In vitro regeneration of Allophylus serratus. Roxb (Kurz), an important medicinal plant

doi:10.21203/rs.3.rs-1989679/v1

Background

Allophylus serratus Roxb (Kurz.) is a medicinal plant with various therapeutic properties. It has been used for the treatment of various alignments such as elephantiasis, edema, fractured bones, in bone dislocations, wound, several gastrointestinal disorders such as dyspepsia, anorexia, and diarrhea. The aim of this study is to establish protocol for surface sterilization, in vitro callus induction and plant regeneration from leaf and nodal segment explants of Allophylus serratus.

Results

From the tested media, the maximum callus induction frequency (81.11%) from leaf and 84.44% from nodal segment explants were achieved on MS medium supplemented with 3 mg l^− 1BAP) combined with 0.5mgl^− 1 NAA. The highest shoot regeneration frequency (57.78%) and root regeneration frequency (65.55%) were achieved on growth regulators free MS medium and MS medium supplemented with 1.5 mg l^− 1IBA respectively. Acclimatization of the regenerated plantlets was established successfully (100%) in a pot containing sand, soil and compost in 1:2:2 ratios.

Conclusions

In this study successful callus induction protocol from leaf and nodal explants of A. serratus using MS media supplemented with different concentrations and combinations of plant growth regulators (2,4-D, NAA, and BAP). MS media containing 3mg/l BAP and 0.5mg/l NAA was the most efficient for callus induction. these callus induction and plant regeneration protocols are very important for mass propagation of the species and also opens a new way to facilitate secondary metabolites production and isolation of pharmaceuticals from callus rather than harvesting the plant.

Allophylus serratus

callus induction

in vitro regeneration

leaf explant

plant regeneration

medicinal plant

Allophylus serratus (Roxb) Kurz, locally known as Tippani in Hindi is a medicinal, dicotyledonous plant of the family Sapindaceae. It is an evergreen, low branching small tree or a large shrub about 3-6m tall. Theplantis found distributed in India, Sri Lanka, and South East Asia. In India; it is found to be growing on the site of Andhra Pradesh, West Bengal, Arunachal Pradesh, Tripura, Orissa, Karnataka, Tamilnadu and Kerala, scrubby forest of Mahabaleshwar, scrubby forests of Kolhapur, Satara, Borbet, Barki, Manoli, Panhala, Patgaon of Kolhapur District (Kero et al. 2015). According to Selvam (2007), the plant is tolerant to drought and high salinity. It is adapted to grow in fine sandy, coarse and nutrient poor soil and it can also grow well in shade too.

Allophylus serratus has strong ethnobotanical and ethnopharmacological background. The plant has been used in Indian system of medicine (Ayurveda) for the treatment of various alignments such as elephantiasis, edema, fractured bones, in bone dislocations, wound, several gastrointestinal disorders such as dyspepsia, anorexia, and diarrhea (Gupta and Tandon 2004). This plant is also used as an anti-inflammatory and as a carminative drug. The plant has edible fruits that are sweet having cooling and nourishing tonic. The leaves are used to reduce fever, to relieve rashes, promote lactation, to treat colic, to relieve stomach aches, as antiulcer and to reduce piles (Maurya and Srivastava 2011; Devi et al. 2013). The roots of this plant contain tannin and are considered astringent and used for treating nose bleeding, diarrhea and rheumatic pains (Maurya and Srivastava 2011).

Due to overexploitation, depletion of habitat and misuse, the world is facing the problem of losing valuable plant resource. This situation is an alarm for efficient and in time conservation measures to enrich our lives with the services of plants. Various research groups across the world have attempted conservation of plants to protect biodiversity (Parabia et al. 2007; Nadeem et al. 2000). Different techniques for conservation of plants have been practiced worldwide, the most important being tissue culture (Pola 2005; Parabia et al. 2007). Plant tissue culture is an important method to overcome the problem connected with medicinal plants conservation and utilization (Bajaj et al.1988).

In vitro conservation of medicinal plants by tissue culture is advantageous in producing multiple copies of a plant species within minimum time and space. This in vitro conservation consists of different techniques such as selection of explants, sterilization of explants, aseptic culture establishment, multiple shoot production; rooting of the shoots followed by acclimatization of the plantlets. From these stages of in vitro conservation techniques, standardization of surface sterilization of explants is fundamental for aseptic culture establishment. In the majority of plant tissue culture laboratories, on average there are 3-15% losses due to contamination (Leifert et al. 1989) the majority of which is caused by bacteria and fungi (Leifert et al.1994). Various factors such as plant species, the source of explants, the age of explants and other climatic conditions are the reasons for explant contaminations (Srivastava et al. 2010). In addition to surface sterilization, standardization of media (concentration and combinations of plant growth regulators) for the callus induction is critical in tissue culture technique (Pola et al. 2007 and 2008). Callus cultures are used to study cell development and to obtain primary and secondary metabolites through cell suspension cultures. In addition suspension cultures can be used to obtain different novel products through bio-transformation. Plant regeneration protocol development through callus has also applications in production of novel products through genetic transformation.

Various studies have been carried out on the biochemical (Priya et al. 2012), ethnobotanical (Kero et al. 2015, Binu and Rajendran 2013 and Trivedi 2006) and medicinal (Dharmani et al.2005 ; Gaikwad and Chavan 2013; Vinay et al. 2005) aspects of Allophylus serratus. Almost all researchers have focused on its medicinal properties. It is sensible to mention that there are no research reports on tissue culture issues (surface sterilization, callus induction and in vitro plant regeneration studies) before in this plant species. Surface sterilization and callus induction studies are the prerequisites for tissue culture and genetic manipulation studies and conservation of this medicinal plant. Therefore, the objectives of this study were to establish surface sterilization and in vitro regeneration protocol through callus induction of this plant.

2.1 Plant materials

Young, green and healthy leaves and nodal segments of Allophylus serratus were used as explants for both surface sterilization and callus induction protocol studies. Explants were collected from mature mother plant from the field at Andhra University compound and Biodiversity park at Visakhapatnam, India. The plant species was authenticated by Dr. Bodaih Padal, taxonomist, department of botany, Andhra University, Visakhapatnam. The voucher specimens (21921) were deposited in the herbarium, college of Science and Technology, Department of Botany, Andhra University.

2.2 Surface sterilization of leaf and nodal segment explants

The explants were collected from the field and taken to the laboratory in a bottle containing water. Once in the laboratory, they were washed under running tap water for 30 minutes. Then the explants were washed with standard detergent powder and kept under running tap water for 30 minutes. They were then washed with Tween -20 and rinsed with sterile double distilled water 3-5 times. In a laminar hood (inside surface was first sterilized by 70 % ethanol and then sterilized by an ultraviolet light for at least 20 min before use), the explants were surface sterilized by using Ethanol, NaOCl and HgCl₂. In the first method, the explants were dipped into 70% Ethanol for 1minute followed by HgCl₂at concentrations of 1% and 0.1% for 3, 4 and 5 minutes. In a second way, 70% Ethanol for one minute followed by NaOCl at 1.0% and 2.0% concentrations for 3, 4 and 5 minutes were used for surface sterilization. The third way involves surface sterilization of explants with70% Ethanol for one minute followed NaOCl at concentrations of 1.0% and 2.0% for 3, 4 and 5 minutes and HgCl₂ (1.0% and 0.1%) for 2 and 3 minutes. After each chemical sterilants treatment, the explants were washed with sterile double distilled water three to four times with gentle shaking under sterile conditions to remove the chemical sterilants and reduce their effect on explants.

2.3 Callus Induction

After sterilization, the explants were cut into the suitable size, dried on sterile tissue paper and were inoculated onto MS (Murashigie and Skoog 1962) medium with 3.0% sucrose and 0.80% bacteriological grade agar (Hi-Media, India) and supplemented with different concentrations and combinations 2,4-D ( 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg/l ), BAP (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg/l) NAA and (0.5 mg/l) alone and in combination. The pH of the medium was adjusted to 5.80 using 1N HCl or 1N NaOH before autoclaving. The media was autoclaved at 121^oC at 1.50 kg cm-2 for 20 minutes. The plant growth regulators were added to the media by filter sterilization (pore size 0.22 micron) after the media was autoclaved and cooled to 50-60 ^oC. Fifteen to Twenty milliliters of the medium was dispensed into culture tubes or Petri dishes under an aseptic condition in a laminar hood. Cultures were then kept at a temperature of 27 ± 3 ^oC, air humidity of 55% and in dark. Growth regulators free MS medium was used as a control. Data on, contamination, survival, and a loss percentage of explants, the rate of callus response, nature of the callus (callus texture, callus color), the day of callusing, callus fresh weight and dry weight were recorded.

2.4 Shoot regeneration from callus

For shoot regeneration, the calli induced on callus induction MS media were sub cultured on same medium every four weeks two times. Then the healthy and embryonic calli were cut into small pieces and inoculated on to shoot regeneration MS media supplemented with BAP (1, 2, 3, 4 mg/ l) and Kinetin (1, 2, 3, 4 mg /l). Plant growth regulators free MS media was used as control. All the cultured calli were incubated under 16/8 hrs photo period (light/dark) with light provided by white fluorescent tube lights (1000 lux) at 27±3 °C for four weeks. After four weeks the data were recorded for percent of shoot formation, (number) and length of shoots.

2.5 Root regeneration

Healthy regenerated shoots with two or more leaves and about 1-3 cm in length were cultured on root induction MS media supplemented with NAA (0.0, 0.5, 1.0, 1.5, 2.0 mg/l) or IBA (0.0, 0.5, 1.0, 1.5, 2.0 mg/l). Hormone free MS media was used as control. The cultures were kept in 16/8 hrs photo period (light/dark) at 27±3 °C temperature. After four weeks of culture, the data were recorded for percent of root induced, number and length of roots.

2.6 Acclimatization

Regenerated plantlets in good conditions (good looking shoot and root) were carefully removed from culture tubes and washed with sterile distilled water to remove the agar. The plantlets were then planted in pots containing sand, soil, and compost in 1:2:2 ratios. The potted plantlets were covered with polyethylene bags with small holes and kept in tissue culture room for 10 days. The polyethylene cover was removed little by little after 7 days. After 30 days the number of survived plants was recorded.

2.7 Experimental Design and Data Analysis

Completely Randomized Design (CRD) with three replicates per treatment and thirteen explants per replicate were arranged. The data of explants surface sterilization (rate of contamination, survival, and loss) were recorded as percentage. The data of effect of different concentrations and combinations of growth regulators on callus induction, shoot and root regeneration were recorded and subjected to one-way ANOVA using statistical data analysis software SPSS version 20.0 at 5% probability level.

3.1 Explant Surface Sterilization

The data obtained on surface sterilization of leaf and nodal segment explants was carried out using Ethanol (70%), NaOCl (1% and 2%) and HgCl₂ (0.1 % and 1%) in different combination at varying length of time intervals given in Table 1. The data reveals that the highest percent survival of leaf explants (80.00%) was achieved with treatment (70% ethanol for 1minute, 1.0% NaOCl for 4 minutes and 0.1% HgCl₂ for 3 minutes) and the highest percent survival of nodal segment explants (90.00%) was with treatment (70% ethanol for 1min, 2% NaOCl for 3 minutes and 1.00% HgCl₂ for 2 minutes. Percentage of contamination was brought down to 30% from 100% for leaf explants and 10% from 100% for nodal segment explants. The least leaf explants survival (10.00%) was recorded with 70% Ethanol for 1 minute followed by 1.00% NaOCl for 3 minutes and for nodal segment explants the least survival (14.4%) was recorded with 70% ethanol for 1 minute followed 1.0% NaOCl for 5 minutes. The result of our study showed that the leaf and nodal segment explants of Allophylus serratus have shown diverse sterility response for different concentrations and combinations of chemical sterilants at the different duration of time.

Table 1. Effect of different Chemical Sterilants on surface sterilization of leaf and nodal segment explants of Allophylus serratus after treatment with 70% Ethanol for 1 min.

Concentrations of Sterilizing Agents (%)		Exposure time (Minutes)	Leaf explants			Nodal segment explants
Concentrations of Sterilizing Agents (%)		Exposure time (Minutes)	Contaminated %	Survival %	Lost %	Contaminated %	Survival %	Lost %
----	0.00	0.00	100	0.00	0.00	100	0.00	0.00
HgCl₂	0.10	2.00	38.89	53.33	7.78	52.22	36.67	11.11
HgCl₂	0.10	3.00	37.78	33.33	28.89	44.44	47.78	7.78
HgCl₂	1.00	2.00	58.89	25.56	15.56	42.22	34.44	23.33
HgCl₂	1.00	3.00	56.67	24.44	18.89	62.22	22.22	13.33
NaOCl	1.00	3.00	72.22	10.00	17.78	60	23.33	16.67
		4.00	58.89	32.22	20.00	57.78	24.44	17.78
		5.00	41.11	38.89	20.00	70.00	14.44	15.56
NaOCl	2.00	3.00	38.89	44.44	16.67	63.33	33.33	3.33
		4.00	41.11	48.89	10.00	52.22	31.11	16.67
		5.00	40.00	50.00	10.00	53.33	32.22	8.89
NaOCl + HgCl₂	1.00+0.10	3.00+2.00	3.33	38.89	50.00	51.11	38.89	10.00
		4.00+2.00	3.67	30	57.78	51.11	38.89	10.00
		5.00+2.00	4.33	25.56	60.00	43.33	38.89	17.78
		3.00+3.00	2.33	37.78	54.44	37.78	47.78	13.33
		4.00+3.00	0.67	40.00	57.78	33.33	44.44	20.00
		5.00+3.00	3.00	21.11	68.89	37.78	48.89	13.33
NaOCl + HgC_l2	2.00+ 0.10	3.00+2.00	20.00	72.22	7.778	30.00	54.44	15.56
		4.00+2.00	18.89	68.89	12.22	25.56	57.78	16.67
		5.00+2.00	12.22	70.00	17.78	33.33	53.33	13.33
		3.00+3.00	18.89	68.89	11.11	33.33	57.78	8.89
		4.00+3.00	15.56	80.00	4.444	23.33	65.56	11.11
		5.00+3.00	13.33	70.00	16.67	15.56	73.33	11.11
NaOCl + HgCl₂	1.00+1.00	3.00+2.00	20.00	72.22	7.778	27.78	60.00	12.22
		4.00+2.00	40.00	42.22	17.78	18.89	68.89	8.89
		5.00+2.00	33.33	55.56	11.11	20.00	67.78	12.22
		3.00+3.00	23.33	66.67	10.00	17.78	70.00	12.22
		4.00+3.00	25.56	47.78	26.67	13.33	72.22	14.44
		5.00+3.00	26.67	57.78	15.56	16.67	71.11	11.11
NaOCl + HgCl₂	2.00 +1.00	3.00+2.00	23.33	62.22	14.44	6.667	90.00	3.33
		4.00+2.00	22.22	67.78	10.00	11.11	72.22	14.44
		5.00+2.00	23.33	51.11	25.56	16.67	61.11	22.22
		3.00+3.00	20.00	47.78	34.44	27.78	56.67	15.56
		4.00+3.00	11.11	50.00	38.89	24.44	61.11	14.44
		5.00+3.00	15.56	53.33	31.11	17.78	71.11	11.11

3.2 Callus induction

The initiation of callus induction from leaves and nodal segment explants of Allophylus serratus was carried out after successful sterilization of the explants. The successfully surface sterilized leaves and nodal segment explants were inoculated onto callus induction media (MS medium supplemented with different concentration and combinations of 2, 4-D, NAA and BAP. Different hormonal regime induced callus from both leaf and nodal segment explants differently. Callus induction frequency and callus type induced varied depending on the type of explant used, type and level of plant growth regulator used.

3.2.1 Effect of 2, 4-D on callus induction

Leaf and nodal segment explants cultured on MS medium supplemented with different concentrations of 2, 4-D responded differently. The highest response of callus induction in leaf explants (68.89 ± 2.94 %) and nodal segment explants (42.22 ± 2.94 %) were observed on the medium supplemented with 3.00mg/l 2, 4-D. Whereas the minimum callus induction response 8.89 ± 4.01% and 10.00 ± 1.92% were observed on MS media with 4.00 mg/l 2,4-D in leaf and nodal segment explants respectively. At low concentration of 2,4-D (below 2.00 mg/l), no callus induction was observed and as the concentration of 2,4-D increases above 3.00mg/l, the percentage of callus induction was declined in both explants (Table 2). When the concentration of 2,4-D is increased from 2.00mg/l to 2.50 mg/l and then to 3.00mg/l, rate of callus induction increased from 42.22 ± 5.88% to 62.22 ± 4.84% and 68.89 ± 2.94% in leaf explant and 30.00 ± 3.83% to 35.56 ± 2.94% and 42.22 ± 2.94% in nodal segment explant.

The calli started to induce at the major veins in leaf explants and the cut end and on upper surface of nodal segment explants then continues to grow until it covered the entire explants. Callus formation was observed earlier in nodal segment explants (15-20 days) than the leaf explants (20 to 25 days). The calli were soft, friable and creamy white in leaf explant but soft, compact and white green in nodal segment explant (Fig 1). In this species, young explants were more likely to produce callus than older explants.

Table 2. Effect of 2,4-D on callus induction from leaf and nodal segment explants of Allophylus serratus

Concentration of 2,4-D (mg/L)	Leaf explants	Nodal Segment explants
Concentration of 2,4-D (mg/L)	Callus induction (%) (Mean ± SE)	Callus induction (%) (Mean ± SE)
0.00	0.00	0.00
2.00	42.22 ± 5.88	30.00 ± 3.83
2.50	62.22 ± 4.84	35.56 ± 2.94
3.00	68.89 ± 2.94	42.22 ± 2.94
3.50	35.56± 2.94	18.89 ± 2.94
4.00	8.89 ± 4.01	10.00 ± 1.92

The highest callus fresh weight 1.30g and dry weight 0.05g (leaf explants) were produced on 2mg/l 2, 4-D containing medium. For the nodal segment explants, the highest callus fresh weight 1.63g and dry weight 0.06g was produced on 2.50mg/l 2,4-D supplemented MS medium (Fig 2). The lowest callus fresh weight (1.00g) were observed with 3.00 mg/l of 2,4- D in both leaf and nodal explants (Fig 2).

3.2.2 Effect of of BAP in combination with NAA on callus induction

From various concentrations of BAP combined with NAA tested, maximum percentage of callusing (81.11%) in leaf explants and (84.44 %) in nodal explants were observed on 3.00mg/l BAP and 0.50 mg/l NAA (Table 3). This medium also frequently produced callus in a shorter period (9.3 days) than that of other hormonal combinations in both leaf and nodal segment explants.

When the concentration of BAP increased from 3.00mg/l to 3.50 mg/l the degree of callus induction reduced to 78.89% in leaf explant and 81.11% in nodal segment explant. Overall, increasing or decreasing the concentration of BAP from 3.00 mg/l keeping the concentration of NAA constant (0.50mg/l) reduces the percentage of callus formation. The nature of the calli produced was soft, compact and light green to white in color (Figure 3).

In both leaf and nodal segment explants, the calli induced on media containing BAP at different concentration combined with NAA at 0.5mg/l are good quality and growth rate is also higher next to the calli produced on BAP alone (Fig 6). This is in agreement with Mohajer et al (2012) who reported that MS medium supplemented with BAP in combination with NAA was the best for callus induction due to high callus percentage and high callus fresh weight. This study also presents nodal segment as the best explant for induction of callus for this species which expressed maximum (84.40%) callusing at 3mg/l BAP and 0.5 mg/L NAA compared to leaf explants (81.10%).

Table 3 Effect of BAP in combination with NAA on callus induction from leaf and nodal segment explants of Allophylus serratus

Concentration (mg/l)		Leaf explant	Nodal Segment explant
BAP	NAA	Callus induction (%) (Mean ± SE)	Callus induction (%) (Mean ± SE)
0.00	0.00	0.00	0.00
1.00	0.50	34.40±2.94	38.90± 2.22
1.50	0.50	45.60±4.01	45.60± 4.01
2.00	0.50	70.00± 3.85	70.00± 1.92
2.50	0.50	68.90± 2.94	70.00± 5.09
3.00	0.50	81.11± 2.22	84.44± 2.94
3.50	0.50	78.89± 1.11	81.11± 2.22
4.00	0.50	72.22± 2.22	77.78± 1.11

The average fresh weight and dry weight of calli from leaf and nodal segment explants were measured after four weeks. The highest callus fresh weight (5.20 g in leaf and 5.80g) and dry weight (0.26 g and 0.26g) in leaf and in nodal segment explants was obtained on media containing 3mg/l BAP + 0.5mg/l NAA (Figure 3 and 4).

3.3.3 Effects of BAP on callus induction

When explants were cultured on MS medium containing BAP alone at various levels, the highest callus induction frequency (71.10% and 73.30% in leaf and nodal segment explants respectively) was obtained on 3mg/l concentration (Table 4). The least callus induction was observed in 1.00mg/l BAP in leaf explant. When the concentration of BAP is lower or higher than 3.00mg/l the formation the percentage of callus induction and callus growth rate decreased in both leaf and nodal segment explants. Overall, better percentage of callus induction on BAP fortified media observed when nodal segment explant used than leaf explants.

The length of time it takes for callus induction is about 15 days in leaf explants and 12 days in nodal segment explants. The nature of calli formed were soft, compact and white gray (Fig 5). Data illustrated in Fig 6, showed the fresh weight and dry weight of callus induced with different concentrations of BAP from leaf and nodal segment explants. In both explants, 3.00mg/l BAP provided better value of callus fresh weight (5.30 g and 6.00g) and the callus dry weight (0.26 g and 0.29g) from leaf and nodal explants respectively than all the other BAP treatments (Fig 6).

Table 4. Effect of different concentrations of BAP on callus induction from leaf and nodal explants of Allophylus serratus

Concentration of BAP(mg/L)	Leaf explant	Nodal Segment explant
Concentration of BAP(mg/L)	Callus induction (%) (Mean ± SE)	Callus induction (%) (Mean ± SE)
0.00	0.00	0.00
1.00	46.60 ±6.67	60.00±1.92
2.00	57.70±2.94	63.30±1.93
3.00	71.10±2.94	73.30±1.93
4.00	64.40±2.94	66.60±0.00

3.3 Shoot regeneration from callus

For shoot regeneration small piece of green and embryonic calli were transferred to shoot regeneration MS medium containing different concentrations of BAP and Kin. MS medium devoid of growth regulators was used as control. There was shooting response in all the tested BAP and Kin containing media as well as MS media devoid of plant growth regulators (Table 5). The calli cultured on shoot regeneration media gradually turned green as they grow and gradually start to develop shoot buds and then shoots within four weeks (Fig 7). Calli from BAP in combination with NAA were superior in shoot regeneration as compared to calli from BAP alone or 2,4-D alone.

Growth regulators free MS media was more effective in obtaining shoots from callus as compared to MS media supplemented with cytokines. The highest percentage 57.78% of shoot regeneration was achieved on growth regulators free MS medium (Table 5). Maximum shoot length (3.30 ± 0.24 cm) and shoot number (1.33±0 .71) were also observed on growth regulator free full strength MS medium. MS media supplemented with BAP and Kin also influenced shoot regeneration from callus. Among all the concentrations of BAP and Kin, BAP (2 mg/l) and Kin 1mg/l were suitable for shoot induction (37.00%) and (28.89%) respectively from callus Table 5. The least shoot regeneration (3.33%) was occurred on MS medium with 3.0mg/l Kin.

Table 5. Effect of BAP and Kinetine on shoot regeneration from callus of Allophylus serratus

Concentration of BAP and Kin (mg/L)		Response
BAP	Kin	% Regeneration	Mean number of shoot/ callus	Mean length of shoot (cm)
0.00	0.00	57.78%	3.33±0.00	1.30±0.24
1.00	0.00	31.11%	1.00 ±0.47	0.90±1.12
2.00	0.00	37.77%	1.33±0.94	1.13±0.93
3.00	0.00	16.67%	1.67±1.14	0.70±1.93
4.00	0.00	12.22%	1.00±0.00	0.60±0.00
0.00	1.00	28.89%	1.00±0.00	1.10±0.81
0.00	2.00	10.00%	1.00±0.00	0.62±0.91
0.00	3.00	3.33%	1.00±0.00	0.82±2.11

3.4 Root regeneration

The small shoots (about 1-3 cm in length) regenerated on shoot induction media were transferred to root induction MS media ( MS media supplemented with IBA and NAA at different levels) to test the response for root induction. The data obtained on root induction from in vitro regenerated shoots is given in Table 6. From all root induction media tested, 1.5 mg/l IBA containing MS medium produced maximum root induction percentage (65.55%). The maximum mean number of roots per shoots (3.33 ± 1.17) and maximum mean root length (4.00 ± 0.00 cm) were also obtained on the same media.

NAA also affect the root induction from shoot. Among all concentrations of NAA tested, 0.50 mg/l NAA produced best root induction (51.00%), mean number of roots (2.75 ± 1.66) and mean root length (2.76 ± 0.13 cm). The least root induction percentage (2.00%) was observed on MS medium supplemented with 2.00mg/l NAA (Table 5).

Table 6. Effect of IBA and NAA on root induction from in vitro shoot of Allophylus serratus

Concentration of IBA and NAA (mg/L)		Response
IBA	NAA	% Regeneration	Mean number of root/ shoot	Mean length of root (cm)
0.00	0.00	0.00	0.00	0.00
0.50	0.00	31.11%	2.33 ±0.67	0.91±1.92
1.00	0.00	36.67%	2.70±1.34	0.93±1.93
1.50	0.00	65.55%	3.33 ± 1.17	1.00 ± 0.00
2.00	0.00	52.22%	1.33±0.94	0.82±0.10
0.00	0.50	51.11%	2.33 ± 1.66	0.76 ± 0.09
0.00	1.00	40.00%	1.33 ± 0.76	0.46 ± 0.11
0.00	1.50	33.33%	2. 67 ± 1.13	0.42 ± 0.15
0.00	2.00	2.22%	0.67 ± 0.36	0.51 ± 0.91

3.5 Acclimatization

The result of acclimatization showed that all plantlets (100%) survived after transferred to a pot containing sand, soil and compost in 1:2:2 ratio. The plantlets were grown normally and no any morphological abnormalities were observed (Fig .8).

4.1 Explant Surface Sterilization

In this study surface sterilization of leaf and nodal segment explants of A. serratus was developed using different chemical sterilants. The highest percent survival of leaf explants (80.00%) was achieved with treatment (70% ethanol for 1minute, 1.0% NaOCl for 4 minutes and 0.1% HgCl₂ for 3 minutes) and the highest percent survival of nodal segment explants (90.00%) was with treatment (70% ethanol for 1min, 2% NaOCl for 3 minutes and 1.00% HgCl₂ for 2 minutes. Our result indicates that different tissue type requires different concentration of sterilants and a time exposure which is in agree with Rezadost et al. (2013) report and with Sharma et al. (2014), which indicated that the exposure time of sterilization depends on the type of tissue; for example, leaf explants require a shorter sterilization time than seeds with a tough seed coat. In this study, leaf explants need shorter sterilization time and a lower concentration than nodal segment explants. When the exposure time becomes shorter or longer, the death of explants was higher which may be due to microbial contamination or effect of chemical sterilants (Tola et al. 2015).

4.2 Callus induction

Callus induction frequency and callus type induced varied depending on the type of explant, type and level of plant growth regulator used. The highest response of callus induction in leaf explants (68.89 ± 2.94 %) and nodal segment explants (42.22 ± 2.94 %) were observed on the medium supplemented with 3.00mg/l 2, 4-D. Callus induction frequency increased as the concentration of 2, 4-D increased. This is in agreement with the report of Xing et al., (2010) who achieved optimum callus induction with increase in 2,4-D. Further increase in concentration of 2,4-D decreased callus production. Many other researchers have been also reported that MS medium supplemented with 2.4-D is suitable for callus induction (Baskaran et al., 2005; Faisal and Anis, 2003; Rani et al., 2003). Liu et al. (2006) also revealed that 2.4-D alone or in combination with cytokinins is commonly used to induce and maintain callus.

From the medium supplemented with BAP and NAA in combination tested for callus induction, highest percentage of callusing was recorded in leaf explants (81.11%) and in nodal explants (84.44 %) on 3.00mg/l BAP and 0.50 mg/l NAA.Akram and Aftab, (2015) also revealed highest callus formation (93.00%) in Tectona grandis on media supplemented with BAP combined with NAA. Similarly, callus induction on MS medium containing BAP combined with NAA were also observed in several plants (Tomar and Tiwari 2006; Panigrahi et al. 2006; Rahmatullah et al. 2012). The nature of the calli produced was soft, compact and light green to white in color. Similar results were also reported by Rajeshwari and Paliwal (2008), Patel and Shah (2009) and Isikalan et al. (2010).

From the different concentrations of BAP alone, the highest callus induction frequency (71.10% and 73.30% in leaf and nodal segment explants respectively) was obtained on 3mg/l concentration. In all tests, leaf and nodal segment explants of A. serratus cultured on MS basal medium without hormone did not induce callus which is in line with previous reports by Zare et al. (2010) and Devendra et al. (2009) on F. asafoetida and cucumber respectively. This confirms that the presence of plant growth regulators is necessary for callus formation from leaf and nodal explants of A. serratus.

Overall, MS media supplemented with BAP combined with NAA is the best for callus induction from leaf and nodal segment explants in Allophylus serratus which confirmed that both cytokinin and auxin are necessary for callus induction. The percentage of callus induction was lower for nodal explants on media with 2,4-D when compared to BAP alone and in combination with NAA (Table 1). The results of this study concluded that callus induction by BAP (3.00mg/l + NAA (0.50 mg/l ) gave best results for callus induction from leaf and nodal segment explants. Other reports such as that of Tola et al. (2015) also found that the combination of auxin with cytokinin (combination of BAP and 2,4-D) gave the high frequency of callusing in Coccinia abyssinica. MS media supplemented with 2, 4-D alone and BAP alone initiated callus induction less frequently than that supplemented with BAP in combination with NAA. BAP alone gave lower percentage of callusing than when it combined with NAA. This indicated that the presence of NAA is important to improve the callusing efficiency of the explants.

Among the leaf and nodal segment explants, nodal segment explants were found to be suitable for callusing in this species. Leaf derived callus showed late induction and retarded growth relatively. Sarin and Bansal (2011) also reported the superiority of nodal segment explant in callusing over leaf explant in Adhatoda vasica and Ageratum conyzoides.

In all tests, leaf and nodal segment explants of Allophylus serratus cultured on MS basal medium without hormone did not induce callus which is in line with previous reports by Zare et al. (2010) and Devendra et al. (2009) on F. asafoetida and cucumber respectively. This confirms that the presence of plant growth regulators such as 2,4-D, NAA and BAP alone or in combination in the medium is necessary for callus formation from leaf and nodal explants of Allophylus serratus. This is because of both hormones auxin (2, 4-D and NAA) and cytokinin (BAP) are essential for stimulating cell division, cell elongation and cell growth in plants.

4.3 Shoot regeneration

In this study, calli produced on MS medium supplemented with BAP in combination with NAA gave highest shoot regeneration as compared to calli from BAP alone or 2,4-D alone which is in agreement with (Teshome and Feyissa, 2015). But growth regulators free MS media was more effective in obtaining shoots from callus as compared to MS media supplemented with cytokines. This is in agreement with Guruchandran and Sasikumar, (2013) and Sen et al., (2014) who reported shoot regeneration on BAP and Kin supplemented media in Stevia rebaudiana and Achyranthes aspera respectively. Al-Taha et al (2012) and Miah et al., (2002) also confirmed our findings.Overall, BAP is better than Kin for shoot regeneration from callus in Allophylus serratus similar to Zare et al. (2010) reportonFerula assa Foetida. Our result, shoot regeneration on hormone free MS media, is in contrast with other reports on shoot regeneration on medium supplemented with plant hormones (Teshome and Feyissa, 2015 on Glinus lotoides ; Guruchandran, and Sasikumar, 2013 on Stevia rebaudiana, Kumar et al,2014 on Swertia chirayita and (Sen et al. 2014) on Achyranthes aspera).

4.4 Root induction and Acclamatization

From all root induction media tested, 1.5 mg/l IBA containing MS medium produced maximum root induction percentage (65.55%), maximum mean number of roots per shoots (3.33 ± 1.17) and maximum mean root length (4.00 ± 0.00 cm). Therefore, IBA was found to be better than NAA for root induction in Allophylus serratus. Other studies also reported that IBA is more effective in root induction than NAA (Fracaro and Echeverrigaray, 2001; Bera et al. 2009; Oluk and Çakyr, 2009; Jitendra et al. 2012; Pandey et al. 2013).

The efficiency of IBA in root induction was also reported in other medicinal plants including Psoralea corylifolia (Pandey et al., 2013), Stevia rebaudiana ( Jitendra et al., 2012), Solanum nigrum ( Rathore et al., 2013) and Operculina turpethum (Sebastinraj et al., 2013). IBA also gave higher response in other parameters such as mean root number, and mean root length than in NAA. But, in contrast to our result, the highest root induction ability of NAA was reported in other plants such as Thymus satureioides (Aicha et al.,2013) and Solanum surattense (Rahman et al., 2011). As the concentration of auxins (IBA and NAA) increased, there was decline in the induction and development of root as previous report (Teshome and Feyissa, 2015).

The rooted plantlets were acclimatized in a pot containing sand, soil and compost in 1:2:2 ratio. The plantlets were grown normally and no any morphological abnormalities were observed.

In general, surface sterilization of explants should guarantee the explants sterility and regeneration capacity which are known to be affected by disinfectant concentration and length of sterilization period. The in vitro surface sterilization protocol reported in this study can be used for surface sterilization of explants from leaf and nodal segments of Allophylus serratus. This result also suggests successful callus induction protocol from leaf and nodal explants of Allophylus serratus using MS media supplemented with different concentrations and combinations of plant growth regulators (2,4-D, NAA, and BAP). MS media containing 3mg/l BAP and 0.5mg/l NAA was the most efficient for callus induction. Callus induction frequency depends on the composition of callus induction media, plant organs, and callus type. Maximum means of shoot and root were observed on MS medium containing 0.5 mg/l BAP and 1.5 mg/l IBA respectively. All the plants were survived after acclimatization in greenhouse. Production of callus from medical plants and regeneration of plant from callus are important for mass propagation of the plant and to facilitate production of secondary metabolites. Allophylus serratus is a medicinal plant which contains different groups of phytochemicals. Therefore, these callus induction and plant regeneration protocols are very important for mass propagation of the species and also opens a new way to facilitate secondary metabolites production and isolation of pharmaceuticals from callus rather than harvesting the plant itself.

ANOVA ; Analysis of Variance

BAP; Benzyl amino Purine

CRD; Completely Randomized Design

IBA; indole-3-butyric acid

MS; Murashige and Skoog

NAA; naphthalene acetic acid

SPSS ;Statistical Package for the Social Sciences

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and material

Available on request

Funding

Not applicable

Competing interests

The authors declare that they have no competing interests

Authors Contribution

The authors K.J, designed, conducted the study, interpreted the results and analyzed the data and wrote and reviewed the manuscript

Acknowledgements

Not applicable

Aicha, N., Rachida, T.C. and El Abdelmalek, M. (2013) Micropropagation of Thymus satureioides Coss. an Endangered Medicinal Plant of Morocco. Journal of Agricultural Technology, 9, 487-501. http://www.ijat-aatsea.com
Akram, M. and Aftab, F. (2015) Efficient Plant Regeneration via Shoot Organogenesis From Explants of in vitro Seedlings of a Recalcitrant Woody Species of Teak (Tectona Grandis L.F.). Acta Hort. (ISHS) 1083:53-60
Al-Taha, H.A.K., Abbas, M.J. and Muayed, F.A. (2012) Somatic Embryogenesis and Plantlet Regeneration from Nucleus Tissues of Local Orange (Citrus sinensis (L.) Osbeck). Acta Agriculture Slovenica, 99, 185-189.
Bajaj, Y.P.S., Furmanowa, M. and Olszowskao, O. (1988) Biotechnology of the Micropropagation of Medicinal and Aromatic plants. In: Bajaj, Y.P.S (Eds), Biotechnology in Agriculture and Forestry. Springer and Verlag , New York, 4:60-103
Baskaran, P.B., Raja,R., Jayabalan,N. (2006). Development of an In vitro Regeneration System in Sorghum (Sorghum bicholar L) Using Root Tranverse Thin Layers (tTCLs). Turk Journal of Botany. 30: 1-9.
Bera, T., Haque, R. and Saha, S. (2009) Micropropagation of an Important Medicinal Plant Chlorophytum borivilianum. International Journal of Physical Sciences, 1, 153-163. www.ijps.info
Binu, T. and Rajendran, A. (2013) Less Known EthnomedicinalPlants Used by Kurichar Tribe of Wayanad District, Southern Western Ghats Kerala, India. Botany Research International, 6 (2): 32-35
Devendra, N.K., Subhash, B., and Y. N. Seetharam, (2009) Callus growth and plant regeneration in Momordica dioica (Roxb.) Willd. Cucurbitaceae : American-Eurasian Journal of Sustainable Agriculture, 3: 743-748
Devi, A. G., Shyma, T. B. and Raghavendra, M. P. (2013) Plants used by the tribes for the treatment of digestive system disorders in Wayanad district, Kerala. Journal of Applied Pharmaceutical Science, 3 (08): 171-175
Dharmani, P., Pushpesh Kumar, M., Rakesh, M., Vinay Singh,C. and Gautam, P. (2005) Allophylus serratus: a plant with potential anti-ulcerogenic activity. Journal of Ethnopharmacolog., 99 : 361–366
Faisal, M., Anis, M. (2003) Rapid mass propagation of Tylophora indica Merrill via leaf callus culture. Plant Cell, Tissue and Organ Culture 75, 125–12.
Fracaro, F. and Echeverrigaray, S. (2001) Micropropagation of Cunila galioides, a Popular Medicinal Plant of South Brazil. Plant Cell, Tissue and Organ Culture, 64, 1-4. http://dx.doi.org/10.1023/A:1010626200045
Gaikwad D.K, Chavan, R.B. (2013) Antibacterial Activityof Medicinally Important Two Species of Allophylus- Allophylus cobbe (L.) Raeusch. & Allophylus serratus (Roxb.) Kurz. Journal of Pharmacognosy and Phytochemistry., 2(1):1-7
Gupta A. K, Tandon N. (2004) Reviews on Indian Medicinal Plants. Indian Council of Medical Research, New Delhi. 2:90-92
Guruchandran, V. and Sasikumar, C. (2013) Organogenic Plant Regeneration via Callus Induction in Stevia rebaudiana Bert. International Journal of Current Microbiology and Applied Sciences, 2, 56-61. http://www.ijcmas.com http://www.actahort.org/books/1083/1083_4.htm
Isikalan C, Abbas F, Namli S, Basaran D. (2010) Adventitous shoot development from leaf and stem explants of Amygdalus communis L. Plant Omics J., 3(3): 92-96.
Jitendra, M., Monika, S., Ratan, S.D., Priyanka, G., Priyanka, S. and Kiran, D.J. (2012) Micropropagation of an Anti Diabetic Plant—Stevia rebaudiana Bertoni, (Natural sweetener) in Hadoti Region of South-East Rajasthan, India. ISCA Journal of Biological Sciences, 1, 37-42. www.isca.in.
Kero Jemal, B. V. Sandeep, Sudhakar Pola (2015) A review on medicinal importance of Allophylus serratus and Premna tomentosa. International Journal of Current Research Vol. 7, Issue, 10, pp.21034-21039
Kumar, A., Kaushal, S., Sharma, S. and Sood, H. (2014) In Vitro Callus Induction and Plantlet Regeneration of Swertia chirayita: A Critically Endangered Medicinal Plant. International Journal of Pharmaceutical Sciences Review and Research, 27, 94-96. www.globalresearchonline.net
Leifert C, Morris C, Waites WM. (1994) Ecology of microbial saprophytes and pathogens in field grown and tissue cultured plants. CRC Crit Rev Plant Sci ; 13: 139-83
Leifert C, Waites WM, Nicholas JR. (1989) Bacterial contaminants of micro propagated plant cultures. J Appl Bacteriol; 67: 353-61
Liu, C., Xia, X., Yin, W., Hung, L. and Zhou, J. (2006) Shoot regeneration and somatic embryogenesis from needles of redwood (Sequoia sempervirens). Plant Cell Rep., 25:621-628.
Maurya Umashanker, Srivastava Shruti, (2011) Traditional Indian herbal medicine used as antipyretic, antiulcer, antidiabetic and anticancer: a review. International Journal of Research in Pharmacy and Chemistry, 1(4)
Miah, M.N., Islam, S. and Hadinzzaman, S. (2002) Regeneration of Plantlets through Somatic Embryogenesis from Nucellus Tissues of Citrus macroptera Mont. var. anammensis (SatKara). Plant Tissue Culture, 12, 167-172.
Murashige T, Skoog F. (1962) A revised medium for rapid growth and bioassay with Tobacco tissue cultures. Physiologia Plantarum. 15:460-497
Nadeem M, Palni L.M.S, Purohit A.N, Pandey H, Nandi S.K. (2000) Propagation and conservation of Podophyllum hexandrum Royle: an important medicinal herb. Biol. Conserv., 92: 121-129
Oluk, E.A. and Çakýr, A. (2009) Micropropagation of Origanum sipyleum L., an Endemic Medicinal Herb of Turkey. African Journal of Biotechnology, 8, 5769-5772. http://www.academicjournals.org/AJB
Pandey, P., Mehta, R. and Upadhyay, R. (2013) In-Vitro Propagation of an Endangered Medicinal Plant Psoralea corylifolia Linn. Asian Journal of Pharmaceutical and Clinical Research, 6, 115-118.
Panigrahi J, Mishra R.R, Behera M. (2006) In vitro multiplication of Asteracantha longifolia (L.) Nees – A medicinal herb. Ind. J. of Biotechnol; 5:562-564
Parabia F.M, Gami B, Kothari I.L, Mohan J.S.S, Parabia MH. (2007) Effect of plant growth regulators on in vitro morphogenesis of Leptadenia reticulata (Retz.) W. and A. from nodal explants. Curr. Sci., 92(9): 1290-1293
Patel RM, Shah RR. (2009). Regeneration of Stevia plant through callus culture. Indian J. Pharm. Sci., 71(1): 46-50.
Pola Sudhakar (2005) Tissue culture and genetic transformation studies of Sorghum bicolor (L.) Moench. Ph.D. Thesis, college of science and technology, Andhra University, Visakhapatnam, INDIA. 4-167. DOI: 10.13140/2.1.1773.9848
Pola Sudhakar, N. Sarada Mani and T. Ramana, (2009) Mature embryo as a source material for efficient regeneration response in Sorghum (Sorghum bicolor L. Moench.) Sjemenarstvo. 26:93-104.http://hrcak.srce.hr/file/75032. ISSN:1330-0121.
Pola Sudhakar., Saradamani, N. and Ramana, T (2007) Enhanced shoot regeneration in tissue culture studies of Sorghum bicolor. Journal of Agricultural Technology 3(2): 275-286 http://www.ijat-rmutto.com/pdf/Nov_V3_no2_07/11-IJAT2007_20-P%20275-286.pdf
Priya, E., Selvan, P. and Tamilselvan, R. (2012) Phytochemical Investigation of Allophylus serratus Kurz Leaves by UV and GC-MS Analysis. Research Journal of Phytochemistry, Vol. 6 Issue 1, p17-24.
Rahman, Md.M., Amin, M.N., Islam, Md.Z. and Sultana, R.S. (2011) Mass Propagation of Solanum surattense Bum. Using Direct Adventitious Shoot Organogenesis from Internode. Acta Agriculturae Slovenica, 97, 11-17. http://dx.doi.org/10.2478/v10014-011-0002-z
Rahmatullah Qureshi1, Mehmooda Munazir, Adel Ahmed, Abul-Soad Mushtaque, Ahmed Jatoi and Ghulam Shabbir, (2012) In vitro callus induction protocol for Leptadenia pyrotechnica using various explants. Journal of Medicinal Plants Research Vol. 6(3), pp. 379-382
Rajeshwari, Paliwal, K. (2008). Callus induction and plant regeneration of red sanders (Pterocarpus santhalinus L.) from cotyledonary node. Indian J. Biotechnol., 7: 541-546.
Rani, G., Virk, G.S., Nagpal, A. (2003) Callus induction and plantlet regeneration in Withamia somnifera (L.) Dunal. In Vitro Cellular and Developmental Biology-Plant 39, 468–474
Rathore, V. and Gupta, D. (2013) In Vitro Clonal Propagation of Solanum nigrum. Research Journal of Chemical and Environmental Sciences, 1, 32-33. http://www.aelsindia.com/rjces.htm
Renu Sarin and Nidhi Bansal, (2011). Impact of growth regulators on Callus production of two medicinal plants viz. Adhatoda vasica and Ageratum conyzoides. International Journal of Research in Plant Science, 1(1) 1-8. www.urpjournals.com
Rezadost Hosein Mohammad, Sohan Mehdi Mohammad , Hatamzadeh Abdollahand Mirzai Reza Mohammad, (2013) In vitro regeneration of sour orange (Citrus aurantium L.) via direct organogenesis: Plant Knowledge Journal; 2:150-156
Sadegh Mohajer , Rosna Mat Taha, Arash Khorasani and Jamilah Syafawati Yaacob, (2012) Induction of different types of callus and somatic embryogenesis in various explants of Sainfoin (Onobrychis sativa). AJCS 6(8):1305-1313
Sebastinraj, J., Britto, S.J. and Kumar, S.R.S. (2013) Micropropagation of Operculina turpethum (Linn.) Silva Manso. Using Cotyledonary Node Explants. Academic Journal of Plant Sciences, 6, 77-81.
Selvam V. (2007) Trees and Shrubs of Maldives. 1st ed. FAO Regional Office for Asia and the Pacific. Thammada Press Co., Ltd., Bangkok
Sen, M.K., Nasrin, S., Rahman, S. and Jamal, A.H.M. (2014) In Vitro Callus Induction and Plantlet Regeneration of Achyranthes aspera L., a High Value Medicinal Plant. Asian Pacific Journal of Tropical Biomedicine, 4, 40-46. www.elsevier.com/locate/apjtbhttp://dx.doi.org/10.1016/S2221-1691(14)60206-9
Sharma Vikas, Srivasta Nidhi , Kamal Barkha, Dobriyal A.K and Jadon S.Vikhas, (2014) Efficient sterilization protocols for different explants of an endangered medicinal herb Swertia chairayita:Trends in life sciences 3:2319-5037 www.sciencejournal.in
Shiferaw Teshome and Tileye Feyissa, (2015)In Vitro Callus Induction and Shoot Regeneration from Leaf Explants of Glinuslotoides (L.) An Important Medicinal Plant. American Journal of Plant Sciences 6, 1329-1340
Srivastava N, Kamal B, Sharma V, Negi YK, Dobriyal AK. (2010) Standardization of Sterilization Protocol for Micropropagation of Aconitum heterophyllum- An Endangered Medicinal Herb. Academ Arena; 2(6): 62-6
Tola Bayisa Guma , Kahia Jane ,Onguso Justus , Peter Njenga Kariuki. (2015) Standardization of in vitro Sterilization and Callus Induction Protocol for leaf explants of Anchote : Coccinia abyssinica. Int. J. Res. Dev. Pharm. L. Sci. Vol. 4, No.2, pp 1427-1433. http://www.ijrdpl.com/docs/v4i2/7.pdf
Tomar R, Tiwari S. (2006) In vitro morphogenesis of Rauwolfia serpentina L. through cotyledons. Plant Cell Biotechnol. Mol. Biol; 7 :53-58
Trivedi, P.C. (2006) Herbal medicine: traditional practices(Ed); Aavishkar Publishers, Jaipur.India :322
Vinay, S.C., Pushpesh, K.M., Rakesh, M., Dharmani, P. andGautam, P. (2005) Allophylus serratus: a plant with potential anti-ulcerogenic activity. Journal of Ethnopharmacology; 99:361–366
Xing, Z.Y., Yuan, Y.H., Wang, L.F. and Zheng, L.P. (2010). Regenerating Plants from in vitro culture of Erigeron breviscapus leaves. African Journal of Biotechnology 9(26): 4022-4024.
Zare, A.R., Solouki, M., Omidi, M., Irvani, N., Nezad, M. Mahdi and Rezazadeh, S.H. (2010) Callus induction and plant regeneration in Ferula assafoetida L. (Asafetida), an endangered medicinal plant. Trakia J. Sci., 8: 11-18

In vitro regeneration of Allophylus serratus. Roxb (Kurz), an important medicinal plant

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

1. Introduction

2. Methods

3. Results

4. Discussion

5. Conclusion

List Of Abbreviations

Declarations

References

Status:

Version 1