Genetic Transformation Mediated by Agrobacterium tumefaciens in Solanum nigrum

Summary

This article provides a detailed protocol of the genetic transformation method mediated by Agrobacterium tumefaciens to produce transgenic Solanum nigrum.

Abstract

Solanum nigrumis an orphan solanaceous relative of potato and tomato, and its fresh fruit is a nutritious and delicious berry. The whole plant of S. nigrum can be used as medicine. However, the wild S. nigrum harbors some undesirable traits. Fortunately, genetic engineering techniques, especially gene-editing technologies like CRISPR/Cas9, offer valuable opportunities to rapidly improve S. nigrum by addressing these deficiencies. In most plants, plant improvement through gene-editing technology is achieved through Agrobacterium tumefaciens-mediated genetic transformation. In this article, we have detailed the genetic transformation protocols mediated by A. tumefaciens in S. nigrum. The cotyledons and hypocotyls of S. nigrum seedlings were used as explants for genetic transformation. Because S. nigrum is resistant to kanamycin, the DsRed gene was used as a visual marker to identify transgenic plants. The genetic transformation of S. nigrum can be efficiently completed according to these operation protocols. The detection of T1 transgenic plants can be directly identified by whether they emit red fluorescence, without the need for PCR identification.

Introduction

Solanum nigrum (known as black nightshade, 2n = 6x = 72) is a solanaceous relative of potato and tomato¹. The fruit is a spherical berry that turns black after ripening. The fresh fruit is a nutritious and delicious berry. The whole plant of S. nigrum can be used as medicine, with the effects of dispersing blood stasis, reducing swelling, clearing heat, and detoxifying. S. nigrum synthesizes two distinct classes of specialized metabolites from a common cholesterol precursor: steroidal saponins in its leaves and steroidal glycoalkaloids (SGAs) in its berries. Uttroside B, a steroidal saponin derived from S....

Protocol

1. Aseptic seedling cultivation

NOTE: The following steps are performed on a super-clean bench.

1. Collect wild seeds of S. nigrum in Liaocheng City, China, and name them Snlc1. Take 50 wild seeds of S. nigrum and place them in a 2 mL centrifuge tube. After soaking in 1 mL of 5% gibberellin for 30 min, discard the gibberellin solution.
2. Add 1 mL of 75% ethanol, shake for 1 min, and then discard the ethanol.
3. Add 1 mL of 2% sodium hypochlorite solution and disinfect the surface of seeds for 10 min. Shake the centrifuge tube thoroughly to sterilize the seed surface. After 10 min, d....

Results

Genetic transformation mediated by Agrobacterium tumefaciens to produce transgenic S. nigrum.
After infecting the cotyledons and hypocotyls of S. nigrum seedlings with A. tumefaciens, we could determine whether the Agrobacterium infection was successful since DsRed was used as a reporter gene. Red fluorescence appeared at the wound site after infec.......

Discussion

So far, genetic transformation methods for S. nigrum mediated by A. tumefaciens have been established in different laboratories^8,9,10,11. Most of these transformation methods use kanamycin as the selection substance^8,9,10,11. However, S. nigrum

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2X SanTaq PCR Master Mix (with Blue Dye)	Sangon Biotech (Shanghai) Co., Ltd.	B532061	The 2X SanTaq PCR Mix includes MgCl?, dNTPs, Taq DNA Polymerase, PCR buffer, loading dye, and PCR enhancer.
6-BA	Sangon Biotech (Shanghai) Co., Ltd.	C14819266	Artificial cytokinins
Agar	Tianjin Damao Chemical Reagent Partnership Enterprise (Limited Partnership)	CAS NO:9002-18-0	Solids
AS	Sangon Biotech (Shanghai) Co., Ltd.	K914BA0002	Induce efficient expression of Agrobacterium
Carb	Sangon Biotech (Shanghai) Co., Ltd.		Antibiotic
Cef	Sangon Biotech (Shanghai) Co., Ltd.	I526BA0010	Antibiotic
gibberellin	Sangon Biotech (Shanghai) Co., Ltd.	K514BA0009	Promotes seed germination
kanamycin	Sangon Biotech (Shanghai) Co., Ltd.	I614BA0013	Antibiotic
LB medium	Sangon Biotech (Shanghai) Co., Ltd.	B540113	Grow the bacteria
MicroPulser ( Gene Pulser Xcell)	Bio-Rad, US	617BR1 06783	Gene Pulser Xcell
MS solid medium	phytotech Co., Ltd., US	HCA0519228A	Murashige & Skoog Basal Medium with Vitamins, M519
plastic box	LiaoSu, China	T909H	Plant planting box
rifampicin	Sangon Biotech (Shanghai) Co., Ltd.	S180305	Antibiotic
sucrose	Sangon Biotech (Shanghai) Co., Ltd.	CAS NO:57-50-1	As a carbon source
Tanon-5200Multi machine	Tanon Co., Ltd., China	5200Multi	Accelerated separation
wild Solanum nigrum			collected in Liaocheng City
ZT	Sangon Biotech (Shanghai) Co., Ltd.	J921BA0014	Plant growth regulators