Table 2.

Genetic transformation of Phalaenopsis by Agrobacterium tumefaciens and particle bombardment.

Method	Phalaenopsis species/ cultivar	Explant	Agrobacterium strain	Genes	Primary findings	Reference
Agrobacterium-mediated	Phalaenopsis [Doritaenopsis Coral Fantasy × Phalaenopsis (Baby Hat × Ann Jessica)]	Cell clumps derived from friable calli	EHA101	hpt, nptII, and gus	- 10 (strain EHA101) and 24 (strain LBA4404) hygromycin-resistant regenerated plants were obtained per gram of cells after 10 h co-cultivation with Agrobacterium - 500 µM of acetosyringone enhanced the infection rate, increasing the number of GUS spots (42.4) compared to the control (10.4)	(Belarmino and Mii 2000)
Agrobacterium-mediated	Phalaenopsis lines (T0, T5, T10, and Hikaru)	PLBs	LBA4404	hpt, nptII and gus	- L1.5-3 mgL−1 hygromycin were suitable for transformed PLBs selection - Percentage of transformed (rooted plantlets) PLBs was 1.5–14.6% - Transversely bisected PLBs were more efficiently transformed (16.2%) than the intact PLBs (8.4%)	(Chai et al. 2002)
Particle bombardment	P. TS444 [(New Eagle × Pinlong Cinderella) × Dtps. Taisuco Red]	Petals	-	F3′5′H P450, gfp	- Phalaenopsis petals bombarded with the F3′5′H gene showed change in color from pink to magenta - The highest frequency of transformation was obtained with bombarding helium pressure of 10.3 MPa. - P450 gene was successfully transformed into Phalaenopsis petals	(Su and Hsu 2003)
Particle bombardment	P. TS340 [P. Taisuco Kochdiam × P. Taisuco Kaaladian]	PLBs	-	CymMV cp, htp, gus, gfp	- 20 transgenic lines were obtained after hygromycin selection - Transgenic Phalaenopsis transformed with cp gene resulted in protection against CymMV infection. - CymMV resistance was RNA-mediated through PTGS	(Liao et al. 2004)
Agrobacterium-mediated Particle bombardment	P. TS97K [P. amabilis W1–10 X P. 0061mabilis W1–22]	PLBs	EHA105	CymMV cp, pflp, gfp, gus, hpt	- Putative transformants with CymMV cp by particle bombardment transformation did not regenerate after hygromycin selection, so PLB were re-transformed with pflp - cp expression increased after CymMV infection - Transgenic lines showed strong resistance to CymMV and Erwinia carotovora - CymMV resistance was transgene-mediated through PTGS	(Chan et al. 2005)
Agrobacterium-mediated	P. S122-2 × S153 and S153 × S119-4	Immature protocorms	EHA101	hpt, nptII, and gus	- The highest transformation efficiency (1.93%) based on hygromycin-resistant plant was obtained when protocorms were subcultured on the medium with acetosyringone 2 days before inoculation - Acetosyringone enhanced the efficiency of Agrobacterium infection at inoculation and during pre-culture - Meropenem (5 mg L−1), was effective to eliminate Agrobacterium. - 88 lines of transgenic plants were obtained from independent protocorms, in which two presented an abnormal leaf shape	(Mishiba et al. 2005)
Agrobacterium-	P. Wataboushi “#6.13”	Embryonic cell suspension culture	EHA101	htp, nptII and wasabi defensin gene	- The use of fine cell aggregates and 25 mg L−1of hygromycin resulted in high yield of hygromycin-resistant calli (19 calli per gram of cell co-cultivated with Agrobacterium) - About 30 transgenic plantlets per gram of embryonic suspension calli were obtained - Transformed Phalaenopsis showed strong resistance to Erwinia carotovora infection - One of 15 (6.7%) transgenic plants tested showed susceptibility and died.	(Sjahril et al. 2006)
Agrobacterium-	P. Wataboushi “#6.13”	Embryonic cell suspension culture	EHA101	htp, nptII and gus	- Cefotaxime at 300 mg L−1 and carbenicillin at 500 mg L−1 suppressed Agrobacterium re-growth while meropenem at 3-5 mg L−1 presented the same effect - The highest transformation efficiency (10.3, 19.7, and 34.8 hygromycin-resistant calli, PLB and plantlet per gram of cell, respectively) was obtained when cells were infected for 2 h with Agrobacterium.	(Sjahril and Mii 2006)
Agrobacterium-	Phalaenopsis derived from the cross between 2 elite clones	Protocorms	EHA101	htp, nptII, gus and gst	- 68 transgenic plants derived independent protocorms were obtained from 6325 mature seeds - Expression of GST was only detected in the plantlets that showed resistance to hygromycin - Regenerated plants grown in greenhouse bloomed within 2 years	(Chin et al. 2007a)
Agrobacterium-	P. amabilis (L.) Blume	Protocorms	LBA4404	nptII and BP/KNAT1 gene	- Transformation frequency of 0.1 and 0.3% was obtained when protocorms were co-cultivated with Agrobacterium (harboring BP/KNAT1 gene) for 4 days on medium containing kanamycin and carbenicillin. - 139 transgenic plantlets were regenerated from 12 starting protocorms producing kanamycin-resistant shoots	(Semiarti et al. 2007)
Agrobacterium-	P. bellina	PLBs	LBA4404	hptII, gfp and gus	- 53% PLB formation frequency was obtained in MS½ medium containing 0.8 µM 2,4-D - Phal. bellina PLBs were sensitive to hygromycin. Percentage of surviving PLBs were 100, 43, 32 and 23% at hygromycin 0, 1, 2 and 3 mg L−1. - The highest percentage of gfp transient expression was obtained for 45–90 min inoculation time	(Maziah and Fern 2008)
Agrobacterium-	P. violacea	PLBs	EHA101, EHA105	gusA hpt mgfp5	- Transformation efficiency of EHA105 was higher than EHA101 - The highest percentage of gus expression was obtained using PLBs (5 mm), EHA105 with OD600nm of 0.6	(Subramaniam et al. 2008)
Agrobacterium-	P. violacea	PLBs	EHA101, EHA105	gusA, gfp, hpt	- Agrobacterium is attracted to Phal. violacea exudates but chemotaxis is not a blocking step in Agrobacterium-mediated transformation	(Subramaniam et al. 2008)
Agrobacterium-	P. violacea	PLBs	EHA101, EHA105	gus, gfp	- Based on the gfp expression, strain EHA105 was better than EHA101 - Strain EHA105 in MS½ supplemented with 5% banana Mas extract, 200 mg L−1 L-cysteine, 60 µM silver nitrate, pH 5.5 increased T-DNA delivery frequency	(Julkifle et al. 2010)
Agrobacterium-	P. amabilis (L.) Blume	Protocorms	LBA4404	gfp and nptII	- Coconut water and tomato extract increased transformation frequency (6.8–16.6%) of regenerated shoots - Tomato extract (100-150 mg L−1) during pre-culture in NP medium improved the transformation efficiency - Of the 210 plantlets examined, 191 were positive for gfp gene fragment	(Semiarti et al. 2010)
Agrobacterium-	P. violacea	PLBs	EHA 101 EHA 105	hptII, gusA and gfp	- Agrobacterium is attracted to exudates from Phal. violacea explants - The highest frequency of transient gus expression was obtained with strain EHA105 co-cultivated in MS½ medium supplemented with 200 mg L−1 L-cysteine and 60 µM silver nitrate at 24 °C	(Subramaniam and Rathinam 2010)
Particle bombardment	P. TS444, Phal. TS440 (P. Taipei Gold × Dtps. Sun-Chen Beauty), P. TS340, P. Hwafeng Red jewel, P. New Cinderella	petals	-	gfp, gusA, P450	- Helium pressure of 220 psi provided the best transformation result	(Su and Hsu 2010)
Particle bombardment	P. aphrodite subsp. formosana, P. “Wedding Promenade”, P. equestris, P. “Luchia Lady”	PLB	-	gusA, gfp, sense-PeUFGT3	- CHS, CHI and ANS might not be the key genes for red color formation since their expression was not significantly different between red and white flowers - Downregulation of PeUFGT3 manipulates flower color development	(Chen et al. 2011)
Agrobacterium-	P. amabilis	Leaf-derived embryogenic calli	LBA4404	nptII and ltp	- P. amabilis transformed with ltp exhibited strong cold stress tolerance at 10/7°C with green, healthy and vivid leaves - The highest transformation efficiency was 12.16%, when infected calli were co-cultivated 0.4 (OD600) A. tumefaciens for 20 min. - Obtention of 470 transgenic plants derived from independent PLBs.	(Qin et al. 2011)
Agrobacterium-	P. Dtps.Tailin Angel	PLBs	LBA4404	gafp and npi,	- The highest frequency of PLB induction (85%) was obtained in MS½ medium with 10.0 mg L−16-BA and 1.0 mg L−1NAA - Cefotaxime sodium concentrations had no significant differences in protocorm induction - Protocorms and roots were not produced at 10 mg L−1 kanamycin - gafp and npi were transferred to Phalaenopsis, indicating its resistance to Colletotrichum gloeosporioides Sacc	(Li et al. 2013)
Agrobacterium-	P. aphrodite cv. M1663 and P. aphrodite subsp. formosana	Protocorms	EHA105	hptII, gfp gus	- 74 transgenic seedlings overexpressing Ubi:eGFP were obtained after three successive hygromycin selections and grown in greenhouse conditions, presenting normal morphology - Transformation frequency was 1.2–5.2% - All 22 transgenic lines showed HptII banding but five plants did not show GFP bands	(Hsing et al. 2016)
Particle bombardment	P. bellina	PLBs	-	Sgfp, hptII, gfp and gusA	- Three hygromycin-resistant lines out of 160 bombarded individual PLBs, achieved an efficiency of 1.88%	(Chew et al. 2019)
Agrobacterium-	P. Sogo Yukidian “SPM313”	PLBs	EHA105	OsGA2ox6, gus	- Out of approximately 400 PLBs on the selection medium, only three PLB groups survived - Transgenic Phalaenopsis displayed green, shorter and wider leaves, thicker roots and much shorter flower spikes (10 cm vs 33 cm) than the nontrans- genic line with a normal flower size and blooming ability	(Hsieh et al. 2020)
Agrobacterium-	P. amabilis (L.) Blume	Protocorms	EHA105	CRISPR/Cas9, hpt	- Transformation efficiency using V2T1 gene is 1.6%, higher than transformation efficiency using V2T2 gene (1.3%) - Agrobacterium-mediated transformation to deliver CRISPR/Cas9 was successfully achieved- A color change from green to pale yellowish was detected in the transformants	(Nopitasari et al. 2020)
Agrobacterium-	P. amabilis	Protocorm	EHA105	CRISPR/Cas9 PDS3T1 sgRNA PDS3T2 sgRNAis	- 0.96% PDS transformants obtained from PDS3T2 lines and 0.9% from PDS3T1 lines. - Transformant showed color changes in the leaf tissue (albino phenotype)	(Semiarti et al. 2020a)
Agrobacterium-	P. equestris	Protocorms	-	hptII, MAD	- The gene-editing efficiencies of the sgRNAs were 100% (MADS8: 7/7; MADS36: 12/12 and MADS44: 2/2). - 46 transformants derived from 20 explants contained triple mutants	(Tong et al. 2020)

Hpt hygromycin phosphotransferase, nptII neomycin phosphotransferase II, gus β-glucuronidase, PLB protocorm-like bodies, CymMV Cymbidium mosaic virus, CymMV cp Cymbidium mosaic virus coat protein, pflp sweet pepper ferredoxin-like protein, gfp green fluorescent protein, PTGS post-transcriptional gene silencing, gst glutathione S-transferases, ltp lipid transfer protein, gafp gastrodia antifungal protein, npi neutrophils peptide-I, OsGA2ox6 gibberellin 2-oxidase 6, PDS3 phyotoene desaturase-3, VAR2 variegate 2, RT-PCR reverse transcriptase polymerase chain reaction, pflp sweet pepper ferredoxin-like protein, F3′5′H flavonoid-3′,5′-hydroxylase, NP New Phalaenopsis.