Protocol for imbibed seed piercing for Agrobacterium-mediated transformation of jute

Summary Here, we present a streamlined Agrobacterium-mediated transformation protocol for jute (Corchorus sp.). We describe steps to pierce and vacuum infiltrate imbibed jute seeds with Agrobacterium suspension. We then detail procedures for selecting transformed seeds by using a hygromycin-B-supplemented medium. This approach can achieve transformation efficiencies of 20.44% ± 1.17% and 15.55% ± 0.58% for tossa (C. olitorius) and white (C. capsularis) jute, respectively. Demanding minimal resources and time, this protocol can elevate genetic engineering research in jute fiber crops. For complete details on the use and execution of this protocol, please refer to Majumder et al. (2020).1


Note:
The final molar concentration (M) of the timentin stock is 0.857 M.
CRITICAL: Use timentin antibiotic to remove bacterial contamination, including Agrobacterium tumefaciens strain LBA4404.
Pause point: Store this stock solution at 4 C and use it within 72 h.
Culture media for Agrobacterium: CRITICAL: The culture of Agrobacterium tumefaciens strain LBA4404, which harbors the pCAMBIA1301 plant transformation vector, is made using Luria broth (HiMedia Lab., India, Cat# M575-500G) supplemented with kanamycin and rifampicin antibiotics.The pCAMBIA1301 vector carries the kanamycin resistance gene, while the Agrobacterium LBA4404 strain possesses the rifampicin resistance gene.
Using the following components, prepare culture media to grow Agrobacterium cells.
CRITICAL: After autoclaving, add kanamycin and rifampicin antibiotics (previously filter sterilized) to the medium, only after it has reached approximately temperature around 45 C.
Pause point: Store this medium at 4 C and use within 2 weeks.
Infiltration & co-cultivation media CRITICAL: This liquid medium facilitates the infiltration of Agrobacterium cells into the seeds and supports their growth during the co-cultivation period.Acetosyringone is an important part of the medium that helps vir genes become active in Agrobacterium cells, which speeds up the transformation process. 11The absence of acetosyringone in the medium leads to necrosis and browning of the explant. 3ing the following components, prepare the 'Infiltration & Co-cultivation' medium.Autoclave the medium at 121 C for 20 min at 15 lbs pressure.
Note: Add acetosyringone (filter sterilized) to the medium after autoclaving and just before use, only when it reaches temperature around 45 C.
Pause point: Store the medium at 4 C and use within 2 weeks.

Agro-washing media
CRITICAL: This medium eliminates Agrobacterium cells after the co-cultivation step in the transformation process.The precise concentration of the timentin antibiotic is key to ensuring the complete removal of Agrobacterium cells, preventing their undesirable overgrowth, which can hinder the subsequent steps.
Using the following components, prepare the 'Agro-washing' medium.
Note: Adjust the pH to 5.6 with 2 M NaOH solution before you autoclave.
Note: Add timentin antibiotic (filter sterilized) to the autoclaved media after it reaches temperature around 45 C.
Pause point: Store the medium at 4 C and use within 2 weeks.

Selection media
CRITICAL: The purpose of this hygromycin-B antibiotic-supplemented medium is to select transgenic jute plants.The transformation vector pCAMBIA1301 carries the hygromycin-B resistance gene (hptII), which is successfully transferred to transgenic jute plants during transformation.These transgenic plants can survive and grow in this medium.Notably, tossa jute (JRO524) selection requires a higher hygromycin-B concentration compared to white jute (JRC212).
Using the following components, prepare the selection medium.

Note:
The same medium is used as rooting medium without the hygromycin-B and timentin antibiotics.

Agarose gel preparation
Mix the following components to prepare the agarose gel.
Dissolve the components by heating (75 C-80 C) using a hotplate or microwave.When solution temperature is around 45 C, add 2 mL of ethidium bromide (from the stock of 100 mg/mL).Keep aside at 22 C-25 C temperature for solidification.

STEP-BY-STEP METHOD DETAILS
Most of the steps require maintaining sterile conditions and using a laminar workbench.Any steps that can be performed outside of the laminar workbench are specifically mentioned.

Seed surface sterilization of jute
Timing: 1.5-2 h This step describes how to remove contamination from the jute seeds.Fungal and bacterial contamination of the seeds needs to be controlled at the very beginning of the experiment without damaging the jute seeds.This step describes the protocol of jute sterilization by the application of Bavistin (carbendazim 50%) fungicides, ethanol, sodium hypochlorite and Tween-20.
1. Examine JRC212 and JRO524 jute seeds to select only those that are filled and healthy.2. Immerse 150 jute seeds in 5 mL of 0.1% Bavistin solution, a commercially available fungicide, for 10 min.
CRITICAL: For steps 3-8, perform the procedures within a laminar workbench to maintain aseptic conditions.Proper care should be taken to avoid any contamination during the process.
3. Remove seeds from the Bavistin solution.4. Wash the selected seeds twice with sterile water for 2 min in a 100 mL conical flask.Note: Seal the flask's mouth with Parafilm to prevent contamination when stirring using a magnetic stirrer outside of the laminar.
Alternatives: Instead of a magnetic stirrer, a shaker incubator can also be used, but with a magnetic stirrer, better results are observed.
CRITICAL: Adjust the stirring speed to prevent excessive seed coat detachment from the jute seeds (Methods video S1).Methods video S1.Surface sterilization of jute seeds-Demonstrates the surface sterilization process for jute seeds in preparation for Agrobacterium-mediated transformation, related to step 7 Note: The change in solution color from pale yellow to light brown is due to the leaching of seed coat color in the solution.
8. Wash seeds 3-4 times with sterile water for 5 min each to thoroughly rinse off the sodium hypochlorite solution.
Note: Continue rinsing thoroughly such that no more foaming (developed by Tween-20 remains) is visible and ensuring that no foam is produced during the final wash, also, getting rid of any residual smell of sodium hypochlorite solution.
9. Place the sterilized seeds on sterile Whatman No. 1 filter paper (GE Healthcare, USA Cat# 1001-090) to remove excess water.
Note: Remove any damaged seeds or any seeds with detached cotyledons in this step.

Timing: 12-24 h
This step describes explant preparation for the Agrobacterium-mediated jute transformation.Here the explant is germinated jute seeds.Only water is required as germination medium for the jute seeds.
CRITICAL: Perform the following steps in a laminar workbench to maintain aseptic conditions.
10. Place the sterilized jute seeds on a filter paper within a petri plate (100 mm).11.Add 3 mL of sterile water to the petri plate containing the seeds.a. Seal the petri plate with Parafilm.b.Cover it with aluminum foil to prevent exposure to light.

Note:
The amount of water required for germination depends on the quantity and size of seeds used in the experiment.For 100-150 seeds, 3 mL is sufficient, and it should be increased as the number of jute seeds increases.
12. Put the petri plate in an incubator (Being Scientific, USA THZ-98A) at 37 C for a duration of 12-24 h.

Note:
The duration of incubation may vary based on the specific seed cultivars.Tossa jute (C.olitorius) typically requires more time compared to white jute (C.capsularis) cultivars.
Alternatives: Incubation can also be carried out at 22 C-25 C temperature.However, in such cases, a slightly longer incubation period might be necessary.

Preparation of bacterial culture media
Timing: 2 h This step describes the process of culture media preparation for Agrobacterium tumefaciens.The Luria broth (LB) powder is weighed, dissolved in water and divided into glass tubes.The tubes are sealed and autoclaved.Later kanamycin is added to the media before it solidifies.At the end of this step, we will receive a medium where the Agrobacterium can be selectively grown.
13. Weigh 2.0 g of LB powder (HiMedia, India).14. Dissolve it in 100 mL of distilled water.15.Distribute 10 mL of the media into each glass tube (Borosil, India Cat# 9820U08).16.Securely seal the tubes with cotton plugs.17.Autoclave the tubes at 121 C for 20 min at 15 lbs pressure (using a Jeio Tech ST-65G).18.After autoclaving, add 10 mL of kanamycin (HiMedia, India) to the 10 mL LB media.
Note: Add the kanamycin antibiotic to the autoclaved media after it reaches around 45 C.
Pause point: Store the autoclaved LB media-containing tubes at 4 C and use them within 2 weeks.

Timing: 8-12 h
This step highlights the process of Agrobacterium tumefaciens LBA4404 culture in the abovementioned LB media.Kanamycin containing LB media is used to grow Agrobacterium cells harboring pCAMBIA1301 plasmid which contain the kanamycin resistant maker gene (nptII).
19. Add 10 mL of Agrobacterium LBA4404 primary culture to 10 mL of LB media containing kanamycin.20.Incubate the mixture in an incubator shaker at 28 C for 12 h.21.Measure the optical density (OD) of Agrobacterium LBA4404 culture at 600 nm using a spectrophotometer (Eppendorf, Germany, Model: Biophotometer Plus).22. Transfer the culture to a sterile 50 mL centrifuge falcon tube (Tarsons, India Cat# 546041).23.Centrifuge it at 2600 3 g for 5 min at 10 C in a centrifuge (Eppendorf, Germany, Model# 5810R) to pellet the Agrobacterium cells.24.Resuspend the pellet in fresh LB media to achieve an OD 0.3; this is equivalent to approximately 1.5 3 10 8 cells/mL.
Alternatives: A full loop of Agrobacterium culture from a 4 C stored culture plate can be directly resuspended in fresh 'infiltration and co-cultivation medium' to achieve OD 0.3.In this case, there is no need for a 12-14 h grown culture, which is a time saver.

Protocol
The imbibed seed piercing method (ISPM) of Agrobacterium-mediated transformation (AMT)

Timing: 2 months
This major step describes Agrobacterium-mediated transformation in jute.Germinated jute seeds are used as explant.A tiny hole is made by piercing the seeds.This facilitates penetration of Agrobacterium cells into the seeds during the vacuum infiltration process.At the end of this step T 0 plants will be developed and grow in the greenhouse.
CRITICAL: Perform the following step 25 to 33, step 35 to 42, and step 44 to 45 in a laminar air flow work bench to maintain aseptic conditions 25. Pierce imbibed jute seeds with a sterilized needle near the apical meristematic zone of the plumule (Methods video S2).
CRITICAL: Needles must be flame sterilized and allowed to cool periodically before each piercing.Each seed can be pierced a maximum of two times without permanent damage.
26. Soak the pierced seeds in the 'infiltration and co-cultivation medium'.27.Cover the soaked seeds with aluminum foil and incubate for 30 min to 1 h in a laminar workbench at 22 C-25 C temperature.

Note:
The 'infiltration and co-cultivation medium' contains Agrobacterium cells (OD 0.3), therefore gentle agitation is necessary during incubation to ensure the cells remain suspended and make contact with the pierced seeds.
Note: Acetosyringone should be added to the 'infiltration and co-cultivation medium' before introducing the pierced seeds.To ensure proper mixing, pipette the medium 10-20 times.
Note: The volume of media required depends on the number of seeds used.Typically, 25 mL is suitable for a 100 mm petri plate (Corning, USA Cat# BP94A-01), while 6 mL is sufficient for a 35 mm plate (Tarsons, India Cat# 460035-35MM).
28.Put the petri plates, which contain pierced seeds submerged in medium, into a vacuum desiccator chamber, and vacuum infiltrate for 10 min at a pressure of 550 mm Hg (Methods video S3).
Note: Ensure that the desiccator is properly cleansed with 70% ethanol and exposed to UV light for 30 min under laminar airflow before use.
CRITICAL: Vacuum infiltration should not exceed 20 min, to avoid the risk of tissue damage.
29. Place the inoculated seeds on sterile Whatman No. 1 filter paper to remove excess Agrobacterium infiltrate.
Alternatives: Sterile blotting paper can also be used in place of filter paper 30.Incubate for 5 min to achieve a semi-dry state (until the excess liquid medium is removed from the seed surface).31.Transfer seeds to a 100 mm petri plate on Whatman No. 1 filter paper.32.Saturate the filter paper with 3 mL of co-cultivation medium.
33. Seal the petri plate with Parafilm and cover with aluminum foil.34.Incubate at 28 C in the dark for 72 h for co-cultivation.

Note:
The co-cultivation medium here is the same as that in step 26, but without Agrobacterium cells.
CRITICAL: Ensure that the amount of media is neither excessive nor scarce.Excess media can lead to seed browning and death, while a shortage of media can cause seeds to dry out and die.
35.After co-cultivation, transfer the seeds to a fresh 100 mm petri plate.36.Wash the seeds four times for 5 min each, using only 'Agro-washing medium' without any antibiotic.
Note: While sterile water can be used for this washing step, using media is recommended as it yields better results.
Alternatives: 1 g/L cefotaxime (HiMedia Lab., India Cat# MB134) can be used as an alternative to timentin for this step.Alternatives: Cultivation can be done in petri plates, but it is preferable to use transparent screw-cap bottles or phyta jars for this step, as plants may touch the lids and become bent in the petri plates.
CRITICAL: The sub-lethal selection concentration of hygromycin-B may vary depending on the cultivar used and should be optimized accordingly.Use 15 mg/L hygromycin-B for JRC212 seeds, and 18 mg/L hygromycin-B for JRO524 seeds.

Note:
The selection media should be freshly prepared, and hygromycin-B should be added after the autoclaved media cools down (around 45 C) but not solidifies.
44. Transfer the surviving seedlings once more to the freshly prepared 'selection media' for an additional 14 days as a second round of selection.
Note: These plants should continue to grow under the same conditions as step 43.
CRITICAL: It is recommended to transfer five or six plants to each magenta box (HiMedia Lab., India Cat# PW1138) during this step.This ensures that the plants have sufficient space and nutrients for optimal growth.
Alternatives: Primers can be ordered from any reliable service provider.
Screening of hptII gene in transformed jute plants though PCR Timing: 3 h This step describes the PCR conditions for the process of amplification of the hptII gene.This is a validation step for the transgenic integration of the hptII gene in the jute genome.
58. a sterile 1.5 mL centrifuge tube, mix the following reagents.Thaw reagents on ice and keep reagents on ice until use.
Note: Prepare a working stock of 100 ng/mL of plant genomic DNA to use as a sample in the PCR reaction mixture.
Note: Mix all the components by repetitive pipetting and give a pulse spin before putting into the thermal cycler.

ProtocolNote:
Adjust pH to 5.8 with 2 M NaOH solution before autoclave.Note: Add hygromycin-B and timentin antibiotics to the autoclaved media after it reaches temperature around 45 C. Pause point: Store the medium at 4 C and use within 2 weeks.

38.
Incubate the seeds for 20 min.39.Vacuum infiltrate the seeds for 5 min at a pressure of 550 mm Hg. 40.Carefully remove the seeds from the antibiotic-containing medium and place them on sterile Whatman No. 1 filter paper.41.Incubate the seeds for 45 min to ensure proper drying.42.Transfer the seeds into round screw-cap phyta jars (HiMedia Lab., India Cat# PW1125) containing hygromycin-B (HiMedia Lab., India Cat# PCT1503) in the 'selection medium'.43.Cultivate them for a duration of 14 days under controlled culture room conditions: a temperature of 27 G 2 C, a photoperiod of 16 h of light, and 8 h of darkness.
5, 102767, March 15, 2024 Protocol 7. Prepare a mixture (50 mL) of 2% sodium hypochlorite with 2-3 drops of Tween-20.a. Immerse seeds in this solution for 15 min with continuous stirring.b.Cover the conical flask with aluminum foil or use an amber-colored conical flask to avoid light exposure.
5. Immerse seeds in 70% ethanol for 4 min with continuous stirring.6. Wash seeds with sterile water for 2 min to remove residual ethanol.
2 mL PCR tubes.60.In each PCR tube, add and mix by pipetting 1 mL of genomic DNA (100 ng/mL) from the tested plant.61.Set the program as follows in a thermal cycler (Applied Biosystems, USA Proflex PCR System) for 35 cycles.62.Run the PCR products on a 1% agarose gel using an electrophoresis system (Bio-Rad, USA Model: PowerPac Basic).