A comprehensive review of in planta stable transformation strategies

Abstract

Plant transformation remains a major bottleneck to the improvement of plant science, both on fundamental and practical levels. The recalcitrant nature of most commercial and minor crops to genetic transformation slows scientific progress for a large range of crops that are essential for food security on a global scale. Over the years, novel stable transformation strategies loosely grouped under the term "in planta" have been proposed and validated in a large number of model (e.g. Arabidopsis and rice), major (e.g. wheat and soybean) and minor (e.g. chickpea and lablab bean) species. The in planta approach is revolutionary as it is considered genotype-independent, technically simple (i.e. devoid of or with minimal tissue culture steps), affordable, and easy to implement in a broad range of experimental settings. In this article, we reviewed and categorized over 300 research articles, patents, theses, and videos demonstrating the applicability of different in planta transformation strategies in 105 different genera across 139 plant species. To support this review process, we propose a classification system for the in planta techniques based on five categories and a new nomenclature for more than 30 different in planta techniques. In complement to this, we clarified some grey areas regarding the in planta conceptual framework and provided insights regarding the past, current, and future scientific impacts of these techniques. To support the diffusion of this concept across the community, this review article will serve as an introductory point for an online compendium about in planta transformation strategies that will be available to all scientists. By expanding our knowledge about in planta transformation, we can find innovative approaches to unlock the full potential of plants, support the growth of scientific knowledge, and stimulate an equitable development of plant research in all countries and institutions.

Keywords: Direct organogenesis; In planta transformation; In situ transformation; In vivo regeneration; Indirect organogenesis; Recalcitrant species.

Fig. 1

Distribution of the publications found in the in planta compendium. This graph shows the distribution of the publications associated with each type of explant

Fig. 2

Classification of the four de novo organogenesis pathways. Regeneration-dependent de novo organogenesis strategies can be performed under in vivo (in planta) or in vitro (not in planta) conditions. The direct regeneration mechanism has many advantages over the indirect mechanism as it is simpler and quicker to perform; however, it leads to the formation of chimeric T₀ mutants that require segregation in the T₁ generation to obtain non-chimeric offspring. Moreover, the direct regeneration mechanism does not suffer from somaclonal variation, unlike callus-based methods. Callus-based methods are generally more challenging to perform but can be useful for specific crops (e.g. plants with a long juvenile phase such as trees) that cannot be transformed efficiently using the direct regeneration mechanism. The in vitro indirect regeneration pathway is generally considered highly genotype-dependent due to the use of multiple growing media, whereas direct regeneration methods are more universal due to their use of simple cultivation medium that are suitable for a larger spectrum of genotypes. The classification of these pathways was inspired by the comparative scheme of bud regeneration avenues developed by Shi et al. [54]

Fig. 3

Gamete-based transformation techniques. (A) Strategies targeting the female gamete (ovule). Several in planta techniques (e.g. the floral dip [59], vacuum-infiltration [60], floral spray [76], and floral painting [67]) targeting the female gametes have been developed and validated. In Arabidopsis, in planta strategies targeting the ovules often lead to the generation of hemizygous offspring in the T₁ generation as the male reproductive organs (i.e. pollen and pollen tubes) remain untouched [137, 243]. A thorough screening must be performed in the T₁ generation and further to identify positive mutants using a selection marker or reporter gene [65, 66]. (B) Male gametes-based in planta approaches. In these strategies, the pollen grains are transformed through various methods such as sonication [83], vacuum infiltration [82], magnetofection [85, 86], Agrobacterium [82, 84], particle bombardment [80, 81], and electroporation [79]. Subsequently, these pollen grains are used to pollinate the recipient plant’s ovules and lead to the generation of putatively transformed T₁ offspring. Following this, screening is performed in the T₁ generation to identify positive transformants

Fig. 4

In planta approaches targeting the embryos at an early stage of development. (A) Pollen-tube pathway [92]. To perform the pollen-tube pathway, the plant’s stigmas are removed and the styles are severed shortly after pollination. Subsequently, exogenous donor DNA is applied to the severed styles and delivered to the recipient plant’s ovaries via the growth of the pollen tube. Following the seed set, the putative transformants are screened to identify positive mutants. (B) Ovary-drip [92]. In this approach, the ovary sac is incised using a sterile scalpel, and exogenous DNA is directly delivered to the ovule drop-by-drop using a micropipette. (C) Pollen-tube agroinjection [113]. In this method, a solution of resuspended Agrobacterium is injected into the plant’s pollen tube using injector needles. To do so, the carina is punctured with the needles and the solution is injected until the wing petals are soaked. (D) Ovary injection [115, 116, 118]. To apply the ovary injection strategy, a solution of resuspended Agrobacterium is injected into the ovaries (i.e. soybean pods in this case) at an early stage of development to infect the developing embryos. Following this step, the mature seeds are further screened to identify positive mutants

Fig. 5

In planta strategies targeting the embryos at a later stage of development. (A) Infection of pre-imbibed embryos with Agrobacterium. The seeds are imbibed with sterile water and either (i) kept uninjured [122] or (ii) injured using pricking, sonication, or vacuum infiltration [121]. Following this treatment, the seeds are infected with a solution of Agrobacterium and grown until the T₁ generation for selection. (B) Agro-imbibition [124]. In this approach, seeds are imbibed with a solution of Agrobacterium instead of sterile water and further selected in the T1 generation. (C) Imbibition of desiccated embryos [125]. To perform this method, seeds are first imbibed with sterile water and subsequently desiccated at room temperature for 9–36 h. The seeds are subsequently infected for 2 h with a solution of Agrobacterium and cultivated until the T₁ generation for selection

Fig. 6

Transformation approaches targeting the apical and adventitious meristems. (A) Shoot apical meristem injury under in vivo conditions [129]. The apical meristematic region is pricked with a needle and subsequently infected with resuspended Agrobacterium. Chimeric T₀ plants are grown under in vivo conditions until seed set. Non-chimeric lines are further selected in the T₁ generation. (B) Plumular meristem approach [22, 148]. In the plumular meristem approach, young seedlings are decapitated and their radicules excised with a sterile scalpel. Following this treatment, the explants are infected with Agrobacterium and co-cultivated on a sterile medium under in vitro conditions. After co-cultivation, the seedlings are moved to greenhouse conditions and allowed to set seeds. The T₁ offspring are then screened to identify positive mutants

Fig. 7

Additional in planta techniques targeting the shoot apical and adventitious meristems. (A) Direct organogenesis of propagules (cut-dip-budding technique) [41]. To perform this method, plants with a high asexual reproduction capacity (e.g. sweet potato) are decapitated and their wounds are treated with a solution of resuspended Agrobacterium rhizogenes. Due to the root-suckering features of these plants, transgenic hairy roots will slowly develop and generate a newly transformed plant. (B) Direct organogenesis of propagules (Regenerative activity-dependent in planta injection delivery technique) [150]. In the RAPID method, a solution of resuspended A. tumefaciens is injected into the stem of plants with a high asexual reproduction capacity such as sweet potato. The plant is subsequently transplanted and transformed roots (pathway #1) or shoots (pathway #2) will subsequently emerge from the wound sites. (C) Direct delivery of exogenous morphogenic regulators [175, 244]. In the Direct delivery approach, the recipient plants’ meristems are removed using a sterile scalpel, and developmental regulators (e.g. WUSCHEL/WUSCHEL2) are subsequently delivered by injecting a solution of resuspended A. tumefaciens into the wound sites. Following this, the wild-type abnormal transgenic offshoots are culled, whereas the normal transgenic shoots are identified for further propagation

Fig. 8

In planta transformation using in vitro direct organogenesis and in vivo callus-based approaches. (A) In vitro direct organogenesis. The shoot apical meristems (SAM) are excised from the growing seedlings and inoculated with resuspended Agrobacterium [57, 58]. Following inoculation, the putatively transformed shoot apical meristems are grown and screened under in vitro conditions to identify positive T₀ mutants. Following the screening process, mutants are rooted and then transferred to in vivo conditions for seed setting. Optionally, embryonic axes from imbibed seeds can be used similarly to shoot apical meristems (details not shown in the figure) [55, 56, 245]. (B) In vivo callus regeneration [16, 17, 19, 210]. Dicot plants are decapitated and their wound sites are injected or rubbed with a solution of Agrobacterium. Subsequently, the wound sites are covered with parafilm and/or aluminum foil to retain moisture and keep the sites under dark conditions to favor callus formation. Optionally, the wounds can be treated with different hormones to promote the formation of a callus. Before or after callus formation, the sites can be treated with a selection marker such as an antibiotic or herbicide to eliminate untransformed calli cells. After the callus is formed, shoot formation is favored by cultivating the callus site under a regular photoperiodic regime. Under these conditions, transformed shoots will emerge from the calli cells surviving the screening process. (C) Shoot apical meristem removal and direct regeneration of adventitious meristems [16, 19, 210]. Plants are decapitated and the wound site is inoculated with Agrobacterium through injection and/or rubbing. The wound site is subsequently covered with parafilm and/or aluminum foil to retain moisture and keep it under dark conditions. Chimeric plants regenerate from the wound site and the adventitious shoot can be maintained on the same plant, grafted on another plant, or rooted in a separate container. Selection is performed in the T₁ generation to retrieve non-chimeric offspring

Fig. 9

Novel transformation techniques used for in planta transformation. (A) Grafting-mediated transformation [227]. Wild-type scion is grafted to a transgenic rootstock containing Cas9 and gRNA sequences. The grafting procedure leads to the formation of chimeric scions containing Cas9 and gRNA sequences due to the movement of tRNA-like sequence motifs that ensure transcript mobility across the plant. The rootstock to scion movement of these sequences causes heritable edits in the germline cells and edited offspring can be retrieved upon selection in the T₁ generation. (B) Viral-based vector using a mobile FT cassette [238]. The leaves of mutant plants overexpressing Cas9 are agroinfiltrated with a viral vector (e.g. tobacco rattle virus vector) containing a gRNA sequence fused to mobile FT sequences. The gRNA sequence reach the germline cells of the Cas9 overexpressing mutants upon the transcription of FT due to its endogenous natural movement to the shoot apical meristem and the edited offspring are retrieved in the T₁ generation upon selection