Developing Highly Efficient Grafting Rootstocks Based on Eucommia ulmoides BGL Gene

doi:10.13560/j.cnki.biotech.bull.1985.2025-1002

Abstract

Abstract:

Objective β-1,4-Glucanase (BGL) is an enzyme that hydrolyzes cellulose in the cell wall and effectively improves graft survival by promoting the healing between rootstock and scion. Elucidating the function of the BGL gene in Eucommiaulmoides is important for establishing an efficient grafting system and further investigating related gene functions. Method Plant expression vectors for the BGL gene, driven by the trauma-inducible promoter FAR6 and the auxin-inducible promoter GH3, were constructed, and transgenic plants were obtained through genetic transformation of E. ulmoides. Micrografting techniques were employed to compare survival rates among different scion-rootstock combinations; RT-qPCR was used to analyze the expression of wound-healing-related genes; and anatomical observations were conducted to evaluate callus formation and healing at the graft union. Result RT-qPCR results indicated that BGL expression was induced by wounding in transgenic FAR6::BGL plants and by auxin in GH3::BGL plants. Micrografting showed that the survival rate of FAR6::BGL scions (63.33%) was significantly higher than that of GH3::BGL (53.33%) and wild-type (WT) scions (33.33%). When FAR6::BGL plants were used as rootstocks grafted with WT scions, the survival rate reached 80%, significantly higher than that with WT rootstocks (30%). Furthermore, FAR6::BGL rootstocks promoted the expression of wound-healing-related genes (EuERF115-like, EuNAC1, and EuWOX13-2). Anatomical observations revealed more rapid callus formation and shorter wound-healing time at the graft junction. Conclusion Transgenic E. ulmoides rootstocks expressing FAR6::BGL significantly enhance micrografting survival rates, promote the expressions of wound-healing-related genes, and accelerate the graft union healing process, thereby providing a novel strategy to overcome challenges such as difficult rooting and low transplant survival in transgenic plants.

Key words: Eucommia ulmoides, β-1,4-glucanase gene, rootstock, micrografting, survival rate, scion, promoter

ZHANG Shan, SUN Zhi-peng, ZHAO De-gang, ZHAO Yi-chen. Developing Highly Efficient Grafting Rootstocks Based on Eucommia ulmoides BGL Gene[J]. Biotechnology Bulletin, 2026, 42(5): 292-301.

Figures/Tables 8

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
GUS-F	GGTGATTGATGAAACTGCTG
GUS-R	GAACATTACATTGACGCAGG
β-1,4--F	ATGGGTTCTCCACTTACACAGTC
β-1,4--R	TTAAGGTGCTAAATGGATATTCTTG
EuActin-F	GTGTTATGGTTGGGATGGG
EuActin-R	TGCTGACTATGCCGTGTTC
β-1,4-qRT-F	GGTTTGGAGACCACTACCCG
β-1,4-qRT-R	GTGTGTTTGGGTTGGGTTCG
EuERF115-like-qRT-F	CATCGTCATCGTCATCATC
EuERF115-like-qRT-R	ACTAAGGTGGTCCATTGAG
EuNAC1-qRT-F	AAGGATCAGTTGGAGCTGCC
EuNAC1-qRT-R	AGACATTCCTGGAAGCTGCC
EuWOX13-2-qRT-F	GGTCTGAGGGCATGTGTTTT
EuWOX13-2-qRT-R	TTGGAGATATGGGTGGTGGT

Fig. 1 Acquisition and identification of transgenic E. ulmoides resistant budsA: E. ulmoides genetic transformation flow chart (a: Suspension culture. b: Co-culture. c: Screening culture. d: Induced callus. e: Resistant bud). B: GUS detection of transgenic E. ulmoides (WT: Wild type E. ulmoides. a: FAR6::BGLE. ulmoides. b: GH3::BGLE. ulmoides). C: PCR amplification of transgenic E. ulmoides (GUS gene:304 bp). M: DNA DL2 000 marker. 1-12: Transfer FAR6::BGL plants. 13-24: Transfer GH3::BGL plants

Fig. 2 Analysis of the relative expression of BGL gene in E. ulmoidesA: Injury treatment; B:10 μmol/L IAA treatment. Different lower letters indicate significant differences at P<0.05 level. The same below

Fig. 3 Statistics of graft survival rate (A) and analysis of BGL gene expression (B)WT: Wild type E. ulmoides; FAR6: FAR6::BGLE. ulmoides; GH3: GH3::BGLE. ulmoides. The same below

Fig. 4 Acquisition and identification of transgenic E. ulmoides rootstocksA: Flow chart of genetic transformation of E. ulmoides by Agrobacterium injection (a: E. ulmoides seedlings. b: Remove the germ of E. ulmoides seedlings. c: Inject bacteria solution into E. ulmoides seedlings. d: E. ulmoides seedlings after 15 d of transformation. e: Converted E. ulmoides seedlings for two months). B: GUS detection of transgenic E. ulmoides (WT: Wild type E. ulmoides. TP: FAR6::BGLE. ulmoides). C: PCR amplification of transgenic E. ulmoides (GUS gene:304 bp) M: DNA DL 2 000 marker. 1-10: Transferring FAR6::BGL plant

Fig. 5 Expression analysis of wound healing related genes in the scions of transgenic E. ulmoides rootstock

Fig. 6 Observation of the anatomy (A) and appearance and morphology (B) of the grafting and healing process of transgenic E. ulmoides rootstockSt: Rootstock. Sc: Scion. IL: Isolation layer. Ci: Callus. Ca: Cambium. V: Vascular

Table 2 Survival rate of E. ulmoides micrografting of different rootstocks

砧木类别

Rootstock type

嫁接株数

Number of grafis （plants）

存活株数

Number of survivors （plants）

成活率

Survival rate （%）

野生型杜仲

WT E. ulmoides

转FAR6::BGL杜仲

FAR6::BGL E. ulmoides

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