Skip to main content
NCBI home page
Physiology and Molecular Biology of Plants logoLink to Physiology and Molecular Biology of Plants
. 2019 Jul 24;25(5):1311–1322. doi: 10.1007/s12298-019-00689-x

The effect of silver nitrate on micropropagation of Moringa oleifera Lam. an important vegetable crop of tropics with substantial nutritional value

R S Drisya Ravi 1, E A Siril 2,✉, Bindu R Nair 2
PMCID: PMC6745574  PMID: 31564791

Abstract

An improved micropropagation protocol facilitating continuous multiplication of elite germplasm of Moringa oleifera has been developed. Initial culture of nodal explant in MS medium supplemented with 2.5 µM BA resulted in the formation of 12.5 shoots per explant with high frequency of leaf fall (84.3%). To confirm whether the leaf fall is due to accumulation of ethylene in the culture vessel, effect of ethylene releasing agent CEPA in the medium was tested. In order to reduce leaf fall and improve multiplication, varying concentration of anti-ethylene agent, AgNO3 was incorporated in the medium. Addition of 2.5 μM AgNO3 in combination with 2.5 μM BA produced maximum number of shoots (17.6) including shoots originated from the base of the explant and shoots from the axillary buds of the primary shoots, where significant reduction in leaf fall (20.6%) was noticed. This enabled sustained multiplication of M. oleifera through continuous subculture without adversely affecting shoot number or shoot quality in terms of shoot length. Microshoots obtained from fourth subculture onwards were used for ex vitro rooting and found that by treating 50 µM NAA for 30 s, maximum numbers of microshoots (83.3%) were rooted. Rooted plants were acclimatized, survived and were successfully transferred to field. Genetic fidelity analysis using 10 ISSR primers revealed more than 95% monomorphic bands among plants raised in MS medium containing low concentration (2.5 µM) of AgNO3 and BA (2.5 µM). The addition of AgNO3 in the medium sustained in vitro growth and effectively prevented leaf fall compared to control, thus demonstrating efficient micropropagation of M. oleifera.

Keywords: 2-chloroethylphosphonic acid, Ethylene, Ex vitro rooting, Moringa oleifera, Silver nitrate

Introduction

Moringa oleifera Lam. (common name: drumstick, horseradish tree) belongs to the family Moringaceae with its origin from sub-Himalayan tracts of North India, distributed all around the world in the tropics and sub-tropics especially in the arid regions. Immature pods, fresh leaves and flowers of M. oleifera are used for culinary purposes. The leaves and young pods contain significant amount of minerals and vitamins A, B, and C (Saini et al. 2014a). M. oleifera is an extraordinarily healthful vegetable tree with a range of potential uses (Saini et al. 2014b, 2016). Extracts from all parts of M. oleifera tree render pharmacological properties including anti-inflammatory, anti-bacterial and anti-asthmatic activity (Mehta and Agrawal 2008). M. oleifera is generally propagated by seeds or cuttings. Drumstick trees obtained from seed propagation vary in genotypes and in their phenotypes leading to variation in fruit production and nutritional values (Ramachandran et al. 1980; Riyathong et al. 2010; Barche et al. 2013). Plantations of M. oleifera raised by using cuttings are excellent for bearing the uniform characters especially growth and yield (Islam et al. 2005). However, conventional ways of propagation often fail to produce large number of superior plants in a short period of time (Thorpe 2007). Selection of superior germplasm based on desirable traits is the primary tool for crop improvement. Selected germplasm often designated as ‘elite material’ can be used for the clonal propagation purpose (Tiwari et al. 2002). In the past, several workers have forwarded micropropagation methods for the M. oleifera, mostly based on seedling materials (Stephenson and Fahey 2004; Saini et al. 2012; Shokoohmand and Drew 2013; Forster et al. 2013; Gayathri et al. 2015; Salem 2016; Avila-Trevino et al. 2017; Jun-jie et al. 2017). Callus based plant regeneration was also reported using various combination of auxin and cytokinins (Mathur et al. 2014). M. oleifera under in vitro cultures is highly sensitive to microenvironment within the culture, where stressful environment lead to defoliation of in vitro shoots and death of culture. This in vitro physiological hindrance is the bottle neck to sustained micropropagation of M. oleifera. Recent report (Hassanein et al. 2018) using in vitro seedling based explant suggests use of salicylic acid to reduce vitrification of shoots and to produce normal shoots.

Development of abnormal shoots is largely due to the culture environment, where concentration of various gases in the micro environment is important. The gaseous phytohormone ethylene regulates growth and development of plants such as germination of seeds, development of root hair, root nodulation, flower senescence, abscission, and ripening of fruit (Johnson and Ecker 1998; Bleecker and Kende 2000). Ethylene is key factor in the sequence of abscission phenomena (Abeles 1973). Biosynthesis of ethylene is strongly regulated by both internal signals and environmental stimuli through abiotic and biotic stresses, such as hypoxia, ozone, wounding, chilling or freezing, pathogen attack  (Wang et al. 2002). However, ethylene is reported to promote growth and shoot proliferation in several ornamentals and perennials such as rose, lavandin, eastern white cedar, petunia and peach (Prakash et al. 1998). Several Brassica genotypes are highly sensitive to ethylene (Pua and Chi 1993). Accumulation of ethylene often hamper sustained growth of in vitro cultures and causes leaf fall (Burg 1968; Kao and Yang 1983), explant and medium browning and consequent deterioration of cultures. Pua and Chi (1993) reported ethylene production by in vitro grown shoots in sealed containers and affecting general plant growth and development. Ethylene can be applied as gas or by using ethephon (ethrel, 2-chloroethylphosphonic acid, CEPA) for experimental purpose (Prakash et al. 1998) and ethephon decomposes to release ethylene at physiological pH or by the addition of ethylene precursor ACC.

To take-up this situation, a class of compounds including AgNO3 were attempted by the several workers. Application of AgNO3 improved shoot multiplication in M. oleifera (Hassanein et al. 2018) and other plant species such as pomegranate (Naik and Chand 2003), black gram (Mookkan and Andy 2014), banana (Tamimi 2015), sunflower (Mirzai et al. 2015) and cherry (Sarropoulou et al. 2016). In processes such as abscission, senescence and growth retardation, silver ion specifically blocks the action of exogenously applied ethylene (Beyer 1976a, b). According to Hassanein et al. (2018), vitrification symptoms decreased at higher concentration of AgNO3 at the stage of culture establishment of M. oleifera. Silver ion, an opponent for ethylene and nitrate salt, has proved to be a very effective inhibitor of ethylene classical responses such as abscission, senescence and growth retardation (Beyer 1976a, b). Because of its effective inhibitor nature, silver ion was used in plant tissue culture (Prakash et al. 1998). Certain properties of AgNO3 such as its availability, solubility and stability allows its various applications in exploiting plant growth regulation and both in vivo and in vitro morphogenesis (Vinod et al. 2009). AgNO3 mediates inhibition of ethylene action and reverses the growth inhibition of cultures (Lemos and Jennet 2015).

Micropropagation through enhanced proliferation of axillary shoots is considered as safe method to produce genetically uniform plants. However, plants produced especially by exposing to heavy metal ions like silver, clonal uniformity of plants are to be duly accessed using molecular markers. In this direction, use of ISSR markers have been well established for high degree of sensitivity, reproducibility, and the dominant representation of polymorphic genetic alleles, thus widely used in the clonal fidelity analysis e.g., M. oleifera (Hassanein et al. 2018), banana (Venkatachalam et al. 2007), Dictyospermum ovalifolium (Chandrika et al. 2008) and Nothapodytes foetida (Chandrika and Rai 2010).

This study was aimed to build up a rapid in vitro regeneration protocol for the elite germplasm of M. oleifera selected on the basis of yield or quality traits. However, major problem faced during the culture establishment of M. oleifera was leaf shedding and consequent deterioration of cultures. Stephenson and Fahey (2004) reported the early shoot senescence that continues to restrict rapid culture proliferation beyond the initial shoot proliferation stage during culturing of M. oleifera. Ethylene that stimulated leaf abscission in cotton plant is blocked by the AgNO3 (Beyer Beyer 1975, 1976a, b, 1979). Lemos and Jennet (2015) reported that AgNO3 was used as an effective inhibitor of leaf abscission in the establishment of Annona squamosa in in vitro culture. Therefore it is envisaged to explore effect of AgNO3 on premature leaf fall of in vitro cultures. The major features of the present protocol include efficient multiple shoot proliferation from mature explants on a medium with reduced concentration of AgNO3, continuous multiplication without growth abnormality of shoots or leaf fall and development of ex vitro rooting.

Materials and methods

Selection of elite germplasm

An exploration survey was carried out in Karnataka, Kerala and Tamil Nadu states of India during May–June, 2014 primarily to record healthy, morphologically and biochemically distinct drumstick trees. Based on fruit yield data and single fruit weight, mean fruit yield and mean fruit weight among surveyed 120 trees, 23 gave more than 50% fruit yield and single fruit weight than average of 120 trees. Each selected tree was designated with CPT number for future identification. Fruit yield based screening continued two more years on CPTs on various yield attributes (Table 1) and recorded CPT 17 as elite tree with maximum yield (163.27 kg/plant/season).

Table 1.

Geographical details and fruit yield of selected CPTs

Collection code Fruit number Fruit weight (g) Fruit yield (kg) Place of collection Location (latitude and longitude)
CPT1 320.0 ± 1.41q 95.58 ± 0.63efg 30.58 ± 0.15l Koramangala, Bangalore 12°55′N and 77°37′E
CPT2 330.0 ± 1.44pq 96.37 ± 0.87efg 31.80 ± 0.14kl Somanahalli, Bangalore 12°39′N and 76°12′E
CPT3 720.0 ± 1.99g 100.78 ± 0.54de 72.56 ± 0.38e Nidaghatta, Bangalore 12°23′N and 77°05′E
CPT4 340.0 ± 1.28p 89.09 ± 0.98k 30.29 ± 0.17m Thuppinakkara, Mysure 12°17′N and 76°38′E
CPT5 552.0 ± 3.52 k 158.24 ± 1.47a 87.34 ± 0.81d Mandya, Mysure 12°31′N and 76°54′E
CPT6 325.0 ± 3.17q 105.88 ± 2.58cd 34.41 ± 0.49kl Sulthanbathery, Wayandu 11°39′N and 76°15′E
CPT7 440.0 ± 3.09n 104.26 ± 1.65cd 45.87 ± 0.39j Adivaram, Calicut 11°29′N and 76°00′E
CPT8 720.0 ± 2.98f 90.01 ± 0.97ghij 64.81 ± 0.70fg Moozhikkal, Calicut 11°17′N and 75°50′E
CPT9 600.0 ± 2.04j 101.59 ± 1.10de 60.95 ± 0.66h Randathanil, Calicut 10°57′N and 76°00′E
CPT10 480.0 ± 1.27m 85.38 ± 0.81ij 40.98 ± 0.14j Puduppadi, Calicut 10°48′N and 75°56′E
CPT11 624.0 ± 5.11i 110.69 ± 1.99c 69.07 ± 1.24ef Panambra, Malappuram 11°07′N and 75°53′E
CPT12 450.0 ± 1.22m 72.22 ± 0.85k 32.49 ± 0.21kl Mundanam, Malappuram 11°03′N and 76°04′E
CPT13 420.0 ± 1.37o 87.98 ± 4.21hij 36.95 ± 1.76k Irinjalakkuda, Trissur 10°20′N and 76°12′E
CPT14 500.0 ± 1.45l 92.84 ± 10.67fgh 46.42 ± 5.31i Alathur, Palakkadu 10°38′N and 76°32′E
CPT15 700.0 ± 9.49h 101.83 ± 4.33ghi 64.50 ± 3.04fg Chittoor, Palakkad 10°40′N and 76°42′E
CPT16 810.0 ± 2.39e 72.53 ± 1.03k 57.20 ± .84h Ernakulam 10°04′N and 76°16′E
CPT17 900.0 ± 4.98c 163.27 ± 1.27a 146.94 ± 1.12a Cherthala, Alleppy 9°41′N and 76°20′E
CPT18 735.0 ± 7.83f 144.23 ± 7.80b 93.10 ± 5.72c Konni, Pathanamthitta 9°13′N and 76°50′E
CPT19 960.0 ± 8.27b 106.34 ± 2.50cd 102.20 ± 2.33b Mundakkal, Kollam 8°5′N and 76°36′E
CPT20 1000.0 ± 6.19a 101.44 ± 2.18de 101.50 ± 2.22b Attingal, Thiruvananthapuram 8°4′N and 76°49′E
CPT21 960.0 ± 2.69b 89.53 ± 0.87ghij 86.10 ± 0.84d Nagercoil, Kanyakumari 8°0′N and 77°24′E
CPT22 735.0 ± 5.17e 99.60 ± 1.96def 73.20 ± 1.42e Ukkadam, Coimbatore 10°59′N and 76°57′E
CPT23 840.0 ± 1.38d 84.48 ± 13.56j 70.96 ± 11.32e Hosur, Krishnagiri 12º44′N and 77°49′E
F value 2187.529*** 101.829*** 296.642***
Open in a new tab

Means with in a column followed by same letters are not significantly (p ≤ .05) different as determined by Duncan’s Multiple Range Test

***Highly significant (p ≤ .001)

Explant selection and sterilization

The limb cuttings derived from elite tree was successfully established in the Department garden (latitude: 8°33′54.13″N, longitude: 76°53′11.55″E, altitude: 46 m) were served as explant source for the experiment. Stem cuttings of 50–100 cm long and 4–5 cm in diameter were typically planted during the onset of wet summer season (mid-May). The collected stem cuttings were planted in the moist soil to a depth of about 15 cm, rooted readily and grown to sizeable trees within few months. Actively growing young branches with 3–4 nodes were collected from elite tree of M. oleifera. The nodal segments from branches were excised and washed thoroughly under running tap water for 30 min, subsequently treated with 1% polysorbitol detergent (Labolene, Mfg. Fischer Scientific Chemicals, Mumbai, India) for 40 min and later immersed in a suspension of 0.2% w/v carbendazim fungicide, Bavistin (BASF, Mumbai, India) for 20 min in a gyratory shaker (100 rpm), followed by several rinses in distilled water. The explants were brought to laminar air flow and then surface sterilized with 0.1% mercuric chloride solution containing Tween 20 (2 drops per 100 ml) for 2 min and 5 times washing in sterile distilled water. The surface sterilized explants were cut into 1 cm segments and were cultured in vitro.

Culture media and culture conditions

MS (Murashige and Skoog 1962) medium containing 3% (w/v) sucrose (SISCO Research Laboratory, Mumbai, India), 0.7% (w/v) agar (Hi Media, Mumbai, India) and various plant growth regulators (Sigma-Aldrich, St. Louis, US) were initially tested for the in vitro culture of M. oleifera. Benzyl adenine (BA) or kinetins (KIN) at different concentrations (0.5, 1.5, 2.5, 3.5 or 4.5 µM) were added in the medium. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C and 108 kPa pressure for 15 min. The medium was poured into culture tubes (25 × 150 mm) and plugged by non-absorbent cotton. Explants were inoculated onto the culture medium and were maintained in a growth chamber at 25 ± 2 °C under 16 h photoperiod at 50 µmol m−2 s−1 irradiance provided by cool white fluorescent tubes (Philips, India) and 55–65% relative humidity.

In vitro culture establishment and shoot proliferation

Nodal segments (1 cm) with a single node were excised and cultured on MS medium supplemented with various cytokinins (BA or KIN) at different concentrations (0.5, 1.5, 2.5, 3.5, and 4.5 μM). Percentage of response, total number of shoots (> 0.5 cm) including shoots originated from the base of the explant and shoots from the axillary buds of the primary shoots were determined. Shoot length (cm) and percentage leaf fall were also recorded after 4 weeks of culture. Leaf fall records were scored by counting number of cultures with fallen leaves respective to treatment. A set of cultures raised on MS medium devoid of plant growth regulators was served as control.

To study the effect of CEPA (2-chloroethylphosphonic acid) and AgNO3 on leaf abscission and shoot multiplication, different concentrations of filter sterilized (AXIVA disposable syringe filter, pore size 0.2 µm) CEPA (0.5, 2.5, 5, 7.5 and 10 µM) or AgNO3 (0.5, 1.5, 2.5, 3.5 and 4.5 µM) was added in MS medium containing 2.5 µM BA. Nodal segments excised from microshoots developed on MS medium supplemented with 2.5 µM BA was used for the culture. Nodal segments planted in medium containing 2.5 µM BA but devoid of CEPA and AgNO3 served as control. After 4 weeks of culture, percentage of response, total numbers of shoots including shoots from the axillary buds of the primary shoots, shoot length (cm) and percentage leaf fall were recorded.

Subculture and shoot multiplication

In vitro raised microshoots were excised in every 4 week interval and these microshoots were segmented having single node and used for further sub culture trials. The in vitro produced shoots were subcultured on to the agar (0.7%) gelled MS medium containing 2.5 µM BA and 2.5 µM AgNO3 and data on shoot number per explants, percentage of response, shoot length and leaf fall percentages in each subculture were scored.

Ex vitro rooting

Microshoots having the length of 3–4 cm were excised from fourth subculture were used for ex vitro rooting experiments. The basal portion of the shoot was trimmed and washed in running tap water. The cut ends were dipped in indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA) solutions (30, 50 and 70 µM) for 30 s. Microshoots planted without any auxin treatment served as control. The auxin treated shoots were planted in nursery pots (diameter 7 cm, height 8 cm) containing soil: sand (1:1) enriched with half strength MS salt solution. The potted plants were initially maintained in a hardening chamber (28 ± 2°, RH 90%; 40 µmol m−2 s−1 irradiance) and were covered with polythene bags. After 4 weeks of planting, percentage rooting, root number and root length were recorded.

Genetic fidelity of in vitro raised plants

Genomic DNA extraction

Genomic DNA was isolated from young leaves of eight in vitro raised plants and mother plant by cetyl-tri-methyl ammonium bromide (CTAB) method (Dellaporta et al. 1983) with modification and 2% polyvinyl pyrrolidone (PVP) was added to extraction buffer for removal of phenolics present in the sample. The DNA concentration was estimated by Biophotometer (Eppendorf Biophotometer Plus, Germany). DNA quality was checked by gel electrophoresis on 0.7% agarose gel (1X TAE buffer, EtBr.). A working DNA concentration of 10 ng/µl was prepared and stored at 4 °C until use.

ISSR-PCR amplification

A total of 15 ISSR primers (BR Biochem Life Sciences) were used for the fidelity survey. ISSR assay was carried out in 25 µl reaction volume, containing 12.5 µl 2 X-PCR smart mixes (Origin Diagnostics & Biochemicals, Karunagapally, India), 3 µl DNA (10 ng/µl), 1 µl primer (20 pmol/µl) and 8.5 µl double distilled water. The polymerase chain reaction (PCR) was performed with a thermo cycler (Eppendorf, Germany). The standardized amplification was performed (Saini et al. 2013) at an initial denaturation at 94 °C for 4 min, followed by 40 cycles of denaturation at 94 °C for 30 s; primer annealing based on Tm for 1 min; primer extension at 72 °C for 2 min and final extension at 72 °C for 10 min. The annealing temperature for each primer was standardized by performing gradient PCR. Based on preliminary screening, 10 ISSR primers were selected to test genetic stability of eight randomly selected hardened plants.

ISSR-PCR products were then analyzed by electrophoresis (V-GEL, UK) with 100 bp DNA ladder ranged from 100 bp to 5000 bp (Origin Diagnostics & Biochemicals, Karunagapally, India) on a 1.8% agarose gel with 1× TAE buffer stained with 2 µl ethidium bromide. DNA banding patterns were then visualized using gel documentation system (BIORAD Gel Doc™ XR+).

Statistical analysis

All the experiments were ordered in a randomized completely block design (RCBD). Every treatment was composed of three replication blocks and each replication block was represented by eight culture tubes. Analysis of variance (ANOVA) followed by mean separation by Duncan’s multiple range test (DMRT, p < 0.05) was carried out using SPSS statistics (SPSS, Version 7.5., for windows, Illinois, Chicago, US) to determine levels of significance and mean separation.

Results and discussion

In vitro culture establishment and shoot proliferation

The axillary buds of nodal explants showed growth response within first week of culture irrespective of concentration of BA tested (Table 2). Among various concentrations of BA tested, the highest response (96%) was observed in 2.5 µM BA (Fig. 1a). The effect of BA on shoot multiplication after 4 weeks of culture was highly significant (p ≤ 0.001). BA seems to be suitable cytokinin to induce shoot proliferation in M. oleifera and was in conformity with M. peregrina (Al Khateeb et al. 2013). Explants cultured on BA produced shoots with white callus from the basal portion of the explant. Callus formation during culture establishment of M. oleifera in MS medium containing BA was reported earlier (Islam et al. 2005). Riyathong et al. (2010) also reported multiplication of M. oleifera in MS medium supplemented with BA at moderately high concentrations (5 mg l−1) with 100% callus formation. The explants cultured on 2.5 µM BA showed maximum number of shoots (12.02) and maximum shoot length (Table 2; Fig. 1b). Kantharajah and Dodd (1991) reported seedling derived nodal explants of M. oleifera in the woody plant medium containing 2% sucrose, solidified with 0.8% agar and 1 mg l−1 BA produced 21 shoots. Shoots developed in this medium grew well initially and after 15 days of culture, leaf fall at the order of expanded lower leaflets first from the basal portion was noticed. However, no data on subculture were available.

Table 2.

Effect of BA and kinetin on shoot multiplication of M. oleifera using nodal explant

Cytokinin type Conc. (µM) % Response (± SE) Total shoot number# (± SE) Shoot length (cm) (± SE) % Leaf fall (± SE)
BA 0.5 83.3 ± 3.33bc 5.0 ± 0.02c 1.3 ± 0.01cde 79.0 ± 2.08b
1.5 86.6 ± 3.33ab 6.2 4 ± 0.16b 1.5 ± 0.02c 82.6 ± 1.45ab
2.5 96.6 ± 3.33a 12.0 ± 0.09a 4.1 ± 0.3a 84.3 ± 1.20a
3.5 73.3 ± 3.33cd 6.2 ± 0.06b 3.0 ± 0.15b 77.6 ± 1.85b
4.5 70.0 ± 0.00d 3.1 ± 0.08d 1.7 ± 0.69c 79.0 ± 0.57b
KIN 0.5 30.0 ± 5.77 g 3.1 ± 0.27d 1.1 ± 0.03cde 77.0 ± 1.52c
1.5 43.3 ± 3.33ef 2.1 ± 0.076e 1.3 ± 0.03cde 82.3 ± 0.88ab
2.5 50.0 ± 5.77e 5.2 ± 0.037c 1.5 ± 0.07 cd 83.0 ± 1.73a
3.5 36.6 ± 3.33g 1.5 ± 0.22f 0.7 ± 0.01de 81.0 ± 0.57ab
4.5 26.6 ± 3.33g 1.3 ± 0.19f 0.6 ± 0.02e 79.0 ± 0.57bc
Control 0.0 0.0 0.0 0.0 0.0
F value df (n − 1) = 9 44.692*** 394.411*** 21.825*** 3.390*
Cytokinin type df (n − 1) (T) 1 345.308*** 1446.651*** 77.352*** 0.006NS
Concentration df (n − 1) 4 12.962*** 440.775*** 18.046*** 6.476***
Type × concentration df (n − 1) 9 1.269NS 84.988*** 11.721*** 1.151NS
Open in a new tab

#Total shoot number–number of shoots emerged from the base of the original explant and shoots from the axillary buds of the primary shoots. Means with in a column followed by same letters are not significantly (p ≤ 0.05) different as determined by Duncan’s Multiple Range Test

***Highly significant (p ≤ 0.001)

Fig. 1.

Fig. 1

Open in a new tab

Micropropagation of Moringa oleifera using nodal segments. a Initiation of multiple shoots in MS medium supplemented with 2.5 µM BA (bar = 0.8 cm), b Multiple shoots in MS medium supplemented with 2.5 µM BA (bar = 0.48 cm), c Multiple shoots in MS medium supplemented with 2.5 µM Kinetin (bar = 0.4 cm), d Premature leaf fall in culture condition (bar = 0.96 cm), e Leaf fall in 2.5 µM BA and 10 µM CEPA ((bar = 0.8 cm), f Multiple shoot formation from the base of the explant and axillary buds of the primary shoots on MS medium containing 2.5 µM BA and 2.5 µM AgNO3 (bar = 0.76 cm), g Hardened plantlets on polyethylene cups (bar = 1 cm), h Ex vitro rooted micro shoots by 50 µM NAA treatment for 30 s (bar = 1 cm), i Acclimatized plant (bar = 1 cm)

Varying concentrations of KIN tested also showed significantly varying response. Of the five KIN concentrations, 2.5 µM KIN showed 50% explant response coupled with significant promotion in leaf fall (83%). The response of kinetin was inferior compared to BA. KIN (2.5 µM) in culture medium resulted in 5.26 number of shoots per explant (Fig. 1c). Marfori (2011) reported highest number of shoots per explant by 5 µM KIN. Higher levels of cytokinin (BA or KIN) leads to callus formation from the basal portion, reduced number of shoots and varying levels of growth abnormalities and deformation of shoots. Similar observations were also made in M. oleifera by previous studies (Riyathong et al. 2010).

Effect of CEPA and AgNO3

Attempts to micropropagate M. oleifera from nodal explants have been hindered by leaf fall during establishment of culture (Fig. 1d). Same problem was observed in the culture establishment of A. squamosa (Lemos and Jennet 2015). Growth and development of cells cultured in vitro largely depend on the presence of phytohormones, including ethylene in the culture environment. Little amount of ethylene can promote abscission of fruits, leaves and buds in many plants (Sisler and Yang 1984). Ethylene action may lead to leaf fall during micropropagation.

To confirm the leaf abscission is due to the production and accumulation of ethylene in the culture, we performed an experiment with ethylene liberating compound, CEPA. Different concentrations (0.5, 2.5, 5, 7.5 and 10 µM) of CEPA were tested in medium containing 2.5 µM BA (Table 3). Among different conc. of CEPA, 7.5 µM and 10 µM CEPA showed 100% leaf fall (Fig. 1e). There was no significant difference in percentage of response. Number of shoots and shoot length were decreased with increase in CEPA concentration. Sung and Huang (2000) reported treatment of ethylene released from CEPA to be negative to both hairy root growth and l-DOPA (L-3, 4-dihydroxyphenylalanine) production in Stizolobium hassjoo. l-DOPA, a neurotransmitter precursor used for treating Parkinson’s disease, has been reported to exist in large amounts in etiolated seedlings of S. hassjoo (Obata-Sasamoto et al. 1981). Gonzalez et al. (1997) used CEPA to enhance the production of ethylene in in vitro organogenesis and plant growth of Populus tremula.

Table 3.

Effect of CEPA in combination with 2.5 µM BA supplemented MS medium on shoot multiplication of M. oleifera

CEPA conc. (µM) % Response (± SE) Total shoot number# (± SE) Shoot length (cm) (± SE) % Leaf fall (± SE)
0.5 96.6 ± 3.33a 12.0 ± 0.09a 3.7 ± 0.31a 87.0 ± 0.67d
2.5 93.3 ± 3.33a 11.5 ± 0.76a 3.8 ± 0.11a 89.0 ± 0.75c
5 91.6 ± 4.41a 10.0 ± 0.28a 3.0 ± 0.00b 93.0 ± 0.63b
7.5 90.0 ± 0.00a 10.6 ± 0.33a 3.0 ± 0.28b 100.0 ± 0.00a
10 90.0 ± 5.77a 9.6 ± 0.33b 2.6 ± 0.10b 100.0 ± 0.00a
F value df (n − 1) = 4 0.51NS 4.7* 7.4** 183.5***
Open in a new tab

#Total shoot number–number of shoots emerged from the base of the original explant and shoots from the axillary buds of the primary shoots. Means with in a column followed by same letters are not significantly (p ≤ 0.05) different as determined by Duncan’s Multiple Range Test

***Highly significant (p ≤ 0.001), *Significant (p ≤ 0.05), NS not significant

To control the leaf fall, we tested different concentrations of AgNO3 in MS medium with 2.5 µM BA (Table 4). Supplementation of AgNO3 in the medium enhanced the shoot regeneration response, the number of shoots per explant and also reduced leaf fall in culture (Fig. 1f). The total shoots counted include number of shoots emerged from the base of the original explant and shoots from the axillary buds of the primary shoots. The explants cultured on MS medium containing 2.5 µM BA and 2.5 µM AgNO3 resulted 96% response and produced highest number of shoots (17 shoots/nodal cutting) and reduced leaf shedding (20.67%) significantly. The apical leaves of the cultures appeared green and healthier than the control. Lemos and Jennet (2015) explained the similar phenomenon as the time of nodal explants of Annona squamosa were placed in culture. They noticed four days were enough for the latter explants to drop 100% of their leaves and after 12 days they had died. At the same time as those with AgNO3 had lost only 50% of their leaves. Cotton plants were grown under controlled conditions had abscised all the leaves on the 7th day in ethylene without AgNO3 (Beyer 1976a, b). Cotton plants treated with increasing concentrations of AgNO3 showed progressively less leaf abscission. Treatment with 25 mg l−1 of AgNO3 reduced the time required to reach 100% leaf abscission by two days in cotton plants (Beyer 1976a, b).

Table 4.

Effect of AgNO3 in combination with 2.5 µM BA supplemented MS medium on shoot multiplication of M. oleifera

AgNO3 conc. (µM) % Response (± SE) Total shoot number# (± SE) Shoot length (cm) (± SE) % Leaf fall (± SE)
Control 96.6 ± 3.33 12.0 ± 0.09b 4.1 ± 0.3a 84.3 ± 1.20a
0.5 96.6 ± 3.33 11.6 ± 0.33b 3.9 ± 0.08b 24.3 ± 3.33a
1.5 96.6 ± 3.33 12.6 ± 0.66b 4.0 ± 0.06a 26.6 ± 4.41a
2.5 96.6 ± 3.33 17.6 ± 0.33a 4.3 ± 0.05a 20.6 ± 0.66a
3.5 90.0 ± 5.77 12.3 ± 0.88b 3.9 ± 0.05b 23.0 ± 1.15a
4.5 86.6 ± 6.667 11.3 ± 0.66b 3.8 ± 0.11b 26.3 ± 0.33a
F value df (n − 1) = 4 1.00NS 17.735*** 5.638** 0.967NS
Open in a new tab

#Total shoot number–number of shoots emerged from the base of the original explant and shoots from the axillary buds of the primary shoots. Means with in a column followed by same letters are not significantly (p ≤ 0.05) different as determined by Duncan’s Multiple Range Test

***Highly significant (p ≤ 0.001), *Significant (p ≤ 0.05), NS not significant

The elongation of the microshoots reached its maximum value (4.30 cm) with the 2.5 µM AgNO3 concentration in the present study. On the other hand, 4.5 µM AgNO3 resulted in a significant decrease of shoot length. As the AgNO3 concentration increased, shoot number and shoot length were diminished. Similar observations were made in Solanum tuberosum (Alva Ticona and Oropeza 2013). In a previous study, a higher concentration of AgNO3 (10 μM) in combination with 2.5 μM BA was used to induce multiple shoots from seedling explants of M. oleifera (Hassanein et al. 2018). Agarwal and Purwar (2013) have reported influence of AgNO3 on in vitro performance of microplants of S. tuberosum cultivar ‘Kufri Himalini’. Sirisom and Te-Chato (2012) explained the effect of AgNO3 on in vitro shoot formation from shoot tips of Hevea brasiliensis seedlings raised ex vitro. Ethylene inhibited shoot regeneration from cotyledon cultures of sunflower and addition of AgNO3 to the medium enhanced regeneration (Chraibi et al. 1991). Naik and Chand (2003) reported the presence of AgNO3 (20 μmol/l) or aminoethoxy vinyl glycine (10 μM) in the shoot regeneration medium (MS + 8.9 μM BA + 5.4 μM NAA + 10% coconut water) was markedly enhanced the percentage of shoot regeneration and number of regenerated shoots per cotyledon explant in Punica granatum. The best medium composition for multiple shoot induction (39 shoots in cotyledonary node and 22 shoots in shoot tip) of Vigna mungo was BA, TDZ combination with adenine sulphate and AgNO3 in MS salts with B5 vitamins (Mookkan and Andy 2014). Cristea et al. (2012) reported, AgNO3 improved the capacity of direct organogenesis in Brassica oleracea. AgNO3 is an inhibitor of ethylene action thereby reduces leaf abscission. Silver ion is capable of specifically blocking the action of ethylene in classical responses such as abscission, senescence and growth retardation (Beyer 1976a, b). Silver ions are capable of generating ethylene insensitivity (Zhao et al. 2002) or perturbing the ethylene binding sites (Rodriguez et al. 1999) in plant cells.

Shoot multiplication and subculture

Established cultures in AgNO3 containing medium were sub cultured after 4 weeks on to the freshly prepared MS medium containing 2.5 µM BA and 2.5 µM AgNO3. The maximum number of shoots (16.97) was noticed in the second sub culture (Table 5). However, ANOVA revealed that there is no significant difference in the percentage of response, number of shoots, shoot length, and multiplication rate from first sub culture to fourth sub culture, proving that there is a stable rate of multiplication over four consecutive subcultures.

Table 5.

Response of in vitro shoots on subculturing of M. oleifera using agar gelled MS medium supplemented with 2.5 µM BA and 2.5 µM AgNO3

Subculture stage % Response (± SE) Total shoot number# (± SE) Shoot length (cm) (± SE)
I 96.6 ± 3.33 16.9 ± 0.37 3.9 ± 0.06
II 100.0 ± 0.00 16.9 ± 0.66 4.1 ± 0.08
III 93.3 ± 3.333 16.4 ± 0.12 4.3 ± 0.05
IV 90.0 ± 0.00 16.5 ± 0.52 3.9 ± 0.21
F value df (n − 1) 3.333NS 0.233NS 1.854NS
Open in a new tab

#Total shoot number–number of shoots emerged from the base of the original explant and shoots from the axillary buds of the primary shoots. Mean separation test DMRT not performed as the F value found non-significant

NS not significant

Most of the previous reports on in vitro propagation of M. oleifera are based on seedling derived explants (Forster et al. 2013; Shokoohmand and Drew 2013; Mathur et al. 2014; Jun-jie et al. 2017). In the present study, explants collected from elite tree were used. The method was advantageous over seedling based technique as it facilitate cloning of superior plants proven productivity. Islam et al. (2005) reported a protocol for the micropropagation of year round fruit bearing tree with 4 shoots per explant as rate of multiplication. In the present study by incorporating AgNO3 in the medium, rate of shoot multiplication improved to 17 shoots per explant, thus achieving efficient propagation of elite plants to meet large scale demand for superior planting materials.

Ex vitro rooting and hardening

Micro shoots obtained from fourth subculture onwards were used for ex vitro rooting trials (Fig. 1g). The micro shoots were treated with different concentrations of NAA and IBA (Table 6). NAA (50 µM) treatment showed maximum response (83.33%) and root length (Fig. 1h). But maximum numbers of roots (3–4) were observed with 30 µM IBA. The rooted plants were transferred to green house after 4 weeks of planting and were survived (75%) (Fig. 1i). In M. oleifera, ex vitro rooting was successfully performed in the present study by using 50 µM NAA pulse treatment for 30 s. In the previous report (Gayathri et al. 2015), in vitro rooting was adopted for the induction of rooting of microshoots. Ex vitro rooting was successfully achieved in several perennial crops and trees e.g., Tectona grandis L. (Tiwari et al. 2002) and Morinda citrifolia L. (Sreeranjini and Siril 2014). The plantlets developed through ex vitro method had lateral roots without any callus at the base of microcuttings (Yan et al. 2010). Development of ex vitro rooting technique is another improvement made in the micropropagation of mature M. oleifera. The technique essentially made rooting and hardening efficient, simple and fast, thus reducing cost, time and labour.

Table 6.

Ex vitro rooting response of micro shoots treated with different types and concentrations of auxins for 30 s

Auxin type Conc. (µM) % Rooting (± SE) Number of roots (± SE) Root length (cm) (± SE)
Control 0.0 16.6 ± 3.33e 1.11 ± 0.14c 3.5 ± 0.18c
NAA
30 53.3 ± 3.33c 1.1 ± 0.11c 3.6 ± 0.33c
50 83.3 ± 3.33a 1.8 ± 0.29bc 8.0 ± 0.57a
70 33.33 ± 3.33d 1.10 ± 0.110c 2.0 ± 0.57d
IBA
30 63.3 ± 0.69b 3.2 ± 0.29a 6.6 ± 0.63ab
50 46.6 ± 0.22c 1.7 ± 0.22bc 2.8 ± 0.13cd
70 36.6 ± 0.105d 2.3 ± 0.19b 6.2 ± 0.11b
F value df (n − 1) = 6 192.530*** 13.408*** 26.596***
Auxin type df (n − 1) 1 0.193NS 36.408*** 3.447NS
Concentration df (n − 1) 3 405.221*** 2.205NS 4.625*
Type × concentration df (n − 1) 5 76.007*** 13.113*** 60.142***
Open in a new tab

Means with in a column followed by same letters are not significantly (p ≤ .05) different as determined by Duncan’s Multiple Range Test

***Highly significant (p ≤ .001), *Significant (p ≤ .05)

Genetic fidelity analysis

To access clonal uniformity among micropropagated plants, 10 ISSR primers were used, where ISSR profile of mother plant and micropropagated plants were compared. Hassanein et al. (2018) suggested ISSR as the best marker to test genome stability in M. oleifera. The number of scored bands for each primer varied from 5 to 11 (Table 7). Ten ISSR primers produced 80 reproducible amplicons with an average of 8 bands per primer. ISSR profile showed monomorphism with the mother plant and band profiles were also similar among the in vitro raised plants (Fig. 2a–c). All ISSR primers produced monomorphic bands except primer UBC834 (Fig. 2d). Primer UBC834 produced one polymorphic band. Mean percentage of polymorphism was estimated (1.23%) and was within the acceptable limit. Presence of monomorphism among the ISSR markers showed the genetic stability of micropropagated M. oleifera plants and is in agreement with reports on M. peregrina using ISSR markers (Al Khateeb et al. 2013). The results suggest safe use of AgNO3 at reduced conc. (2.5 µM) in the culture medium for the sustained production of normal microshoots without leaf fall.

Table 7.

Details of ISSR primers with various parameters revealing the discriminatory power of each primer

Primer code Primer sequence Tm NB NPB NMB PP
Primer 1 UBC 810 GAG AGA GAG AGA GAG AT 50 11 0 11 –
Primer 2 UBC 811 GAG AGA GAG AGA GAG AC 50 9 0 9 –
Primer 3 UBC 812 GAGAGAGAGAGAGAGAA 50 8 0 8 –
Primer 4 UBC 813 CTC TCT CTC TCT CTC TT 50 7 0 7 –
Primer 5 UBC 814 CTC TCT CTC TCT CTC TA 50 10 0 10 –
Primer 6 UBC 815 CTCTCTCTCTCTCTCTG 50 7 0 7 –
Primer 7 UBC 817 CACACACACACACACAA 50 10 0 10 –
Primer 8 UBC 824 TCT CTC TCT CTC TCT CG 50 5 0 5 –
Primer 9 UBC 827 ACA CAC ACA CAC ACA CG 50 7 0 7 –
Primer 10 UBC 834 AGA GAG AGA GAG AGA GYT 50 7 1 6 14.28
Mean 8.1 0.1 8.0 1.23
Open in a new tab

Tm annealing temperature, NB number of bands, NPB number of polymorphic bands, NMB number of monomorphic bands, PP percentage of polymorphism

Fig. 2.

Fig. 2

Open in a new tab

Genetic fidelity analysis of micropropagated plants using ISSR markers. a ISSR profile of primer UBC813, b ISSR profile of primer UBC815, c ISSR profile of primer UBC827, d ISSR profile of primer UBC834, L-100 bp ladder (100–5000), M-Mother plant, 1–8-regenerants

Previously, numbers of authors have reported the micropropagation of M. oleifera from different explants such as seedling derived nodal segments (Forster et al. 2013; Saini et al. 2013), indirect organogenesis (Mathur et al. 2014), multiplication using immature seeds (Stephenson and Fahey 2004) and regeneration of axillary cotyledons and buds (Steinitz et al. 2009). Compared to previous reports, higher rate of multiplication was achieved except report by Kantharajah and Dodd (1991), where seedling derived explant was used thus cannot achieve elite cloning. Our results differ from the Stephenson and Fahey (2004), where after 1 week of culture, leaves undergone rapid necrosis in medium containing 5 mg l−1 AgNO3 with or without plant growth regulators. Hassanein et al. (2018) reported culture establishment of seedling based explant of M. oleifera, with reduced vitrification symptoms when cultured on MS medium containing 2.5 μM BA in combination with 10 μM AgNO3 or 50 μM salicylic acid (SA) and recommended SA as good anti-ethylene compound for in vitro multiplication without severe vitrification. In the present study, confirmation of ethylene accumulation and consequent leaf fall was confirmed by using ethylene releasing compound CEPA. In addition, we showed that a lower concentration of AgNO3 (2.5 µM) along with BA was sufficient to induce multiple shoot formation. The number of shoots that emerged from base of the original explant and shoots from the axils of the primary shoots in the presence of AgNO3 (2.5 μM) was significantly high coupled with reduced leaf fall (20.6%) over control (84.3%), suggesting the effectiveness of AgNO3 to promote shoot formation and facilitated sustained multiplication.

Conclusion

Present study outlines in vitro multiplication of elite M. oleifera without adversely affecting nature of microshoots. AgNO3 treatment saved cultures from leaf fall and consequent deterioration and this approach can be applied to many other plants exhibiting similar problems when cultured in vitro. The forwarded protocol was efficient to produce 17.6 shoots per explant, including shoots from axillary buds of primary shoots in MS medium supplemented with BA (2.5 µM) and AgNO3 (2.5 µM). The newly produced microshoots transferred to fresh medium in every 4 week interval can efficiently produce large number of shoots within 4 months of subcultures, and subsequently half of the total number of microshoots in every subculture was used for rooting and plant production. In vitro shoots were rooted ex vitro by applying 50 µM NAA treatment (83.3%) and were successfully transferred to field. Ex vitro rooting adopted in the present study enabled to combine rooting and hardening and thus reduced steps in micropropagation process.

Acknowledgements

The authors are grateful to Dr. Suhara Beevy S, Professor and Head, Department of Botany, University of Kerala for the facilities provided. DRS wish to thank University of Kerala for granting fellowship (No. Ac. EI/A2/10625/2016-I) to undertake the present work.

Author’s contributions

DRS conducted the experiments. DRS and EAS analyzed the data. DRS drafted the manuscript. EAS and BRN designed the experiments. EAS, BRN and DRS revised the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Abeles FB. Ethylene in plant biology. New York: Academic Press; 1973. [Google Scholar]
  2. Agarwal A, Purwar JP. Influence of silver nitrate on in vitro performance of microplants of Potato Cultivar ‘Kufri Himalini’. Vegetos Int J Plant Res. 2013;26:96–100. doi: 10.5958/j.2229-4473.26.1.014. [DOI] [Google Scholar]
  3. Al Khateeb W, Bahar E, Lahham J, Schroeder D, Hussein E. Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR) Physiol Mol Biol Plants. 2013;19:157–164. doi: 10.1007/s12298-012-0149-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alva Ticona S, Oropeza M. Effect of culture medium consistence and silver nitrate on micropropagation of two potato (Solanum tuberosum) cultivars. Revista Colombiana de Biotecnologia. 2013;15:55–62. doi: 10.15446/rev.colomb.biote.v15n2.41265. [DOI] [Google Scholar]
  5. Avila-Trevino JA, Muñoz-Alemán JM, Perez-Molphe-Balch E, Rodríguez-Sahagun A, Morales-Domínguez JF. In vitro propagation from bud and apex explants of Moringa oleifera and evaluation of the genetic stability with RAMP marker. South Afr J Bot. 2017;108:149–156. doi: 10.1016/j.sajb.2016.10.003. [DOI] [Google Scholar]
  6. Barche S, Sharma KSKA, Mishra PK. Standardization of propagation method in drumstick cv. PKM-1. Nat Sci. 2013;11:141–143. [Google Scholar]
  7. Beyer EM. Abscission: the initial effect of ethylene is in the leaf blade. Plant Physiol. 1975;55:322–327. doi: 10.1104/pp.55.2.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beyer EM. A potent inhibitor of ethylene action in plants. Plant Physiol. 1976;58:268–271. doi: 10.1104/pp.58.3.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Beyer EM. Silver ion: a potent anti-ethylene agent in cucumber and tomato. HortScience. 1976;11:175–196. [Google Scholar]
  10. Beyer EM. [14C] Ethylene metabolism during leaf abscission in cotton. Plant Physiol. 1979;64:971–974. doi: 10.1104/pp.64.6.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bleecker AB, Kende H. Ethylene: a gaseous signal molecule in plants. Annu Rev Cell Dev Biol. 2000;6:1–18. doi: 10.1146/annurev.cellbio.16.1.1. [DOI] [PubMed] [Google Scholar]
  12. Burg SP. Ethylene, plant senescence and abscission. Plant Physiol. 1968;43:1503–1511. doi: 10.1104/pp.43.7.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chandrika M, Rai VR. ISSR marker based analysis of micropropagated plantlets of Nothapodytes foetida. Biol Plant. 2010;54:561–565. doi: 10.1007/s10535-010-0100-5. [DOI] [Google Scholar]
  14. Chandrika M, Rai VR, Kini KR. Assessment of genetic stability of in vitro grown Dictyospermum ovalifolium. Biol Plant. 2008;52:735–739. doi: 10.1007/s10535-008-0142-0. [DOI] [Google Scholar]
  15. Chraibi BKM, Latche A, Raustan JP, Fallot J. Stimulation of shoot regeneration from cotyledons of Helianthus annuus by ethylene inhibitors silver and cobalt. Plant Cell Rep. 1991;10:204–207. doi: 10.1007/BF00234296. [DOI] [PubMed] [Google Scholar]
  16. Cristea TO, Leonte C, Brezeanu C, Brezeanu M, Ambarus S, Calin M, Prisecaru M. Effect of AgNO3 on androgenesis of Brassica oleracea L. anthers cultivated in vitro. Afr J Biotechnol. 2012;11:13788–13795. [Google Scholar]
  17. Dellaporta SL, Wood J, Hicks JB. A plant DNA minipreparation: version II. Plant Mol Biol Rep. 1983;1:19–22. doi: 10.1007/BF02712670. [DOI] [Google Scholar]
  18. Forster N, Mewis I, Ulrichs C. Moringa oleifera-establishment and multiplication of different ecotypes in vitro. Gesunde Pflanzen. 2013;65(1):21–31. doi: 10.1007/s10343-013-0291-8. [DOI] [Google Scholar]
  19. Gayathri M, Kumar PS, Prabha AML, Muralitharan G. In vitro regeneration of Arachis hypogaea L. and Moringa oleifera Lam. using extracellular phytohormones from Aphanothece sp. MBDU 515. Algal Res. 2015;7:100–105. doi: 10.1016/j.algal.2014.12.009. [DOI] [Google Scholar]
  20. Gonzalez A, Arigita L, Majada J, Tames RS. Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L. Plant Growth Regul. 1997;22:1–6. doi: 10.1023/A:1005751017498. [DOI] [Google Scholar]
  21. Hassanein AM, Salem JM, Faheed FA, El-Nagish A. Effect of anti-ethylene compounds on isoenzyme patterns and genome stability during long term culture of Moringa oleifera. Plant Cell Tissue Organ Cult. 2018;132:201–212. doi: 10.1007/s11240-017-1326-0. [DOI] [Google Scholar]
  22. Islam S, Jahan MAA, Khatun R. In vitro regeneration and multiplication of year-round fruit bearing Moringa oleifera L. J Biol Sci. 2005;5:145–148. doi: 10.3923/jbs.2005.145.148. [DOI] [Google Scholar]
  23. Johnson PR, Ecker JR. The ethylene gas signal transduction pathway: a molecular perspective. Annu Rev Genet. 1998;32:227–254. doi: 10.1146/annurev.genet.32.1.227. [DOI] [PubMed] [Google Scholar]
  24. Jun-jie Z, Yue-sheng Y, Meng-fei L, Shu-qi L, Yi T, Han-bin C, Xiao-yang C. An efficient micropropagation protocol for direct organogenesis from leaf explants of an economically valuable plant, drumstick (Moringa oleifera Lam.) Ind Crops Prod. 2017;103:59–63. doi: 10.1016/j.indcrop.2017.03.028. [DOI] [Google Scholar]
  25. Kantharajah AS, Dodd WA. Rapid clonal propagation of Moringa oleifera Lam., using tissue culture. South Indian Hortic. 1991;39:224–228. [Google Scholar]
  26. Kao CH, Yang SF. Role of ethylene in the senescence of detached rice leaves. Plant Physiol. 1983;73:881–885. doi: 10.1104/pp.73.4.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lemos EEP, Jennet B. Control of leaf abscission in nodal cultures of Annona squamosa L. J Hortic Sci Biotechnol. 2015;71:721–728. doi: 10.1080/14620316.1996.11515452. [DOI] [Google Scholar]
  28. Marfori EC. Clonal micropropagation of Moringa oleifera L. Philipp Agric Sci. 2011;93:454–457. [Google Scholar]
  29. Mathur M, Yadav S, Katariya PK, Kamal R. In vitro propagation and biosynthesis of steroidal sapogenins from various morphogenetic stages of Moringa oleifera Lam., and their antioxidant potential. Acta Physiol Plant. 2014;36:1749–1762. doi: 10.1007/s11738-014-1549-1. [DOI] [Google Scholar]
  30. Mehta A, Agrawal B. Investigation into the mechanism of action of Moringa oleifera for its anti-asthmatic activity. Orient Pharm Exp Med. 2008;8:24–31. doi: 10.3742/OPEM.2008.8.1.024. [DOI] [Google Scholar]
  31. Mirzai F, Uliaie ED, Hagh AB. Stimulation effect of AgNO3 and CoCl2 as ethylene inhibitors on in vitro organogenesis of sunflower (Helianthus annuus L.) J Agric Sci. 2015;25:113–118. [Google Scholar]
  32. Mookkan M, Andy G. AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper. Plant Signal Behav. 2014;9(10):1–5. doi: 10.4161/psb.32165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Murashige T, Skoog F. A revised medium for rapid growth and bioassays for tobacco tissue cultures. Physiol Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. [DOI] [Google Scholar]
  34. Naik SK, Chand PK. Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.) J Plant Physiol. 2003;160:423–430. doi: 10.1078/0176-1617-00949. [DOI] [PubMed] [Google Scholar]
  35. Obata-Sasamoto H, Nishi N, Komamine A. Mechanism of suppression of DOPA accumulation in a callus culture of Stizolobium hassjoo. Plant Cell Physiol. 1981;22:827–835. [Google Scholar]
  36. Prakash PK, Prakash L, Trevor AT. Review: regulation of morphogenesis in plant tissue culture by ethylene. Vitro Cell Dev Biol Plant. 1998;34:94–103. doi: 10.1007/BF02822771. [DOI] [Google Scholar]
  37. Pua EC, Chi GL. De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiol Plant. 1993;88:467–474. doi: 10.1111/j.1399-3054.1993.tb01361.x. [DOI] [Google Scholar]
  38. Ramachandran C, Peter KV, Gopalakrishnan PK. Drumstick (Moringa oleifera) a multipurpose Indian vegetable. Econ Bot. 1980;34:276–283. doi: 10.1007/BF02858648. [DOI] [Google Scholar]
  39. Riyathong T, Dheeranupattana S, Palee J, Shank L (2010) Shoot multiplication and plant regeneration from in vitro cultures of drumstick tree (Moringa oleifera Lam.). In: The 8th international symposium on biocontrol and biotechnology. King Mongkut’s Institute of Technology Ladkrabang and Khon Kaen University, Nongkhai Campus, Thailand, pp 99–104
  40. Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB. A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science. 1999;283:996–998. doi: 10.1126/science.283.5404.996. [DOI] [PubMed] [Google Scholar]
  41. Saini RK, Shetty NP, Giridhar P, Ravishankar GA. Rapid in vitro regeneration method for Moringa oleifera and performance evaluation of field grown nutritionally enriched tissue cultured plants. 3 Biotech. 2012;2:187–192. doi: 10.1007/s13205-012-0045-9. [DOI] [Google Scholar]
  42. Saini RK, Saad KR, Ravishankar GA, Giridhar P, Shetty NP. Genetic diversity of commercially grown Moringa oleifera Lam. cultivars from India by RAPD, ISSR and cytochrome P450-based markers. Plant Syst Evolut. 2013;299:1205–1213. doi: 10.1007/s00606-013-0789-7. [DOI] [Google Scholar]
  43. Saini RK, Manoj P, Shetty NP, Srinivasan K, Giridhar P. Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats. Nutr Res. 2014;34:630–638. doi: 10.1016/j.nutres.2014.07.003. [DOI] [PubMed] [Google Scholar]
  44. Saini RK, Shetty NP, Giridhar P. Carotenoid content in vegetative and reproductive parts of commercially grown Moringa oleifera Lam. cultivars from India by LC–APCI–MS. Eur Food Res Technol. 2014;238:971–978. doi: 10.1007/s00217-014-2174-3. [DOI] [Google Scholar]
  45. Saini RK, Manoj P, Shetty NP, Srinivasan K, Giridhar P. Relative bioavailability of folate from the traditional food plant Moringa oleifera L. as evaluated in a rat model. J Food Sci Technol. 2016;53:511–520. doi: 10.1007/s13197-015-1828-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Salem JM. In vitro propagation of Moringa oleifera L. under salinity and ventilation conditions. Genet Plant Physiol. 2016;6:54–64. [Google Scholar]
  47. Sarropoulou V, Dimassi-Theriou K, Therios I. Effect of the ethylene inhibitors silver nitrate, silver sulfate, and cobalt chloride on micropropagation and biochemical parameters in the cherry rootstocks CAB-6P and Gisela 6. Turk J Biol. 2016;40:670–683. doi: 10.3906/biy-1505-92. [DOI] [Google Scholar]
  48. Shokoohmand A, Drew RA. Micropropagation of Moringa oleifera. Acta Hort. 2013;988:149–160. doi: 10.17660/ActaHortic.2013.988.17. [DOI] [Google Scholar]
  49. Sirisom Y, Te-chato S. The effect of peptone and silver nitrate on in vitro shoot formation in Hevea brasiliensis Muell Arg. J Agric Technol. 2012;8:1509–1516. [Google Scholar]
  50. Sisler EC, Yang SF. Ethylene the gaseous plant hormone. Bioscience. 1984;34:234–238. doi: 10.2307/1309461. [DOI] [Google Scholar]
  51. Sreeranjini S, Siril EA. Field performance and genetic fidelity evaluation of micropropagated Morinda citrifolia L. Indian J Biotechnol. 2014;13:121–130. [Google Scholar]
  52. Steinitz B, Tabib Y, Gaba V, Gefen T, Vaknin Y. Vegetative micro-cloning to sustain biodiversity of threatened Moringa species. Vitro Cell Dev Biol Plant. 2009;45:65–71. doi: 10.1007/s11627-008-9162-x. [DOI] [Google Scholar]
  53. Stephenson KK, Fahey JW. Development of tissue culture methods for the rescue and propagation of endangered Moringa spp. germplasm. Econ Bot. 2004;58:116–124. doi: 10.1663/0013-0001(2004)58[S116:DOTCMF]2.0.CO;2. [DOI] [Google Scholar]
  54. Sung LS, Huang SY. Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3, 4-dihydroxyphenylalanine. Biotechnol Lett. 2000;22:875–878. doi: 10.1023/A:1005679800393. [DOI] [Google Scholar]
  55. Tamimi SM. Effects of ethylene inhibitors, silver nitrate (AgNO3), cobalt chloride (CoCl2) and aminooxy acetic acid (AOA), on in vitro shoot induction and rooting of banana (Musa acuminata L.) Afr J Biotechnol. 2015;14:2511–2516. [Google Scholar]
  56. Thorpe TA. History of plant tissue culture. Mol Biotechnol. 2007;37:169–180. doi: 10.1007/s12033-007-0031-3. [DOI] [PubMed] [Google Scholar]
  57. Tiwari SK, Tiwari KP, Siril EA. An improved micropropagation protocol for teak. Plant Cell Tissue Organ Cult. 2002;71:1–6. doi: 10.1023/A:1016570000846. [DOI] [Google Scholar]
  58. Venkatachalam L, Sreedhar RV, Bhagyalakshmi N. Genetic analyses of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers. Vitro Cell Dev Biol Plant. 2007;43:267–274. doi: 10.1007/s11627-007-9028-7. [DOI] [Google Scholar]
  59. Vinod K, Giridhar P, Ravishankar GA. Review: AgNO3—a potential regulator of ethylene activity and plant growth modulator. Electron J Biotechnol. 2009;12(2):1–15. [Google Scholar]
  60. Wang KLC, Hai L, Ecker JR. Ethylene biosynthesis and signaling networks. Plant Cell. 2002;14:31–51. doi: 10.1105/tpc.001768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yan H, Liang C, Yang L, Li Y. In vitro and ex vitro rooting of Siratia grosvenorii, a traditional medicinal plant. Acta Physiol Plant. 2010;32:115–120. doi: 10.1007/s11738-009-0386-0. [DOI] [Google Scholar]
  62. Zhao XC, Qu X, Mathews DE, Schaller GE. Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis. Plant Physiol. 2002;130:1983–1991. doi: 10.1104/pp.011635. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Physiology and Molecular Biology of Plants are provided here courtesy of Springer

RESOURCES