGGG	TCCAACGACGTATTCGGTGA
	Solyc02g022850.1.1	GCAAGTCCAAGGGTGCAAAG	CGGTTCAACAACGCTTCTGG
	Solyc08g066840.3.1	TTGCTGTTGGAAGTAGCCGT	AGGTGCCCACCAGAAATACG
	Solyc10g052510.2.1	TCGAGCCTGCTAAAACAGCA	TTTCAATGGAACCGGGGCTT
内参基因Reference gene	UBI	TCGTAAGGAGTGCCCTAATGCTGAA	CAATCGCCTCCAGCCTTGTTGTA

Fig.1 Dedifferentiation of cotyledons and hypocotyls under different hormone combinations A, The cotyledons of 10 days under CK treatment; B, The hypocotyls of 10 days under CK treatment; C, The cotyledons of 10 days under A2 treatment; D, The hypocotyls of 10 days under B1 treatment; E, The cotyledons of 20 days under B1 treatment; F, The hypocotyls of 20 days under B1 treatment.

Table 2 Effects of different hormone combinations on dedifferentiation rate of cotyledon and hypocotyl of J6

处理 Treatments	外植体数 Number of explants	子叶脱分化率 Dedifferentiation rate of cotyledon/%		下胚轴脱分化率 Dedifferentiation rate of hypocotyl/%
处理 Treatments	外植体数 Number of explants	10 d	20 d	10 d	20 d
CK	60	0 b	0 b	0 f	0 e
A1	60	100 a	100 a	70.00±0.01 bc	71.67±2.89 b
A2	60	100 a	100 a	88.33±2.89 a	90.00±5.00 a
A3	60	100 a	100 a	78.33±7.64 ab	81.67±5.77 a
A4	60	100 a	100 a	81.67±5.77 ab	83.33±2.89 a
B1	60	100 a	100 a	35.00±5.00 e	36.67±2.89 d
B2	60	100 a	100 a	65.00±5.00 cd	66.67±2.89 bc
B3	60	100 a	100 a	56.67±2.89 d	58.33±5.77 c
B4	60	100 a	100 a	65.00±5.00 cd	66.67±2.89 bc

Fig.2 Redifferentiation of cotyledons and hypocotyls treated with different hormone combinations A, The hypocotyls of 60 days under CK treatment; B, The cotyledons of 60 days under B1 treatment; C, The cotyledons of 60 days under B4 treatment; D, The hypocotyls of 60 days under A3 treatment.

Table 3 Effects of different hormone combinations on the redifferentiation rate of cotyledon and hypocotyl of J6

处理 Treatments	外植体数量 Number of explants	子叶再分化率 Redifferentiation rate of cotyledon/%	下胚轴再分化率 Redifferentiation rate of hypocotyl/%
CK	60	0 d	0 d
A1	60	22.00±2.89 b	1.67±1.15 c
A2	60	13.00±2.89 bc	0 d
A3	60	0 d	15.00±0.01 a
A4	60	0 d	0 d
B1	60	67.00±2.89 a	1.67±0.58 c
B2	60	58.00±2.89 a	6.67±2.89 b
B3	60	15.00±0.58 bc	6.67±2.89 b
B4	60	3.00±0.58 c	1.67±0.01 c

Fig.3 Relative expression level of dedifferentiated genes in callus of cotyledons treated with different hormone combinations at the 10th day 1-6 represented Solyc09g066260.3.1, Solyc06g083930.2.1, Solyc01g091420.2.1, Solyc04g072900.1.1, Solyc12g056980.1.1 and Solyc04g054910.3.1, respectively. Data marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

Fig.4 Relative expression level of dedifferentiated genes in callus of cotyledons treated with different hormone combinations at the 20th day

Fig.5 Relative expression level of dedifferentiated genes in callus of hypocotyls treated with different hormone combinations at the 10th day

Fig.6 Relative expression level of dedifferentiated genes in callus of hypocotyls treated with different hormone combinations at the 20th day

Fig.7 Relative expression level of redifferentiation genes in callus of cotyledons treated with different hormone combinations at the 60th day 7-12 represented Solyc05g013540.1.1, Solyc03g082980.3.1, Solyc12g042210.2.1, Solyc02g022850.1.1, Solyc08g066840.3.1 and Solyc10g052510.2.1, respectively. The same as below.

Fig.8 Relative expression level of redifferentiation genes in callus of hypocotyls treated with different hormone combinations at the 60th day

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Effects of different hormone combinations on tomato in vitro regeneration and related gene expression

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