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Organogenesis from Leaf-derived Calli of S. potatorum L. f.: A Vulnerable Medicinal Tree Species

P.G. & Research Department of Botany, The Madura College (Autonomous), Madurai - 625 011, Tamil Nadu, India

Article Info:

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Article History:

Received 29 April 2022

Reviewed 04 June 2022

Accepted 09 June 2022

Published 15 June 2022

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Cite this article as:

Vijaya Kumar V, Saralla RP, Kannan P, Organogenesis from Leaf-derived Calli of S. potatorum L. f.: A Vulnerable Medicinal Tree Species, Journal of Drug Delivery and Therapeutics. 2022; 12(3-S):169-174

DOI: http://dx.doi.org/10.22270/jddt.v12i3-s.5528 _____________________________________________

*Address for Correspondence:

Palanisamy Kannan, P.G. & Research Department of Botany, The Madura College (Autonomous), Madurai - 625 011, Tamil Nadu, India

Abstract

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An efficient protocol for in vitro regeneration of Strychnos potatorum L. f. through indirect organogenesis was developed using mature leaf disc explants. The explants were selected from twenty years old tree. Cultured on Murashige and Skoog’s medium supplemented with 2,4-dichloro phenoxy acetic acid (2,4-D) was used for the induction of organogenic calli. A pale yellow coloured friable callus developed at 2 mg/L^-1 2,4-D where showed higher morphogenic potential. Further de-novo shoots formation and multiplication in optimal concentrations of cytokinins with different combinations of auxins. An average mean shoots number (14.2±0.42) and shoots height (3.5±0.27cm) was obtained from piece of calli in 6-benzylaminopurine(BAP) 1 mg/L^-1with 0.5 mg/L^-1α-naphthalene acetic acid (NAA). BAP in combinations with NAA was found to be superior to shoot development than other combination used. The well developed shoots were transferred to root induction medium for effective rooting. The rooting medium consisted of in half-strength MS medium augmented with 0.5 mg/L^-1 NAA was used. The maximal mean number of roots per shoots 6.5±0.53 was recorded in 4 weeks. The rooted plantlets were removed from the culture tubes, gently washed with sterile double distilled water to remove agar adhering on the plant surface. The regenerated plants were transferred to plastic cups filled with a potting mixture containing sterile sand and soilrite (1:1 ratio) and allowed to grow in the greenhouse. The slowly acclimatized plants were further transferred to substrate containing red soil and farmyard manure (1:1) for effective hardening. 75% survival rate was recorded. This protocol can be useful for the ex-situ conservation and rehabilitation of this vulnerable medicinal tree S. potatorum.

Keywords: Indirect organogenesis, friable callus, shoot development, 6-benzylaminopurine, α-naphthalene acetic acid

Strychnos potatorum L. f. (Loganiaceae) is a medium sized deciduous tree found in India and other parts of the world ¹. It is naturally growing in forests up to 1200 m altitude in the forest ². Seeds are popularly known as ‘clarifying nuts’ and are utilized by rural household for generations to clarify turbid water ³. This property was attributed to the presence of anionic polyelectrolytes having -COOH and free -OH surface groups that are present in the seed proteins ⁴. It is a tropical tree species used in traditional medicinal systems. The roots are used to cure leucoderma. The ripe fruit is emetic, diaphoretic, alexiteric, cures inflammation, anemia, jaundice ⁵. Its bark and root is used to control microbial infections. In Ayurveda, the plant is used for treating eye and urinary tract infections ⁶. Due to indiscriminate collection and over-exploitation for medicinal and commercial uses leads to decline its numbers in the natural habitat. Further, the plant fruits severely damaged by the invasion of fungal mycelium as a result seeds showed a hollow without embryos. Moreover, fewer seed germination rate and poor vegetative cuttings due to erratic rooting behavior are also encompasses major bottlenecks of this tree species. It is one of the fast disappearing vulnerable medicinally important forest tree species ^7-8. To the best of our knowledge, only a single report describing in-vitro regeneration of S. potatorum was documented ⁸. Till date, no reports are available on in-vitro organogenesis from leaf disc explants. Hence, the present study was under taken to demonstrate efficient protocol for organogenesis of S. potatorum.

The tender leaves were collected from natural habitat (latitude 10°16'01.4"N and longitude 77°59'57.6"E 750 feet elevation) in Eastern Ghats, Tamil Nadu, India during autumn season. The leaves were thoroughly washed with running tap water for 1 hr. to remove debrices. The leaves were surface disinfected with 0.1% bavistin and 2-3 drops of Tween 20 followed by 0.03% (w/v) mercuric chloride HgCl₂ (Himedia Mumbai, India) for 5 minutes. The explants were rinsed for 5 or 6 times with sterile double distilled water, in order to remove chemical residues. Aseptically, the leaves were made into leaf-discs 1cm using cork borer and used further for in-vitro studies.

The nutrient medium consisted of Murashige and Skoog’s media ⁹, with 3% (w/v) sucrose (Nice, Chemicals, India) and 0.8% (w/v) agar (Himedia, Mumbai, India). The pH of the medium was adjusted to 5.8 by 0.1 N NaOH or 0.1 N HCl after adding the plant growth regulators (PGRs) (Himedia, Mumbai, India). The medium was autoclaved at 1.05 kg/cm² pressure and 121 °C temperature for 15 minutes. The culture was maintained at 25 ± 2 °C for 16 h under a light intensity supplied by cool white fluorescent lamps (Philips, India) and > 80% relative humidity.

The effect of various concentrations of 2,4-dichloro phenoxy acetic acid (2,4-D) was tested for callus induction. The calli were subcultured after the interval of 30 days in the same medium. After 30 days, the colour and texture, were observed and recorded. Percentage of callus formation was also calculated. The suitable ideal calli were selected for the regeneration through organogenic pathways of this species. Further, shoot development and multiplication on MS medium was supplemented with various concentrations (0.5 to 3.0 mg/L^-1) of 6 benzylaminopurine (BAP), thidiazuron (TDZ), and kinetin (Kn) with Naphthalene acetic acid (NAA) was studied. Subcultures were made at four weeks intervals. The frequencies of shoots numbers and shoot heights was recorded after 8-weeks incubation.

The healthy regenerated shoots from multiple shoot clusters were separated and transferred to a rooting medium. The rooting medium contains half-strength MS medium supplemented with different concentration (0.2 to 2.0 mg/L^-1) of α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA), and Indole-3-acetic acid (IAA) individually. Well-developed rooted plantlets were removed from the culture tube, gently washed with sterile distilled water to removed agar adhering on plant surface. The plantlets were transferred to plastic cups filed with soilrite. The cups were covered with polythene bags to maintain relative humidity in the controlled environmental condition. The slowly acclimatized plants were transferred to polythene cups containing potting mixture consisted of sterile sand and soilrite (1:1 ratio) and allowed to grow in the greenhouse. The survival rate was recorded 4 weeks after transplantation.

Experiments have been set up indefinitely in a randomized design. Data had been collected at the end of the 8-weeks for callusing and formation of multiple shoots. The rooting was evaluated 4-weeks from culture initiation. Each experiment were repeated at least twice consisted of 30 explants. Significance was once determined by using calculating the Mean ± Standard Deviation in One-way ANOVA using SPSS statistical software package (Trial version: 16) was used.

The present investigation describes high-frequency shoot induction from the callus-derived from tender leaf disc were taken as an explants. Leaves selected from mature trees were amenable to obtain calli and effective for organogenesis. Moreover, initially plant regeneration using young leaves may reduce the chances of genetic variation ¹⁰. A reddish- pale brown, friable callus formation was seen at the edges of the leaf lamina. No callus formation was noticed in PGRs-free control medium. Explants were turned necrotic after few days of inoculation. In this study the microbial contaminants are found on both surface as well as inside the leaflets. Similar observation was made in ¹¹. At the early stages of the culture period, the proliferation frequency rate of calli was very low. At the cut end of the explants became blackened due to phenolic exudation. Following successive for two or three subcultures to control browning of explants and calli rapidly proliferation frequency increased. These calluses were distinguished into three types based on their color and surface texture (Fig. 1 a & b). Callus formation decreased with increasing concentrations of the plant hormones.

A white friable callus was induced on MS medium supplemented with 2,4-D. Interestingly, no callus formation was observed on MS medium supplemented with low 2,4-D. MS medium containing 2,4-D at 1.0 mg/L^-1 produces compact callus whereas soft friable callus was produced in MS medium containing 2,4-D (2.0 mg/L^-1). Among the different concentrations of 2,4-D tested, the best callus production (71%) was achieved at 2.0 mg/L^-1 2,4-D (Table 1). Similarly callus induction from leaf-discs were reported from earlier studies on de-novo organogenesis in Gymnema sylvestre ¹². Furthermore, 2,4-D influenced the molecular and physiological processes of calli by activating proteins, regulating endogenous IAA metabolism and regulating DNA methylation ^13-14. In contrast, in other trees the combination of auxin with cytokinins obtained regenerable green callus was also reported ¹⁵.

Organogenic green calli were transferred to regeneration medium for shoot development. The medium for calli-derived shoot differentiation was achieved with different combinations of plant growth regulators. Organogenic calli was considered to be one of the most efficient regeneration and transformation system for plants ^16-17. The shoot differentiation started 4-weeks after transferring the calli to regeneration medium (Fig.1 c). The plant regeneration was characterized by the development of different concentrations of cytokinins in combinations with NAA. In the above medium improved the shoot induction both in terms of height and number of shoots per calli piece. The TDZ with NAA combination showed low frequency of shoot formation was observed (Fig 1 d & Table 2). However, BAP 1.0 mg/L^-1 with NAA at 0.5 mg/L^-1 produced maximum mean shoots (14.2 ± 0.42) with average mean shoot height (3.5 ± 0.27 cm) was recorded (Fig. e & f). The induction frequency and number of shoots per calli was low in other cytokinins used in this study. The synergistic effect of BAP & NAA combinations on shoot organogenesis reported for several woody species ^18-20. Different concentrations of TDZ in combination with NAA and Kn (Table 2) were also tested in this study aforesaid. The combination showed, the maximum average mean value for callus induction frequency (57% and 64%) was recorded.

The in vitro regenerated healthy shoots were excised from the base and transferred to half-strength MS medium supplemented with different concentration of IBA,NAA and IAA (0.2 and 3.0 mg/L^-1) individually. Rooting was observed within 4 weeks of culture as shown in Fig. 1 g. A significant rooting with maximum mean shoot and root length were observed with the increasing in concentration NAA. The highest rooting frequency (70%) were achieved on ½ MS medium supplemented with 0.5 mg/L^-1 NAA the maximum number of root per shoots (6.5 ± 0.53) and highest root length (3.0 ± 0.24cm) was observed (Table 3). Auxins are known to promote adventitious root formation and further development. NAA is the best auxins showed effective rhizogenesis. The similar findings of high root induction were reported in Neolamarkia cadamba ²¹, Coccoloba uvifera ²². This may be due to an appropriate concentration of auxin acting on cells, which is beneficial for binding with ATP enzyme on the plasma membrane. This acidifies the cell wall environment, and some unstable hydrogen bonds are easy to break, so that the molecular structure of cell wall polysaccharides is intertwined. The cell wall tends to relax, making the cells that form the adventitious roots easily broken through. Well-rooted plantlets were transplanted into sterile plastic cups containing sterile sand and soilrite (1:1) mixture, acclimatized in the controlled condition then the plants were transferred to green house after 30 days. The survival percentage of such plants was 75%.

Figure 1: In-direct organogenesis from leaf-derived calli of S. potatorum. a - Callus induction from mature leaf disc, b – Callus formation after 10 days, c – Initiation of shoots from callus, d - Multiple shoot formation in TDZ at 1.0 mg/L^-1 0.5 mg/L^-1NAA, e - Multiple shoot formation in BAP 1.0 mg/L^-1 0.5 mg/L^-1NAA, f – Shoots elongation, g – Root formation in 0.5 mg/L^-1NAA, h – Plant hardening in paper cups containing sand and soilrite.

2,4-D (mg/L^-1)	% callusing response	Degree of callus formation	Colour and texture of callus
0.0	00	0.0	0.0
0.5	12	-	-
1.0	29	+	White, friable
1.5	42	++	Yellowish brown and friable
2.0	71	+++	Pale yellow and friable
2.5	59	++	Yellow and compact
3.0	40	+	Yellow and compact
Induction of callus given in different grades viz. (-) Nil, (+) Minute, (++) Moderate, (+++) High

Table 2: Effect of adventitious shoots formation and regeneration of S. potatorum leaf cultured on MS medium.

PGRs (mg/L^-1)				Shooting response (%)	Mean number of shoots	Height of the shoot (cm)
TDZ	BAP	Kn	NAA	0.0	0.0	0.0
0.5			0.5	23	4.8±0.42^d	2.5±0.46^d
1.0			0.5	64	10.6±0.52^g	2.4±0.43^c
1.5			0.5	52	8.3±0.48^ef	2.3±0.35^c
2.0			0.5	47	6.6±0.52^e	2.3±0.22^c
2.5			0.5	41	4.3±0.48^d	1.9±0.41^ab
3.0			0.5	36	2.5±0.53^b	1.7±0.32^a

	0.5		0.5	35	8.5±0.53^f	2.4±0.36^b
	1.0		0.5	76	14.2±0.42^h	3.5±0.27^c
	1.5		0.5	67	10.4±0.52^gh	2.8±0.31^b
	2.0		0.5	54	9.5±0.53^g	2.2±0.48^b
	2.5		0.5	45	8.3±0.48^ef	1.8±0.08^a
	3.0		0.5	30	7.5±0.53^ef	1.6±0.41^a

		0.5	0.5	18	2.7±0.48^b	2.1±0.09^b
		1.0	0.5	57	6.6±0.52^ef	2.5±0.29^b
		1.5	0.5	50	5.8±0.42^e	2.1±0.16^b
		2.0	0.5	44	3.7±0.48^c	1.9±0.45^ab
		2.5	0.5	39	2.9±0.32^b	1.8±0.41^ab
		3.0	0.5	28	1.7±0.48^a	1.7±0.50^a

*Mean ± SD; mean for each experiment marked with the same letter does not differ significantly (P< 0.05)

Table 3: In vitro root formation of S. potatorum in different auxins on half-strength MS medium.

Auxin (mg/L^-1)			Rooting response (%)	Mean number of roots per explant	Length of the roots (cm)
NAA	IBA	IAA	0.0	0.0	0.0
0.2			42	4.0±0.47^e	1.9±0.29^ab
0.5			70	6.5±0.53^f	3.0±0.24^c
0.7			59	4.9±0.57^cd	2.2±0.15^b
1.0			43	3.3±0.48^c	2.1±0.41^b
1.5			36	2.0±0.47^b	1.8±0.08^a
2.0			28	1.3±0.48^a	1.6±0. 50^a

	0.2		39	3.4±0.52^c	2.1±0.18^ab
	0.5		63	4.5±0.53^cd	2.6±0.10^b
	0.7		51	3.8±0.42^d	2.4±0.29^b
	1.0		39	2.9±0.32^b	2.1±0.18^b
	1.5		32	1.8±0.42^a	1.9±0.41^a
	2.0		29	1.3±0.48^a	1.7±0.25^a

		0.2	35	2.2±0.42^bc	2.0±0.12^b
		0.5	58	3.3±0.48^bc	2.5±0.29^bc
		0.7	49	2.5±0.53^b	2.1±0.46^b
		1.0	36	2.2±0.42^b	1.9±0.66^ab
		1.5	27	1.3±0.48^a	1.8±0.41^a
		2.0	25	1.1±0.32^a	1.7±0.50^a

*Mean ± SD; mean for each experiment marked with the same letter does not differ significantly (P< 0.05)

In conclusion, this study demonstrated a complete feasible protocol for indirect-organogenesis of S. potatorum under in vitro using leaf-derived callus. BAP at 1.0 mg/L^-1along with 0.5 mg/L^-1NAA was found best combination was optimized for effective de-novo shoot formation. Effective root formation was achieved using NAA. This protocol can be useful for large scale propagation in a short span of time and also helpful in ex-situ conservation of this vulnerable medicinal tree species.

The authors are grateful to Prof. S. Karuppusamy, Department of Botany, The Madura College, Madurai, Tamil Nadu, India for helping in plant collection and identification. Dr. P. Kannan thank University Grants Commission [UGC, MRP (42/936) 2013-2017] New Delhi for financial assistance.

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