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. nigrum, has recently been approved by the US Food and Drug Administration as an orphan drug for the treatment of hepatocellular carcinoma². The knockout of the GAME15 gene in S. nigrum resulted in plants lacking both saponins and steroidal alkaloids, and also revealed that steroidal saponins control serious agricultural insect pests². Polyphenols in S. nigrum reduce weight and body fat by affecting adipocytes and lipid metabolism³. S. nigrum can enrich cadmium and is used as a model plant for studying cadmium enrichment^4,5. S. nigrum is an undomesticated plant, and its research foundation is relatively weak. It is inspiring that the research on S. nigrum has been gradually increasing in recent years. The genome of the hexaploid S. nigrum has recently been assembled, and S. nigrum might have evolved from its diploid Solanum americanum (2n = 2x = 24)^6,7. Therefore, S. nigrum is a plant that possesses both medicinal and edible value. Research on S. nigrum not only contributes to a deeper understanding of its potential applications in the treatment of human diseases but also provides valuable insights into the enhancement of agricultural production. So, establishing a genetic transformation system in S. nigrum is the basis for genetic improvement through transgenic and gene editing technology.

There are several reports that focus on the Agrobacterium tumefaciens-mediated genetic transformation of S. nigrum^8,9,10,11. In our previous work, we established an A. tumefaciens LBA4404-mediated genetic transformation system for S. nigrum, and transformed AcMYB110 into S. nigrum, obtaining new germplasms of S. nigrum with increased anthocyanin content¹. Transgenic and gene-edited S. nigrum plants were obtained with A. tumefaciens EHA105-mediated genetic transformation¹⁰. Different fluorescent proteins have green, red, yellow, orange, and blue fluorescence emissions and are widely used in indicating cellular life activities¹². In the genetic transformation process of plants, red fluorescent genes are often used as reporter genes to distinguish genetically modified events from non-genetically modified events¹³. Using the DsRed gene as a reporter gene allows for convenient discrimination of transgenic events, while using the R2R3-MYB transcription factor as a reporter gene may be influenced by the genotype of the plants¹⁴. In transgenic progeny, transgenic plants can also be easily distinguished from non-transgenic plants using optical instruments (e.g., fluorescence microscopes or imaging systems), eliminating the need for PCR-based identification. By integrating the fluorescent protein reporter gene with CRISPR gene-editing technology, the fertility genes in maize were precisely deleted, resulting in the creation of a nuclear sterile line and a controlled nuclear sterile maintainer line¹⁵. The self-pollination and fruiting of maintainer plants will produce 1:1 offspring of maintainer and sterile lines. Based on different luminescent characteristics, non-destructive sorting of maintainer and sterile line seeds can be achieved through visual or machine recognition¹⁵. Thus, employing the fluorescent protein gene as a reporter gene simplifies the identification of transgenic seeds or plants without the need for PCR analysis. This represents a significant advantage in the field of genetic transformation. This method aims to describe the protocol of Agrobacterium tumefaciens-mediated genetic transformation in S. nigrum, using the red fluorescent gene, DsRed, as a reporter gene.

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, discard the sodium hypochlorite.
4. Wash the seeds 5x with sterilized water to remove residual sodium hypochlorite. Place the seeds in Petri dishes with a diameter of 90 mm containing 1/2 MS solid medium. Culture the plates in a plant growth chamber under a photoperiod of 16 h light/8 h dark at 25 °C (Figure 1A).

2. Culture of A. tumefaciens LBA4404

1. Transform the binary vector pRed1305¹⁶ harbored with a red fluorescent gene, DsRed2,into A. tumefaciens LBA4404 by electroporation with MicroPulser with parameters: Voltage 1800 V, Capacitance 25 µF, Resistance 200 Ω, Cuvette 1 mm. Culture the bacteria on solid LB medium with 50 mg/L kanamycin and 50 mg/L rifampicin at 28 °C for 48 h.
2. Pick up one clone of LBA4404 harboring pRed1305 and culture it in 1 mL of liquid LB medium with 50 mg/L kanamycin and 50 mg/L rifampicin in a 15 mL centrifuge tube in a table concentrator with 180 rpm at 28 °C for 12 h.
3. Add 50 mL of liquid LB with the same antibiotic to 1 mL of bacterial solution and then culture in a table concentrator with 200 rpm at 28 °C for 6 h.
4. Stop culturing when the OD₆₀₀ of the bacterial culture medium is 0.4, and then centrifuge the bacterial solution at 2200 x g for 10 min at 25 °C.
5. Resuspend the centrifuged bacteria with infection solution (IS: M519 4.43 g/L, sucrose 30 g/L, 6-BA 2 mg/L, AS, 100 µmol/L, pH 5.8) and adjust bacterial solution concentration to OD₆₀₀= 0.6 with a spectrophotometer.

3. Explant infection with A. tumefaciens

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

1. Grow the seeds for 7 days until the seedling cotyledons have unfolded (Figure 1B). Cut the two cotyledons and hypocotyl with a length of approximately 1 cm from the seedlings with a sterilized, sharp scalpel in a culture dish with filter paper moistened with bacterial IS and discard the primary roots. Be careful when using a scalpel.
2. Infuse the explants in the infection solution for 30 min. Place the explants on dry filter paper to allow the bacterial solution on the surface of the explant to be absorbed.
3. Place the explants in IS solid medium (0.7% Agar; Figure 1C). Place the explants in a plant growth chamber without light at 19 °C and culture for 3 days.

4. Differentiation of explants

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

1. Pick the explants up from the plant growth chamber at 19 °C. Wash the explants using sterilized water 5x and place the explants on dry, sterilized filter paper.
2. Place the explants in shoot differentiation (SD) medium (SD: M519 4.43 g/L, sucrose 30 g/L, 6-BA 2 mg/L, ZT 0.5 mg/L, Cef 100 mg/L, Carb 100 mg/L, 0.7 g/L Agar, pH 5.8), and culture at 25 °C for 4 weeks (Figure 1D,E).
3. Cut off the callus/buds of explants showing red fluorescence under the green excitation light of a handheld fluorescent lamp using a scalpel, and then transfer them to a new SD medium, and continue to culture for 4 weeks at 25 °C (Figure 1F,G).

5. Rooting and transplant

1. Cut off the buds showing red fluorescence with a handheld fluorescent lamp, transfer them to the rooting medium (RM: M519 4.43 g/L, sucrose 30 g/L, 6-BA 2 mg/L, ZT 0.5mg/L, Cef 100 mg/L, Carb 100 mg/L, 0.7 g/L Agar, pH 5.8), and culture them for 2-4 weeks at 25 °C to promote root production.
2. Transplant the seedlings showing red fluorescence with root length of more than 2 cm to soil ( Figure 1H-K). Before transplantation, remove the agar from the roots by hand to avoid excessive growth of bacteria in the roots.

6. Testing genetically modified T0 plants with PCR amplification

1. Extract the genomic DNA of an independent transgenic line using the Cetyl Trimethyl Ammonium Bromide (CTAB) method¹⁷.
2. Amplify DsRed gene with primer set, RedF (5'-TGGCCTCCTCCGAGAAC-3') and RedR (5'-CGGAGGGGAAGTTCACG-3') using 10 ng genomic DNA as a template in a 15 µL reaction volume containing 7.5 µL of 2x PCR Master Mix and 0.3 µL of each primer (10 µmol/L). The expected DNA fragment was 393 bp. Subject the PCR products to 1% agarose gel electrophoresis to determine whether they are transgenic plants.

7. Identification of transgenic plants in the T1 generation with a fluorescent lamp

1. Harvest the fruit of T0 transgenic plants of S. nigrum that have completely turned black-purple in color and then squeeze out the seeds inside the fruit by hand. Naturally air dry the seeds at room temperature for about 1 week.
2. Culture the dried seeds in 1/2 MS medium according to steps 1.1 to 1.4 of this protocol and place in a plant growth chamber for 1 week. Distinguish transgenic seedlings and non-transgenic seedlings with a handheld fluorescent lamp.

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