Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana

Abstract

There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.

Fig. 1 |. Overview of procedures.

a, Procedure 1, Golden Gate assembly of the T-DNA vector. Four modules (A–D) encode genes of interest, such as reporters (A), gene-editing reagents (A and/or B) and DRs (C′ and D). The modules are cloned into T-DNA vectors using Golden Gate assembly and used in downstream DD or Fast-TrACC procedures. b, Procedure 2, DD: (1) All meristems are removed from mature N. benthamiana plants grown in soil. (2) Agrobacterium cultures are delivered to trimmed plants. (3) De novo meristems are induced, and new shoots contain genetic modifications of interest. c, Procedure 3, Fast-TrACC: (1) N. benthamiana seeds are sown into each well of a six-well plate. (2) Five days after germination, the seedlings are co-cultured with Agrobacterium strains. (3) The seedlings are rinsed, and individual seedlings are transferred to wells in a 12-well plate. Growths form on the cotyledons due to DR overexpression; some of these growths produce shoots. (4) Shoot-like growths are excised from parent seedlings and transferred to solid medium. (5) Shoots from the solid medium are transferred to rooting medium. (6) Fully regenerated and genetically modified plants are transferred to soil.

Fig. 2 |. Golden Gate assembly of T-DNA vectors.

Golden Gate modular assembly takes advantage of type IIs restriction enzymes, which cleave DNA outside recognition sites. The type IIs digest key describes the directionality of enzyme cutting with respect to position on cloning vectors used in Procedure 1. Enzymes (E) digest destination vectors away from CcdB and toward inserts, which prime vectors for assembly in Golden Gate reactions. a–c, Golden Gate cycles from Procedure 1, where assembly of inserts into destination vectors changes antibiotic selectable markers (spectinomycin, ampicillin and kanamycin) and type IIs enzymes used in subsequent reactions. Changing selectable markers and type IIs enzymes is important in reducing background and changing overhangs used in downstream assemblies, respectively. Each panel outlines all overhangs in each Golden Gate reaction and assembled vectors with new type IIs enzymes used in downstream assemblies. Positional cloning of two sgRNAs (a). Even numbered (two, four or six) sets of sgRNAs are individually assembled into pTAG vector backbones. Complementary oligos for sgRNA spacer sequences are annealed, leaving compatible overhangs for vector entry. The sgRNA vector backbones contain tRNA processing sequences that release individual sgRNAs when expressed in the cell (TAG vectors: pTAG1-pTAG6E). The sgRNAs are assembled into the backbone using the type IIs restriction enzyme, Esp3I. Assembly of the sgRNA-tRNA array in module B (b). Previously assembled sgRNA vectors (a) are positionally cloned between a RNA Pol II promoter (Pro) and terminator (Term) of choice (Supplementary Table 2) using the type IIs restriction enzyme, BsaI. Individual sgRNAs in the array are separated by tRNA processing sequences contributed by the backbones in the previous cloning step. The expression cassette is assembled into a B module vector (typically) for downstream modular cloning. Assembly of the transformation vector (c). Inserts from modules A, B, C′ and D are sequentially assembled into a final T-DNA backbone using the type IIs enzyme, PaqCI. Activity of PaqCI removes the bacterial negative selectable marker, CcdB, from the transformation vector backbone. The T-DNA contains BeYDV replicon components, previously described in ref. , between the left (LB) and right (RB) T-DNA borders.

Fig. 3 |. DD procedure and sample outcomes.

a, Individual N. benthamiana plants are grown in soil in at least 3-inch pots. Plants are sufficiently mature when axillary shoots have developed. b, All existing meristems (shoot apical and axillary meristems) are removed using a scalpel. c, After trimming, plants have two to three nodes and a few supporting leaves with no observable shoot meristems. d,e, Agrobacterium cultures are delivered using a syringe to leaf axils and all wound sites (shoot apices and sites of axillary meristems, white arrows in e). f–h, Examples of shoots that emerge after a 20 day culling period. Among these are wild-type (f) and edited (g) shoots. Some shoots may have morphological abnormalities due to constitutive overexpression of the DRs (h). Reproduced with permission from ref. , Springer Nature Limited.

Fig. 4 |. Fast-TrACC procedure and sample outcomes.

a, N. benthamiana seedlings are germinated in sterile six-well plates. b, Once the cotyledons emerge, as shown in this single-well close-up, the liquid medium is removed and replaced with the treated Agrobacterium culture. c, After 2 d of co-culture, the seedlings are rinsed in water and individual seedlings are transferred to wells in a 12-well plate. d, After about 20 d, green growths begin to form on the seedlings (white arrow). These growths can be maintained on the seedling (e) or removed and transferred to solid 1/2 MS medium (f). Shooting has been observed to occur directly on seedlings (e, white arrow); however, most de novo growths continue to increase in mass without shooting (e, black arrow). f, Transfer of de novo growths to 1/2 MS medium can stimulate shoot development. g, Upon shooting, the growths can be transferred to root-inducing medium to promote root formation. h, Finally, the plantlets with established root systems can be transferred to soil for further growth.

Fig. 5 |. Timeline for DD (Procedure 2).

Steps 1–5, N. benthamiana seeds are planted in soil and allowed to grow to maturity as indicated by formation of axillary shoots. Step 14, observable shoot meristems are removed from mature plants. T-DNA vectors are assembled in Procedure 1 that contain editing reagents, reporters or transgenes of interest. These T-DNA vectors are transformed into Agrobacterium to create −80 °C freezer stocks. Steps 6–13, Agrobacterium and liquid cultures are prepared for injections. Steps 15 and 16, the Agrobacterium cultures are injected into trimmed plants at sites where meristems were removed. Step 17, shoots that form in the first 20 d are removed. Step 18, de novo shoots form that are induced by DRs. Steps 19–21, shoots are screened for editing or transgenesis by analyzing DNA prepared from a leaf punch.

Fig. 6 |. Timeline for Fast-TrACC (Procedure 3).

Steps 1–8, sterile seeds are plated in 1/2 MS liquid medium. Step 9, the seeds are left to germinate until the cotyledons have emerged. T-DNA vectors are assembled in Procedure 1 that contain editing reagents, reporters or transgenes of interest. These T-DNA vectors are transformed into Agrobacterium to create −80 °C freezer stocks. Steps 10–18, Agrobacterium and liquid cultures are prepared for co-cultivation steps. Steps 19–27, Agrobacterium infection of seedlings in co-cultivation medium. Steps 28–33, after two d of co-culture, the seedlings are rinsed and moved to a new plate. Seedlings are left to grow for 2 weeks, during which time the ectopic expression of the DRs initiates de novo growths. Steps 34–37, newly emerging true leaves are trimmed, and new medium is added to the plates. Step 38, after roughly 20 d, de novo growths should be visible. Steps 39–46, once shoot-like structures form, they can be maintained on the seedling until they reach a size amenable for transfer to root-inducing medium. Alternatively, the shoot-like growths can be transferred to solid 1/2 MS medium until they reach a size amenable for transfer to root-inducing medium. Steps 47–49, shoot-like growths with leaflets are moved to root-inducing medium. Steps 50–54 plantlets with roots are transferred to soil. Steps 55–57, Shoots are screened for genetic modifications.