The effect of the plant growth regulators benzyl amino purine (BAP), 1-naphthaleneacetic acid (NAA) and kinetin (KIN) on in vitro shoot induction and proliferation of <i>Plectranthus amboinicus </i> was examined. Explants obtained from lateral shoots and apical shoots of <i>P. amboinicus </i> were inoculated on Murashige and Skoog (MS) culture medium supplemented with different concentrations of BAP, NAA and KIN. When the effect of each growth regulator was considered singly, the highest rate of shoot induction (80% of explants producing shoots) and highest number of shoots produced (2.4 shoots per explant) were obtained from lateral shoot explants cultured on MS media supplemented with 3.0 mg/L BAP within 6 - 7 weeks. Better results were obtained using MS medium supplemented with 1 mg/L BAP + 5 mg/L NAA. Shoot proliferation rose to 85%, while 5.7 shoots per explants were recorded. Among the different media tested for rooting, MS medium supplemented with 1.0 mg/L IBA was the most effective for root induction. The quality of the roots obtained was better than that obtained using MS media supplemented with NAA or IAA.
Plectranthus amboinicus (Lour.) Spreng, a plant native to south and east Africa, belongs to family Lumiaceae. The plant is known by different names in various languages, such as country borage, Indian borage, Patta ajavayan or Pathacur in Hindi, and Karpuravalli or Malyalam-Kannikkaurkka in Sanskrit [
Healthy Plectranthus amboinicus plants (about one month-old) were maintained in a net house for two weeks prior to explants excision and establishment in vitro. Shoots and stem segments of plants collected were washed in running tap water for 1 h. Three centimetre pieces of stem carrying vegetative buds were prepared from either lateral shoots or apical shoots and washed with detergent (Teepol) solution for 30 min, followed by rinsing in distilled water. The explants were transferred to a laminar air flow chamber where they were surface sterilized with 10% - 20% Clorox® containing drops of Tween-20 for 5 - 20 min on a rotary shaker. After rinses with sterile water, the explants (apical shoots and lateral shoots) were cut into 2 - 3 cm, and then cultured on Murashige and Skoog (MS) medium supplemented with the plant growth regulators benzyl amino purine (BAP), 1-naph- thaleneacetic acid (NAA) and kinetin (KIN) at concentrations of 0, 0.5, 1.0, 3.0 or 5.0 mg/L. The basal MS medium that also contained 30 g/L sucrose was adjusted to pH 5.7 to 5.8 before adding 3 g/L gelrite agar for gelling. Sterilization of the culture medium was performed by autoclaving at 12˚C for 20 min. Explants were inoculated on to 40 mL of medium contained in 150 mL flasks. The cultures were incubated in a plant growth room at a temperature of 25˚C ± 1˚C with a 16 h photoperiod provided by cool-white fluorescent lamps (1000 - 2000 lux). The cultures were checked regularly for contamination and observations were recorded at weekly intervals. Twenty replicated flasks were used in each treatment to compare the effects of the growth regulators. Results were expressed as percent shoot induction, the number of shoots per culture and also the length of the shoots after 45 days of culture.
Separate experiments were conducted for shoot multiplication. Cut segments of explants were cultured on basal MS medium supplemented with either BAP (0, 0.5, 1.0, 3.0 and 5.0 g/L), NAA 0, 0.5, 1.0, 3.0 and 5.0 g/L or combinations of both growth regulators. Ten replicated flasks were used in each treatment and observations were recorded at weekly intervals. The results were expressed as percent explants showing shoot proliferation, as the number of shoots per culture and the length of the shoots after 45 days of culture. Shoots (>2.0 cm long) from the best treatment were individually placed inside the flasks for root initiation. The explants were cultured on MS medium supplemented with IBA, IAA or NAA at concentrations of 0, 0.5, 1.0, or 2.0 mg/L. Twenty replicated flasks were used in each treatment. The number of shoots that produced roots, as well as the number and length of the induced roots were recorded after 30 days. Complete plantlets produced in vitro were removed from the culture medium and the roots were washed to remove the agar. The plantlets were then transferred into pots containing organic soil mixed with garden soil (1:1) and placed in the net house under controlled conditions with 75% shading and temperature at 28˚C - 32˚C. To maintain humidity, the plants were watered periodically twice a day. Observations were recorded on the percent survival of rooted and acclimatized plants.
A simple and effective protocol was developed for the in vitro micropropagation of Plectranthus amboinicus. Two different types of explants (apical shoots and lateral shoots) were cultured on MS media containing different concentrations of BAP, NAA, or KIN to evaluate their effects on shoot initiation. Explants grown on plant growth regulator-containing media showed varying success in shoot initiation depending on the type of explant and the growth regulators added. The response of explants cultured in MS media supplemented with BAP, NAA, and KIN are shown in
Explant segment | Growth regulator concentration (mg/L) | Shoot induction (%) | Average number of shoots/explant |
---|---|---|---|
Apical shoots | BAP | ||
0 | 20 | 0.3 ± 0.04 | |
0.5 | 30 | 0.5 ± 0.03 | |
1.0 | 20 | 0.5 ± 0.01 | |
3.0 | 30 | 0.6 ± 0.05 | |
5.0 | 35 | 0.6 ± 0.09 | |
NAA | |||
0.5 | 30 | 0.3 ± 0.02 | |
1.0 | 30 | 0.4 ± 0.01 | |
3.0 | 25 | 0.4 ± 0.04 | |
5.0 | 40 | 0.7 ± 0.06 | |
KIN | |||
0.5 | 30 | 0.5 ± 0.04 | |
1.0 | 35 | 0.5 ± 0.07 | |
3.0 | 30 | 0.5 ± 0.02 | |
5.0 | 30 | 0.4 ± 0.02 | |
Lateral shoots | BAP | ||
0 | 40 | 0.5 ± 0.03 | |
0.5 | 60 | 1.2 ± 0.21 | |
1.0 | 80 | 1.9 ± 0.31 | |
3.0 | 80 | 2.4 ± 0.43 | |
5.0 | 75 | 0.9 ± 0.09 | |
NAA | |||
0.5 | 45 | 0.4 ± 0.03 | |
1.0 | 55 | 0.5 ± 0.01 | |
3.0 | 70 | 1.0 ± 0.11 | |
5.0 | 60 | 1.0 ± 0.21 | |
KIN | |||
0.5 | 30 | 0.4 ± 0.04 | |
1.0 | 35 | 0.4 ± 0.03 | |
3.0 | 45 | 0.5 ± 0.01 | |
5.0 | 40 | 0.8 ± 0.07 |
to those from the shoot apices on all the media tested. Generally, the addition of BAP resulted in significantly higher shoot initiation and the number of shoots at the initiation stage, as compared with other growth regulators. The highest rate of shoot induction (80%) (
Different concentrations of BAP or NAA added to MS medium singly or in combination affected shoot proliferation rate, the number of shoots produced and the average length of the shoots. The highest rate of shoot proliferation (85%) and number of shoots per explant (5.7) was obtained on media supplemented with 1 mg/L BAP + 5 mg/L NAA after six weeks of culture (
Amiri et al. (2011) reported that the maximum shoot regeneration and maximum number of regenerated shoots in Datura stramonium were obtained in the treatment containing 2 mg/L BAP + 1 mg/L NAA. The percentage of shoot regeneration in Artimisia was highest with a combination of BAP and NAA, both at the concentration of 0.5 mg/L [
Roots were produced in all media, including the medium that was free of growth regulators (
BAP (mg/L) | NAA (mg/L) | % of explants showing shoot proliferation | Average number of shoots per explant (mean ± SD) | Average length of shoot (cm) |
---|---|---|---|---|
0 | 0 | 0 | 1.0 ± 0.01 | 2.6 |
0.5 | 0 | 0 | 1.0 ± 0.01 | 2.5 |
1.0 | 0 | 5 | 1.0 ± 0.01 | 1.9 |
3.0 | 0 | 30 | 1.5 ± 0.12 | 2.2 |
5.0 | 0 | 30 | 2.5 ± 0.02 | 1.5 |
0 | 0.5 | 0 | 1.0 ± 0.01 | 3.1 |
0 | 1.0 | 5 | 1.2 ± 0.51 | 3.7 |
0 | 3.0 | 50 | 3.5 ± 0.11 | 1.5 |
0 | 5.0 | 60 | 3.7 ± 0.42 | 1.2 |
0.5 | 0.5 | 0 | 1.0 ± 0.01 | 2.7 |
0.5 | 1.0 | 20 | 2.1 ± 0.02 | 2.8 |
0.5 | 3.0 | 40 | 3.1 ± 0.45 | 1.7 |
0.5 | 5.0 | 40 | 3.4 ± 0.13 | 1.4 |
1.0 | 0.5 | 10 | 1.3 ± 0.07 | 1.6 |
1.0 | 1.0 | 40 | 3.0 ± 0.12 | 2.0 |
1.0 | 3.0 | 55 | 3.1 ± 0.05 | 2.4 |
1.0 | 5.0 | 85 | 5.7 ± 0.32 | 2.3 |
BAP (mg/L) | IAA (mg/L) | NAA (mg/L) | % of explants rooted | No. of roots/explant | Average root length (cm) |
---|---|---|---|---|---|
0 | 0 | 0 | 100 | 9.7 ± 0.39 | 3.1 ± 0.9 |
0.5 | 0 | 0 | 100 | 12.5 ± 0.91 | 2.1 ± 0.4 |
1.0 | 0 | 0 | 100 | 10.8 ± 0.72 | 2.0 ± 0.3 |
0 | 0.5 | 0 | 80 | 8.3 ± 1.09 | 1.4 ± 0.1 |
0 | 1.0 | 0 | 85 | 9.5 ± 0.34 | 1.5 ± 0.4 |
0 | 0 | 0.5 | 75 | 8.6 ± 0.51 | 0.6 ± 0.04 |
0 | 0 | 1.0 | 75 | 7.8 ± 0.44 | 0.4 ± 0.03 |
Among the growth regulators, IBA had the largest effect on root formation (
In this study on the in vitro micropropagation of the medicinal plant Plectranthus amboinicus, MS culture medium containing 3.0 mg/L BAP gave the best rate of shoot initiation using explants derived from lateral shoots. The highest rate of shoot proliferation was obtained using BAP at 1.0 mg/L in combination with NAA at 5.0 mg/L. BAP at 0.5 - 1.0 mg/L was most suited for root initiation. The plantlets obtained survived and grew normally in the greenhouse. This procedure was recommended for rapid in vitro shoot micropropagation of P. amboinicus.
The authors would like to thank MARDI for supporting this project under the project WRM.