Skip to main content
PMC home page
Physiology and Molecular Biology of Plants logoLink to Physiology and Molecular Biology of Plants
. 2016 Jan 16;22(1):143–151. doi: 10.1007/s12298-016-0337-3

Mass propagation of Plectranthus bourneae Gamble through indirect organogenesis from leaf and internode explants

R Thaniarasu 1, T Senthil Kumar 2,✉, M V Rao 1
PMCID: PMC4840149  PMID: 27186028

Abstract

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.

Keywords: Callus induction, Organogenesis, Plectranthus bourneae, Endemic plant, Growth regulators

Introduction

The implements of contemporary biotechnology are being progressively applied for plant diversity depiction, indisputably they have a major role in assisting plant conservation programmes. Consequently, special solicitude should be remunerate to protect and preserve the endemic flora that is normally found in limited areas (Fay 1992; Sarasan et al. 2006). Conservation of endemic species by plant tissue culture has attracted the attention of many researchers (Mallon et al. 2010; Piovan et al. 2010). Lamiaceae herbs namely basil, oregano, sage, marjoram and rosemary are popular aromatic herbs. The food industry is becoming increasingly interested in aromatic herbs, mainly of the Lamiaceae family due to growing consumer demands for healthy natural foods. In addition to the food industry, Lamiaceae herbs are also of high demand in dyeing, fragrances, cosmetics, beverages, confectionary and Pharmaceutical industries (Zaidi and Dahiya 2015). The genus Plectranthus, comprising of 300 species, belongs to around 40 % of the Lamiaceae genera, possess aromatic properties (Lawrence 1992). Several Plectranthus species are used to treat skin infections, peptic ulcers, constipation, flatulence and stomach ache (Kokwaro 1993; Lukhoba et al. 2006). Plectranthus leaves contain essential oils, flavonoids, cinaminics and terpene derivatives, all of which have antineoplastic properties (Brandao et al. 2013).

Plectranthus bourneae Gamble is an endemic plant species, has very restricted distribution only in the Pambar Shola of Kodaikanal (Plalni hills), Western Ghats of Tamil Nadu, India (Matthew, 1993). It is a well branched, compact shrub, branchlets subsucculent. Our previous study on antibacterial effect of this plant extract showed prominent results on different human pathogenic bacteria (Thaniarasu et al. 2015). Matthew (1999) has reported this species as to the plant become threatened and highly vulnerable in position due to habitat destruction at Palni hills. Peoples settled from overseas since the mid-19th century, introduced many plants from the temperate countries, by which the percentage of alien plants now is 20 % of the total and these have also become a standing threat to the native species, by altering and disturbing the very visage of the hills and their primary vegetation (Matthew 1999).

Low seed germination and slow growth rate impends threat to this species hence required an alternative conservation strategies viz. tissue culture technique to ensure its restoration. Tissue culture methods offer highly efficient tools for germplasm conservation and mass multiplication of many threatened plant species (Salehi et al. 2014; Ozel et al. 2015). Sreedevi et al. (2013) and Thangavel et al. (2014a) have previously reported successful organogenesis from Plectranthus barbatus using leaf explants. In the present study, an exertion has been made to develop an efficient in vitro organogenesis method of multiple shoot formation through callus culture from leaf and internode explants of Plectranthus bourneae.

Materials and methods

Plant material and surface sterilization

The plants of P. bourneae collected from Pambar Shola (10°5′-10°25′ N, 77°50′ E at an altitude of 2020 m) in the Western Ghats of Tamil Nadu, India, being maintained in the glass house of Bharathidasan University, Tiruchirappalli, were used as the explant source. Leaf and internode explants (4 cm length) were washed under running tap water for 20 to 30 min. The internode explants were sterilized with 70 % ethanol for 30 s followed by 0.1 % bavistin treatment for 5 min and finally 0.1 % mercuric chloride treatment for 2 min. Surface sterilization of the leaf explants were similar to the internodes, but the ethanolic treatment was reduced to 10 s, since over exposure of ethanol to the leaf tissue caused early browning of explants. The explants were rinsed with sterile distilled water thrice after each treatment. These sterilized explants were inoculated on culture medium. The sterilized leaves (about 2 cm2) and internodes (2 cm) after excision were inoculated (leaves - adaxial side facing upwards) on the callus induction medium. This whole process was carried out under the laminar air flow chamber.

Culture conditions

The basal MS (Murashige and Skoog 1962) medium fortified with 30 g/l sucrose (Himedia, India) and gelled with 0.8 % (w/v) agar (Himedia, India), and the pH of the medium was adjusted to 5.7 ± 0.2 using 0.1 N NaOH or 0.1 N HCl after addition of the plant growth regulators. The medium was autoclaved at 121 °C and pressure for 30 min and maintained. All the cultures were maintained in culture room at 26 ± 2 °C, under 16/8 h light regime provided by cool white fluorescent light (60 μmol−2 s−1 light intensity) and with 55 to 60 % relative humidity.

Callus induction and regeneration of shoots

Sterilized, leaf and internode explants were cultured on MS medium supplemented with auxins (NAA, IAA, IBA, 2, 4-D, PIC - 0.1 mg/l to 2.0 mg/l) (Himedia, India) and cytokinin BA (0.5 mg/l) for callus induction. The cultures were initially kept in dark for 7 days and then transferred to light for 4 weeks. The fully matured green compact calluses (1.0 mg/l NAA with 0.5 mg/l BA) obtained from both the explants were transferred to regeneration medium (BA, KN, and TDZ 0.1, 0.5, 1.0, 1.5 and 2.0 mg/l) (Himedia, India). The number of regenerated shoots from each explant callus was recorded after 4 weeks.

Effect of auxins on shoot regeneration

The fully matured green compact calluses were further cultured on KN (1.0 mg/l) supplemented MS medium with different auxins (NAA, IBA, and IAA) at 0.1, 0.5, 0.7, 1.0, 1.5, and 2.0 mg/l for shoot multiplication. The multiplied plantlets continued to grow on the same medium for shoot elongation. The data on multiple shoot formation, shoot number and shoot lengths were recorded after 4 weeks of culture.

Influence of additives on shoot multiplication

The effect of various additives on improved shoot multiplication was studied after determining the optimum cytokinin combination for the shoot bud induction and multiplication from callus. The leaf and internode derived fully matured green compact callus were further cultured on MS medium containing KN (1.0 mg/l), NAA (0.7 mg/l) along with different concentrations of (25, 50, 75, 100 mg/l) AA, CH, AC, and AdS (Himedia, India). After 6 weeks of culture, the percentage of shoot formation, shoot number and the length of newly formed shoots were recorded.

Root induction and acclimatization

In vitro raised, elongated shoots (~ 3-4 cm) obtained from MS medium supplemented with KN (0.1 - 2.0 mg/l) + NAA (0.1 - 2.0 mg/l) + CH (25 - 100 mg/l) were transferred to root induction on half strength MS medium supplemented with auxins IBA, IAA and NAA (0.1, 0.5, 1.0, 1.5 and 2.0 mg/l). Rooting percentage, number of roots and root length were recorded after 3 weeks of culture.

The well rooted healthy plantlets were gently picked up from culture vessels without any damage to the roots and washed carefully with distilled water and subsequently transferred to paper cups (6.5 cm diameter) containing red soil, sand and coconut coir (1:1:1). Potted plantlets were covered with transparent polythene bags and maintained under controlled growth conditions of 26 ± 2 °C, 16 h photoperiod, 80–85 % relative humidity and 60 μmol−2 s−1 light intensity. The plantlets were frequently irrigated with sterile water every three days for four weeks. Well established plantlets were finally transplanted to the field.

Statistical analysis

All the treatments were consisted a minimum of 7 test tubes with single explant and each experiment was repeated three times. The data on frequency of explants responding callus, average number and length of shoots and roots were recorded regularly at weekly intervals. The mean values of the repeated experiments were compared by DMRT test at 5 % level of significance, using statistical software SPSS Ver.17.0.

Results and discussion

Callus induction was observed from cut margins of leaf and internode explants of P. bourneae after 2 weeks of incubation, cultured on MS medium supplemented with auxins viz. 2, 4-D, IAA, IBA, NAA and PIC (0.1–2.0 mg/l) along with BA (0.5 mg/l), under a partial incubation in dark (Table 1). Initial responses like leaf curl followed by swelling of explants were observed from the 6th day of culture period (Fig. 1a and d). Of the various treatments, NAA along with BA gave the best callus initiation and proliferation followed by 2, 4-D, IAA, IBA and PIC (Table 1, Fig. 1b and e). Depending upon the concentration and combination of plant growth regulators used, a wide range of variation in frequency of callus formation and nature of callus was observed. Fresh weight and dry weight of callus biomass for leaf and internode explants of P. bourneae are shown in Table 1. The highest fresh weight was achieved on 0.5 mg/l BA and 1.0 mg/l NAA in both of the explant.

Table 1.

Callus induction from leaf and internode explants of P. bourneae cultured on MS medium supplemented with different concentrations of auxins with BA, after 4 weeks of culture

PGR’s (mg/l) Percentage of response Callus weight mg/explant
Leaf Internode Leaf callus fresh weight (mg) Leaf callus dry weight Internode callus fresh weight Internode callus dry weight
Control 00.0 ± 0.0 00.0 ± 0.0 00.0 ± 0.0 00.0 ± 0.0 00.0 ± 0.0 00.0 ± 0.0
2,4-D+ BA (0.5)
 0.1 49.0 ± 1.0gh 35.0 ± 1.6ghi 328.5 ± 0.90j 81.0 ± 0.53i 308.9 ± 0.38k 74.4 ± 0.65m
 0.5 79.0 ± 1.0b 39.0 ± 1.0fg 338.4 ± 2.45i 86.7 ± 0.59h 301.1 ± 0.76l 69.3 ± 0.65n
 1.0 55.0 ± 1.0ef 46.0 ± 1.6de 504.7 ± 1.33e 105.9 ± 2.15d 323.8 ± 0.55i 77.6 ± 0.58l
 1.5 43.0 ± 1.5jk 44.0 ± 1.6de 406.1 ± 1.59f 99.0 ± 2.09f 230.2 ± 0.41p 52.4 ± 0.71p
 2.0 42.0 ± 1.3k 33.0 ± 1.5hi 400.4 ± 1.64g 94.5 ± 0.96g 228.4 ± 0.49p 47.5 ± 0.52q
NAA+ BA (0.5)
 0.1 63.0 ± 1.5c 55.0 ± 1.6c 405.9 ± 1.20f 85.2 ± 1.05h 553.2 ± 0.51ef 128.8 ± 0.35e
 0.5 78.0 ± 1.3b 75.0 ± 1.6b 508.5 ± 1.26d 102.9 ± 1.50e 567.5 ± 0.52d 130.9 ± 0.27d
 1.0 98.0 ± 1.3a 89.0 ± 1.0a 1698.6 ± 1.90a 255.6 ± 1.46a 885.3 ± 0.42a 211.6 ± 0.61a
 1.5 58.0 ± 1.3de 53.0 ± 1.5c 815.7 ± 0.68b 173.3 ± 0.77b 874.8 ± 0.26b 202.9 ± 1.01b
 2.0 48.0 ± 1.3hi 38.0 ± 1.3gh 803.6 ± 2.63c 169.9 ± 0.78c 783.6 ± 1.03c 184.0 ± 0.73c
IAA + BA (0.5)
 0.1 32.0 ± 1.3l 37.0 ± 1.5ghi 235.3 ± 0.44o 54.6 ± 0.90n 346.8 ± 0.38g 86.2 ± 0.35k
 0.5 43.0 ± 1.5jk 48.0 ± 1.3d 275.8 ± 0.66m 52.3 ± 0.77o 343.7 ± 0.55h 90.9 ± 0.23i
 1.0 47.0 ± 1.5hij 55.0 ± 2.2c 280.9 ± 1.40l 64.1 ± 0.56l 356.0 ± 0.63e 103.7 ± 0.98f
 1.5 44.0 ± 1.6ijk 46.0 ± 1.6de 276.0 ± 0.68m 61.1 ± 037no 341.9 ± 0.40h 98.6 ± 0.47g
 2.0 34.0 ± 1.6l 37.0 ± 1.5ghi 268.1 ± 0.64n 59.1 ± 0.54n 308.2 ± 1.08k 95.7 ± 0.30h
IBA + BA (0.5)
 0.1 47.0 ± 1.5hij 35.0 ± 1.6ghi 145.2 ± 0.90q 34.9 ± 0.62q 143.0 ± 0.53q 38.8 ± 0.38s
 0.5 42.0 ± 1.3k 37.0 ± 1.6ghi 187.7 ± 0.97p 40.6 ± 0.45p 154.3 ± 0.55p 43.0 ± 0.53r
 1.0 32.0 ± 1.3l 43.0 ± 1.5f 130.1 ± 0.80r 31.1 ± 0.52r 160.7 ± 0.68o 46.4 ± 0.71q
 1.5 - 34.0 ± 1.6ghi - - 142.4 ± 1.21q 37.0 ± 0.68t
 2.0 - 32.0 ± 1.3i - - 127.4 ± 0.49r 36.3 ± 0.76t
PIC + BA (0.5)
 0.1 43.0 ± 1.5jk 33.0 ± 1.5hi 327.3 ± 0.42j 85.0 ± 0.57h 293.7 ± 0.57m 73.1 ± 0.37m
 0.5 61.0 ± 1.0cd 44.0 ± 1.6de 331.3 ± 1.07jk 79.9 ± 0.99i 308.2 ± 1.00k 66.8 ± 0.53o
 1.0 53.0 ± .15fg 38.0 ± 1.3gh 353.2 ± 0.98h 73.4 ± 0.22j 319.9 ± 0.43j 88.8 ± 0.51j
 1.5 45.0 ± 1.6hij 35.0 ± 1.6ghi 311.6 ± 2.02jk 69.6 ± 0.52k 286.0 ± 0.33n 86.8 ± 0.53k
 2.0 42.0 ± 1.3k 33.0 ± 1.5hi 275.0 ± 0.67m 63.5 ± 0.76mn 282.8 ± 0.48o 70.6 ± .40n
Open in a new tab

Values are mean ± S.E. from 7 replicates per treatment and all the experiments were repeated thrice. Means followed by the same letters in each column are not significantly different (P = 0.05) using Duncan’s multiple range test

Fig. 1.

Fig. 1

Open in a new tab

Callus induction and plant regeneration from leaf and internode explants of P. bourneae. a leaf explant showing callus initiation, after 2 weeks of inoculation, b Callus proliferation on MS + NAA (1.0 mg/l) + BA (0.5 mg/l), after 4 weeks, c Shoot proliferation on MS + KN (1.0 mg/l), after 4 weeks of culture, d Internode explant showing callus initiation, after 2 weeks of inoculation, e Callus proliferation on MS + NAA (1.0 mg/l) + BA (0.5 mg/l), after 4 weeks, f Shoot proliferation on MS + KN (1.0 mg/l), after 4 weeks of culture, g & h Shoot multiplication and elongation on MS + KN (1.0 mg/l) + NAA (0.7 mg/l) + CH (50 mg/l) from internode and leaf derived callus, after 6 weeks of culture, i Rooting on MS + IBA (1.0 mg/l), after 3 weeks, j Hardening of in vitro raised plantlets, after 1 month. The bar represents 1.0 cm in a-f, 1.0 cm in g, 1.5 cm in h, 0.4 cm in i and 1.0 cm in j

2, 4-D at lower and higher concentrations produced light yellow to brown friable or nodular callus from both internode and leaf explants, which were failed to respond further (Table 1). In contrast 2, 4-D was reported to develop embryos with irregular morphology (Passinho-Soares et al. 2013). Medium containing 1.0 mg/l IAA and 0.5 mg/l BA produced green organogenic callus was obtained from leaf explants, whereas IBA (1.0 mg/l) with BA (0.5 mg/l) produced dark brown callus with rooting. Similarly Soh et al. (1998) suggest that the modified callus did not form adventitious roots on medium with auxins but only with cytokinins, therefore it is suggested that cytokinin have stimulate effect on root formation from callus. The highest frequencies of 98 % and 89 % of well developed, dark green organogenic calluses were induced from leaf and internode explants respectively on MS medium supplemented with 1.0 mg/l NAA and 0.5 mg/l BA (Fig. 1b and e). Lower concentrations of NAA (0.1–1.0 mg/l) along with BA (0.5 mg/l) formed profuse amount of callus in contrast to higher concentrations (Table 1).

Among the different concentrations of auxin tested, NAA with BA proved to be the best for high frequency of greenish compact callus induction. Earlier reports by Erisen et al. (2010); Bakar et al. (2014); Thangavel et al. (2014b) confirmed the same. All the callus cultures were sub-cultured every 15 days on to a fresh medium containing the same Plant growth regulators (PGR) composition. Most of the calli induced and developed as yellow in colour become green compact organogenic callus in 4 weeks. Only green compact callus was examined for further study.

In comparison, leaf explants produced significantly more frequency of callus (32.0–98.0 %) than the internode explant (32.0–89.0 %). It has been revealed from the present study that explant nature is a significant feature and responsible for the rate of achievement in callus induction.

Effect of cytokinins on shoot regeneration

The well-established callus transferred to the shoot regeneration medium induced multiple shoots within two weeks of culture. The leaf derived callus produced a highest number of 10.4 shoots than the internode derived callus (6.2 shoots) on KN (1.0 mg/l) (Table 2, Fig. 1c). Above this optimum concentration (1.0 mg/l) of KN decrease in shoot development was observed similar to Plectranthus rotundifolius (Asha et al. 2013). KN has been most frequently used for the in vitro shoot regeneration and multiplication of several plants (Akbar et al. 2003; Libin et al. 2012). MS medium supplemented with BA exhibited noticeable effect on shoot development in both explants but was less effective in shoot multiplication than KN. Similar effect of BA was previously recorded on Mentha piperita (Sunandakumari et al. 2004). TDZ responded similar to BA on shoot induction, while shoot development was comparatively low (Table 2).

Table 2.

Shoot regeneration from callus of P. bourneae on MS medium supplemented with different concentrations of cytokinins, after 4 weeks of culture

Plant growth regulators (mg/l) Regeneration frequency (%) Mean number of shoots per callus Shoot length (cm)
control BA KN TDZ Leaf Internode Leaf Internode Leaf Internode
0.0 00.00 00.00 00.00 ± 0.00 00.00 ± 0.00 00.00 ± 0.00 00.00 ± 0.00
0.1 57.14bcd 48.97cde 3.85 ± 0.14f 3.14 ± 0.26g 1.30 ± 0.21h 0.97 ± 0.35de
0.5 69.38ab 63.26b 7.85 ± 0.14b 4.42 ± 0.20de 2.12 ± 0.35c 1.38 ± 0.40b
1.0 57.14bcd 55.10bcd 4.71 ± 0.18e 4.57 ± 0.20cd 1.68 ± 0.26e 1.08 ± 0.93cd
1.5 53.06bcd 44.89def 3.71 ± 0.18f 3.42 ± 0.36fg 1.52 ± 0.18fg 0.87 ± 0.42ef
2.0 48.97cd 40.81ef 3.14 ± 0.14g 3.28 ± 0.14fg 1.45 ± 0.36g 0.77 ± 0.42fg
0.1 57.14bcd 48.97cde 5.28 ± 0.18d 4.28 ± 0.18de 1.50 ± 0.21fg 1.11 ± 0.40cd
0.5 61.22bcd 61.22b 6.57 ± 0.20c 5.57 ± 0.36b 2.05 ± 0.29c 0.90 ± 0.40ef
1.0 83.67a 75.51a 10.42 ± 0.29a 6.42 ± 0.42a 2.84 ± 0.42a 1.70 ± 0.61a
1.5 67.34b 57.14bc 7.42 ± 0.20b 4.00 ± 0.21ef 2.35 ± 0.42b 1.28 ± 0.12bc
2.0 65.30bc 55.10bcd 5.42 ± 0.20d 3.14 ± 0.14g 1.85 ± 0.36d 0.71 ± 0.10gh
0.1 46.93d 36.73f 3.42 ± 0.20f 2.14 ± 0.26h 1.04 ± 0.29i 0.42 ± 0.40i
0.5 57.14bcd 48.97cde 4.71 ± 0.18e 3.28 ± 0.18fg 1.25 ± 0.29h 0.51 ± 0.55hi
1.0 63.26bcd 61.22b 7.57 ± 0.20b 5.28 ± 0.18bc 1.58 ± 0.55f 1.31 ± 0.79bc
1.5 57.14bcd 53.06bcd 5.71 ± 0.18d 4.28 ± 0.28de 1.34 ± 0.29h 1.10 ± 0.11cd
2.0 55.10bcd 48.97cde 3.85 ± 0.14f 3.71 ± 0.18fg 1.08 ± 0.40i 0.81 ± 0.96fg
Open in a new tab

Values are mean ± S.E. from 7 replicates per treatment and all the experiments were repeated thrice. Means followed by the same letters in each column are not significantly different (P = 0.05) using Duncan’s multiple range test

Effect of auxins on shoot regeneration

The occurrence of cytokinin along with auxin is required for indirect adventitious shoot multiplication. Accordingly, the effect of auxins NAA, IAA, and IBA were tested along with the established best KN concentration (1.0 mg/l) for shoot multiplication. KN at 1.0 mg/l in combination with 0.7 mg/l NAA responded highest shoot regeneration (89 %) with highest number of shoots (25.57) and shoot length (3.87) on leaf derived callus (Table 3). Similar results have also been observed in leaf explants of in Peperomia obtusifolia (Naggar and Osman 2014) and Ziziphora tenuior (Dakah et al. 2014). Likewise, internodal explants cultured on MS medium supplemented with KN (1.0 mg/l) and NAA (0.7 mg/l) showed higher regeneration frequency (85 %) and highest number (16.42) of shoots and shoot length (2.94). This result agreement with the report by Tsegaw and Feyissa (2014) who obtained the highest number of shoot multiplication on combination of higher concentration of KN and lower concentration of NAA in Plectranthus edulis. IAA was less effective in shoot production and the quality of shoots were also poor (Table 3). IBA combination on MS + KN 1.0 mg/l formed less number of shoots. The interaction between various growth regulators and the two explants revealed that, the 1.0 mg/l KN with 0.7 mg/l NAA was the best combination for shoot multiplication and elongation in P. bourneae. The consistency of inducing shoot elongation by NAA was previously reported in other plants such as, Fuchsia magellanica (Parveen and Rasheed 2013) and Cajanus cajan (Lakshmi Sita and Venkatachalam 2008). The proficiency to regenerate shoots from callus has various benefits. Numerous shoots can be formed from an explant through callus induction and shoot development.

Table 3.

Shoot regeneration from callus of P. bourneae on MS medium supplemented with KN (1.0 mg/l) in combination with auxins, after 4 weeks of culture

Plant growth regulators (mg/l) Regeneration frequency (%) Mean number of shoots per callus Shoot length (cm)
control NAA IAA IBA Leaf Internode Leaf Internode Leaf Internode
0.0 38.00f 32.00h 5.60 ± 0.30 3.28 ± 0.43 1.50 ± 0.43h 1.36 ± 0.20gh
0.1 55.10de 51.02fg 12.42 ± 0.48def 8.28 ± 0.96ef 3.22 ± 0.11bcd 1.98 ± 0.22def
0.5 67.34bcd 53.06ef 13.57 ± 0.75def 12.42 ± 0.84bc 3.14 ± 0.20cde 2.42 ± 0.09bc
0.7 89.79a 85.71a 25.57 ± 1.04a 16.42 ± 1.28a 3.87 ± 0.15a 2.94 ± 0.09a
1.0 69.38bcd 75.51bc 22.00 ± 0.61ab 11.71 ± 0.77cd 3.45 ± 0.19ab 2.40 ± 0.08bc
1.5 65.30cd 65.30cd 18.42 ± 1.34bc 9.28 ± 1.18def 3.02 ± 0.16cde 2.11 ± 0.15cd
2.0 59.18cde 63.26de 15.85 ± 0.88cde 8.28 ± 0.80ef 2.85 ± 0.11de 1.81 ± 0.10fg
0.1 48.97e 46.93g 9.57 ± 0.42f 6.85 ± 0.55f 3.14 ± 0.19bcd 1.87 ± 0.21ef
0.5 71.42bc 61.22de 11.28 ± 0.77def 11.28 ± 1.08cde 3.07 ± 0.10bcd 2.40 ± 0.07bc
0.7 63.26cd 63.26de 13.57 ± 0.61def 11.14 ± 0.88cde 2.95 ± 0.13de 2.12 ± 0.04cd
1.0 59.18cde 77.55ab 15.42 ± 2.36cde 8.85 ± 0.93def 2.70 ± 0.12ef 1.92 ± 0.10def
1.5 55.10de 65.30cd 13.28 ± 2.11def 7.85 ± 0.88f 2.34 ± 0.11fg 1.77 ± 0.14fg
2.0 55.10de 57.14ef 10.00 ± 1.36f 7.00 ± 0.61f 2.15 ± 0.12g 1.57 ± 0.10g
0.1 63.26cd 51.55fg 11.71 ± 0.71f 9.85 ± 0.88cde 2.87 ± 0.17de 2.31 ± 0.07cd
0.5 79.59ab 75.51ab 15.71 ± 2.63cde 15.00 ± 1.17ab 3.25 ± 0.23bc 2.71 ± 0.13ab
0.7 67.34bcd 67.34cd 13.57 ± 1.98def 11.57 ± 0.84cd 2.94 ± 0.18de 2.31 ± 0.07cd
1.0 63.26cd 61.22de 13.57 ± 0.84def 11.71 ± 1.06cd 3.11 ± 0.20bcd 2.14 ± 0.08cde
1.5 67.34bcd 55.10fg 12.57 ± 1.25def 9.00 ± 0.57de 2.84 ± 0.14de 1.82 ± 0.10ef
2.0 59.18cde 51.02fg 10.28 ± 0.64f 8.42 ± 0.86ef 2.62 ± 0.12ef 1.71 ± 0.07fg
Open in a new tab

Values are mean ± S.E. from 7 replicates per treatment and all the experiments were repeated thrice. Means followed by the same letters in each column are not significantly different (P = 0.05) using Duncan’s multiple range test

Influence of additives on shoot regeneration

Shoot multiplication and development of P. bourneae was highly influenced by additives. Four different additives AA, AC, AdS and CH were tested further on callus derived shoots to enhance the shoot multiplication frequency. The maximum number of 29.71 and 19.71 shoots were obtained from leaf and internode derived callus, at 50 mg/l of CH supplemented medium (Table 4, Fig. 1g and h). CH is an intricate assortment of amino acids and ammonium salts, which together with the combination of KN (1.0 mg/l) and NAA (0.7 mg/l) supported to increase the shoot multiplication rate in P. bourneae. Similar effect of CH have also been reported in Stevia rebaudiana (Sridhar and Aswath 2014), Digitalis lanata (Fatima et al. 2009). The shoots cultured on medium containing AC, failed to develop more number of multiple shoots, because AC is known to absorb high concentrations of growth regulators and may reduce shoot regenerative response (Constantin et al. 1977; Weatherhead et al. 1978). The AA also failed to enhance the multiple shoots, a maximum number of 15.42 and 14.85 shoots were produced from the shoot explant derived from internode and leaf callus respectively. Even though, AdS did not enhance the multiple shoots, at 75 mg/l it was marginally contributed to shoot multiplication (21.00) from leaf callus derived shoots.

Table 4.

Influence of Adenine sulphate, Casein hydrolysate, Citric acid and Yeast extract along with KN (1.0 mg/l) + NAA (0.7 mg/l) on shoot regeneration from callus, after 6 weeks of culture

Additives (mg/l) Percentage of response Mean number of shoots Shoot length (cm)
control AC AA AdS CH Leaf Internode Leaf Internode Leaf Internode
00 40.10h 33.46g 6.32 ± 0.57j 3.40 ± 0.62hi 2.00 ± 0.10j 1.30 ± 0.13ef
25 51.02g 40.81f 9.14 ± 0.67ef 5.00 ± 0.57h 2.60 ± 0.61gh 1.92 ± 0.05e
50 63.26de 53.06de 10.57 ± 0.84hi 12.14 ± 0.50c 2.88 ± 0.10e 2.25 ± 0.06d
75 65.30cd 59.18de 12.57 ± 0.64hi 8.28 ± 0.64fg 2.31 ± 0.82ij 1.91 ± 0.09e
100 53.06fg 53.06ef 10.28 ± 0.68i 7.28 ± 0.77g 2.08 ± 0.76j 1.90 ± 0.07e
25 51.02g 46.10ef 11.14 ± 0.55hi 8.85 ± 0.55fg 2.40 ± 0.12hi 1.90 ± 0.04e
50 61.22ef 57.14de 13.85 ± 1.18ef 10.85 ± 0.50cd 2.34 ± 0.61ij 2.88 ± 0.07b
75 65.30cd 67.34bc 14.85 ± 0.45ef 15.42 ± 0.89b 3.21 ± 0.55e 2.54 ± 0.13c
100 57.14ef 55.10de 13.71 ± 0.47fg 7.85 ± 0.79f 2.62 ± 0.13fg 2.21 ± 0.09de
25 63.26de 51.02ef 16.42 ± 0.99de 11.28 ± 0.56cd 2.57 ± 0.74gh 1.94 ± 0.12e
50 71.42cd 59.18de 13.85 ± 0.63ef 14.85 ± 0.76b 3.65 ± 0.81d 3.74 ± 0.14a
75 75.51bc 65.30cd 21.00 ± 0.69c 9.14 ± 0.59ef 3.35 ± 0.92e 2.68 ± 0.14bc
100 63.26de 53.06de 10.28 ± 0.52i 4.85 ± 0.55h 2.75 ± 0.11fg 1.92 ± 0.06e
25 65.30cd 55.10de 18.42 ± 1.92d 11.14 ± 0.45cd 3.25 ± 0.81e 2.71 ± 0.14bc
50 91.83a 81.63a 29.71 ± 0.52a 19.71 ± 0.68a 4.95 ± 0.71a 3.75 ± 0.08a
75 79.59b 69.38ab 23.71 ± 1.04b 12.57 ± 0.52c 4.70 ± 0.69b 2.90 ± 0.09b
100 71.42cd 63.26cd 17.85 ± 0.40d 9.57 ± 0.48de 4.22 ± 0.11c 2.68 ± 0.06bc
Open in a new tab

Values are mean ± S.E. from 7 replicates per treatment and all the experiments were repeated thrice. Means followed by the same letters in each column are not significantly different (P = 0.05) using Duncan’s multiple range test

The higher frequency of callus formation and shoot bud regeneration ability of leaf over internode explant is related to the densely packed meristamatic cells possessing higher proliferation ability. Similar result of high callus formation and shoot bud regeneration was recorded on the leaf explant in P. barbatus (Thangavel et al. 2014b). Subculture of leaf and internode derived callus on 1.0 mg/l KN, 0.7 mg/l, NAA and 50 mg/l CH yielded a higher number of adventitious shoots which increased gradually from subculture to subculture. On this medium, from the initial stage of 4–6 regenerated shoot buds (2 weeks), a cluster of 25–30 shoots were produced from leaf and 15–20 shoots were produced from internode derived callus within 6 weeks.

Overall, leaf derived callus showed more regeneration ability than the internode. This may be attributed to the variations of endogenous growth regulator levels in the explant or the difference in tissue sensitivities to these plant growth regulators (Lisowska and Wysokinska 2000).

Rooting of regenerated shoots

In vitro root induction of shoots occurred on half MS medium containing IBA, NAA and IAA, within a week period followed by efficient root system formation in 15 days. The maximum rooting response (97.75 %) and number of roots per shoot (19.14) was recorded on ½ MS medium containing 1.0 mg/l IBA (Table 5, Fig. 1i). The superiority of IBA over other auxins on in vitro rooting has been reported in Plectranthus ambonicus (Rhaman et al. 2015) and Glinus lotoides (Teshome and Feyissa 2015). The number of roots per shoot was increased with increasing concentration of IBA from 0.1 to 1.0 mg/l, whereas subsequent concentrations decreased the frequency of rooting. NAA at 1.5 mg/l presented second highest rooting response (81.63 %) as good as IBA. IAA at all concentrations, showed lower response of root formation, whereas highest length of roots (9.02 cm) formed only in IAA at 1.0 mg/l. Of the three auxins used, IBA was the most effective for rooting in P. bourneae. The probable reason could be that, IBA is more insistent than IAA or NAA to chemical degradation in tissue culture media, both during auto-claving and at room temperature (Cuenca et al. 1999).

Table 5.

In vitro rooting of callus derived shoots of P. bourneae on MS medium supplemented with different concentrations of auxins, after 3 weeks of culture period

Growth regulators (mg/l) Percentage of response Mean number of roots Mean root length (cm)
control IAA IBA NAA
0.0 18.04f 1.82 ± 0.10k 1.00 ± 0.22i
0.1 51.02de 5.42 ± 0.36i 4.05 ± 0.25g
0.5 55.10de 7.57 ± 0.36h 6.77 ± 0.47d
1.0 63.26cde 8.71 ± 0.18f 9.02 ± 0.30b
1.5 53.06de 6.00 ± 0.72fg 6.31 ± 0.26de
2.0 48.97e 4.28 ± 0.35j 4.74 ± 0.35gh
0.1 59.18cde 8.00 ± 0.30gh 6.38 ± 0.19de
0.5 67.34bcd 15.71 ± 0.35b 6.58 ± 0.19de
1.0 97.75a 19.14 ± 0.34a 10.85 ± 0.29a
1.5 65.30cde 14.85 ± 0.26b 7.77 ± 0.15c
2.0 59.18cde 10.28 ± 0.35e 6.97 ± 0.12d
0.1 53.06de 5.42 ± 0.20i 5.80 ± 0.16ef
0.5 59.18cde 8.14 ± 0.34gh 5.91 ± 0.06ef
1.0 75.51abc 11.57 ± 0.36d 6.31 ± 0.11de
1.5 81.63ab 13.14 ± 0.34c 5.85 ± 0.22ef
2.0 67.34bcd 9.71 ± 0.28ef 5.35 ± 0.45fg
Open in a new tab

Values are mean ± S.E. from 7 replicates per treatment and all the experiments were repeated thrice. Means followed by the same letters in each column are not significantly different (P = 0.05) using Duncan’s multiple range test

Acclimatization of regenerated plants

Well rooted plantlets (70 plantlets) were successfully hardened and acclimatized with a maximum survival of 90 % (63 plantlets) in vitro. After a month of hardening plantlets were transferred to greenhouse condition, where 56 (80 %) of the rooted plantlets survived and showed no morphological variations (Fig. 1j).

Conclusion

This is the first report describing a protocol for indirect organogenesis of P. bourneae. The present study demonstrates a reliable method for shoot regeneration through leaf and internode derived callus cultures. Prominent callus response from leaf explant was observed under the influence of BA and NAA combinations. A combination of 1.0 mg/l KN, 0.7 mg/l, NAA and 50 mg/l CH was effective for multiple shoot production from leaf and internode derived callus. Best rooting response was achieved on half-strength MS medium supplemented with 1.0 mg/l IBA and the plantlets were successfully hardened and established in the field. The organogenesis and plant regeneration system developed in this study could be utilized in future for the in vitro production, transformation and secondary metabolites production studies for this genus.

Acknowledgment

Authors are thankful to University Grants Commission, New Delhi for financial support in the form of major research project (UGC REF. NO: 40-325/2011(SR) dated 30.06.2011), and Mr. R.W. Stewart and Mrs. Tanya Balcar of Vattakanal Conservation Trust, Kodaikanal, Tamil Nadu, India for the help rendered during the field survey.

Abbreviations

MS

Murashige and Skoog medium

BA

6-Benzyl adenine

IAA

Indole-3 acetic acid

IBA

Indole-3-butyric acid

KN

Kinetin

NAA

α-Naphthalene acetic acid

2,4-D

2,4-Dichlorophenoxyacetic acid

PIC

Picloram

AA

Ascorbic acid

CH

Casein hydrolysate

AC

Activated charcoal

AdS

Adenine sulphate

References

  1. Akbar MA, Karmakar BK, Roy SK. Callus induction and high-frequency plant regeneration of pineapple (Ananas comosus (L.) Merr.) Plant Tiss Cult. 2003;13:109–116. [Google Scholar]
  2. Asha KI, Dwivedi NK, Devi AI, Nair RA (2013) In vitro propagation of Chinese potato (Plectranthus rotundifolius (Poir.) J.K. Morton) through axillary shoot bud culture. 39(2):62–67
  3. Bakar AD, Ahmed BA, R.M T. In vitro callus induction and plant regeneration of Celosia argentea- an important medicinal plant. Braz Arch Biol Technol. 2014;57:860–866. doi: 10.1590/S1516-8913201402611. [DOI] [Google Scholar]
  4. Brandao EM, Brandao PHDM, Souza IA, Pavia GS, Carvalho MC, Lacerda CM. Antineoplastic effect of aqueous extract of Plectranthus ambonicus in Ehrlich ascites carcinoma. J Cancer. 2013;4(7):573–576. doi: 10.7150/jca.6730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Constantin MJ, Henke RR, Mansur MA. Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. In Vitro. 1977;13:293–287. doi: 10.1007/BF02616173. [DOI] [PubMed] [Google Scholar]
  6. Cuenca S, Amo-Marco JB, Parra R (1999) Micropropagation from inflorescence stem of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae). Plant Cell Rep 18:674–679
  7. Dakah A, Zaid S, Suleiman M, Abbas S. In vitro propagation of the medicinal plant Zizphora tenuior L. and evaluation of its antioxidant activity. Saudi J Biol Sci. 2014;21:317–323. doi: 10.1016/j.sjbs.2013.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Erisen S, Yorgancilar M, Atalay E, Babaoglu M, Duran A. Callus induction and plant regeneration of the endemic Astragalus nezaketae in Turkey. Electron J Biotechn. 2010;13:1–7. [Google Scholar]
  9. Fatima Z, Mujib A, Fatima S, Arshi A, Umar S (2009) Callus induction, biomass growth, and plant regeneration in Digitalis lanata Ehrh.: influence of plant growth regulators and carbohydrates. Turk J Bot 33:393–405
  10. Fay MF. Conservation of rare and endangered plant using in vitro methods. In Vitro Cell Dev Biol. 1992;28:1–4. doi: 10.1007/BF02632183. [DOI] [Google Scholar]
  11. Matthew KM. Precursory notes on the flora of the palni hills (pulney) hills, south India: II. Kew Bull. 1993;48:757–765. doi: 10.2307/4118854. [DOI] [Google Scholar]
  12. Kokwaro JO. Medicinal plants of east Africa. Second. Nairobi: Kenya Literature Bureau; 1993. [Google Scholar]
  13. Lakshmi Sita G, Venkatachalam P (2008) Genetic Transformation as a tool for improvement of Pigeon pea, Cajanus cajan (L.) Mills P. Handbook of New Technologies for Genetic Improvement of Legumes edited by P. B. Kirti p.(131), Chapter 8
  14. Lawrence BM (1992) Chemical components of Labiate oils and their exploitation. In- Advances in Labiate Science, ed. Royal Botânic Gardens KEW, Whitstable, pp. 399–436
  15. Libin A, King PJH, Ong KH, Chubo JK, Sipen P. Callus induction and plant regeneration of Sarawak rice (Oryza sativa L.) variety. Biris. Afr J Agric Res. 2012;7:4260–4265. doi: 10.5897/AJAR12.587. [DOI] [Google Scholar]
  16. Lisowska K, Wysokinska H. In vitro propagation of Catalpa ovata G. Don. Plant Cell Tissue Organ Cult. 2000;60:171–176. doi: 10.1023/A:1006461520438. [DOI] [Google Scholar]
  17. Lukhoba CW, Simmonds MSJ, Paton AJ (2006) Plectranthus: a review of ethanobotanical uses. J Ethanopharmacol 103:1–24 [DOI] [PubMed]
  18. Mallon R, Rodríguez-Oubiña J, González ML. In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tissue Organ Cult. 2010;101:31–39. doi: 10.1007/s11240-009-9659-y. [DOI] [Google Scholar]
  19. Matthew KM. A report on the conservation status of south Indian plants. Biodivers Conserv. 1999;8:779–796. doi: 10.1023/A:1008804029859. [DOI] [Google Scholar]
  20. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. [DOI] [Google Scholar]
  21. Naggar HME, Osman AR. Micropropagation and organogenesis of Peperomia obtusifolia. Asian J Crop Sci. 2014;6(1):58–66. doi: 10.3923/ajcs.2014.58.66. [DOI] [Google Scholar]
  22. Ozel CA, Khawar KM, Unal F (2015) Factors affecting efficient in vitro micropropagation of Muscari muscarini Medikus using twin bulb scale. Saudi J Biol Sci 22:132–138 [DOI] [PMC free article] [PubMed]
  23. Parveen A, Rasheed S. In vitro clonal propagation of Fuchsia magellanica Lam. Afr J Biotechnol. 2013;12(7):670–678. [Google Scholar]
  24. Passinho-Soares HC, Meira PR, David JP, Mesquita PRR, Vale AE, Rodrigues FM, Pereria PAP, Santana JRF, Oliveira FS, Andrade JB, David JM (2013) Volatile organic compounds obtained by in vitro callus cultivation of Plectranthus orantus Codd. (Lamiaceae). Molecules 18:10320–10333. doi:10.3390/molecules180910320 [DOI] [PMC free article] [PubMed]
  25. Piovan A, Caniato R, Cappelletti EM, Filippini R (2010) Organogenesis from shoot segments and via callus of endangered Kosteletzkya pentacarpos (L.) Ledeb. Plant Cell Tissue Organ Cult 100:309–315
  26. Rhaman ZAB, Noor ESM, Ali MSM, Mirad R, Othman AN. In vitro micropropagation of a valuable plant, Plectranthus ambonicus. Am J Plant Sci. 2015;6:1091–1097. doi: 10.4236/ajps.2015.68113. [DOI] [Google Scholar]
  27. Salehi M, Hosseini B, Jabbarzadeh Z (2014) High-frequency in vitro plantlet regeneration from apical bud as a novel explant of Carum copticum L. Asian Pac J Trop Biomed 4(1):5424–5428 [DOI] [PMC free article] [PubMed]
  28. Sarasan V, Cripps R, Ramsay MM, Atherton C, McMichen M, Prendergast G, Rowntree JK. Conservation in vitro of threatened plants progress in the past decade. In Vitro Cell Dev Plant. 2006;42:206–214. doi: 10.1079/IVP2006769. [DOI] [Google Scholar]
  29. Soh WH, Choi PS, Cho DY (1998) Effects of cytokinin on adventitious root formation in callus cultures of Vigna unguiculata (L.) Walp. In Vitro cell dev Biol Plant 34:189–195
  30. Sreedevi E, Anuradha M, Pullaiah T. Plant regeneration from leaf-derived callus in Plectranthus barbatus Andr. [Syn.: Coleus forskohlii (Wild.) Briq.] Afr J Biotechnol. 2013;12:2441–2448. [Google Scholar]
  31. Sridhar TM, Aswath CR. Influence of additives on enhanced in vitro shoot multiplication of Stevia rebaudiana (Bert.)-an important anti diabetic medicinal plant. Am J Plant Sci. 2014;5:192–199. doi: 10.4236/ajps.2014.51025. [DOI] [Google Scholar]
  32. Sunandakumari C, Martin KP, Chithra M, Sini S, Madhusoodanan PV. Rapid axillary bud proliferation and ex vitro rooting of herbal spice, Mentha piperita L. Indian J Biotechnol. 2004;3:108–112. [Google Scholar]
  33. Teshome S, Feyissa T. In vitro callus induction and shoot regeneration from leaf explants of Glinus lotoides (L.)- an important medicinal plant. Am J Plant Sci. 2015;6:1329–1340. doi: 10.4236/ajps.2015.69132. [DOI] [Google Scholar]
  34. Thangavel P, Prabhu S, Britto SJ (2014a) High frequency shoots regeneration from nodal explants of Plectranthus barbatus Andrews belong to the Lamiaceae. J Andaman Sci Assoc 19(2):126–135
  35. Thangavel P, Britto SJ, Prabhu S, Senthil Kumar S. In vitro callus induction and plantlets regeneration from leaf explants of Plectranthus barbatus Andrews- a valuable medicinal plant. Int J Adv Sci Eng. 2014;2:51–57. [Google Scholar]
  36. Thaniarasu R, Senthil Kumar T, Abubacker MN, Rao MV (2015) Preliminary phytochemical screening and evaluation of antibacterial activity of different solvent extracts of Plectranthus bourneae Gamble (Lamiaceae). Asian J Pharm Clin Res 8(1):79–82
  37. Tsegaw M, Feyissa T (2014) Micropropagation of Plectranthus edulis (Vatke). Agnew from Meristem Culture. Afr J Biotechnol 13(36):3682–3688
  38. Weatherhead MA, Burdon J, Henshaw GG. Some effects of activated charcoal as an additive to plant tissue culture media. Z P Flanzenphysiol. 1978;89:141–147. doi: 10.1016/S0044-328X(78)80054-3. [DOI] [Google Scholar]
  39. Zaidi S, Dahiya P. In vitro antimicrobial activity, phytochemical analysis and total phenolic content of essential oil from Mentha spicata and Mentha piperita. Ifrj. 2015;22(6):2440–2445. [Google Scholar]

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

RESOURCES