Genotype-independent plant transformation

Abstract

Plant transformation and regeneration remain highly species- and genotype-dependent. Conventional hormone-based plant regeneration via somatic embryogenesis or organogenesis is tedious, time-consuming, and requires specialized skills and experience. Over the last 40 years, significant advances have been made to elucidate the molecular mechanisms underlying embryogenesis and organogenesis. These pioneering studies have led to a better understanding of the key steps and factors involved in plant regeneration, resulting in the identification of crucial growth and developmental regulatory genes that can dramatically improve regeneration efficiency, shorten transformation time, and make transformation of recalcitrant genotypes possible. Co-opting these regulatory genes offers great potential to develop innovative genotype-independent genetic transformation methods for various plant species, including specialty crops. Further developing these approaches has the potential to result in plant transformation without the use of hormones, antibiotics, selectable marker genes, or tissue culture. As an enabling technology, the use of these regulatory genes has great potential to enable the application of advanced breeding technologies such as genetic engineering and gene editing for crop improvement in transformation-recalcitrant crops and cultivars. This review will discuss the recent advances in the use of regulatory genes in plant transformation and regeneration, and their potential to facilitate genotype-independent plant transformation and regeneration.

Figure 1

Key steps and factors in exogenous hormone-induced plant regeneration. Aerial explants go through the sequential steps of elimination of leaf identity, establishment of root identity, establishment of shoot identity, followed by organogenesis (step ①) or somatic embryogenesis (steps ② and ③). Wounding, epigenetic modifications, growth regulatory genes, and developmental regulatory genes are the four classes of crucial regeneration-promoting factors. Boxed arrows, key steps. Dark red, regeneration-promoting factors. Blue, key genes for each factor. Green, plant growth hormones. Orange circle, somatic embryo.

Figure 2

Shoot apical meristem (SAM), the WUS-CLV3 negative feedback loop, and the growth and developmental regulatory genes currently known to improve plant transformation efficiency. LP, leaf primordia; PZ, peripheral zone; CZ, central zone; SC, stem cells; OC, organizing center; RZ, rib zone; L1 – L3, cell layer 1–3.

Figure 3

Structures and mechanisms of the expression vectors that utilized growth and developmental regulatory genes in plant transformation. (A) Estradiol-inducible AtWUS expression for transformation of Coffea canephora [26]. The XVE fusion gene driven by the constitutive P_G10–90 promoter contains the DNA-binding domain of the bacterial repressor LexA, the activation domain of the herpes viral protein VP16, and the carboxyl region of the human estrogen receptor. The binding of the estrogen hormone to the estrogen receptor in XVE enables XVE to bind to O^LexA, the eight copies of the LexA operator sequence, leading to expression of AtWUS driven by O^LexA and a minimal 35S promoter. (B) A heat shock inducible-excision system to control BcBBM expression in transgenic Chinese white poplar [37]. Heat shock treatment on the stem cuttings of transgenic Chinese white poplar activates expression of the yeast FLP recombinase that is driven by the heat shock-inducible promoter AtHSP18.2, leading to the removal of the AtHSP18.2:FLP and 35S:BcBBM cassette with one footprint (a single FRT recombination site) left in the transgenic genome. (C) Low expression of ZmWUS2 under the control of the weak Agrobacterium nopaline synthase promoter (Nos:ZmWUS2) and high expression of ZmBBM driven by the strong maize Ubiquitin promoter (ZmUbi:ZmBBM) for transformation of maize and sorghum [15]. (D) A desiccation-inducible excision system to control Nos:ZmWUS2 and ZmUbi:ZmBBM expression in transgenic maize and sorghum [15]. Desiccation of the embryogenic calli activates the expression of the CRE recombinase driven by the desiccation-inducible ZmRab17 promoter, leading to the removal of Nos:ZmWUS2, ZmUbi:ZmBBM and ZmRab17:CRE, with one footprint (a single LoxP recombination site) left in the transgenic genome. (E) Conditional expression of the ZmWUS2 and ZmBBM by the auxin-inducible promoter ZmAxig1 and the maize embryo/leaf-specific promoter ZmPLTP, respectively, for maize transformation [38]. (F) A selectable marker-free transformation system in tobacco and hybrid aspen [57]. The 35S:IPT-containing Ac transposase gene can automatically jump out of the chromosome, leaving no footprint in the transgenic genome. (G) Dexamethasone (Dex)-inducible IPT expression for transformation of tomato and lettuce [58]. The GVG fusion gene driven by the 35S promoter contains the DNA-binding domain of the yeast transcription factor GAL4, the activation domain of VP16, and the hormone-binding domain of the rat Glucocorticoid Receptor (GR). Exogenous application of the synthetic glucocorticoid DEX releases GVG into the nucleus, where it binds to the 6× UPS binding sites of GAL4 and activates the expression of AtWUS driven by 6× UPS and a minimal 35S promoter. (H) Low expression of ZmWUS2 (Nos:ZmWUS2) plus high expression of the Agrobacterium IPT (ZmUbi:IPT) for organogenesis in the seedling leaves of Arabidopsis, tobacco, and tomato, and in the mature plants of tobacco, potato and grape [59].