Genetic modification of Water spinach (Ipomoea aquatica), a genoprotective perennial leafy green

Fayas Thayale Purayil; Mariam Alzaabi; Shina Sasi; Saranya Krishnan; Zarreen Badar; Ling Li; Martin Kottackal; Khaled M A Amiri

doi:10.1111/ppl.70257

. 2025 May 5;177(3):e70257. doi: 10.1111/ppl.70257

Genetic modification of Water spinach (Ipomoea aquatica), a genoprotective perennial leafy green

Fayas Thayale Purayil ¹, Mariam Alzaabi ¹, Shina Sasi ¹, Saranya Krishnan ¹, Zarreen Badar ^1,², Ling Li ¹, Martin Kottackal ^1,^✉, Khaled M A Amiri ^1,^3,^✉

PMCID: PMC12052931 PMID: 40325599

Abstract

Improvement of leafy greens, especially perennials with year‐round harvesting, is binding to the food security drive. “Food for All” by WHO demands the improvement of regional crops due to the agroclimatic specificity to ensure regional food security. Water spinach (Ipomoea aquatica) is a perennial nutritious leafy green with regional/ethnic cultivation. We accomplished organogenesis and somatic embryogenesis from different explants of I. aquatica, and transgenesis and genome editing through Agrobacterium‐mediated transformation. The Ipomoea Basal (CLC‐CP) medium was superior to the Murashige and Skoog medium. Hypocotyl explants produced a mean of 12.4 shoots on CLC‐CP containing 4.5 μM thidiazuron and 8.7 μM gibberellic acid (GA₃), 50 mg l⁻¹ ascorbic acid (AA), and 100 mg l⁻¹ adenine hemi‐sulfate (AdS). Leaf and root explants induced the highest somatic embryos on a medium containing AdS, AA, 4.4/4.7 μM 6‐benzyladenine/kinetin (KIN), and 0.45 μM 2,4‐dichlorophenoxyacetic acid. CLC‐CP medium with 4.7 μM KIN, 8.7 μM GA₃, AA, and AdS exhibited elongation of hypocotyl‐derived shoots and maturation of somatic embryos. A. tumefaciens‐mediated transformation of hypocotyl developed a mean of 3.7 GFP expressing shoots per explant; leaf and root produced 4.3 and 3.1 somatic embryos, respectively. A. rhizogenes infection induced a mean of 4.1 and 3.4 hairy roots from leaf and root explants, respectively. Western blotting of the GFP protein validates water spinach to express human therapeutic proteins. Genome editing of IaNAP1 using hypocotyl explants confirmed the reproducibility of transformation. The plantlets exhibited 100% survival in soil. The present protocol is useful for improving this ethnic leafy green with traits‐of‐interest.

1. INTRODUCTION

Leafy green vegetables are a prime choice for a healthy diet as they are rich in minerals and nutrients essential for growth. They are mostly short‐lived annual herbs, while few are perennials that provide edible leaves. There are thousands of leafy greens, which are storehouses of vitamins, minerals, and fiber. A healthy diet warrants a mixture of leafy greens, which serve as a low‐calorie food source packed with essential nutrients, especially antioxidants, including vitamins that are mandatory for life's well‐being. Indigenous vegetables have contributed to food and nutritional security and enhanced the livelihoods of marginal and smallholder farmers (Dubey et al., 2020).

Ipomoea, the megadiverse genus of the family Convolvulaceae, with over 600 species, consists of annual and perennial herbs, lianas, shrubs, and small trees (I. arborescence), though mostly twining climbers. The genus is mainly distributed in the tropical and subtropical regions of the world. The genus is well known for sweet potato (I. batatas) – the edible root tuber; the morning glory (I. nil) – an ornamental; and beach morning glory (I. pes‐caprae), the salt‐tolerant species. Ipomoea aquatica Forssk., known as water spinach, water morning glory, or kangkong, is considered to have originated in China (Edie and Ho, 1969; Umar et al., 2007) and is generally believed to have been first domesticated in Southeast Asia. Water spinach is widely cultivated for its tender shoots and leaves in Southeast Asia, East Asia, and South Asia (Austin, 2007). It is a well‐known culinary component that spans Eastern Asia and the warmer regions of the Americas as a key component of dishes such as canh chua rau muống (Mekong sour soup) or callaloo. It is rich in vitamins such as vitamin B1, vitamin C, and vitamin A (Dubey et al., 2020; Yudhistira et al., 2021). Kangkong is also electrolytically dense and contains minerals like calcium, iron, phosphorus, and potassium (Igwenyi et al., 2011). It is a good source of protein, crude fiber, and amino acids (Selamat et al., 2012). In short, it is a nutritious vegetable that provides vitamins, minerals, protein, and fiber and is an add‐on as the choice for a healthy diet. Water spinach is a fast‐ and easy‐growing, highly branched aquatic or semi‐aquatic perennial herbaceous vine requiring 5–7 weeks of cultivation at an appropriate temperature after sowing. Harvesting the tender shoot tips facilitates new flushes from each stem node, making it available irrespective of the season and reserving a place in supermarkets as fresh greens along with others such as spinach, lettuce, kale, etc. It is the primary source of nutrients due to its ability to convert nitrogen into edible protein (Zhang et al., 2014; Xiao et al., 2015). The absorption rates of nitrogen and phosphorus are as high as those of water hyacinth (Furukawa and Fujita, 1993), and they have become one of the major vegetables in Thailand and nearby countries. Besides being a leafy green, it has several medicinal properties (Dubey et al., 2020). Saikia et al. (2023) performed the chemical and biochemical characterization and reported the genoprotective and antidiabetic potentials, nutritional parameters with calorific value, and amino acid profile of water spinach. They identified more than 65 different compounds and 36 important secondary metabolites, viz., polyphenol glycosides, phenolic acids, alkaloids, and terpenoids, having antibacterial, antiviral, antitumor, hepatoprotection, and anti‐depressant effects. They reported the presence of euphoria, lucidenic acid, myricetin glycosides, squalene, and α‐linolenic acid, an essential unsaturated fatty acid. An aqueous extract of I. aquatica was reported as effective as the drug tolbutamide in reducing the blood glucose levels of glucose‐challenged Wistar rats (Malalavidhane et al., 2001). Anti‐breast cancer properties of Merromoside isolated from hydroalcoholic extract of water spinach have been emphasized (Sasikala et al., 2022). Aquaterins (I to XIX) present in water spinach were evaluated for aquaterin II‐induced apoptosis (Fan et al., 2015). Isolation of a free radical‐scavenging antioxidant from water spinach has been reported (Parimala and Sukumar, 2013). Anticancer activity of water spinach in prostate cancer has been documented (Octaviani et al., 2013). Besides the functional phytochemicals, Omi et al. (2020) isolated two plant growth inhibitors from the roots of water spinach. Water spinach accumulates significant amounts of toxic metals such as Fe, Cu, Cr, Mn, and Pb in its leaves (Rai and Shina, 2001) as well as arsenic (Ke et al., 2022) and microplastics at the micron level (Zhao et al., 2024). The capacity of water spinach to grow in polluted water makes it a choice for phytoremediation (Furukawa and Fujita, 1993).

I. aquatica, usually propagated vegetatively, is self‐pollinating and amenable to cross‐pollination. Of the two cultivars of water spinach, land kale (cultivated in fields) and Lombok (cultivated in ponds or swamps), the cultivar Lombok is valued for its good quality, with vivid light green leaves and crunchy stems. Simarmata et al. (2016) suggest that Lombok water spinach has unique characteristics that set it apart from other varieties of kale and might be improved further. VRWS‐1 is an Indian cultivar suitable for upland cultivation (Dubey et al., 2020). Since the domestication of water spinach, it has progressed from an orphan crop status to a large cultivated crop and has become an over‐the‐counter leafy green.

Fast improvement of crops through genetic transformation and genome editing requires an efficient regeneration system via organogenesis and/or somatic embryogenesis. Plant regeneration in various species of Ipomoea through organogenesis: I. batatas (Lou et al., 2006), and somatic embryogenesis: I. batatas (Jarret et al., 1984; Chee et al., 1992; Otani and Shimada, 1996; Zheng et al., 1996), I. obscura (Otani et al., 1996, 1998), I. nil ( Jia and Chua, 1992; Shimizu et al., 2003, 2005); I. purpurea and I. tricolor (Ishikuro et al., 2014) has been accomplished. The amenability of genetic modification in many species of Ipomoea, especially I. batatas, using Agrobacterium‐mediated transformation has been documented (review Imbo et al., 2016; Otani et al., 2021; Zhang et al., 2023; Cao et al., 2023; Mie et al., 2024). Overexpression of genes (Ono et al., 2000; Kikuchi et al., 2005; Hoshino et al., 2019) and genome editing in I. nil has been reported (Watanabe et al., 2017, 2018b). While in vitro regeneration via organogenesis has been reported in water spinach using seedling tissues, it is with very low frequency (Mori et al., 1999; Akaracharanya et al., 2001). Somatic embryogenesis has not been reported in water spinach to date. In addition, the procedures of transformation using the gus gene (Khamwan et al., 2003) and expressing rice cysteine synthase (Phokrai et al., 2006) demonstrated <1% frequency. Genome editing is worthwhile to improve agronomic traits of this leafy green, e.g., shelf life increase by knocking down genes like staygreen. So far, no genome editing has been reported in water spinach. High efficiency transgenesis and genome editing are essential to improve water spinach. It warrants a genetic transformation procedure followed by high‐frequency plant regeneration. This study establishes a robust transformation and genome editing procedure by optimizing plant regeneration through organogenesis and somatic embryogenesis using various explants of water spinach

2. MATERIALS AND METHODS

2.1. Seed germination in vitro

The seeds of water spinach (Ipomoea aquatica Forssk.) collected from local farmers in Al Ain, Abu Dhabi, the United Arab Emirates, were used in the present study. The seeds were washed with 70% (v/v) ethanol for 10 min, followed by two washes with sterile water, and kept in water for 2–3 h. The seeds were placed on a wet sterile filter paper in a sterile Petri dish (100 × 20 mm; Corning), and the plates were incubated in the dark at 25 ± 1°C in a plant growth chamber (Percival). The germinating (5–7 d) embryos (Figure S1A), after gently removing the seed coat (pressing the germinating seeds using thumb and forefinger; Figure S1B), were disinfected using 10% (w/v) Clorox with Tween 20 (0.1%, v/v) for 10–15 min. Some disinfected germinated embryos were cultured on half‐strength MS (Murashige and Skoog, 1962; PhytoTech Labs) medium containing 2% (w/v) sucrose for cotyledon explants. The shoots were maintained on half‐strength MS medium by subculturing the shoot tips to fresh media at intervals of 60 d.

2.2. Shoot tip/node culture establishment

Shoot tips and nodes (2–4 cm) were excised from plants grown in the plant room (25 ± 2°C; 70% humidity; 400 μmol m⁻² s⁻¹‐ Heliospectra LED Lights; 16 h light) in pots (10 × 10 cm) containing potting soil and sand (1:2 ratio). The shoot tips and nodes were disinfected using 10% (w/v) Clorox for 10–15 min and were followed by three washes with sterile water. The shoot tips and nodes blot‐dried on sterile filter paper were cut to 1–2 cm and cultured on half‐strength MS medium containing 2% (w/v) sucrose and were maintained by subculturing the shoot tips to fresh media at intervals of 60 d. Roots and leaves from in vitro‐grown shoots from node/shoot tip culture and germinated embryos were used as an explant source for somatic embryogenesis and transformation.

2.3. Shoot organogenesis

The disinfected germinated embryos were blot‐dried on a sterile filter paper, and the hypocotyls were cut to a size of 0.5–1.5 cm (excised off the shoot meristem by cutting just below it and removing the epicotyl region). Organogenesis was attempted by culturing the hypocotyl explants (without the shoot apex), petiole‐cotyledon, and also the leaf (1.0–1.5 cm²; the small leaves as used as such) and root segments (1.0–1.5 cm) from the in vitro‐derived plants were cultured on MS and CLC‐CP (Ipomoea basal medium; Chee et al., 1992; SKU# 30630011, PlantMedia) media with 3% (w/v) sucrose as described elsewhere in Petri dishes (90 × 15 or 90 × 20 mm; Corning). The media were fortified with various plant growth regulators (PGRs) viz., 6‐benzyladenine (BA), kinetin (KIN), thidiazuron (TDZ), trans‐zeatin (ZN), indole‐3‐acetic acid (IAA), α‐naphthaleneacetic acid (NAA), gibberellic acid (GA₃), ascorbic acid (AA), and adenine hemi‐sulfate (AdS) (all PGRs were purchased from PhytoTech Labs) at different concentrations either alone or in combinations (Table 1).

TABLE 1.

Shoot regeneration from hypocotyl and petiole‐cotyledon explant explants of Ipomoea aquatica on media with different concentrations of PGRs.

Media	PGRs (μM)				Ascorbic acid (mg l⁻¹)	Adenine hemi‐sulfate (mg l⁻¹)	Response (%)	No. of shoot buds (Mean ± SE)
Media	TDZ	NAA	IAA	GA₃	Ascorbic acid (mg l⁻¹)	Adenine hemi‐sulfate (mg l⁻¹)	Response (%)	No. of shoot buds (Mean ± SE)
MS*	2.3						50^g	2.1 ± 0.6^L
	4.5						75^d	6.1 ± 1.7^hi
	6.8						80^c	5.8 ± 1.9ⁱ
	9.0						80^c	4.3 ± 1.1^j
	11.3						65^f	4.0 ± 1.5^j
	4.5	0.29					65^f	4.2 ± 1.0^j
	4.5	0.57					80^c	3.1 ± 1.2^k
	4.5		0.27				65^f	5.7 ± 1.4ⁱ
	4.5		0.54				65^f	4.6 ± 1.6^j
	4.5			1.5			65^f	6.1 ± 2.1^h
	4.5			2.9			65^f	6.3 ± 1.9^h
	4.5			4.3			70^e	6.5 ± 1.6^gh
	4.5			5.8			70^e	6.8 ± 1.7^g
	4.5			7.3			75^d	7.1 ± 2.2^g
	4.5			8.7			80^c	7.9 ± 2.0^fg
	4.5			10.2			80^c	6.1 ± 2.7^h
	4.5			11.6			75^d	5.3 ± 2.9ⁱ
	4.5			8.7	50		80^c	8.1 ± 2.5^ef
	4.5			8.7	100		85^b	8.9 ± 2.3^de
	4.5			8.7	150		85^b	8.7 ± 2.6^e
	4.5			8.7	50	25	80^c	8.4 ± 2.1^e
	4.5			8.7	50	50	90^a	10.3 ± 2.8^bc
	4.5			8.7	50	100	90^a	10.7 ± 3.1^c
	4.5			8.7	50	150	65^f	9.1 ± 3.5^d
MS*^#	4.5			8.7	50	100	60^a	3.1 ± 1.2^k
CLC‐CP*	4.5			7.3	50	100	90^a	9.8 ± 3.7^cd
	4.5			8.7	50	100	90^a	12.4 ± 2.8^a
	4.5			10.2	50	100	85^b	12.1 ± 3.4^a
CLC‐CP*^#	4.5			8.7	50	100	70^e	4.2 ± 1.0^j
CLC‐CP**	4.5			8.7	50	100	90^a	11.1 ± 3.3^b
CLC‐CP**^#	4.5			8.7	50	100	65^f	4.1 ± 0.9^j

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Data represent the mean of 20 explants. Values followed by different letters in the column are significantly different at the 5% level (P > 0.05, DMRT). PGRs – Plant Growth Regulators; MS – Murashige and Skoog (1962) medium; CLC‐CP – Chee et al. (1992) medium; TDZ – thidiazuron; NAA – α‐naphthalene acetic acid; IAA – indole‐3‐acetic acid; GA₃ – gibberellic acid; * – 3% sucrose; ** – 3% maltose; # – petiole‐cotyledon explant. Growth period 40 d.

2.4. Somatic embryogenesis and plant regeneration

Plant regeneration through somatic embryogenesis was attempted using the hypocotyl, cotyledon (with and without petiole), in vitro‐derived leaf and root explants of plants maintained on half‐strength MS media. The explants were cultured on MS and CLC‐CP media (3% sucrose) with various levels of KIN, BA, 2,4‐dichlorophenoxyacetic acid (2,4‐D), and NAA alone and in combinations (Table 2).

TABLE 2.

Somatic embryogenesis on leaf and root explants of Ipomoea aquatica.

Media	PGRs (μM)			Response (%)		Embryos per explant (Mean ± SE)
Media	BA	KIN	2,4‐D	Leaf	Root	Leaf	Root
MS*	2.2		0.45	85^b	75^d	6.7 ± 0.5^bc	4.6 ± 0.7^bc
	2.2		0.90	90^a	85^b	5.1 ± 0.7^d	2.9 ± 0.6^e
	4.4		0.45	80^c	80^c	7.3 ± 0.6^b	5.1 ± 0.5^b
	4.4		0.90	90^a	85^b	5.2 ± 0.5^d	4.7 ± 0.6^bc
		2.3	0.45	85^b	80^c	6.8 ± 0.6^bc	4.6 ± 0.5^bc
		2.3	0.90	90^a	85^b	5.6 ± 0.6^d	2.8 ± 0.5^e
		4.7	0.45	80^c	80^c	7.4 ± 0.4^b	5.2 ± 0.4^b
		4.7	0.90	85^b	90^a	6.0 ± 0.4^c	4.5 ± 0.7^bc
	2.2	2.3	0.45	80^c	75^d	6.1 ± 0.6^c	4.3 ± 0.8^c
	2.2	2.3	0.90	85^b	85^b	5.2 ± 0.8^d	2.5 ± 0.7^e
CLC‐CP*	2.2		0.45	80^c	75^d	7.1 ± 0.6^ab	4.9 ± 0.5^b
	2.2		0.90	90^a	80^c	5.3 ± 0.8^d	3.3 ± 0.7^de
	4.4		0.45	80^c	85^b	7.9 ± 0.7^a	5.7 ± 0.7^ab
	4.4		0.90	90^a	85^b	5.8 ± 0.8^cd	4.8 ± 0.6^b
		2.3	0.45	80^c	75^d	7.8 ± 0.8^a	5.7 ± 0.5^ab
		2.3	0.90	90^a	90^a	5.9 ± 0.7^cd	3.6 ± 0.6^d
		4.7	0.45	80^c	85^b	8.1 ± 0.5^a	6.2 ± 0.7^a
		4.7	0.90	85^b	90^a	6.3 ± 0.8^c	5.7 ± 0.9^ab
	2.2	2.3	0.45	80^c	80^c	6.9 ± 0.8^bc	4.4 ± 0.8^c
	2.2	2.3	0.90	90^a	90^a	6.2 ± 0.9^c	2.8 ± 1.0^e
CLC‐CP**	2.2		0.45	80^c	70	6.2 ± 0.7^c	4.0 ± 0.6^c
	4.4		0.90	85^b	85^b	5.0 ± 0.5^d	2.6 ± 0.9^e
		2.3	0.45	85^b	75^d	6.3 ± 0.5^c	4.4 ± 0.7^c
		4.7	0.90	85^b	80^c	5.3 ± 0.8^d	2.8 ± 1.2^e
	2.2	2.3	0.45	85^b	80^c	5.9 ± 0.9^cd	3.9 ± 1.0^cd

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Data represent the mean of 20 explants. Values followed by different letters in the column are significantly different at the 5% level (P > 0.05; DMRT). All media were supplemented with 50 mg l⁻¹ ascorbic acid and100 mg l⁻¹ adenine hemi‐sulfate. MS – Murashige and Skoog (1962) medium; CLC‐CP – Chee et al. (1992) medium, BA – 6‐benzyladenine; KIN – kinetin; 2,4‐D – 2,4‐dichlorophenoxyacetic acid; * – 3% sucrose; ** – 3% maltose. Growth period 40 d.

The effect of 25–150 mg l⁻¹ adenine hemi‐sulfate (AdS) and 50–150 mg l⁻¹ ascorbic acid (AA), alone (both from Sigma) or in combination tested for shoot organogenesis and somatic embryogenesis by adding to the media with optimal PGRs (Table 1 & 2) before adjusting the pH. Subcultures were carried out on different media as specified elsewhere. The effect of maltose was tested by adding 3% (w/v) it in place of sucrose. Germination of somatic embryos was carried out on basal half‐ or full‐strength MS, CLC‐CP, and CLC‐EP (Chee et al., 1992; SKU# 30630011, PlantMedia) media with different levels of PGRs (Table 3). Media with 1–3% sucrose or maltose were also tried. The germinated embryos were transferred onto a half‐strength MS basal medium for further growth.

TABLE 3.

Elongation of shoot buds and maturation of somatic embryos developed on SIM‐H and SEM, respectively.

Media	PGRs (μM)				Shoot bud elongation (%)	Embryo maturation (%)
Media	BA	KIN	ZN	GA₃	Shoot bud elongation (%)	Embryo maturation (%)
MS*				2.9	5.0^L	00^j
				4.3	10^k	00^j
				5.8	10^k	00^j
				7.3	20^j	5.0ⁱ
				8.7	40^g	10^h
				10.2	25ⁱ	12.5^h
	2.2			8.7	30^h	10^h
	4.4			8.7	20^j	5.0ⁱ
		2.3		8.7	60^d	67.5^e
		4.7		8.7	70^b	80^c
		6.6		8.7	55^e	75^b
			2.3	8.7	50^f	45^g
			4.6	8.7	55^e	50^f
CLC‐CP*		2.3		8.7	65^c	77.5^c
CLC‐CP*		4.7		8.7	75^a	85^a
CLC‐CP**		2.3		8.7	60^d	65^e
CLC‐CP**		4.7		8.7	70^b	72.5^d

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Data represent the mean of 20 shoot clumps and 40 somatic embryos. Shoot elongation means the per cent of shoot clumps showed elongation. Values followed by different letters in the column are significantly different at the 5% level (P > 0.05; DMRT). MS – Murashige and Skoog (1962) medium; CLC‐CP – Chee et al. (1992) medium, BA – 6‐benzyladenine; KIN – kinetin; ZN – trans‐zeatin; GA₃ – gibberellic acid; * – 3% sucrose; ** – 3% maltose. All media were supplemented with 50 mg l⁻¹ ascorbic acid and100 mg l⁻¹ adenine hemi‐sulfate. Growth period 40 d. SIM‐H – CLC‐CP medium with 4.5 μM thidiazuron, 8.7 μM GA₃, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS; SEM ‐ CLC‐CP medium with 4.4 μM BA, 0.45 μM 2,4‐dichlorophenoxyacetic acid.

The pH of all plant tissue culture media was adjusted to 5.7–5.8 before gelling with 0.8% (w/v) Phytoagar (PlantMedia). All the PGRs were filter sterilized using a 0.22 μm filter (Millex‐GP, Millipore) and were added to the media after autoclaving. The cultures on solid media were incubated in a growth room at 25 ± 1°C in 16 h light (40 μmol m⁻² s⁻¹)/8 h dark or dark as specified. Suspension cultures prepared in 50 mL media in 250 mL conical flasks (Corning) were incubated in the dark or light on a rotary shaker (Innova 44, Eppendorf) at 100 rpm (25 ± 1°C; 16/8 h light/dark). All the media were autoclaved at 121°C and 15 lb. for 20 min.

2.5. Antibiotic sensitivity test

The antibiotic sensitivity of the hypocotyl, leaves, and root explants was tested by culturing 20 explants (repeated once) on optimal media hygromycin sensitivity of hypocotyl on SIM‐H and leaf and root on SEM, and kanamycin sensitivity of leaf and root on half‐strength MS medium at different levels of kanamycin (10–75 mg l⁻¹; PhytoTech Labs) dissolved in sterile water and hygromycin (5–20 mg l⁻¹) solution (PlantMedia). The antibiotics were added after autoclaving. The cultures were incubated in the culture room under the conditions described above.

2.6. Plant transformation vectors

Binary vectors pH7m24‐35S:mgfp (spectinomycin for plasmid selection; Dutta et al., 2013) and pCAMBIA1305.2‐35S:DsRed (kanamycin for plasmid selection) were used for transformation. Both vectors contained hpt (for plant selection) under CaMV35S promoter and nos terminator. The binary vector p35S:mgfp kanamycin under CaMV35S for plasmid and plant selection.

2.7. Transformation and transgenic plant regeneration

A. tumefaciens EHA105 strains with pH7m24‐35S:mgfp and pCAMBIA1305.2:DsRed were streaked on LBA (Luria‐Bertani Agrobacterium) agar plates (see Sasi et al., 2024) with 25 mg l⁻¹ rifampicin and 100 mg l⁻¹ spectinomycin for pH7m24‐35S:mgfp or 25 mg l⁻¹ kanamycin for pCAMBIA1305.2:DsRed. Single colonies of these bacteria were inoculated into a 25 mL liquid LBA medium with rifampicin and respective antibiotics and were incubated overnight on a shaker (250 rpm) at 28°C until the optical density was 0.4–0.6 (OD₆₀₀). The bacterial cells were pelleted by centrifuging at 8,000 g for 10 min and resuspended in an equal volume of liquid CLC‐CP medium containing 3% (w/v) sucrose supplemented with 200 μM acetosyringone (AS; Arcos Organics).

A. rhizogenes strain ATCC15834 containing the p35S:mgfp was streaked on Yeast Mannitol (YM) medium containing 50 mg l⁻¹ kanamycin (antibiotics of PhytoTech Labs). A loopful of A. rhizogenes bacteria from solid YM medium (containing 15 g l⁻¹ Bacto Agar, SRL) was transferred into YM liquid medium with kanamycin and grown overnight on a shaker (250 rpm) at 28°C until the optical density reached 0.6–0.8 OD (OD₆₀₀). The bacterial culture was pelleted by centrifuging at 10,000 g for 10 min and resuspended in an equal volume of half‐strength MS liquid medium containing 2% (w/v) sucrose and 200 μM AS. The bacterial infection cultures (A. tumefaciens and A. rhizogenes) were incubated in the dark for 1.30–2 h at 25 ± 1°C on a shaker at 50 rpm.

The hypocotyl (from germinated seeds), leaf, and root explants (derived from in vitro maintained shoot cultures) were infected with A. tumefaciens, while only leaves and roots explants with A. rhizogenes. The explants (1–1.5 cm hypocotyl and leaves, 1–3 cm roots) were immersed in the agrobacterial suspension for 20 min, followed by 10 min of vacuum infiltration. The explants were subjected to sonication before vacuum infiltration by placing the tubes in a bath‐type sonicator (Branson 5800). The explants were sonicated for 0–40 s at 60 kHz. After blot drying on sterile filter paper, the agro‐infected explants were cultured on the co‐cultivation medium (CLC‐CP medium containing 3% sucrose with 4.5 μM TDZ, 8.7 μM GA₃ for hypocotyl; 4.4/4.7 μM BA/KIN, and 0.45/0.9 μM 2,4‐D leaf and root explants) supplemented with 200 μM AS. A. rhizogenes infected explants were cultured on a half‐strength MS medium (2% sucrose) containing 200 μM AS. The cultures were incubated in the dark at 25 ± 1°C for 48–72 h. The infected explants, after washing with 300 mg l⁻¹ timentin, were blot‐dried on sterile filter paper and cultured on optimal medium with PGRs (CLC‐CP medium containing 3% sucrose with 4.5 μM TDZ, 8.7 μM GA₃ for hypocotyl; 4.4/4.7 μM BA/KIN, and 0.45/0.9 μM 2,4‐D leaf and root explants) and, 300 mg l⁻¹ timentin (PhytoTech Labs) and hygromycin (10 mg l⁻¹) or kanamycin at 50 mg l⁻¹ (for A. rhizogenes with p35:mgfp). The cultures incubated in conditions described for organogenesis and somatic embryogenesis were subcultured subsequently on CLC‐CP medium containing 4.7 μM KIN and 8.7 μM GA₃ for hypocotyl explants, and CLC‐CP medium having 4.4/4.7 μM BA/KIN, and 0.45 μM 2,4‐D for progression of the somatic embryos from root and leaf explants. These media were supplemented with 200 mg l⁻¹ timentin and 10 mg l⁻¹ hygromycin. The hairy roots induced by A. rhizogenes with p35:mgfp were grown in the dark on half‐strength MS (liquid and solid) media with timentin and kanamycin (50 mg l⁻¹). The somatic embryogenesis from the hairy root was performed by culturing root segments (1.5–2.0 cm) on CLC‐CP medium containing 4.4/4.7 μM BA/KIN and 0.45/0.9 μM 2,4‐D and 100 mg l⁻¹ timentin and 50 mg l⁻¹ kanamycin. Maturation/germination of somatic embryos and shoot elongation were carried out on the CLC‐CP medium containing 4.7 μM KIN and 8.7 μM GA₃ and the same concentration of timentin and kanamycin. The transformation efficacy was calculated based on the shoot or embryo formation of the transformed explants.

2.8. Rooting and acclimatization of shoot/plantlets

The rooting of regenerated shoots (transgenic and non‐transgenic) grown over >4 cm was carried out in solid or liquid (10 mL in 15 mL test tubes, immersing ~2 cm of the base of the shoot) either on PGR‐free half‐strength MS medium (2% sucrose) or with 0.49 μM indole‐3‐butyric acid (IBA). In the case of transgenic plants, the medium was supplemented with 100 mg l⁻¹ timentin and 10 mg l⁻¹ hygromycin. Direct transfer of the healthy shoots from the shoot growing media (without rooting) to water in plastic cups (25 mL) and growing in the plant room was also attempted.

In vitro‐derived plantlets and those directly rooted ex vitro in water were transplanted into pots (10 × 10 cm) containing potting soil and sand (1:2 ratio) and grown in a plant growth room (25 ± 2°C; 70% humidity; 400 μmol m⁻² s⁻¹–Heliospectra LED Lights; 16 h light)

2.9. Expression of GFP and DsREd

The expression of GFP and DsREd were documented at different stages after transformation (transient, shoot buds, shoots, somatic embryos, roots, and floral parts of regenerated plants) using GFP (excitation at 395 nm and emission at 475 nm) and for DsRED (RFP, excitation at 558 and emission at 583 nm) filters in a fluorescence microscope (Thunder Imager Model Organisms, Leica).

2.10. Confirmation of transformation

Genotyping of the transgenic plants was performed by PCR using the genomic DNA extracted from the leaves of independent lines (10 each through shoot organogenesis and somatic embryogenesis) of the ex vitro established plants. DNA was extracted from ten independent hairy root lines. DNA of the transformed and the untransformed (WT) plants was extracted following Sasi et al. (2024). The extracted DNA was dissolved in 50 μL of 10 mM Tris buffer (pH 8.0) and was quantified using NanoDrop 2000 (Thermo Scientific). The quality of extracted DNA was determined by resolving it in a 1% (w/v) agarose gel with 1× Hydragreen (nucleic acid dye) in TAE buffer

PCR was carried out in a thermocycler (Applied Biosystems) using the HotStar Taq DNA Polymerase kit (Qiagen) in a 20 μL volume using the primers for the mgfp and DsRed genes (Table S1). The PCR mixture contained 1× reaction buffer, 50 ng of DNA template, 0.2 mM of each dNTP, 0.2 μM of forward and reverse primer, and 2.5 units of HotStar Taq DNA polymerase. The PCR amplification program was: 95°C for 15 min, 25 cycles of 94°C for 30 s, 57°C (48°C for DsRed) for 20 s, 72°C for 30 s, and a final extension of 72°C for 5 min. Reactions with DNA of the untransformed plants, no template DNA (negative control), and the respective plasmid DNAs (positive controls) were included. The PCR products, along with 1 kb or 1 kb plus DNA ladder (Thermo Scientific), were resolved on a 1% (w/v) agarose gels in TAE containing Hydragreen and were documented using Geldoc (EZ Imager, Bio‐rad).

2.11. Western blotting

Total protein from the GFP‐expressed plants was extracted using the buffer (10% glycerol, 25 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl) with a freshly added protease inhibitor cocktail (, ). 250 μl of the extract was mixed with an equal amount of dilution buffer (10 mM Tris pH 7.5, 150 mM NaCl. 0.5 mM EDTA) and centrifuged at 20,000 g for 10 min at 4°C. The supernatant collected was resolved in 4–15% mini‐PROTEAN®TGX™ precast gel (Bio‐rad). The gels were blotted nitrocellulose membrane (Bio‐rad). Western blot was performed following the Pierce™ Fast Western Blot Kit (ThermoFisher) protocol using the GFP (A11122) and ACTIN (MA1‐744) antibodies (Invitrogen), and the blots were documented using C‐DiGit Western blot scanner (Li‐Cor).

2.12. Plasmid construction for genome‐editing

A twenty‐base target sequence for editing the IaNAP1, an ortholog of the AtNAP, gene was designed from transcriptome data of I. aquatica (unpublished) using CRISPR‐GE (http://skl.scau.edu.cn/). The 20‐base target of IaNAP1 was synthesized with adaptors on the forward and reverse primers (Table S1) and inserted into the Cas9 (under the 35S promoter) vector pHSE401 (https://www.addgene.org/62201/) carrying kanamycin for bacterial selection and hygromycin for plant selection. The prepared target duplex was inserted into the vector by restriction‐ligation method using BsaI and T4 Ligase (New England Biolabs). The reaction mixture was incubated in a thermocycler at 37°C for 5 h, 50°C for 5 min, and 80°C for 10 min (Xing et al., 2014). The positive colonies after transformation to DH5α were selected on LB plates with kanamycin and confirmed by colony PCR. PCR was carried out in a 20 μL reaction using Hotstar Taq DNA polymerase kit (Qiagen) using the pHSE401‐F and pHSE401‐R primers of the target (Table S1) with the following program: 95°C for 15 min, 25 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s and a final extension of 72°C for 5 min. The target insert was confirmed by sequencing the plasmid extracted from the positive colonies using the plasmid extraction kit (Qiagen). The confirmed plasmid mobilized into the A. tumefaciens strain EHA105 (Gene Pulsar, Bio‐rad), and confirmed positive colonies were used to transform hypocotyl explants.

2.13. Confirmation of genome‐edited plants

PCR primers were designed from the IaNAP1 genomic sequence using Primer3 (https://primer3.ut.ee/). Ten genome‐edited plants were established ex vitro in the plant room. Genomic DNA from those plants was extracted as described in 2.10, followed by PCR using a Phusion Hi‐Fidelity polymerase kit (New England Biolabs) with the forward and reverse primers of IaNAP1 (Table S1) with 50 ng of template DNA following the manufacturer's protocol (New England Biolabs). The PCR program was: 98°C for 30 s, 30 cycles of 98°C for 10 s, 62°C for 30 s, and 72°C for 30 s, and a final extension of 72°C for 5 min. The PCR reactions were resolved in 1% agarose gel, and the amplified bands were extracted using the QIAquick gel extraction kit (Qiagen) and sequenced by Sanger sequencing (Macrogen).

2.14. Hairy root culture

The confirmed hairy root segments were transferred onto half‐strength MS (2% sucrose) basal medium and CLC‐CP medium with or without 2.3 μM KIN. The biomass of the hairy root lines was recorded by growing the roots in a 50 mL half‐strength liquid medium (in 250 mL Corning conical flasks) in the light and the dark. The roots developed from untransformed explants (hypocotyl and root) subcultured in half‐strength MS liquid basal medium grown in conditions similar to those of the hairy roots used as the control. The liquid cultures were incubated on a rotary shaker (Innova 44, Eppendorf) at 100 rpm (25 ± 1°C) in 16 h light (40 μmol m⁻² s⁻¹)/8 h dark. The culture flasks were covered with aluminum foil for dark. The fresh weight (FW) of the transformed and control roots grown for 30 d were determined after blot drying on filter paper. The dry weight (DW) was recorded after drying samples in a hot‐air oven at 50°C till the samples showed no difference in weight in subsequent weighing.

2.15. Statistical analysis

All the regeneration and transformation experiments were repeated twice. The data represents the mean ± SE of replicates as described in the footnotes. The experiments were performed in a completely randomized design. Data were analyzed using the Microsoft Excel program, and the significance was determined using Duncan's Multiple Range Test (DMRT; p < 0.05).

3. RESULTS

3.1. Shoot tip/node culture

The disinfected shoot tips and nodes cultured on half‐strength MS medium showed the emergence of shoot tips and axillary buds, which grew well. The shoots branched well and produced roots later. The cultures were maintained on half‐strength MS media by subculturing shoot tips/the node periodically as the source of leaf and root explants.

3.2. In vitro regeneration

In vitro regeneration was accomplished through shoot organogenesis and somatic embryogenesis.

3.2.1. Shoot organogenesis

Hypocotyl and petiole‐cotyledon explants were cultured on MS and CLC‐CP medium with different PGRs at various concentrations (Table 1). The hypocotyl explants were superior in the induction of shoots (Table 1). TDZ at 4.5 μM was the cytokinin that induced shoots. The hypocotyl explants on medium with other PGRs (ZN, KIN, and BA) did not induce shoot buds in 40 d. A combination of TDZ and auxins (NAA, IAA) at different levels decreased the shoot buds and induced callus, while adding GA₃ increased the shoots (Table 1). The addition of AdS at 50 and 100 mg l⁻¹ did not show a significant difference in the induction of shoot buds (Table 1). Ascorbic acid at 50 and 100 mg l⁻¹ supplemented with the medium effectively prevented the browning, especially as to the progression of the culture and in the induction of shoot buds. AdS was beneficial in inhibiting the growth of roots from the explants. MS medium containing 4.5 μM TDZ, 8.7 μM GA₃, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS produced a mean of 10.7 shoot buds per explant (Table 1; Figure 1A) in a 40 d growth period. The petiole‐cotyledon explants induced 4.2 shoot buds in 40 d. (Table 1; Figure 1B), but induction was delayed compared to hypocotyl. Of the shoot buds on hypocotyl explants, 2–4 shoots grew over 1.5 cm. CLC‐CP medium was superior to MS medium in the induction of shoot buds (Table 1). The hypocotyl explants on CLC‐CP medium with 4.5 μM TDZ, 8.7 μM GA₃, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS (herein after SIM‐H) displayed 90% response and developed the highest number of shoot buds (Table 1). Hypocotyl explants produced a mean of 12.4, though the shoot buds varied from 8 to 16, and 25% of the induced shoot buds in 40 d (Figure 1C, D). However, along with the induction of more shoot buds, 40% of shoot buds emerged over 3 cm in 50 d (Figure 1E). In all cases, the cut ends proliferated as nodular and continued to develop shoot buds (Figure 1A‐H). The elongated shoots later initiated roots. SIM‐H with sucrose was superior to 3% maltose (Table 1). Subculture of the shoot buds onto MS or CLC‐CP medium with 4.7 μM KIN and 8.7 μM GA₃ 50 mg l⁻¹ AA and 100 mg l⁻¹ AdS (Elongation/Germination Medium, EGM) facilitated shoot bud proliferation initially and growth further (Figure 1 F‐H). The proliferation of buds and growth of the shoots was better on CLC‐CP medium than on MS. The shoots grew healthy and produced roots when retained without subculture (Figure 1H).

Shoot regeneration in water spinach. (A) Hypocotyl explant (30 d); (B) Petiole‐cotyledon (40 d); (C) Hypocotyl on shoot induction medium (SIM‐H; 30 d); (D) Enlarged view of one explant from C; (E) Hypocotyl (40 d); (F) Subculture showing numerous shoot buds' development; (G) & (H) Subcultured on Elongation/Germination Medium (EGM), 30 and 45 d, respectively. (A – on MS medium with 4.5 μM thidiazuron (TDZ), 8.7 μM gibberellic acid (GA₃), 50 mg l⁻¹ ascorbic acid (AA), and 100 mg l⁻¹ adenine hemi‐sulfate (AdS); SIM‐H – CLC‐CP (Chee et al., 1992) medium with 4.5 μM TDZ, 8.7 μM GA₃, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS; (G) & (H) – EGM ‐ CLC‐CP medium with 4.7 μM kinetin and 8.7 μM GA₃, 50 mg l⁻¹ AA and 100 mg l⁻¹ AdS. (Scale = 10 mm).

3.2.2. Somatic embryogenesis and plant regeneration

The hypocotyl, cotyledon, in vitro‐derived leaf and root explants were cultured on MS or CLC‐CP media with different auxins (2,4‐D, NAA) singly and combined with cytokinins (BA, KIN, ZN, and TDZ) at various concentrations to induce somatic embryos and plant regeneration. All types of explants on MS medium with 2,4‐D (0.45–18.0 μM) and NAA (0.57–17.1 μM) alone and in combination with lower levels of cytokinins favored only callus induction in the case of 2,4‐D, and green callus and roots with NAA. As in shoot organogenesis, the CLC‐CP medium was superior to MS (Table 2). The medium with cytokinin alone also induced callus; KIN alone induced roots, but BA‐supplemented was inferior in root induction. Leaf and root explants displayed the highest somatic embryo induction on CLC‐CP medium containing 3% sucrose 4.7/4.4 μM KIN/BA and 0.45/0.9 μM 2,4‐D, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS (Somatic Embryogenesis Medium, SEM; Table 2). The hypocotyl explants induced roots and calluses only. AdS enhanced the induction of somatic embryos from leaf and root explants, and it reduced the tendency to form roots even from the calluses induced. Lower concentration of KIN/BA (2.2/2.3 μM) showed a similar result (Table 2). On leaf explants, the callus proliferation was less, while the root explants formed callus initially and later formed embryos. The explants occasionally displayed the formation of embryos directly after 30 d. The rootlets during the culture slightly bulged and later produced embryos after slight callusing. The combination of BA and 2,4‐D displayed less root initiation tendency than KIN and 2,4‐D. The increase of 2,4‐D concentration decreased the induction of embryos and the root initial formation. The cotyledon and hypocotyl explants induced two types of calluses: buffy translucent white and embryogenic‐like calluses on SEM, but were delayed compared to root and leaf explants. The concentration of 2,4‐D above 0.9 μM induced only callus formation. AA reduced the browning of embryos/calluses formed from the explants. Subculture on the SEM medium induced more embryos and was higher in suspension cultures. On SEM, the embryos progressed to the torpedo or rarely to the early cotyledonary stage. The somatic embryos induced on leaf and root explants were transferred onto media with different KIN/BA/ZN levels alone and in combination with GA₃ (Table 3). Transfer of the embryos to the basal half‐ or full‐strength MS or CLC‐CP and CLC‐EP medium (with PGRs also) did not facilitate embryo maturation. CLC‐CP medium containing AA, AdS, 4.7 μM KIN, and 8.7 μM GA₃ displayed the highest maturation of somatic embryos, which later showed conversion (Figure 2A‐D). The embryogenic calluses transferred into liquid SEM medium in light in a rotary shaker favored the proliferation of embryogenic calluses and induction of embryos (Figure E). The somatic embryos were bipolar with distinct root and shoot poles (Figure 2B‐D), while some of the embryos produced adventitious roots from the root pole (Figure 2F). The liquid cultures of the embryos exhibited profuse rooting. Of the early cotyledonary embryos, 85% showed germination (Table 3). The germination of transferred torpedo or earlier stages was <35% and was delayed. The mature embryos/germinated embryos grew well upon transfer to half‐strength MS medium (2% sucrose) with profuse roots (Figure 2G). In all stages, incubation in the dark was inhibitory for the induction of somatic embryos.

Somatic embryogenesis in water spinach. (A) somatic embryos on leaf explants subcultured on Elongation/Germination Medium (EGM); (B) & (C) Embryos on root‐derived callus on EGM; (D) Stages of somatic embryos; (E) Suspension of somatic embryos in Somatic Embryogenesis Medium (SEM); (F) Germination of somatic embryos; (G) Embryo‐derived plantlet on half‐strength MS; (H) & (I) Shoot organogenesis from SEM‐derived calluses on CLC‐CP (Chee et al., 1992) medium with 4.4 μM 6‐benzyladenine (BA), 4.9 μM indole‐3‐butyric acid, 50 mg l⁻¹ ascorbic acid (AA), and 100 mg l⁻¹ adenine hemi‐sulfate (AdS); EGM ‐ CLC‐CP medium with 4.7 μM kinetin and 8.7 μM gibberellic acid, 50 mg l⁻¹ AA and 100 mg l⁻¹ AdS; SEM – CLC‐CP medium with 4.4 μM BA, 0.45 μM 2,4‐dichlorophenoxyacetic acid, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS. (Scale = 10 mm). The Blue and red arrows point to the callus and embryos, respectively, on leaf explants.

The calluses induced from root explants on CLC‐CP/MS medium containing 3% sucrose 4.4 μM BA and 0.9 μM 2,4‐D, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS when transferred onto medium with 2.5/4.9 μM IBA in place of 2,4‐D resulted in callus proliferation, which later induced shoots buds (Figure 2H, I). Transfer to the root calluses onto CLC‐CP/MS medium with 4.4 μM BA and 0.57 μM NAA, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS favored the root pole growth of the embryos, while the shoot pole rarely developed.

3.3. Antibiotic sensitivity of different explants

Culture of the explants on media with 5–20 mg l⁻¹ hygromycin (SIM‐H for hypocotyl and SEM for leaf and roots) resulted in no response of hypocotyl explants at 15 mg l⁻¹, and the leaves and roots showed the death of the explants (Figure S1C). The lethal dose of kanamycin was 50 mg l⁻¹ for leaves and root explants (Figure S1C). Hygromycin and kanamycin at 10 and 50 mg l⁻¹, respectively, were used to select the transformed shoots/calluses/embryos/roots after Agrobacterium‐mediated transformation, depending on the plant selection maker in the vector as specified.

3.4. Agrobacterium‐mediated transformation and transgenesis

Agrobacterium‐mediated transformation was performed using A. tumefaciens (binary vectors with mgfp/DsRed and A. rhizogenes harboring mgfp). The transformation efficiency was significantly increased by sonication (Figure 3). Sonication of 10–15 s was optimal for leaf explants, while 30 s was for roots and hypocotyl explants (Figure 3). More than 15 s sonication of leaf explants, though, showed high transient expression of GFP/DsRED, but the explants became dead on selection media (SEM containing 300 mg l^‐l timentin and 10 mg l^‐l hygromycin). In all cases, the percentage of infection was 100%. However, 80% of hypocotyl explants produced shoots on SIM‐H containing 300 mg l^‐l timentin and 10 mg l^‐l hygromycin, while leaf and root explants (on SEM with timentin and hygromycin) were 65 and 70%, respectively. Shoot buds on hypocotyl and embryos on leaf/root explants appeared after 20 d of incubation (Figure 4A‐F). The root explants displayed a slight initial callusing (Figure 4C & D), followed by embryos that appeared after 20 days (Figure 4E & F). Subculture of the calluses with the embryos in liquid or solid SEM favored the proliferation of embryogenic calluses and the induction of more embryos. Hypocotyl explants developed a mean of 3.7 transformed shoots per explant (Figure 3). The transformation efficiency in terms of shoots or embryos is shown in Figure 3. The developed shoots from hypocotyl (Figure 4G) and embryos from leaf and root were transferred on EGM medium supplemented with 300 mg l^‐l timentin and 10 mg l^‐l hygromycin for elongation of shoots and maturation/germination of embryos, respectively.

Efficacy of sonication in transformation. Hypocotyl infected with *Agrobacterium tumefaciens* on CLC‐CP (Chee et al., 1992) medium with 4.5 μM thidiazuron, 8.7 μM gibberellic acid, 50 mg l⁻¹ ascorbic acid and 100 mg l⁻¹ adenine hemi‐sulfate; Leaf and root infected with *A. rhizogenes* on half‐strength MS medium. Letters a‐e, m‐p, and u‐z were for hypocotyl, leaf, and root, respectively, and different letters concerned are significantly different at the 5% level (p > 0.05; DMRT).

(A) Hypocotyl explants expressing GFP; (B) GFP expressing early shoot buds (20 d) on shoot induction medium (SIM‐H) with timentin and hygromycin; (C) Callus developed on infected root explant; (D) Callus on root expressing GFP; (E) Embryogenic callus expressing GFP on Somatic Embryogenesis Medium (SEM) with timentin and hygromycin; (F) Embryogenic callus expressing RFP on SEM with timentin and hygromycin; (G) Developed shoot expressing GFP on SIM‐H; (H) & (I) GFP expressing hairy roots on leaf and root explant: (J) Well‐grown transformed roots expressing GFP; (K) Well‐grown hairy roots in the light (30 d). (H)‐(K) On half‐strength MS medium with timentin and 50 mg l⁻¹ kanamycin; SIM‐H – CLC‐CP (Chee et al., 1992) medium with 4.5 μM TDZ, 8.7 μM GA₃, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS; SEM – CLC‐CP medium with 4.4 μM BA, 0.45 μM 2,4‐D, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS. Timentin 300 mg l⁻¹; Hygromycin 10 mg l⁻¹. (Scale = 5 mm).

3.5. Hairy root induction and plant regeneration

The stable transformation efficiency of root and leaf explants by A. rhizogenes harboring the plasmid with 35S:mgfp was 80% on half‐strength MS solid medium containing 300 mg l^‐l timentin and 50 mg l^‐l kanamycin. Leaf and root explants developed a mean of 4.1 and 3.4 transformed roots, respectively (Figure 4H & I). The root initials appeared after 12 d. Initially, the root's growth was slow, but after 25 d, the roots showed faster growth and branched well (Figure 4J). The hairy roots grew faster and branched well when subcultured on half‐strength MS basal medium (Figure 4K). The control explants showed no root emergence on media containing 300 mg l^‐l timentin and 50 mg l^‐l kanamycin.

Hairy root segments cultured on an SEM medium containing 300 mg l^‐l timentin and 50 mg l^‐l kanamycin showed growth of roots initially. The hairy root explants thickened and displayed a slight initial callusing; the callus phase was slightly longer than the untransformed root explants. The embryos were initiated after 25 d. Subsequent cultures on SEM with antibiotics increased the number of embryos, especially in suspension cultures. The embryos were matured and germinated after transferring onto EGM medium containing timentin and kanamycin, as described above.

3.6. Shoot tip necrosis or dieback

The retaining of the shoots on SIM‐H and the shoots transferred on half‐strength MS media for rooting and maintenance exhibited shoot‐tip necrosis or dieback. The symptoms appeared after 25 days of incubation and became severe as the period continued. Nevertheless, following the dieback of the shoot tip, the axillary bud at the below node emerged and grew well (Figure S2A). Severe necrosis resulted in the emergence of most of the axillary buds below it (Figure S2B). The emerged shoot buds after elongation and growth exhibited shoot necrosis and the emergence of axillary buds below. The necrosis and shoot emergence continued cyclically. No additives to the media were tried to alleviate the necrosis due to the cyclic growth. It facilitated the maintenance and periodic transfer of individual shoots onto rooting medium and subsequent acclimatization, especially the transgenic shoots.

3.7. Rooting and acclimatization of shoot/plantlets

The regenerated shoots from hypocotyl explants (transgenic and non‐transgenic) were initiated roots in solid or liquid (10 mL in 15 mL test tubes, immersing ~2 cm of the base of the shoot) half‐strength MS basal medium after 10 d. They produced a mean of 7.2 roots per shoot in 20 d and branched well (Figure 5A). While the shoots were transferred in liquid or solid half‐MS medium containing 0.49 μM IBA, roots were developed in 5–7 d. A mean of 8.1 roots per shoot was developed in 15 d and grew faster with branches than in the case of an IBA‐free medium (Figure 5A). In both cases, all the shoots induced roots. The shoots on half‐strength MS solid medium grew well with many branches (Figure 5B). The somatic embryo‐derived transgenic and non‐transgenic plantlets on half‐strength MS basal medium grew well with roots. The hairy root‐derived developed more roots and were highly branched.

(A) Rooting of shoots in half‐strength MS liquid media (Left: 20 d in basal media; Right: 10 d in basal media with 0.49 μM indole‐3‐butyric acid; (B) Rooting and growth of shoots on half‐strength MS (50 d); (C)‐(G) Expression of GFP on floral parts: (C) Flower; (D) Petal; (E) Stamen and stigma; (F) Anther; (G) Carpel; (H) Western blot of GFP; (I) Hardened *IaNAP1* edited plant. (Scale = 10 mm).

Shoots and somatic embryos with roots on SEM transplanted directly in the soil, and the plantlets, after in vitro rooting, displayed cent percent survival following acclimatization. Transferring healthy shoots from the shoot growing media (without rooting) to water in plastic cups (25 mL) in the plant room produced a mean of 4.5 roots per shoot in 20 d, and subsequently transplanted in soil, they showed 100% survival. The acclimatized transgenic and non‐transgenic plants grew well. The plants were transferred to large pots, and they flowered and set seeds.

3.8. Expression of GFP and DsREd

The expression of GFP and DsRED at different stages of transformation through organogenesis and somatic embryogenesis (Figures 4A, B, D‐J), and on the floral parts such as petal, stigma, and stamen of plants established ex vitro was shown in Figure 5C‐G.

3.9. Western blotting

Western blot of the GFP protein from plants transformed with GFP (through hypocotyl and hairy roots) using total protein extracted from their leaves showed a single band of 27 kDa (Figure 5H) of GFP protein.

3.10. Genome editing

Genome editing of the IaNAP1 gene by the transformation protocol using hypocotyl explants showed an efficiency of 70%, and two independent lines were developed per explant. The plants established (Figure 5I) did not display any phenotypic difference to that of the non‐edited. Of the genome‐edited plants established, 20% showed deletion of G (Figure 6) from the target (GAAAGCCACAGGCACA–ACA). Figure 7 shows the flow chart of the Agrobacterium‐mediated transformation using hypocotyl, leaf, and root explants with the timeline.

Sequence of the target *IaNAP1* (Orthologous to AT1G69490 (ANAC029/*AtNAP1*) showing the base deletion (red highlight).

Flow chart of *Agrobacterium‐*mediated transformation. Shoot induction medium‐hypocotyl (SIM–H ‐ CLC‐CP (Chee et al., 1992) medium with 4.5 μM thidiazuron, 8.7 μM gibberellic acid (GA₃), 50 mg l⁻¹ ascorbic acid (AA), and 100 mg l⁻¹ adenine hemi‐sulfate (AdS); Somatic Embryogenesis Medium (SEM) ‐ CLC‐CP medium with 4.4 μM 6‐benzyladenine, 0.45 μM 2,4‐dichlorophenoxyacetic acid, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS; Elongation/Germination Medium (EGM) ‐ CLC‐CP medium with 4.7 μM kinetin and 8.7 μM GA₃, 50 mg l⁻¹ AA and 100 mg l⁻¹ AdS; AS – Acetosyringone; ½MS – half‐strength MS medium. Timentin and selection antibiotics were added to the media as mentioned in the text. (using https://www.biorender.com).

3.11. Confirmation of transformation

All transgenic plants developed through organogenesis and somatic embryogenesis established ex vitro were positive and confirmed the integration of the marker genes. The PCR using the mgfp/ DsRed primers showed expected amplicons: 750 bp for mgfp and DsRed (Figure S2C & D). The PCR of the hairy root lines with mgfp produced 750 bp (Figure S2E). The untransformed plants and roots showed no amplification (Figure S2C‐E).

3.12. Hairy root culture

The confirmed hairy root segments transferred onto half‐strength MS (2% sucrose) medium and CLC‐CP medium with or without KIN favored faster growth with high branches (Figure S3A‐D). The hairy roots grew well in the light and were green, unlike off‐white in the dark (Figure S3C & D). Liquid media showed faster growth of the roots than on solid medium. The biomass (FW and DW) of hairy roots grown in the light and the dark in half‐strength MS liquid medium and those of the control (developed from hypocotyl and roots subcultured on half‐strength MS basal medium) was shown in Figure S3E. The hairy root segments were cultured and maintained on half‐strength MS solid media containing 2% sucrose.

4. DISCUSSION

Leafy vegetables are sources of essential nutrients for the well‐being of all humans. Rapid improvement of crops, especially neglected crops, through modern biotechnology tools is mandatory to contribute to the food security drive by providing quality/enriched food. The improvement of leafy greens, especially the regionally important ethnic ones, is inevitable in meeting the escalated demand. Water spinach is a perennial leafy green, unlike most annuals like spinach. Though a regionally important leafy green, its improvement by genetic modification warrants an efficient protocol of genetic transformation and genome editing.

A reliable and reproducible plant regeneration in vitro protocol is obligatory to accomplish the genetic modification. The flexibility of explants to in vitro morphogenesis is one of the important features in the rapid propagation and genetic modification of crops in particular. We have established an efficient in vitro regeneration protocol via organogenesis using hypocotyl and somatic embryogenesis from root and leaf explants of in vitro maintained shoots.

The hypocotyl explants of 5–7 d old seedlings in the present study displayed high‐efficiency shoot bud regeneration on CLC‐CP medium. Though TDZ was the superior cytokinin in the present study, supplementation of the media with 4.5 μM TDZ, 8.7 μM GA₃, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS yielded the highest frequency (90%) number of shoot buds and the highest (mean of 12.4 shoots per explant) on CLC‐CP medium. The culture of the petiole‐cotyledon in the present study produced green nodular development and induced a mean of 4.2 shoots per explant. In vitro regeneration from petiole‐cotyledon segments of water spinach through organogenesis (Akaracharanya et al., 2001) or callus (Mori et al., 1999) has been reported. They reported the shoot regeneration frequency in percentage, and the number of shoots per explant was not provided. In their study, the shoots were regenerated only from petiole‐cotyledon segments. The basal media used for their studies was half‐strength MS media. As to Akaracharanya et al. (2001), direct shoot regeneration from petiole‐cotyledon explants showed differential response to the age of the seedling with the best from 7‐d on TDZ supplemented medium, but as with BA in the case of 14‐d seedlings. Direct organogenesis was achieved by Mori et al. (1999) on media with 5 mg l⁻¹ BA. BA and KIN alone favored callusing, though KIN induced roots depending on the concentration. Akaracharanya et al. (2001) reported the effectiveness of TDZ in the induction of shoot buds in petiole‐cotyledon segments of water spinach. Shoot organogenesis by TDZ has also been documented in I. batatas (Gosukonda et al., 1995a; Sefasi et al., 2013; Kumar et al., 2013; Masekesa et al., 2016). The efficacy of TDZ, an N,N′‐diphenylurea derivative with its mode of action in morphogenesis, has been well emphasized in African violet as a model system (Erland et al., 2020). The potential of TDZ in shoot induction has also been documented in Scaevola sericea (Liang et al., 2020). GA₃ generally facilitates shoot elongation; however, in the present study, GA₃ was mandatory to induce shoot buds and their elongation. The efficacy of GA₃ in shoot induction has been emphasized in spinach (Al‐Khayri et al., 1992) and tea (Gonbad et al., 2014). Plant regeneration in various species of Ipomoea through organogenesis has been reported: I. batatas (Prakash et al., 1993; Dessai et al., 1995; Gosukonda et al., 1995a, b; Sihachakr et al., 1997; Vollmer et al., 2023), I. nil (Shimizu et al., 2003), I. obscura (Mungole et al., 2009), and I. sepiaria (Cheruvathur et al., 2015).

Somatic embryogenesis, for the first time in water spinach, was accomplished from leaf and root explants. In line with the organogenesis from hypocotyl explant, MS and CLC‐CP media were efficient in the induction of somatic embryos. The CLC‐CP media known as Ipomoea Basal Medium CP was originally developed for the somatic embryogenesis of the sweet potato (I. batatas) by Chee et al. (1992). They have formulated the media for sweet potato by evaluating the effects of K⁺, NO₃ ⁻ and NH₄ ⁺ ions ratio. CLC‐CP differs from MS in high K⁺ ions and organic supplements and is devoid of glycine. MS contains 18.8 mM, KNO₃ 20.6 mM NH₄NO₃, 26.6 μM glycine, 555 μM myo‐inositol, 0.29 μM thiamine HCl, 4.06 μM nicotinic acid, 2.42 μM pyridoxine HCl. In comparison, CLC‐CP consists of 20 mM KNO₃, 20 mM NH₄NO₃, 30 mM KCl, 500 μM myo‐inositol, 5 μM thiamine HCl, 10 μM nicotinic acid, and 5 μM pyridoxine HCl. The basal CLC‐CP medium contains 20 mM and 50 mM K⁺ with a total N of 60 mM (Chee et al., 1992). In the case of MS, the available ions are 39.4 mM NO₃ ⁻, 61.25 mM N, 0.97 mM NH₄ ⁺/K⁺, 0.52 mM NH₄ ^+/K⁺, and 5.98 Cl⁻, and in CLC‐CP, it is 51.25 mM, 60 mM, 0.5 mM, 0.4 mM, and 35.98 mM Cl⁻, respectively. The ionic forms of N affect growth and nutrient uptake by altering cation‐anion balance, changing organic anion accumulation, and ultimately affecting medium and cytosol pH values (Marschner, 1986; Poole, 1978; Spanswick, 1981). In the MS medium, the uptake of K⁺ and NH₄ ⁺ ions regulated the pH of the medium. Ammonium directly competes for uptake with K, the major cation that maintains cation‐anion balance and the most common counterion for organic anions (Marschner, 1986). Potassium is essential to the cytoplasm for the active conformation of numerous enzymes and the maintenance of osmotic potential (Clarkson and Hanson, 1980). As depicted by Chee et al. (1992), the increase of shoot buds and somatic embryogenic potential of leaf and root explants of water spinach in the present study is probably because the readily exchangeable K⁺ and its role in neutralizing organic and inorganic anions by an increase of K⁺ concentration from 18.8 to 60 mM in the presence of 20 mM NH₄ ⁺. High K⁺ concentration in CLC‐CP reduced the non‐embryogenic callus in leaf and root explants, as reported in sweet potato (Chee et al., 1992). Nevertheless, the reduction of non‐embryogenic callus and increase of embryogenic potential was less in the case of cotyledon and hypocotyl explants, probably due to the physiological status, and it necessitates further study.

Irrespective of the basal medium, the PGRs, and the additives played significant roles in the induction of shoot buds' induction and somatic embryogenesis. CLC‐CP or MS with 4.7/4.4 μM KIN/BA and 0.45/0.9 μM 2,4‐D, 50 mg l⁻¹ AA, and 100 mg l⁻¹ AdS facilitated somatic embryogenesis. As in organogenesis, AdS and AA influenced the organogenesis and somatic embryogenesis. The stimulatory effect of AdS and AA has been documented in in vitro morphogenesis of several plant species (Rathore et al., 2011; Shekhawat et al., 2021). The KIN‐supplemented medium favored the emergence of the root pole of the embryos. The concentration of the 2,4‐D determined the somatic embryogenesis efficacy. The somatic embryo induction potential of 2,4‐D alone or in combination with cytokinins at different ratios has been emphasized well (Raghavan, 2004; Kumar et al., 2017). However, the somatic embryogenesis in the present study relied on a high cytokinin to auxin ratio, as has been reported in other plants (Sagare et al., 2000; Liang et al., 2020; see review Asghar et al., 2023), which is probably pointed out to be due to the explant's physiological status. The hypocotyl and cotyledon explants on media KIN induced roots than with BA. These explants did not induce embryos in the 40 d period, which may be because of the physiological status of the explants. Maturation and germination of somatic embryos in the present study required the removal of 2,4‐D and the addition of GA₃ along with KIN. The influence of GA₃ in the progression of the embryos to maturation and germination as in water spinach has been reported in other plants as well (Sagare et al., 2000; Martin, 2004; Montero‐Córtes et al., 2010; Kumar et al., 2017). The culture of embryogenic calluses on CLC‐EP medium did not favor somatic embryogenesis unlike as in sweet potato (Chee et al. 1992). In the case of water spinach, all stages of the somatic embryogenesis were light inducive, and the influence of light has been emphasized in many plant species (Chen et al., 2014; Chen et al., 2016; Chan and Stasolla, 2023). Somatic embryogenesis and plant regeneration using various explants has reported in the Ipomoea spp: I. batatas (Jarret et al., 1984; Chee et al., 1992; Otani and Shimada, 1996), I. obscura (Otani et al., 1998), I. nil (Jia and Chua, 1992; Shimizu et al., 2003), I. purpurea and I. tricolor (Ishikuro et al., 2014).

Genetic modification by transgenesis and genome editing is highly valued in empowering basic science and improving crops. As a prerequisite for genetic modification, we determined the antibiotic sensitivity of different explants. The present study generated transgenic plants via Agrobacterium‐mediated transformation of hypocotyl explants by shoot development and root and leaf explants through somatic embryogenesis. Water spinach transformation with gus (Khamwan et al., 2003) and with rice cysteine synthase (rcs1) gene (Phokrai et al., 2006) was reported previously with <1% transformation efficiency. The transformation efficiency of the expression of rcs1 was 0.83% (2/240 regenerated shoots out of 908 cotyledons transformed). The present study using different explants exhibited a frequency with a mean of >3 plants (Figure 4) per explant. The induction of hairy roots and subsequently transformed plants through somatic embryogenesis in the present study is a facile and easy strategy to accomplish transgenesis in water spinach. The amenability of genetic modification in many species of Ipomoea, especially I. batatas, using Agrobacterium‐mediated transformation, has been documented (see review Imbo et al., 2016; Otani et al., 2021; Zhang et al., 2023). Overexpression of carotene biosynthetic pathway genes was reported in I. nil (Ono et al., 2000; Kikuchi et al., 2005; Watanabe et al., 2018a; Hoshino et al., 2019).

Shoot tip necrosis in a cyclic manner, with the emergence of shoot buds followed by shoot tip necrosis, was observed in the present study. Shoot tip necrosis is a physiological condition in vitro that results in the death of the shoot tip. It appears as basipetal and affects the emergence of axillary shoots from buds lower down the stem because of the cessation of apical dominance. As da Silva et al. (2020) reviewed, it occurs in shoot multiplication and rooting stages. The use of calcium chloride (Martin et al., 2007; Surakshitha et al., 2019), silver nitrate (Martin, 2002), and periodic subculturing at short intervals (Surakshitha et al., 2019) were demonstrated to alleviate the necrosis. Subculture at the onset of the necrosis controlled it in the present study. The shoot tip necrosis was not fatal to the shoots of water spinach in vitro, and the apical dominance is assumed by the next closest axillary bud assured the growth, as reported in sweet chestnut and oak (Vieitez et al., 1989).

Western blot of the GFP protein in transgenic water spinach in the present study confirmed the expression of proteins in leaves. The expression of human therapeutic proteins has been accomplished in many plants (see review Kulshreshtha et al., 2022). As a perennial leafy green, water spinach will be a choice for expressing proteins of therapeutic interest.

Genome editing through CRISPR introduced a new wave in the improvement of crops. It is highly valuable in improving the crops' ability to grow in adverse environmental conditions and to increase the nutrient profile. Genome editing in water spinach was accomplished for the first time. NAP/ANAC029 (a NAC‐like transcription factor activated by APETALA 3/PISTILLATA) is a key member of the NAC transcription family and was initially discovered to play a role in the cell division and expansion of stamens and petals in Arabidopsis (Sablowski and Meyerowitz, 1998). The functional analysis of AtNAP (AT1G69490) revealed its crucial role in regulating leaf senescence in Arabidopsis, with delayed senescence in knockout plants and accelerated senescence in overexpressing plants (Guo and Gan, 2006). AtNAP acts as a negative regulator by repressing the expression of AREB1, a key gene in the ABA‐dependent stress pathway (Seok et al., 2017). In their study, overexpression of AtNAP increased the sensitivity to salt, while the loss‐of‐function mutants enhanced salt tolerance. We have selected IaNAP1 from our transcriptome data (unpublished) for genome editing to delay the senescence, thereby increasing the shelf‐life of water spinach as a leafy green. Also, as an aquatic plant, enhanced salt tolerance is beneficial for cultivating in saline water. The studies related to evaluating the IaNAP1 edited plants are in progress. Nevertheless, the present study proves the reliability of the transformation protocol in generating genome‐edited lines of water spinach. Genome editing of DFR‐B in I. nil (Watanabe et al., 2017, 2018b) and I. batatas has been well‐documented (Wang et al., 2019) for improving Starch quality.

Besides developing transgenic plants, hairy roots facilitate large‐scale production of pharmaceutically important secondary metabolites in vitro. In the present study, we have accomplished hairy root culture in solid and suspension cultures. Water spinach contains several metabolites of pharmaceutical importance. It is the source of euphoria (anticancer; Li et al., 2018), lucidenic acid (tetracyclic triterpenoid that possesses anticancer, anti‐inflammatory, antioxidant, antiviral, antihyperlipidemic, antihyperglycemic, neuroprotective, and immunomodulatory properties; Zheng et al., 2023), and myricetin glycosides, squalene and α‐linolenic acid (Sasikala et al., 2022). Antidiabetic (Malalavidhane et al., 2001) and anti‐breast cancer properties by merromoside (Sasikala et al., 2022) and antiprostrate cancer (Octaviani et al., 2013) of water spinach have been emphasized. The roots of water spinach are a source of umbelliferone(7‐hydroxy‐2H‐1‐benzopyran‐2‐one) and scopoletin(7‐hydroxy‐6‐methoxy‐2H‐1‐benzopyran‐2‐one), two plant growth inhibitors (Omi et al., 2020). Induction of hairy roots has been accomplished on other species of Ipomoea, I. trichocarpa (Otani et al., 1996), and I. batatas (Otani et al., 1993; Zhang et al., 2023). Mei et al. (2024) accomplished in planta hairy root induction in I. batatas and I. pes‐caprae.

5. CONCLUSION

The accomplishment of an efficient plant regeneration protocol and reliable transformation methodology is valuable in improving water spinach, especially from a biofortification and phytoremediation perspective. Ipomoea Basal (CLC‐CP) medium fortified with plant growth regulators was superior to those supplemented with MS medium for organogenesis and somatic embryogenesis. Transformation of hypocotyl explants requires a shorter timeframe for transgenesis than leaf and root. Western blot of the GFP protein confirms its use to express therapeutic proteins of interest. Genome editing using hypocotyl explants of water spinach serves as a platform to improve the agronomic traits. As water spinach is amenable to self‐pollinating, it is easy to produce homozygous transgenic/genome‐edited lines and propagate vegetatively by stem cuttings.

AUTHOR CONTRIBUTIONS

FP: Preformed the experiments, analysis, and writing draft; MA: Preformed the experiments, reviewed the manuscript; SS, SK, & SB: Preformed the experiments, reviewed the manuscript; MK & LL: designed and supervised the experiments, reviewed the manuscript; KMAA: reviewed the manuscript, and editing.

FUNDING INFORMATION

The research project (No. LL0124) was funded by The Presidential Court, United Arab Emirates.

Supporting information

Appendix S1: Supporting information

PPL-177-e70257-s001.pdf^{(647.8KB, pdf)}

ACKNOWLEDGMENTS

We acknowledge the financial support of The Presidential Court, United Arab Emirates.

Purayil, F.T. , Alzaabi, M. , Sasi, S. , Krishnan, S. , Badar, Z. , Li, L. et al. (2025) Genetic modification of Water spinach (Ipomoea aquatica), a genoprotective perennial leafy green. Physiologia Plantarum, 177(3), e70257. Available from: 10.1111/ppl.70257

Edited by M.T. Islam

Contributor Information

Martin Kottackal, Email: martin@uaeu.ac.ae.

Khaled M. A. Amiri, Email: k.amiri@uaeu.ac.ae.

DATA AVAILABILITY STATEMENT

All data supporting the findings of this study are included in the paper.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1: Supporting information

PPL-177-e70257-s001.pdf^{(647.8KB, pdf)}

Data Availability Statement

All data supporting the findings of this study are included in the paper.

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PERMALINK

Genetic modification of Water spinach (Ipomoea aquatica), a genoprotective perennial leafy green

Fayas Thayale Purayil

Mariam Alzaabi

Shina Sasi

Saranya Krishnan

Zarreen Badar

Ling Li

Martin Kottackal

Khaled M A Amiri

Abstract

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Seed germination in vitro

2.2. Shoot tip/node culture establishment

2.3. Shoot organogenesis

TABLE 1.

2.4. Somatic embryogenesis and plant regeneration

TABLE 2.

TABLE 3.

2.5. Antibiotic sensitivity test

2.6. Plant transformation vectors

2.7. Transformation and transgenic plant regeneration

2.8. Rooting and acclimatization of shoot/plantlets

2.9. Expression of GFP and DsREd

2.10. Confirmation of transformation

2.11. Western blotting

2.12. Plasmid construction for genome‐editing

2.13. Confirmation of genome‐edited plants

2.14. Hairy root culture

2.15. Statistical analysis

3. RESULTS

3.1. Shoot tip/node culture

3.2. In vitro regeneration

3.2.1. Shoot organogenesis

FIGURE 1.

3.2.2. Somatic embryogenesis and plant regeneration

FIGURE 2.

3.3. Antibiotic sensitivity of different explants

3.4. Agrobacterium‐mediated transformation and transgenesis

FIGURE 3.

FIGURE 4.

3.5. Hairy root induction and plant regeneration

3.6. Shoot tip necrosis or dieback

3.7. Rooting and acclimatization of shoot/plantlets

FIGURE 5.

3.8. Expression of GFP and DsREd

3.9. Western blotting

3.10. Genome editing

FIGURE 6.

FIGURE 7.

3.11. Confirmation of transformation

3.12. Hairy root culture

4. DISCUSSION

5. CONCLUSION

AUTHOR CONTRIBUTIONS

FUNDING INFORMATION

Supporting information

ACKNOWLEDGMENTS

Contributor Information

DATA AVAILABILITY STATEMENT

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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