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. 2021 Feb 19;27(2):429–443. doi: 10.1007/s12298-021-00955-x

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree

R Raji 1, E A Siril 1,✉
PMCID: PMC7907408  PMID: 33707879

Abstract

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White’s, Gamborg’s B5 or woody plant medium (WPM) supplemented with 2.5 µM benzyl adenine (BA) and 0.1 µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5 µM) and NAA (0.1 µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83 cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13 cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2 mM indole-3-butyric acid (IBA) for 5 min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.

Keywords: Culture media, Elaeocarpus serratus, Ex vitro rooting, In vitro culture, Micropropagation

Introduction

Elaeocarpus serratus L. (family Elaeocarpaceae) commonly known as Ceylon olive is a less known fruit tree distributed in Southern peninsula of India and all over the Sri Lanka. All parts of E. serratus have specific uses in traditional medicine and used as a potent cardiovascular stimulant and diuretic. Leaves are used in the treatment of rheumatism and as an antidote to poison, while the fruits are locally prescribed for the treatment of diarrhea and dysentery. The fruits of E. serratus are regarded as rich source of minerals, vitamins, fiber, and phenolic compounds. Most of these constituents are nutritionally or medicinally valued (Ghani 1998; Jayasinghe et al. 2011). Several phytochemical constituents present in fruit are proved to effectively prevent ailments such as diabetes, cancer and heart diseases. Fruit extract also exhibits antioxidative, antimicrobial, anti-inflammatory and anti-ulcer properties. Besides its medicinal application, tree deserves due attention in the industry for the value-added fruit-based products. The fruits especially the fruit pulp, which is used in the preparation of jam, jelly, etc.… (Peiris 2007). The fruit juice of E. serratus is given for stimulating secretions from taste buds, thus increasing appetite in patients (Ghani 1998). The fruits are the source of nutraceutically significant pulp, rich in sugar, protein, vitamin C, other antioxidants, minerals like calcium, phosphorus and magnesium and minor levels of fat, thus having the potential to utilize in the formulation of health drink (Awodoyin et al. 2015). The fruit pulp of Ceylon olive was successfully incorporated to produce ice cream. Its mildly sweet and sour taste, characteristic aroma makes the fruit to promote it as a table fruit and also in the preparation of ready to serve drinks. Mature, unripened fruits are pickled. The organic acid profiling of fruit pulp revealed a significant amount of citric acid and tartaric acid (Peiris 2007).

While considering the prioritized economic value and quality trait of this fruit tree, fruit pulp content seems most important. To ensure fixation of such traits to the next generation, cloning of identified germplasm with high pulp and sugar content will be the promising option. A comprehensive literature survey revealed that no in vitro propagation had been reported in E. Serratus from mature trees.

In the natural stands, E. serratus is regenerated through seeds. Germination capacity, however, was recorded < 10% under experimental conditions (Raji and Siril 2018). Alternatively, E. serratus can be propagated through softwood cuttings, layering and grafting (Raji and Siril 2016). Application of conventional vegetative propagation are limited to achieve large scale multiplication of superior germplasm. Recent reports from Assam, India recognized E. serratus as a threatened tree species in the region (Baruah et al. 2019) underlines the need for biotechnological intervention on conservation of elite germplasm.

In most of the micropropagation methods of tree species, alteration is limited to plant growth regulators, but nutrients and other factors followed as remains as standard. Optimization of media by addressing ionic concentration of macro and micronutrients are therefore often required versatile, elegant experimentation that ultimately leads to the identification of exact requirement of various mineral nutrient for the sustained growth of in vitro cultured species (Phillips and Garda 2019). Apart from the molar concentration of nutrients, other factors such as the synergic and antagonistic effect of ions, reactions between supplied salts and consequent formation of complexes and precipitation can influence nutrient uptake by in vitro cultured tissues. The concentration of mineral ions in the medium when inappropriate, normal metabolism of the plant gets affected, and it may symptomize by morphological and growth abnormalities (Lozzi et al. 2019). Thus, it is suggested that for the normal physiology, growth and development of in vitro cultured tissues, optimized level of macro and micronutrients is to be used (Nas and Read 2004; Rahman et al. 2009). Though micropropagation protocols were reported for other species of Elaeocarpus (Arshad and Kumar 2006; Rahman et al. 2009), no previous reports are available with micropropagation of E. serratus. Therefore, present investigation was undertaken to establish a protocol allowing micropropagation of E. serratus based on explants derived from a 17-year-old tree. A preliminary trial using agar gelled, full-strength MS medium showed poor growth response and elongation of axillary shoots formed in vitro. Thus, different media formulations viz., woody plant medium (WPM) (Lloyd and McCown 1980), White’s medium (White 1939) and B5 (Gamborg et al. 1968), along with varying ionic strength of Murashige and Skoog medium (¾ MS, ½ MS) were compared with the full-strength MS medium (Murashige and Skoog 1962).

Materials and methods

Source of explant and sterilization

A seventeen-year-old tree growing at Varkala Sivagiri (8° 45′ 0.60″ N; 76° 43′ 34.98″ E; 17.98 m asl) was used as explant source. Healthy shoots (5–6 cm) of wet summer season’s (May–August) growth were excised, leaves were removed, and explants were made into 2–2.5 cm sized segments having a single node bearing a rudiment axillary bud. Nodal explants were then successively exposed to running tap water for 15 min, 1% polysorbitol detergent (Labolene, Mfg. Fischer Scientific Chemicals, Mumbai, India) for 10 min and then rinsed in distilled water for 3 times. Surface sterilization of explants was performed in a laminar airflow cabinet using 0.2% mercuric chloride solution for 3 min, followed by 4–5 rinses in sterile distilled water. These explants were segmented into 1–1.5 cm size and cultured on agar (0.8%) gelled, ½ MS medium supplemented with varying levels of cytokinins (BA or Kn).

Culture media and incubation conditions

As explant response and growth of new shoots in full strength MS medium was poor, MS medium was modified into ½ strength of macro and micronutrients, organic nutrients, 3% sucrose and 0.8% agar were used for standardization growth regulators. The ½ MS medium supplemented with various concentrations (0.5, 1.0, 1.5, 2, 2.5, 3 or 5 μM) of benzyl adenine (BA) or Kn (Sigma–Aldrich, St. Louis, US) singly or in combinations of BA and Kn at various concentrations (0.5, 1, 1.5, 2 or 2.5 μM) was used for maximum proliferation. Growth regulator free ½ MS medium was used as control. The pH of medium prior to autoclaving (108 kPa, 121 °C for 15 min) was adjusted at 5.8 ± 0.02.

To study the combined effect of BA and α-naphthalene acetic acid (NAA), nodal explants were cultured on medium containing 2.5 μM BA along with 0.05, 0.1, 0.25, or 0.5 μM NAA. After 4 weeks of culture, nascent shoots were subcultured to optimized growth regulator supplemented medium.

The surface-sterilized explants after trimming both ends were inoculated in a culture tube (25 × 150 mm) containing agar gelled medium (15 ml). The cultures were incubated at 26 ± 2 °C, 55–60% relative humidity maintained by using humidifier (Model NH 809; Novita Instruments, Mumbai) and dehumidifier (Model ND 320 Dehumidifier, Novita Instruments, Mumbai), monitored by a digital hygrometer. The culture racks provided with cool and white fluorescent tubes (Philips, Mumbai, India) having a photon flux density (PFD) of 40–50 μmol m−2 s−1 for 16 h photoperiod.

Effect of different media

Effect of different media formulations viz., WPM, B5 and White’s medium on in vitro growth of explant was tested. To evaluate the effect of varying molar strength of MS basal medium, nodal explants were inoculated on full strength MS, ¾ MS or ½ MS media. All media were supplemented with 3% sucrose, 2.5 µM BA and 0.1 µM NAA and were gelled with 0.8% agar.

Effect of modified MS media

Culture media optimization was further extended to test the effectiveness of following modifications in MS media; Elaeocarpus serratus medium 1 (ESM1) containing ½ strength of major and minor salts, the full strength of vitamins, ESM2 provided with ½ strength of major salts, full strength of minor salts, ½ of vitamins and ESM3 containing ½ strength major salts, full strength of minor salts and vitamins. In all media formulations, uniform Fe EDTA level was maintained (65.1 mM). All salts used for the experiments were made off analytical grade (Merck, Darmstadt, Germany or Merck India, Bengaluru). The media were supplemented with 3% (w/v) sucrose (Hi Media, Mumbai, India) and pH was adjusted (5.8) prior to adding 0.8% agar for gelling. After four weeks of incubation, percentage of response, number of shoots, the average length of the shoot (cm), average leaf number and leaf area were recorded.

Reculture and multiplication

Reculturing of original explant to fresh medium at every 4-week interval led to multiplication of microshoots. To facilitate sustained multiplication, optimized media augmented with a standardized level of growth regulators was used. The subculture of nodal segment excised from in vitro raised shoots or reculture of original explant was done using media prepared in glass bottles (350 ml) provided with transparent polypropylene screw cap. Cultures raised in the optimized medium were maintained over 3 years by regular subculturing at an interval of 5–6 week, without any loss of growth and vigour.

Effect of explant collection month

Effect of explant sampling months on culture response was studied by collecting explants from January to December and cultured on ESM3 medium containing 2.5 μM BA and 0.1 μM NAA.

Ex vitro rooting of microshoots

Actively growing in vitro shoots (~ 4 cm) from IV subculture onwards were excised and used for ex vitro rooting experiments. Microshoots were thoroughly washed in tap water and arranged into bundles (n = 12 per bundle). The basal portion of microshoots was then dipped in different concentrations (1, 2, 3, 4 or 5 mM) of auxins viz., indole-3-butyric acid (IBA) or NAA for 5 min. Each auxin solution was prepared by dissolving the required quantity in 100% (v/v) ethanol and diluting this to 50% (v/v) with distilled water. Micro shoots devoid of auxin treatment served as control. The auxin treated microcuttings were then planted in garden pots (12 cm height × 7 cm diameter) filled with the potting mixture (1:1:1 sand, soil, and farmyard manure) and covered with transparent polyethylene bags. Potted microshoots were maintained in a glass house (28 ± 2 °C, RH 90%; 40 µmol m−2 s−1 PFD) and irrigated at one-week interval. The polyethylene bags were removed after 6 weeks of planting and % rooting, root number and mean root length were recorded. The plantlets after 8 weeks were replanted in nursery pots and maintained in the greenhouse for 3 months and then transferred to fruit tree experimental garden of the Department.

Morphological and molecular marker-based evaluation of micropropagated plants

Three-month-old plants were transferred to the experimental plot (25 m × 10 m). Plants were carefully removed from nursery pots and planted in nursery pits (30 cm length × 30 cm width × 30 cm depth) prepared at 5 × 5 m spacing, where 10 plants were planted with three replication blocks. The morphometric characters (plant height, collar diameter, number of branches, petiole length, leaf length, and leaf width) were analyzed after 1 year. Genetic uniformity analysis was conducted based on 2 markers. Genomic DNA was extracted from young leaves of 10 randomly sampled micropropagated plantlets by using the method of Dellaporta et al. (1983). Molecular marker-based uniformity analysis was conducted using 8 RAPD primers, screened among 12 primers (Integrated DNA Technologies Inc., India) on the basis of amplification. The PCR reaction was carried out in a total volume of 25 µl containing 50 ng of genomic DNA. The 25 µl reaction mixture consisted of 12.5 µl 6X DNA loading buffer (Taq 6X Smart Mix, Origin Diagnostics, India), 2 µl of RAPD primers, 50 ng of template DNA and the final volume was adjusted to 25 µl with sterile double distilled water. The reactions were carried out in a thermal cycler programmed with initial denaturation of DNA at 95 °C for 5 min followed by 40 cycles of denaturation at 94 °C for 1 min.; primer annealing based on Tm for 1 min, and extension at 72 °C for 2 min with the final extension at 72 °C for 8 min. The annealing temperature was standardized by performing a gradient PCR at temperature regime of 34 to 42 °C for the selection of primers.

To determine ISSR based uniformity, six ISSR primers UBC815, UBC830, UBC897, UBC864, UBC814 and UBC836 (UBC series 800, University of British Columbia, Vancouver, Canada) were selected. PCR reaction master mix composition and temperature conditions were standardized for ISSR markers. The PCR reaction mix (25 µl) containing the same composition as described above was used. The polymerase chain reaction was carried out in a thermal cycler (Eppendorf master cycler gradient, SI NO 533149527, Germany). Amplification conditions using ISSR primers were performed as initial DNA denaturation at 95 °C for 5 min, followed by 39 cycles of 30 s denaturation step at 94 °C, an annealing step for 1 min at the respective annealing temperatures of each primer in the range of 50–66 °C, an extension of 2 min at 72 °C and the last cycle of final extension at 72 °C for 5 min.

The amplified products of both RAPD and ISSR fragments were separated along with 100 bp DNA ladder ranged from 100 to 1500 bp (Cat No. 3422A, Takara Bio USA, Inc.) on agarose (1.8% w/v; SRL, Mumbai) gel using 1X Tris-Boric acid- EDTA (TBE) buffer (pH 8.0) at a constant voltage (100 V) and stained with ethidium bromide. The gel was photographed and analyzed using a gel documentation system (BIO RAD molecular imager, Gel Doc ™ XR with image lab ™ software, BIO RAD, Hercules, California, US). Amplification reaction of each primer was repeated twice for every plant to ensure reproducibility.

Experimental design and statistical analysis

Randomized complete block design (RCBD) was adopted for all experiments. For in vitro culture establishment, media standardization and subsequent subculture experiments, each treatment, 20 explants with three replication blocks were done. At the end of the initial 4-week culture period, percentage of explants response, number of shoot buds, number of shoots and shoot length were recorded. In the case of ex vitro rooting, for every treatment, three replication blocks each consisted of 12 microcuttings was planted. After 6 weeks of planting, percentage rooting, root number and mean root length were recorded. The recorded data were subjected to analysis of variance, and Duncan’s multiple range test (p < 0.05) was conducted to compare the mean values (SPSS ver. 20, SPSS Inc., Chicago, IL, US).

Results and discussion

Effect of growth regulators on establishment and multiplication

The nodal explants (Fig. 1a) cultured on ½ MS medium supplemented with different concentrations (0.5, 1, 2, 2.5, 3 and 5 μM) of BA and Kn alone or in combinations (0.5–2.5 μM) showed bud break within six days and subsequent in vitro growth and elongation. In hormone-free ½ MS medium (control), nodal explants were failed to produce new shoots. Among the different concentrations of BA, 2.5 μM gave the highest response (Table 1). After 4 weeks of culture on ½ MS medium supplemented with 2.5 μM BA, 6.26 new shoots per culture was recorded. Response and multiplication efficiency of Kn at varying concentration seems to be significantly (p < 0.05) inferior to BA (Table 1). Medium containing 2.5 μM Kn showed 36.67% response, where 2.93 shoots were noticed. The combined effect of 1 μM BA and 1 μM Kn produced 3.34 shoots/culture with 1.88 cm shoot length. Significant reduction in the number of shoots per explants and inhibition of shoot growth was observed in relatively low levels of cytokinin added medium. BA showed superior effect over Kn on explant response indicates effective uptake and metabolization of BA that leads to the synthesis of natural cytokinins within the tissue (Lodha et al. 2014). In the previous reports on micropropagation of E. tuberculatus (Arshad and Kumar 2006), BA was used as preferred cytokinin over Kn.

Fig. 1.

Fig. 1

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Multiple shoots produced in three different formulations of MS medium. a Nodal explants collected from 17- years-old mature E. serratus tree, b Multiple shoot with leaf chlorosis in ESM1 containing 2.5 μM BA and 0.1 μM NAA (Scale bar = 1.8 cm), c Multiple shoots developed in ESM2 medium supplemented with 2.5 μM BA and 0.1 μM NAA (Scale bar = 1.8 cm), d Shoot multiplication in ESM3 with 2.5 μM BA and 0.1 μM NAA (Scale bar = 1.6 cm), e Initial response of nodal explants in ESM3 with 2.5 μM BA and 0.1 μM NAA, f Six week old culture in ESM3 medium containing 2.5 μM BA and 0.1 μM NAA (Scale bar = 2.1 cm)

Table 1.

Effect of BA and Kn supplemented half strength MS medium on in vitro response of nodal segments of E. serratus

Cytokinin Type Conc. (µM) Percentage of explant response Shoot number/explant No. of shoot buds/explant Shoot length (cm)
Control 0.0 0.00 ± 0.00 h 0.00 ± 0.00j 0.00 ± 0.00 k 0.00 ± 0.00i
BA 0.5 18.33 ± 1.67efg 1.81 ± 0.17fgh 2.68 ± 0.35cde 0.56 ± 0.03 h
1.0 31.67 ± 1.67 cd 2.18 ± 0.03f 1.97 ± 0.11fgh 0.83 ± 0.01gh
1.5 38.33 ± 4.41bc 4.36 ± 0.04c 3.14 ± 0.12bc 0.89 ± 0.05fgh
2.0 46.67 ± 4.41ab 5.61 ± 0.13b 3.66 ± 0.12b 0.98 ± 0.01efg
2.5 55.00 ± 2.89a 6.26 ± 0.04a 4.44 ± 0.06a 1.10 ± 0.05def
3.0 40.00 ± 5.00bc 5.45 ± 0.12b 3.17 ± 0.10bc 1.08 ± 0.10def
5.0 16.67 ± 3.33efg 3.51 ± 0.09d 2.35 ± 0.09def 0.58 ± 0.02 h
Kn 0.5 10.00 ± 2.89 g 1.11 ± 0.11i 2.01 ± 0.08fgh 0.73 ± 0.08hi
1.0 16.67 ± 1.67efg 1.55 ± 0.29ghi 2.04 ± 0.14fgh 1.08 ± 0.08def
1.5 25.00 ± 2.89de 1.90 ± 0.05 fg 2.37 ± 0.19def 1.32 ± 0.09d
2.0 33.33 ± 4.41d 2.88 ± 0.12e 2.92 ± 0.05 cd 1.83 ± 0.06b
2.5 36.67 ± 4.41c 2.93 ± 0.19e 3.10 ± 0.19bc 2.29 ± 0.12a
3.0 21.67 ± 1.67ef 2.11 ± 0.21f 1.72 ± 0.36ghi 1.73 ± 0.03c
5.0 11.67 ± 1.67 g 1.19 ± 0.10i 1.07 ± 0.09j 0.93 ± 0.04efgh
BA + Kn 0.5 + 0.5 21.67 ± 1.67ef 2.14 ± 0.32f 1.47 ± 0.35hij 1.16 ± 0.17de
1 + 1 35.00 ± 2.89c 3.34 ± 0.11de 2.13 ± 0.15efg 1.88 ± 0.08b
1.5 + 1.5 31.67 ± 1.67 cd 1.49 ± 0.23ghi 1.08 ± 0.21j 1.04 ± 0.04efg
2 + 2 21.67 ± 1.67ef 1.32 ± 0.09hi 1.17 ± 0.17ij 1.01 ± 0.10efg
2.5 + 2.5 13.33 ± 3.33 fg 1.50 ± 0.29ghi 1.50 ± 0.29hij 0.74 ± 0.09hi
Treatment Df (n − 1) 19 20.498*** 104.124*** 29.735*** 48.527***
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Means ± SE with in a column followed by same letters are not significantly (p < 0.05) different as determined by Duncan’s multiple range test.*significant at p < 0.05 level; ***significant at p < 0.001 level

The percentage of explant response and mean length of shoots in the medium containing BA and NAA was significantly (p < 0.001) higher than medium containing BA alone (Table 2). The maximum number of shoots (6.82) were noticed in a combination of 2.5 µM BA with 0.1 µM NAA which resulted in twofold higher elongation than 2.5 µM BA alone. Auxin-cytokinin balance plays an important role in multiple shoot formation and elongation (Wang and Jiao 2018). Previous reports on various woody species revealed that the combination of BA and NAA induced better multiplication of shoots and internodal elongation than BA alone from nodal explants of mature trees (Sreeranjini and Siril 2014; Ahmed et al. 2017; Ahmad and Anis 2019).

Table 2.

Effect of different concentrations of NAA in combination with 2.5 µM BA in agar gelled half strength MS medium on multiple shoot induction of E. serratus

NAA (µM) Percentage of explant response Shoot number/explant Shoot bud/explant Shoot length (cm)
0.0 55.00 ± 2.89b 6.13 ± 0.04b 4.24 ± 0.06b 1.18 ± 0.05c
0.05 51.67 ± 1.67ab 6.23 ± 0.06ab 3.41 ± 0.21b 2.06 ± 0.07b
0.10 58.33 ± 4.41a 6.82 ± 0.12a 4.88 ± 0.66a 2.66 ± 0.09a
0.25 48.33 ± 1.67bc 5.78 ± 0.12b 2.96 ± 0.20c 1.98 ± 0.07b
0.5 40.00 ± 2.89c 3.91 ± 0.33c 2.09 ± 0.05c 1.86 ± 0.13b
F Value Df (n − 1) = 4 6.972** 43.681*** 10.247*** 11.520***
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Means ± SE with in a column followed by same letters are not significantly (p < 0.05) different as determined by Duncan’s multiple range test.**significant at p < 0.01 level; ***significant at p < 0.001 level

Effect of different media formulations

MS medium in its original form is usually not used in tree tissue culture. However, in the past, reports on the use of the modified level of various macronutrients in the MS medium for the tree tissue culture is available (Bell and Reed 2002; Phillips and Garda 2019). In the previous report, a series of experiments aimed to evolve the most suitable ionic level of the different macronutrient constituent to achieve increased response over widely using standard formulations was conducted (Greenway et al. 2012). In the present study, various media formulations tested (full strength MS, ¾ MS, ½ MS, B5, WPM and White’s) had significant (p < 0.001) effect on percentage response, shoot number and other parameters recorded. Malformed shoots were noticed when nodal explants were cultured on full strength MS medium containing 2.5 µM BA and 0.1 µM NAA (Table 3). In the case of ¾ MS medium, noticeable explant response (65%) and shoot length (2.92 cm) were recorded over other media formulations. However, shoot multiplication (6.6) was high in the ½ MS medium fortified with 2.5 µM BA and 0.1 µM NAA, where at the axillary node region of explant, the emergence of an average of 5 shoot buds was noticed. Explants cultured on B5 and White’s medium showed an inferior response (23.3%) coupled with less number of shoots. The inferior response of B5 medium is explained in light of high concentrations of nitrate present in this medium (Greenway et al. 2012). In the case of White’s medium, probably due to the very low concentration of macro salts, and omission of some important nutrients, the response reduced over ½ or ¾ MS. The response of explants in full strength MS (35.0%) and WPM (31.67%) medium was also inferior to ½ MS or ¾ MS medium. WPM medium is featured with higher sulphate and lower nitrate content (Hand et al. 2014) compared to other basal media. Reduction in explant response in high ionic concentration media such as full-strength MS was noticed in the present study is in conformity with the reports on Eucalyptus dunnii (Oberschelp and Gonçalves 2016). Further, it is reasonable to infer that high levels of nitrogen in the MS medium may hinder shoot elongation (Lymperopoulos et al. 2018). The present result is in agreement with the reports of other woody species (Timofeeva et al. 2014; Kabylbekova et al. 2020), where half-strength MS or reduced ionic concentration of nitrogen were used to achieve better in vitro growth and multiplication.

Table 3.

In vitro response of nodal segments of E. serratus cultured on different media supplemented with 2.5 µM BA and 0.1 µM NAA

Media type Percentage of explant response Shoot number/explant No. of shoot buds/explant Shoot length (cm)
WPM 31.67 ± 1.67bc 2.53 ± 0.07c 3.90 ± 0.22c 1.93 ± 0.12b
White’s 23.33 ± 1.67c 1.33 ± 0.10d 4.55 ± 0.55abc 0.47 ± 0.03d
MS 35.00 ± 2.8b 0.00 ± 0.00e 5.33 ± 0.04a 0.48 ± 0.02d
¾MS 65.00 ± 5.77a 4.95 ± 0.26b 4.10 ± 0.36bc 2.92 ± 0.17a
½ MS 58.33 ± 3.33a 6.60 ± 0.18a 5.02 ± 0.39ab 2.61 ± 0.06a
B5 23.33 ± 1.67c 1.30 ± 0.03d 0.56 ± 0.11d 0.96 ± 0.14c
F value Df (n − 1) = 5 31.60*** 324.26*** 27.57*** 100.84***
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Means ± SE with in a column followed by same letters are not significantly (p < 0.05) different as determined by Duncan’s multiple range test. ***significant at p < 0.001 level

Effect of modified MS media

Modifications of MS medium (ESM1, ESM2, or ESM3) significantly (p < 0.001) influenced explant response. The nodal explants cultured on ESM3 (½ strength of major salts, original strength of minor salts, original strength of vitamins and 65.1 mM Fe EDTA) produced significantly (p < 0.05) higher number of shoots (8.88) as compared to other modified media used (Table 4; Fig. 1d, e). Other experimental parameters viz., shoot length, and leaf area was greater in ESM3 than other media formulations and were statistically significant (p < 0.05). Explants cultured on ESM3 medium responded moderately well (61.67%) with a comparable number of leaves (Table 4). The overall outcome of the experiment suggests the suitability of ESM3 medium for the in vitro culture of E. serratus which is in agreement with previous reports on E. robustus (Rahman et al. 2009) where ½ strength of major salts, the full strength of minor salts and vitamins along with 4 µM BA, 4 µM Kn and 15% coconut water was used. An important characteristic of the full strength of MS medium is the high ionic concentration of nitrogen (NH4+ and NO3−). In woody plants, high ionic concentration of nitrogen often affects normal in vitro growth of cultures. Ammonium assimilation in woody species possibly block cytokinin synthesis or translocation to the shoot axis, ultimately inhibits the shoot morphogenesis (Endres et al. 2002). Intake and further transport of different major and minor nutrients are an interlinked process. In view of this, growth morphology, shoot length, and multiplication of shoots could be improved suitably by modifying the level of minor nutrients of the medium. In ESM3 medium, the original strength of minor salts was used thus resulted in a marginal increase in shoot number and elongation. In addition to these, shoots formed in this medium had superior shoot quality in terms of leaf number and leaf area over other media types compared (Table 4).

Table 4.

Effect of three different altered MS media supplemented with 2.5 µM BA and 0.1 µM NAA on establishment and axillary bud proliferation of nodal segments of E. serratus

Modified media % Response Shoot number Shoot length (cm) Leaf number Leaf area (cm2)
ESM1 58.33 ± 1.67 7.10 ± 0.21b 2.69 ± 0.23b 4.48 ± 0.21 3.2 ± 0.6b
ESM2 60.00 ± 5.00 7.72 ± 0.45b 3.28 ± 0.16ab 4.06 ± 0.09 4.0 ± 0.2b
ESM3 61.67 ± 4.41 8.88 ± 0.15a 3.83 ± 0.11a 4.33 ± 0.18 4.8 ± 0.4a
F value df (n − 1) = 2 0.176NS 9.223* 10.623* 1.652NS 11.375*
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Means ± SE with in a column followed by same letters are not significantly (p < 0.05) different as determined by Duncan’s multiple range test. *significant at p < 0.05 level; NS Non significant

The microshoots formed in ESM1 medium appeared yellowish (Fig. 1b) than those raised on ESM3. ESM2 showed developmental inferiority in terms of shoot length as well as leaf area (Fig. 1c), compared to ESM3. The greatest shoot proliferation with least visually scored abnormality such as yellowing and chlorosis of leaves was recorded in ESM3 medium. It is to be explained on the recognition that in the ESM3 medium adequate amount of Cu and Zn ions are present, which may help normal growth of microshoots (Oberschelp and Gonçalves 2016). Role of Cu and Zn ions implicated to mitochondria and other membrane-bound cell organelles, thus take part in electron transport and other physiological activities (Morgan et al. 2008). Cultures growing in the high amount of ammonium containing medium such as full-strength MS may lead to ammonium toxicity as evident in E. serratus where inhibition of shoot growth associated with shoot chlorosis has been related to oxidative stress or ionic imbalance. In contrast, in ESM3 medium where ammonium concentration reduced to half of the original MS strength, the microshoots developed were free from symptoms of oxidation and chlorosis (Esteban et al. 2016; Liu and Wirén 2017).

Minor nutrients are components of enzymes which are required only in minute quantities. While considering the role of micronutrients in cell physiology, complex interactions between iron and other micronutrients is well documented (Ramage and Williams 2002). Such interaction reported to greatly influence shoot quality and growth of shoot (Niedz et al. 2014). When micronutrients at an optimal level were included in conjunction with macronutrient formulations, normal development of adventitious shoot buds occurred. The presence of an optimal level of iron in the chelated form in combination with growth regulators is particularly important for the adventitious shoot and root formation (Phillips and Garda 2019).

Reculture and multiplication

Reculturing the original explants in ESM3 containing BA (2.5 µM) and NAA (0.1 µM) produced an almost stable rate of multiplication from III subculture onwards (Figs. 1f; 2), where at V passage, the highest number of shoots (16.08) were achieved. Shoots from actively growing cultures were excised, made into segments and sub cultured on ESM3 medium augmented with BA (2.5 µM) and NAA (0.1 µM). The reculturing of original explant to the fresh nutrient medium is an alternate route, successfully applied to produce large number of new shoots at an interval of 4 weeks. This practice essentially rejuvenates and reinvigorates the basal dormant meristematic cells (Tripathi and Kumari 2010; Amirchakhmaghi et al. 2019). This practice was successfully employed in several woody plant species (Rathore et al. 2005; Lodha et al. 2014; Patel et al. 2014) to ensure sustained supply of microshoots for rooting.

Fig. 3.

Fig. 3

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Influence of explant collection month on in vitro response of E. serratus cultured on ESM3 medium containing 2.5 μM BA and 0.1 μM NAA

Effect explant collection month on culture establishment

The response of explants in culture was significantly influenced by the collection month (Fig. 3). Explants cultured during the month of May gave the maximum (65%) response. May to August is the wet summer season in Kerala state, South India is the active growth period of the plant in the region (Raji and Siril 2016), and at this phenophase, endogenous cytokinin get elevated, and lowered stress conditions characterized by minimal levels of phenolics thus promote bud break and growth of shoot buds (Phulwaria et al. 2011; Patel et al. 2016). The response (Fig. 3) was low during the months of December and January, possibly due to the cessation of vegetative growth in the winter months and consequent bud dormancy. The influence of phenophase and physiological conditions of source tree in the establishment of cultures observed in the present study is in agreement with other reports (Dhavala and Rathore 2010; Goyal et al. 2012; Siril and Joseph 2013).

Fig. 4.

Fig. 4

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Ex vitro rooted microshoots. a Control, b Ex vitro rooted microshoots in different length class (< 0.5–1.0, 1.1–2.0, 2.1–3.0, 3.1–4.0 cm) treated with 2 mM of IBA (Scale bar = 0.8 cm), c Ex vitro rooted microshoots by 2 mM of NAA treatment (Scale bar = 3 cm), d, e Plantlet produced by ex vitro rooting (Scale bar = 0.5 cm), f Acclimatized plantlets after three month of potting (Scale bar = 5 cm), g Field grown plant (Scale bar = 5.2 cm)

Ex vitro rooting and acclimatization

Rooting of microshoots through ex vitro method was significantly influenced by auxin type and concentrations. Microshoots planted without an auxin treatment (control) failed to produce roots (Fig. 4a). Among various auxin treatments, 2 mM IBA treatment for 5 min produced 86.11% rooting, maximum number of roots per cuttings and elongation of roots (Table 5; Fig. 4b, d, e). Whereas microshoots treated with 2 mM NAA gave 41.66% rooting along with the reduced number of roots (Table 5; Fig. 4c). The development of plantlets through ex vitro rooting method is advantageous as it combines rooting and hardening stage of micropropagation (Tiwari et al. 2002). Rhizogenesis through ex vitro method offers the opportunity to improve the biological as well as economic efficiency of micropropagation; to save time, labour and resources (Krishnan and Siril 2016). Ex vitro rooting promote the development of lateral roots directly from the cut end and basal portion of the microshoots (Lozzi et al. 2019) and was reported in several woody plants such as Morinda citrifolia (Sreeranjini and Siril 2014), Tectona grandis (Tiwari et al. 2002), Bixa orellana (Siril and Joseph 2013), Embelia ribes (Dhavala and Rathore 2010). The role of IBA in ex vitro rooting was documented in various woody species (Lodha et al. 2014; Patel et al. 2014, 2016; Cuenca et al. 2017). Roots developed under in vitro conditions often leads to abnormal development and structural differences such as short roots and hairs, which in turn affect the successful establishment of plantlets in the field. As per previous reports in strawberry, plantlet rooted ex vitro showed superior growth behavior and improved root system in the field than in vitro rooted plantlets (Borkowska 2001). In the present study, the proportion of sand was high in potting media. Thus, ample aeration was facilitated. Conversely, reduced accumulation of ethylene in the soil environment that promoted a significant increase in rooting (Martin 2003; Newell et al. 2005). In agar gelled medium, accumulation of ethylene in the basal cut end of microshoot may inhibit gentle rooting (De Klerk 2002). For acclimatization, plantlets were kept under nursery conditions for three months (Fig. 4f) and successfully transferred to the field.

Fig. 2.

Fig. 2

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Effect of reculturing of mother explants of E. serratus on ESM3 (½ strength major salts, original strength of minor salts and vitamins) medium containing 2.5 µM BA and 0.1 µM NAA

Table 5.

Effect of pulse treatment (5 min) of different concentrations of IBA and NAA on ex vitro rooting of microshoots of E. serratus

Auxin Conc. (mM) % Rooting Root number/shoot Root length (cm)
Control 0.0 0.00 ± 0.00e 0.00 ± 0.00 g 0.00 ± 0.00 h
IBA 1.0 44.44 ± 2.78c 2.25 ± 0.07bc 2.31 ± 0.12b
2.0 86.11 ± 2.78a 3.84 ± 0.22a 3.35 ± 0.10a
3.0 55.54 ± 7.35b 2.64 ± 0.04bc 1.63 ± 0.17c
4.0 16.66 ± 4.81d 1.94 ± 0.34 cd 0.79 ± 0.10ef
5.0 0.00 ± 0.00e 0.00 ± 0.00 g 0.00 ± 0.00 h
NAA 1.0 19.44 ± 2.78d 1.39 ± 0.20de 1.37 ± 0.19 cd
2.0 41.66 ± 4.81c 1.71 ± 0.11cde 1.12 ± 0.06de
3.0 11.11 ± 2.78de 1.17 ± 0.17ef 0.68 ± 0.07 fg
4.0 5.55 ± 2.78e 0.67 ± 0.33f 0.37 ± 0.19 g
5.0 0.00 ± 0.00e 0.00 ± 0.00 g 0.00 ± 0.00 h
Treatment Df (n − 1) 10 62.300*** 45.916*** 88.904***
Auxin type (T) Df (n − 1) 1 88.794*** 83.855*** 170.129***
Auxin conc. (C) Df (n − 1) 4 95.076*** 63.351*** 129.840***
TXC Df (n − 1) 9 15.579*** 9.465*** 27.697***
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Means ± SE with in a column followed by same letters are not significantly (p < 0.05) different as determined by Duncan’s multiple range test; ***significant at p < 0.001 level

Clonal fidelity evaluation

Morphological evaluation

Three-month-old, in vitro raised plantlets in three replication blocks each having 10 plants were transferred to the field. Field transferred plants survived and resumed their active growth within five weeks of transfer. Morphometric characters of these plants were evaluated after one year of transfer. The morphological evaluation revealed non-significant differences between replication blocks, indicates the uniformity among the individuals within three replication blocks. All the plants attained ~ 40 cm height and produced ~ 3 branches at the age of one year of field transfer (Table 6; Fig. 4g). The quantitative characters studied such as plant height, the number of branches, leaf length, leaf width, collar diameter and petiole length were insignificant between replication blocks.

Table 6.

Growth performance of field transferred micropropagated plants of E. serratus

Plantation blocks Plant height (cm) Collar diameter (cm) Number of branches Petiole length (cm) Leaf length (cm) Leaf width (cm)
1 44.00 ± 1.00 2.13 ± 0.07 2.67 ± 0.33 3.27 ± 0.08 11.50 ± 0..29 4.20 ± 0.11
2 33.33 ± 1.76 1.92 ± 0.08 2.33 ± 0.33 3.53 ± 0.15 12.67 ± 0.33 4.50 ± 0.08
3 36.33 ± 1.20 2.13 ± 0.09 3.00 ± 0.58 3.97 ± 0.09 12.00 ± 0.93 4.53 ± 0.03
Main effect F Df (n − 1) = 2 0.004NS 0.167NS 0.579NS 0.023NS 0.394NS 0.092NS
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NS non significant F value

Genetic uniformity analysis

Out of 12 RAPD primers, 8 primers produced clear and reproducible bands. Total of 50 bands produced by 8 RAPD primers. The number of bands produced ranged from 4 to 8, and an average of 6.25 bands per primer was recorded (Table 7; Fig. 5a–d). All the bands produced were monomorphic in nature, and band size ranged from 100 to 1000. The monomorphic banding pattern indicates the high degree of uniformity between the in vitro raised plants and mother plants. Six ISSR primers tested gave 33 bands, and all are monomorphic in nature. The average number of bands produced per primer was 5.5, and the band size ranged from 100 to 967 bp (Table 8; Fig. 6a–d). The uniform banding pattern produced both RAPD and ISSR profile among micropropagated and mother plants indicates that there is no variation emerged due to in vitro maintenance of cultures. RAPD has a selective advantage over other molecular markers to check genetic uniformity because of its high polymorphic nature within a genome (Purohit et al. 2017; Ahmad and Anis 2019). At the same time, ISSR showed higher reproducibility over RAPD (Purohit et al. 2017) and also showed a comparative advantage over RFLP, SSR and AFLP (Goyal et al. 2014). Thus, we used two marker system (RAPD and ISSR) to reveal the genetic identity of in vitro raised plantlets of E. serratus that enabled foolproof analysis of clonal uniformity among micropropagated plants.

Table 7.

List of RAPD primer sequences with the number and size of amplified products in E. serratus mother plant and micropropagated plantlets

S. No Primer code Primer sequence (5′–3′) Annealing temperature (OC) No. of scorable bands Band size (range in bp)
1 OP A-02 TGCCGAGCTG 38 7 100–910
2 OP A-04 AATCGGGCTG 42 6 100–750
3 OP A-06 GGTCCCTGAC 35 7 100–710
4 OP A-07 GAAACGGGTG 38 4 200–1000
5 OP A-08 GTGACGTAGG 36 7 100–920
6 OP A-10 GTGATCGCAG 35 7 510–900
7 OP A-14 TCTGTGCTGG 38 8 100–900
8 OP A-17 GACCGCTTGT 34 4 150–790
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Fig. 5.

Fig. 5

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Analysis of genetic stability of in vitro propagated plants through RAPD primers. a OPA -02, b OPA-04, c OP A-08, d OP A-17, M-100 bp DNA ladder, S-Source plant, 1–9 in vitro propagated plants

Table 8.

List of ISSR primer sequences with the number and size of amplified fragments generated in E. serratus mother plant and micropropagated plants

Sl. No Primer code Primer sequence (5′–3′) Annealing temperature (°C) No. of scorable bands Band size (range in bp)
1 UBC815 CTC TCT CTC TCT CTC TG 52 5 100–789
2 UBC830 TGT GTG TGT GTG TGT GG 52 3 350–734
3 UBC897 CCG ACT CGA GNN NNN NAT GTG G 66 7 229–967
4 UBC864 ATG ATGATGATGATGATG 47 5 350–750
5 UBC814 CTC TCT CTC TCT CTC TCTA 50 6 320–955
6 UBC836 AGA GAG AGA GAG AGA GYA 53 7 139–820
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Fig. 6.

Fig. 6

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Analysis of genetic stability of in vitro propagated plants through ISSR primers. a UBC815, b UBC830, c UBC864, d UBC836, M-100 bp DNA ladder, S-Source plant, 1–9 in vitro propagated plants

Conclusion

Our finding is the first report on the in vitro induction and shoot multiplication from the mature nodal explants of E. serratus through the modified MS medium. The optimized medium (ESM3) composed of ½ strength MS major salts, the full strength of MS minor salts, 65.1 mM Fe EDTA and vitamins of original MS formulation. The ESM3 medium supplemented with BA (2.5 µM) and NAA (0.1 µM) produced 8.8 shoots per explants with 3.83 cm shoot length. Reculturing of original explant on this medium increased the number of shoots (16.08) and vigour. Ex vitro rooting of microshoots was achieved by pulse treatment with 2 mM IBA for 5 min. The ex vitro rooting coupled with acclimatization has reduced the cost and time of plant production. The hardened plantlets were morphologically evaluated under field conditions, and genetic uniformity was confirmed with ISSR and RAPD markers. The developed method, therefore, can be efficiently used for the micropropagation of elite germplasm of E. serratus.

Acknowledgements

The authors are grateful to Dr. Suhara Beevy S, Professor and Head, Department of Botany for providing the facilities and Kerala State Council for Science Technology and Environment (KSCSTE), Government of Kerala, Thiruvananthapuram, India, for the financial assistance (P.1409/2014/KSCSTE) through Junior Research Fellowship.

Author contributions

RR and EAS designed the experiments. RR performed the experiments and data collection. EAS and RR analyzed data. RR prepared the first draft of the manuscript and EAS edited and communicated for publishing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Informed consent

Informed consent not obtained as the article does not contain any studies with human participants performed by any of the authors.

Footnotes

Publisher's Note

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

References

  1. Ahmad A, Anis M. Meta-topolin improves in vitro morphogenesis, rhizogenesis and biochemical Analysis in Pterocarpus marsupium Roxb.: a potential drug-yielding tree. Plant Growth Regul. 2019;38:1007–1016. [Google Scholar]
  2. Ahmed MR, Anis M, Alatar AA, Faisal M. In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant. Agro Forest Syst. 2017;91:637–647. [Google Scholar]
  3. Amirchakhmaghi N, Hosseinpour B, Yousefzadeh H. Development of a micropropagation protocol for Malus orientalis using axillary buds. In Vitro Cell Dev Biol Plant. 2019 doi: 10.1007/s11627-019-09992-4. [DOI] [Google Scholar]
  4. Arshad SM, Kumar A. Tissue culture investigation of Elaeocarpus tuberculatus a highly valued rudraksha. Vegetos-An Int J Plant Res. 2006;19:111–114. [Google Scholar]
  5. Awodoyin RO, Olubode OS, Ogbu JU, Balogun RB, Nwawuii JU, Orji KO. Indigenous fruit trees of tropical Africa: Status, opportunity for development and biodiversity management. Agri Sci. 2015;6:31–41. [Google Scholar]
  6. Baruah PS, Deka K, Lahkar L, Sarma B, Borthakur SK, Tanti B. Habitat distribution modelling and reinforcement of Elaeocarpus serratus L.: A threatened tree species of Assam, India for improvement of its conservation status. Act Ecol Sinica. 2019 doi: 10.1016/j.chnaes.2018.06.002. [DOI] [Google Scholar]
  7. Bell RL, Reed BM. In vitro tissue culture of pear: advances in techniques for micropropagation and germplasm preservation. Acta Hortic. 2002;596:412–418. [Google Scholar]
  8. Borkowska B. Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro. Sci Hort. 2001;89:195–206. [Google Scholar]
  9. Cuenca B, Sánchez C, Aldrey A, Bogo B, Blanco B, Correa B, Vidal N. Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa × C. crenata) in liquid medium in a continuous immersion system. Plant Cell Tiss Organ Cult. 2017;131:307–320. [Google Scholar]
  10. De Klerk GJ. Rooting of microcuttings: theory and practice. In Vitro Cell Dev Biol Plant. 2002;38:415–422. [Google Scholar]
  11. Dellaporta SL, Wood J, Hicks JB. A plant DNA minipreparation: version II. Plant Mol Biol Rep. 1983;1:19–21. [Google Scholar]
  12. Dhavala A, Rathore TS. Micropropagation of Embelia ribes Burm F. through proliferation of adult plant axillary shoots. In Vitro Cell Dev Biol Plant. 2010;46:180–191. [Google Scholar]
  13. Endres L, Souza BM, Mercier H. In vitro nitrogen nutrition and hormonal pattern in bromeliads. In Vitro Cell Dev Biol Plant. 2002;38:481–486. [Google Scholar]
  14. Esteban R, Ariz I, Cruz C, Moran JF. Review: mechanisms of ammonium toxicity and the quest for tolerance. Plant Sci. 2016;248:92–101. doi: 10.1016/j.plantsci.2016.04.008. [DOI] [PubMed] [Google Scholar]
  15. Gamborg OL, Miller RA, Ojima K. Nutrient requirement of suspension cultures of soyabean root cells. Exp Cell Res. 1968;50:151–158. doi: 10.1016/0014-4827(68)90403-5. [DOI] [PubMed] [Google Scholar]
  16. Ghani A (1998) Monographs: Elaeocarpus serratus Linn. In: Medicinal plants of Bangladesh: chemical constituents and uses. Asiatic Soc Bangla Publ, pp. 167–168
  17. Goyal P, Kachhwaha S, Kothari SL. Micropropagation of Pithecellobium dulce (Roxb.) Benth a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants using molecular markers. Physiol Mol Biol Plants. 2012;18:169–176. doi: 10.1007/s12298-012-0112-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goyal AK, Pradhan S, Basistha BC, Sen A. Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) Nees using RAPD and ISSR markers. 3 Biotech. 2014;5:473–482. doi: 10.1007/s13205-014-0244-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Greenway MB, Phillips IC, Lloyd MN, Hubstenberger JF, Phillips GC. A nutrient medium for diverse applications and tissue growth of plant species in vitro. In Vitro Cell Dev Biol Plant. 2012;48:403–410. [Google Scholar]
  20. Hand C, Maki S, Reed MB. Modeling optimal mineral nutrition for hazelnut micropropagation. Plant Cell Tiss Organ Cult. 2014;119:411–425. [Google Scholar]
  21. Jayasinghe L, Amarasinghe RN, Arundathie BGS, Rupasinghe GK, Jayatilake NHAN, Fujimoto Y. Antioxidant flavonol glycosides from Elaeocarpus serratus and Filicium decipiens. Nat Prod Res. 2011;26:717–721. doi: 10.1080/14786419.2010.551514. [DOI] [PubMed] [Google Scholar]
  22. Kabylbekova B, Kovalchuk I, Mukhitdinova Z, et al. Reduced major minerals and increased minor nutrients improve micropropagation in three apple cultivars. In Vitro Cell Dev Biol Plant. 2020 doi: 10.1007/s11627-019-10019-1. [DOI] [Google Scholar]
  23. Krishnan SSR, Siril EA. Enhanced in vitro shoot regeneration in Oldenlandia umbellata L. by using quercetin: a naturally occurring auxin-transport inhibitor. Proc Natl Acad Sci India Sect B Biol Sci. 2016;21:271–278. [Google Scholar]
  24. Liu Y, Wirén NV. Ammonium as a signal for physiological and morphological responses in plants. J Exp Bot. 2017;68:2581–2592. doi: 10.1093/jxb/erx086. [DOI] [PubMed] [Google Scholar]
  25. Lloyd GB, McCown BH. Commercially feasible micropropagation of mountain laurel (Kamalia latifolia) by use of shoot tip culture. Comb Proc Int Plant Propagators Soc. 1980;30:421–427. [Google Scholar]
  26. Lodha D, Patel AK, Rai MK, Shekhawat NS. In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs. Acta Physiol Plant. 2014;36:3071–3079. [Google Scholar]
  27. Lozzi A, Abdelwahd R, Mentag R, Abousalim A. Development of a new culture medium and efficient protocol for in vitro micropropagation of Ceratonia siliqua L. In Vitro Cell Dev Biol Plant. 2019 doi: 10.1007/s11627-019-09990-6. [DOI] [Google Scholar]
  28. Lymperopoulos P, Msanne J, Rabara R. Phytochrome and phytohormones: working in tandem for plant growth and development. Front Plant Sci. 2018 doi: 10.3389/fpls.2018.01037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martin KP. Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant. Plant Cell Rep. 2003;21:415–420. doi: 10.1007/s00299-002-0547-8. [DOI] [PubMed] [Google Scholar]
  30. Morgan MJ, Lehmann M, Schwarzländer M, et al. Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis. Plant Physiol. 2008;147:101–114. doi: 10.1104/pp.107.113613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murashige T, Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant. 1962;15:473–497. [Google Scholar]
  32. Nas MN, Read PE. A hypothesis for the development of a defined tissue culture medium of higher plants and micropropagation of hazelnuts. Sci Hortic. 2004;101:189–200. [Google Scholar]
  33. Newell C, Growns DJ, Mc Comb JA. A novel in vitro rooting method employing an aerobic medium. Aust J Bot. 2005;53:81–89. [Google Scholar]
  34. Niedz RP, Hyndman SE, Evens TJ, Weathersbee AA. Mineral nutrition and in vitro growth of Gerbera hybrida (Asteraceae) In Vitro Cell Dev Biol Plant. 2014;50:458–470. [Google Scholar]
  35. Oberschelp GPJ, Gonçalves AN. Assessing the effects of basal media on the in vitro propagation and nutritional status of Eucalyptus dunnii Maiden. In Vitro Cell Dev Biol Plant. 2016;52:28–37. [Google Scholar]
  36. Patel AK, Lodha D, Ram K, Shekhawat S, Shekhawat NS. Evaluation of physiochemical factors affecting high-frequency plant regeneration of Blyttia spiralis (synonym: Pentatropis spiralis), a threatened climber of medicinal value. In Vitro Cell Dev Biol Plant. 2016;52:10–19. [Google Scholar]
  37. Patel AK, Phulwaria M, Rai MK, Gupta AK, Shekhawat S, Shekhawat NS. In vitro propagation and ex vitro rooting of Carallumaedulis (Edgew.) Benth. and Hook. f.: an endemic and endangered edible plant species of the Thar Desert. Sci Hortic. 2014;165:175–180. [Google Scholar]
  38. Peiris KHS (2007) Veralu, Elaeocarpus serratus L. In: Pushpakumara DKNG, Gunasena HPM and Singh VP (eds), Underutilized fruit in Sri Lanka, vol I, Asian Center for underutilized fruit crops, Sri Lanka
  39. Phillips GC, Garda M. Plant tissue culture media and practices: an overview. In Vitro Cell Dev Biol Plant. 2019 doi: 10.1007/s11627-019-09983-5. [DOI] [Google Scholar]
  40. Phulwaria M, Ram K, Gahlot P, Shekhawat NS. Micropropagation of Salvadora persica: a tree of arid horticulture and forestry. New For. 2011;42:317–327. [Google Scholar]
  41. Purohit S, Jugran AK, Bhatt ID, Palni LMS, Bhatt A, Nandi SK. In vitro approaches for conservation and reducing juvenility of Zanthoxylum armatum DC: an endangered medicinal plant of Himalayan region. Trees Struct Funct. 2017;31:1101–1108. [Google Scholar]
  42. Rahman MM, Amin MN, Ishiguri F, Yokota S, Sultana RS, Takashima Y, Iizuka K, Yoshizawa N. In vitro plantlet regeneration of ‘‘dwarf’’ Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh. Plant Biotechnol Rep. 2009;3:259–266. [Google Scholar]
  43. Raji R, Siril EA. Assessment of different pretreatments to breakage dormancy and improve the seed germination in Elaeocarpus serratus L.: an underutilized multipurpose fruit tree from South India. For Sci Technol. 2018;14:160–168. [Google Scholar]
  44. Raji R, Siril EA. Cloning of Ceylon olive (Elaeocarpus serratus L.) using conventional methods. J Hortic Sci Biotech. 2016;91:292–298. [Google Scholar]
  45. Ramage CM, Williams RR. Mineral nutrition and plant morphogenesis. In Vitro Cell Dev Biol Plant. 2002;38:116–124. [Google Scholar]
  46. Rathore JS, Rathore MS, Shekhawat NS. Micropropagation of Maerua oblongifolia: a liana of arid areas. Phytomorphology. 2005;55:241–247. [Google Scholar]
  47. Siril EA, Joseph N. Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers. Physiol Mol Biol Plants. 2013;1:147–155. doi: 10.1007/s12298-012-0150-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sreeranjini S, Siril EA. Field performance and genetic fidelity evaluation of micropropagated Morinda citrifolia L. Indian J Biotech. 2014;13:121–130. [Google Scholar]
  49. Timofeeva SN, Elkonin LA, Tyrnov VS. Micropropagation of Laburnum anagyroides Medic. through axillary shoot regeneration. In Vitro Cell Dev Biol Plant. 2014;50:561–567. [Google Scholar]
  50. Tiwari SK, Tiwari KP, Siril EA. An improved micropropagation protocol for teak. Plant Cell Tiss Organ Cult. 2002;71:1–6. [Google Scholar]
  51. Tripathi M, Kumari N. Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis. Acta Physiol Plant. 2010;32:1011–1015. [Google Scholar]
  52. White PR. Potentially unlimited growth of excised plant callus in an artificial medium. Am J Bot. 1939;26:59–64. [Google Scholar]
  53. Wang Y, Jiao Y. Auxin and above-ground meristems. J Exp Bot. 2018;69:147–154. doi: 10.1093/jxb/erx299. [DOI] [PubMed] [Google Scholar]

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