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Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

Published: November 20th, 2018



1Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, 2State Key Lab Breeding Base for Sustainable Control of Plant Pest and Disease, Zhejiang Academy of Agricultural Sciences, 3Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, 4Shanghai Biozeron Biotechnology Co., Ltd
* These authors contributed equally

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Grafting has long been employed as an agricultural technique to improve crop production and tolerance to biotic and abiotic stresses1,2,3. In heterografting systems, elite rootstocks can enhance water and nutrients uptake of plants, strengthen resistance to soil pathogens, and limit the negative effects of metal toxicity4,5, which may confer the grafts an enhanced growth vigor and increased tolerance to environmental stresses. In many cases, heterografting can also impact fruit qualities in horticultural plants, leadi....

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1. Seed Sterilization and Germination

  1. For surface sterilization, soak the bottle gourd seeds in a 500-mL beaker filled with water at 58 °C with occasional stirring, until the water temperature drops to 40 °C.
  2. Meanwhile, put 3 kg of peat soil into a nylon bag and, to sterilize it, autoclave it at 120 °C/0.5 MPa for 20 min.
  3. Keep soaking the bottle gourd seeds for 4 - 5 h more with no stirring.
    1. Once the water reaches room temperature, rinse the seeds 2x - 3x with di.......

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Figure 2
Figure 2: Phenotypes of various grafts at room temperature and cold-stressed conditions. (a) This panel shows homo- and heterografted seedlings at room temperature as the control. (b) This panel shows homo- and heterografted seedlings after 48 h of cold treatment.

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In this protocol, we described in detail a highly efficient and reproducible method to make homo- and heterografts between watermelon and bottle gourd. This method, requiring no specific equipment, is very easy to operate and typically has a very high survival rate of grafting. The method can also be used to make grafts for other cucurbits, such as between watermelon, cucumber, and pumpkin.

It is worth noting that the relative size (age) of the rootstock and scion is critical to making a succe.......

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This work was supported by the National Natural Science Foundation of China (31772291), the Research Project for Public Interest in Zhejiang Province (2017C32027), the Key Science Project of Plant Breeding in Zhejiang (2016C02051), and the National Program for the Support of Top-notch Young Professionals (to P.X.).


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Name Company Catalog Number Comments
TRIzol Reagent Invitrogen 15596026
RNA-free DNase I Takara D2270A
Truseq Small RNA sample prep Kit Illumina RS-200-0012
2100 Bionalyser Agilent 5067
DNA Polymerase Thermo Fisher Scientific F530S
UEA sRNA workbench 2.4-plant version (software) NA NA
Rfam 11.0 database (website) NA NA
miRBase 22.0 (website) NA NA
MIREAP(software) NA NA
TargetFinder (software) NA NA

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