An efficient root transformation system for CRISPR/Cas9-based analyses of shoot–root communication in cucurbit crops

Abstract

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot–root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K+ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock, and by overexpressing the pumpkin tonoplast Na+/H+ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.

CsAKT1 is a key gene for the CeO2 nanoparticle's improved cucumber salt tolerance: a validation from CRISPR-Cas9 lines

“…Previously, we developed a highly efficient root transformation system for Cucurbitaceae that can be used to verify the function of cucumber genes. 37 In this study, in order to verify whether CsAKT1 is a key gene for nanoceria-improved cucumber salt tolerance, we used Cas9 gene editing technology to obtain cucumber seedlings with root deletion of CsAKT1 . Our results showed that root deletion of CsAKT1 significantly reduced the enhancement of nanoceria-improved cucumber salt tolerance (no matter with leaf or root application mode).…”

“…K + and Na + contents were measured using an atomic absorption spectrophotometer (6300C, Shimadzu). 37…”

“…The PCR products were subjected to Hi-TOM sequencing to detect gene editing efficiency. 37 The transformed cucumber seedlings were obtained, cultivated to 2 leaves, and subjected to 75 mM NaCl treatment. The morphological and physiological indices of cucumber were measured at 75 mM NaCl treatment for 7 d. The statistical analysis and sketching of the images were performed using Rstudio 4.03 software.…”

“…K + and Na + contents were measured using an atomic absorption spectrophotometer (6300C, Shimadzu). 37 2.7. Determination of ROS content, MDA, and REC After 7 days of salt stress (75 mM NaCl), H 2 O 2 , O 2 ˙−, and MDA (malondialdehyde) content and REC (relative electrical conductivity) in leaf and root samples were measured as described in our previous study.…”

The essential role of jasmonate signaling in Solanum habrochaites rootstock-mediated cold tolerance in tomato grafts

“…However, investigations on the molecular mechanism of enhancing tomato cold tolerance through grafting are rare. Plants’ abiotic stress tolerance is greatly influenced by root–shoot communication [ 13 ]. Rootstock can improve scion tolerance through long-distance communication signals, such as Ca 2+ signals, reactive oxygen species (ROS), plant hormones, water and nutrients, RNA, and peptides [ 14 , 15 ].…”