Phalaenopsis orchids are popular potted ornamental plants around the world due to
their beauty, floral diversity and long indoor blooming period. Orchids are also important
in plant research, having tiny, dust‐like seeds, crassulacean acid metabolism photosynthesis,
complex deletion of the genes encoding NADH dehydrogenase subunits and mycorrhizal
symbiosis.
Previously, we investigated the orchid MADS gene family, which encodes DNA‐binding
proteins that are highly expressed in floral organs and may be important for flower
initiation and development (Lin et al., 2016). The Phalaenopsis MADS gene family includes
more than 50 members (Chao et al., 2018). As it is challenging to obtain different
combinations of mutants in perennial plants such as Phalaenopsis using traditional
crosses, there is great interest in developing alternative techniques for gene family
studies. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR‐associated
endonuclease (CRISPR/Cas) genome editing provides a convenient tool to obtain null
and multiple mutants in nonmodel organisms, may prove useful for breeding and plant
research (Li et al., 2019).
Here, we used two CRISPR/Cas strategies to generate multiple mutants in Phalaenopsis
equestris MADS genes. First, using a vector containing a hygromycin selection marker
(HPTII) and SpCas9 genes, we introduced three MADS target sites (MADS44, MADS36 and
MADS8) together in one vector (pYLMADS8_36_44; Ma et al., 2015) or separately in individual
vectors (P1300_MADS8, P1300_MADS36, P1300_MADS44; Lin et al., 2018) to produce single‐guide
RNAs (sgRNAs). We used Agrobacterium tumefaciens‐mediated transformation (Hsing et al.,
2016) to transfect explants with pYLMADS8_36_44 (the 3sg1C strategy) or a mixture
of P1300_MADS8, P1300_MADS36 and P1300_MADS44 (the 3X1sg strategy). The transformed
Agrobacteria were co‐cultured with Phalaenopsis explants (Figure 1a), and the explants
were incubated on hygromycin medium for selection (Figure 1b, c). We numbered the
antibiotic‐resistant explants (3sg1C#1 to 21 and 3X1sg#1 to 21) and incubated the
regenerated transformants (e.g. 3sg1C#17‐1 to 3sg1C#17‐4) individually (Figure 1d).
The genomic DNA of 3sg1C and 3X1sg transformants was isolated. Specific primers were
used to amplify the target genes, SpCas9, sgRNA and HPTII.
Figure 1
Strategies and results of CRISPR/Cas targeted mutagenesis of Phalaenopsis MADS
genes. Homozygous, harbours the same edited sequences in both alleles; biallelic,
both alleles were edited but the sequences were different; chimera, more than two
alleles in the transformants; heterozygous, one wild type and one edited allele. In
(g) and (j), letters in red, mutation; – in red, deletion. Numbers in parentheses
indicate the number of bases deleted; ‘Hetero’, heterozygous. The coloured blocks
in (h) and (k) indicate the genotypes of transformants that were edited in the genes
shown above the column. Red, homozygous; orange, biallelic; yellow, chimera; purple,
heterozygous and green, mutated in the sequences in
MADS44 that are similar to the
MADS8 target site. (a). One‐month‐old protocorms were incubated with 3sg1C Agrobacteria.
Bar = 1 cm. (b). Transfected protocorms were incubated in hygromycin medium. The green
protocorms are putative transformed explants. Bar = 1 cm. (c). Transformants with
green true leaves. Bar = 1 cm. (d). Rooted 3sg1C#17‐1 to ‐4 incubated in hygromycin
medium after 1‐month of subculture. Bar = 1 cm. (e). Two‐month‐old 3sg1C#13‐1 transformant.
Bar = 0.5 cm. (f). In the 3sg1C#8‐3 transformant, three
MADS
gene target‐site regions were amplified and sequenced. Blue bar, target site; red
bar, protospacer‐adjacent motif (PAM). Multiple peaks start from the sequences near
the PAM indicating that there were mutated PCR products. (g). 3sg1C#8‐3 PCR products
of three
MADS
gene target‐site regions were cloned, and eight clones from each construct were sequenced
to determine the genotype. (h).
MADS8,
MADS36 and
MADS44 target gene analysis in 3sg1C transformants. The explants are distinguished
by lines (#1 to #21). Each row indicates one transformant (‐1 to ‐4). (i). DNA was
isolated from each transformant derived from the 3X1sg strategy for PCR, including
the target regions of
MADS
genes (
MADS8,
MADS36 and
MADS44); sgRNAs of each construct (sgRNA8, sgRNA36 and sgRNA44); Cas9 and actin as
an internal control. The
MADS
gene PCR products were combined with wild‐type
MADS
DNA and tested using a T7 Endonuclease I assay. Cleavage of the PCR product indicates
the presence of a mutation in this transformant. (j). These mutated
MADS
gene PCR products in (i) were cloned, and eight clones were sequenced for each construct.
(k).
MADS8,
MADS36 and
MADS44 target gene and sgRNA analysis in 3X1sg transformants. The coloured blocks
indicate the genotype of transformants that were edited in the genes shown above the
column.
The 51 3sg1C transformants were incubated in a growth chamber (Figure 1e). The DNAs
of target gene regions were amplified and sequenced (Figure 1f, g, h). All except
3sg1C #14 (no sgRNA) contained the target MADS insertion/deletion (indel) mutation(s).
Aside from 3sg1C#8‐2, 46 transformants derived from 20 explants contained triple mutants
of all three target sites (97.9%, 46/47, Figure 1h). In comparison, a study in Dendrobium
orchid produced an indel rate of only 10% (15/150) transformates in 15 target sites
from five genes and only 33.3% target sites had mutations (Kui et al., 2017). Furthermore,
60.0% (12/20, Figure 1h) of explants were homozygous or biallelic for triple MADS
gene mutations. Thus, all the transformants from these 12 explants were nonchimeric
triple MADS‐null mutants. This feature makes the technique especially useful for gene
editing of long‐juvenile‐phase, heterozygous and vegetative crops such as Phalaenopsis.
In the 3X1sg experiment, genotyping of 45 transformants derived from 21 explants indicated
that except for 3X1sg#1 (no sgRNA) and 3X1sg#18, which carried two sgRNAs (MADS8 and
MADS44), each carried only one sgRNA (Figure 1i, j, k). The numbers of transformants
with one sgRNA in each of the indicated genes were 6, 12 and 1 for MADS8, MADS36 and
MADS44, respectively (Figure 1k). The gene‐editing efficiencies of the sgRNAs were
100% (MADS8: 7/7; MADS36: 12/12 and MADS44: 2/2). The Cas9 codons in pYL‐derived and
P1300‐derived transformants have been modified in different fashions—plant‐optimized
in pYL‐derived (Ma et al., 2015) and human‐optimized in P1300‐derived (Lin et al.,
2018)—but did not differ in their gene‐editing efficiencies in Phalaenopsis. These
results indicate that multiple sgRNAs can be combined into a library and transformed
to create an edited plant library. The mutants can be selected on the basis of phenotype,
and the sgRNA sequenced to identify the gene(s) or DNA region(s) associated with the
phenotype.
Compared with pYL‐derived (3sg1C) transformants, P1300‐derived (3X1sg) transformants
had a higher proportion of chimerism (33.3%; 14/42, Figure 1k). This contrasts with
the situation in protoplasts that were gene‐edited before cell division, which resulted
mostly in homozygous or biallelic mutations. Therefore, high‐efficiency gene editing
is important for vegetatively propagated crops for which there is no protoplast regeneration
system. However, in some CRISPR/Cas9 systems, dicot plants are mutated less efficiently
than monocots (Endo et al., 2019). Resolving the issue of chimerism in vegetatively
propagated dicots will require increased efficiency of both CRISPR/Cas and protoplast
regeneration.
Given current public concerns about genetically modified organisms (GMOs), methods
to perform gene editing without transgenic gene integration are highly desirable.
A recent Agrobacterium‐mediated transient expression experiment in tobacco (Nicotiana
tabacum) using Cas9 and sgRNA yielded 8% nontransgenic gene‐editing mutants (Chen
et al., 2018). The transformants from 3X1sg#20 had mutations in the target site of
MADS36 without integration of MADS36 sgRNA and 3X1sg#5 had mutations in the target
site of MADS44 without integration of MADS44 sgRNA into their genomes, indicating
that nontransgenic targeted mutagenesis occurred in Phalaenopsis at an efficiency
of 4.8% (2/42, Figure 1k).
The protospacer‐adjacent motif (PAM) neighbouring the target sequence is essential
for CRISPR/Cas gene editing. The PAM sequence of SpCas9 is NRG. In Arabidopsis thaliana,
the average efficiency was 52.7% when following an NGG PAM sequence but 0%–1.1% when
following NGA, NGT or NGC (Ge et al., 2019). In rice (Oryza sativa), the same target
site had a 65.5% efficiency when following a TGG PAM but 0% when following TGA, TGT
or TGC (Endo et al., 2019). Notably, MADS44 contains a sequence similar to the MADS8
target sequence but with a CGA PAM. In our 3sg1C experiment, this PAM CGA target site
was edited (Figure 1h). In the 3X1sg experiments, however, only the transformants
with MADS8 sgRNA constructs had PAM CGA mutations in MADS44 (Figure 1k). Mutation
to the PAM CGA target site in MADS44 created a sequence that could act as a MADS8
sgRNA.
In this study, single, double and triple Phalaenopsis mutants can be obtained by different
sgRNA construction and transformation strategies, reducing the labour required for
transformation. We obtained MADS‐null mutants of Phalaenopsis, a crop plant with a
heterozygous genome and long juvenile period. This protocol has potential applications
for gene family studies in other perennial plants.
Competing interests
The authors declare that they have no competing interests.
Author contributions
CGT performed Phalaenopsis transformation. FHW, YHY and YRC performed molecular biology
experiments. CSL designed the experiments, interpreted the data and wrote the manuscript.