A successful protocol was developed for mass propagation of Lawsonia inermis Linn., an important medicinal plant. Multiple shoots were induced in apical and axillary meristems derived from mature explants of L. inermis on Murashige and Skoog (1962) medium supplemented with 0.25 mg/l 6-benzylaminopurine (BA), 0.25 mg/l Kinetin (Kn), 0.5 mg/l ascorbic acid and 3% (w/v) sucrose. The rate of multiplication was higher when the cultures were incubated under continuous light rather than the 14h photoperiod. Rooting was readily achieved upon transferring the microshoots onto MS basal semi-solid medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA) after ten days of culture. Micropropagated plantlets were acclimatized and successfully grown in soil.
Lawsonia inermis Linn (Lythraceae) is an important medicinal plant, distributed in northern Africa and south-west Asia (Anonymous 1962). The leaves are used as a prophylactic against skin diseases. They are used externally in the form of a paste or decoction against boils, burns, bruises and skin inflammations. A decoction is used as gurgle against sore throat. Alcoholic extracts of Lowsonia leaves show mild antibacterial activity against Micrococcuspyogenes var. and aureusEscherichia coli (Kritikar and Basu 1981). The flower of Lawsonia has a strong aroma with high commercial value. It is extensively used as a dye in silk and wool industry. Conventional methods of propagation of L. inermis, sexual as well as vegetative, are beset with many problems that restrict their multiplication on a large scale. Propagation through seed is unreliable because of disease and pest problems, short viability and heavy rains during the seeding season in the natural habitat. An unplanned exploitation by the ever growing human population has resulted in the rapid depletion of plant resources particularly the economically important plants. Cosmetic and pharmaceutical companies largely depend upon materials procured from naturally occurring stands raising concern about possible extinction and providing justification for development of in vitro techniques for mass propagation of L. inermis . Preservation of genetic stability in germplasm collections and micropropagation of elite plants is of the utmost importance and propagation of plants through apical or axillary meristem culture allows recovery of genetically stable and true to type progeny (Hu and Wang 1983, George and Sherrington 1984). There is no report on micropropagation of L. inermis. The present communication describes a successful protocol for mass propagation of L. inermis Linn. ]]>
Materials and methods
Plant material and explant source: Actively growing young stems (4-5 cm) of L. inermis were collected from greenhouse grown plants at Regional Plant Resource Centre, Bhubaneswar and washed with 2% (v/v) detergent ‘Teepol’ (Qualigen, India) and rinsed several times with running tap water. The explants were surface sterilized in 0.1 % (w/v) aqueous mercuric chloride solution for 15 min followed by four washings with sterile distilled water. The apical and axillary meristems (~ 0.5 mm) were isolated and used as explants.
Culture medium and condition : The meristem (apical and axillary) was placed on semi-solid basal MS (Murashige and Skoog 1962) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BA: 0.0, 0.25, 0.5 and 1.0 mg/l), kinetin (Kn: 0.0, 0.25, 0.5 and 1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) (0.0, 0.10, 0.25 and 0.5 mg/l) for shoot proliferation and multiplication. The pH of the media was adjusted to 5.8 using 0.1N NaOH or 0.1N HCl before autoclaving. Routinely, 25 ml of the molten medium was dispensed into culture tubes (25 x 150 mm), plugged with non-absorbent cotton wrapped in one layer of cheese cloth and sterilized at 121 ºC and 1.06 Kg/cm2 pressure for 15 min. The cultures were maintained at 25 ± 2 ºC either under 14 rh photoperiod or continuous light (55 mmol m-2s-1) from cool, white fluorescent lamps. The cultures were maintained by regular subcultures at 4-week intervals on fresh medium with the same compositions. To avoid blackening, the medium was supplemented with 0.5 mg/l ascorbic acid.
Induction of rooting and acclimatization: For root induction, excised microshoots (1-2 cm length) were transferred to MS basal medium supplemented with different concentrations of IAA or IBA (0.0, 0.1, 0.25 and 0.5 mg/l) and 2% (w/v) sucrose. One excised shoot was placed in each tube (25 x 150 mm) having 15 mL of the culture media. All the cultures were incubated at 25 ± 2 ºC under 16h photoperiod with cool, white fluorescent lamps. Rooted micro-propagules were thoroughly washed to remove the adhering gel and planted in 2.5 cm earthern pots containing a sterile mixture of sand: soil and cow-dung manure in the ratio of 1:1:1 (v/v) and kept in the greenhouse for acclimatization.
Observation of cultures and presentation of results: Twenty cultures were used per treatment and each experiment was repeated at least three times. The data pertaining to mean percentage of cultures showing response, number of shoots/culture and mean percentage of rooting were statistically analysed by the Post-Hoc Multiple Comparison test (Marascuilo and McSweency 1977). Between the treatments, the average figures followed by the same letters were not significantly different at P < 0.05 level.
]]>
Figs 1A-D. Micropropagation of Lawsonia inermis. A. Proliferation of shoot from apical meristems on MS medium sup-plemented with 0.25 mg/l BA, 0.25 mg/l Kn, 0.5 mg/l ascorbic acid and 3 % sucrose. (Bar = 5 mm). B. Multiple shoots from apical meristems on MS medium supplemented with 0.25 mg/l BA, 0.25 mg/l Kn, 0.5 mg/l ascorbic acid and 3 % sucrose after 4 weeks of culture. (Bar = 10 mm). C. Rooting in the in vitro derived shoots after 10 days of culture on MS medium sup-plemented with 0.25 mg/l IBA and 2 % (w/v) sucrose. (Bar = 10 mm). D. Plant grown in the pot. (Bar = 50 mm).
Results
Meristem proliferation and multiplication: Meristem proliferation and multiplication was initiated from apical and axillary explants of L. inermis within 8-10 days of inoculation onto MS basal medium supplemented with BA, Kn and 0.5 mg/l ascorbic acid. Of the different cytokinins tested, BA + Kn was the most effective for shoot proliferation and multiplication. The maximum shoot proliferation and multiplication was observed both in apical and axillary meristems cultured on MS medium supplemented with 0.25 mg/l BA, 0.25 mg/l Kn and 0.5 mg/l ascorbic acid within 4 weeks of culture under 14h photoperiod (Table 1). The apical and axillary shoots proliferated and elongated to 1.0-1.5 cm within 4 weeks of culture (Fig. 1A). There was no sign of shoot proliferation when explants were cultured in media devoid of cytokinin. At higher concentrations of BA or Kn, the rate of shoot proliferation declined. Inclusion of either IAA or IBA in the culture medium did not help in proliferation and multiplication of shoot. In most of the cases, the growth was inhibited and only 1-2 shoots would elongate; some produced compact callus at the base of the explants. Prolonged culture on the proliferation and multiplication media resulted in the blackening of the basal ends of the developing shoots. There were differences among the treatments for both the percentage of cultures with multiple shoots and the mean number of shoots/culture. The axillary meristems produced more number of shoots (4.57) than the apical meristems (3.25) (Fig. 1B) (Table 1). The study also revealed that the continuous light (24h) was more conducive to higher rate of shoot multiplication than 14h photoperiod (Table 2). The highest percentage of cultures with multiple shoots (86.4) was observed on media containing 0.25 mg/l BA, 0.25 mg/l Kn and 0.5 mg/l ascorbic acid when the cultures were incubated in the continuous light for 4 weeks. The frequency of multiple shoots per culture varied from 1.24 to 4.42 in case of the 14h photoperiod and 2.01 to 4.78 in case of the continuous light incubation (Table 2). The rate of multiplication was high and stable upto 5th subculture and declined in subsequent subcultures (Fig.2).
Table 1.
Effect of BA, kinetin and 0.5 mg/l ascorbic acid on shoot growth from apical (A) and axillary (B) meristems of Lawsonia inermis after 4 weeks of culture under 14h photoperiod.
MS + Growth regulators (mg/l)
Percent of cultures with ]]>
multiple shoots (Mean ± S.E.)*
Number of shoots/explant (Mean ± S.E.)*
BA
Kn
A
B
A
B
0
0
]]>
0
0
0
0
0.25
0
32.6 ± 0.4 d
40.4 ± 0.3 d
2.41 ± 0.2 e
2.97 ± 0.5 e
]]>
0.50
0
40.2 ± 0.5 e
44.8 ± 0.6 d, e
2.11 ± 0.4 d
2.35 ± 0.4 d
1.0
0
28.6 ± 0.7 c+
24.5 ± 0.4 b+
]]>
1.01 ± 0.5 a+
1.34 ± 0.6 a+
0
0.25
38.7 ± 0.3 e
46.7 ± 0.3 e
2.72 ± 0.5 f
2.31 ± 0.2 d
0
0.50
]]>
42.2 ± 0.5 f
47.3 ± 0.7 f
1.27 ± 0.4 b
1.76 ± 0.5 b
0
1.0
18.7 ± 0.4 a+
22.3 ± 0.6 a+
1.80 ± 0.3 c+
1.32 ± 0.6 a+
]]>
0.25
0.25
62.8 ± 0.5 h
75.7 ± 0.3 h
3.25 ± 0.2 h
4.57 ± 0.4 g
0.25
0.50
51.7 ± 0.4 g
58.6 ± 0.5 g
]]>
3.16 ± 0.4 g
3.23 ± 0.7 f
0.50
0.25
50.3 ± 0.6 g
57.4 ± 0.3g
2.26 ± 0.7 e
2.18 ± 0.6 c
0.50
0.50
]]>
39.4 ± 0.4 e
42.8 ± 0.5d
1.86 ± 0.6 c
1.83 ± 0.5 b
0.5
1.0
21.6 ± 0.4 b+
20.4 ± 0.5a+
1.14 ± 0.5 a+
1.18 ± 0.3 a+
]]>
1.0
0.5
23.7 ± 0.5 b+
27.5 ± 0.4c+
1.07 ± 0.6 a+
1.23 ± 0.6 a+
* Mean of 20 cultures per treatment; repeated thrice. + Callusing at the basal end. a-h Means having the same letter in a column were not significantly different by Post-Hoc Multiple Comparison test P < 0.05 level.
Table 2.
Effect of photoperiod on shoot multiplication of Lawsonia inermis cultured on MS medium supplemented with various concentrations of BA, kinetin and 0.5 mg/l ascorbic acid after 4 weeks of culture. ]]>
Growth regulators (mg/l)
Percent of cultures with multiple shoots (Mean ± S.E.)*
Number of shoots/explant (Mean ± S.E.)*
BA
Kn
A
B
A
]]>
B
14 hr Photoperiod
0
0
0
0
0
0
0.25
0
]]>
30.4 ± 0.3 a
38.2 ± 0.3 a
2.31 ± 0.3 f
2.94 ± 0.4 f
0.50
0
38.2 ± 0.6 d
43.6 ± 0.4 b
2.14 ± 0.6 d
2.23 ± 0.5 c
]]>
0
0.25
36.5 ± 0.4 c
45.8 ± 0.3 c
2.62 ± 0.4 h
2.36 ± 0.3 e
0
0.50
41.5 ± 0.7 f
46.3 ± 0.5 c
]]>
1.24 ± 0.7 a
1.66 ± 0.5 a
0.25
0.25
61.2 ± 0.5 l
73.4 ± 0.6 g
3.20 ± 0.4 m
4.42 ± 0.3 l
0.25
0.50
]]>
52.4 ± 0.3 k
59.4 ± 0.3 f
3.03 ± 0.5 l
3.18 ± 0.7 i
0.50
0.25
48.2 ± 0.4 h
56.5 ± 0.4 e
2.20 ± 0.6 e
2.23 ± 0.6 c
]]>
0.5
0.50
37.3 ± 0.6 c
44.5 ± 0.3 b
1.76 ± 0.5 b
3.82 ± 0.4 k
Continuous light
0
0
0
]]>
0
0
0
0.25
0.25
33.1 ± 0.5 b
39.2 ± 0.4 a
2.71 ± 0.6 i
3.06 ± 0.4 g
0.50
]]>
0.50
42.2 ± 0.4 g
45.6 ± 0.2 c
2.41 ± 0.5 g
2.91 ± 0.2 f
0
0.25
39.6 ± 0.3 e
51.4 ± 0.4 d
2.92 ± 0.5 k
]]>
3.12 ± 0.4 h
0
0.50
43.4 ± 0.7 g
46.2 ± 0.2 c
2.17 ± 0.2 d, e
2.27 ± 0.5 d
0.25
0.25
70.4 ± 0.5 m
]]>
86.4 ± 0.4 h
3.41 ± 0.3 o
4.78 ± 0.6 m
0.25
0.50
50.3 ± 0.4 d, I
57.5 ± 0.4 e
3.21 ± 0.8 n
3.53 ± 0.3 j
0.50
]]>
0.25
51.6 ± 0.6 d, j
58.3 ± 0.3 e, f
2.82 ± 0.4 j
2.91 ± 0.4 f
0.5
0.50
42.5 ± 0.4 g
45.6 ± 0.6 c
2.01 ± 0.5 c
]]>
2.13 ± 0.6
A-Apical meristem; B-Axillary meristem. * Mean of 20 cultures per treatment; repeated thrice. a-o Means having the same letter in a column were not significantly different by Post-Hoc Multiple Comparison test P < 0.05 level.
Induction of rooting from microshoots: Elongated shoots (1-2 cm long) were rooted on MS basal medium supplemented with various concentrations of either IAA or IBA (Table 3). The rooting in the microshoots was inhibited in the medium devoid of growth regulator. Root initiation took place within 10-12 days of transfer to MS basal medium supplemented with 0.1-0.25 mg/l IAA or IBA. However, optimal rooting (85.6%) and growth of microshoots were observed on medium containing 0.25 mg/l IBA with 2% (w/v) sucrose (Fig. 1C). The rooting ability was reduced with the increase in the concentration of IAA or IBA in the culture medium. The percentage of shoots forming roots and days to rooting significantly varied with different concentrations of IAA or IBA.
Table.3.
Effect of IAA and IBA on rooting from excised shoots of Lawsonia inermis cultured on MS basal medium supplemented with 2% (w/v) sucrose.
MS + Growth regulators (mg/l)
Percentage of shoot rooted (Mean + S.E)*
]]>
Days to rooting
IAA
IBA
0
0
0
0
0.1
0
48.4 + 0.6
]]>
11-12
0.25
0
52.8 + 0.3
12-13
0.50
0
36.4 + 0.4
13-14 +
0
]]>
0.1
60.3 + 0.6
11-
0
0.25
75.6 + 0.7
10-
0
0.50
52.8 + 0.4
]]>
12 +
Acclimatization and field establishment: Rooted plantlets grown in vitro were washed thoroughly to remove the adhering gel, transplanted to 2.5cm earthern sterile pots containing garden soil, sand and cow-dung at the ratio of 2: 1: 1 (v/v). About 96% of the rooted plantlets established in the greenhouse within 2-3 weeks of transfer. The plant grew well and attained 6-8 cm height within 4 weeks of transfer (Fig. 1D). The acclimatized plants were established in the field condition and grew normally without morphological variation.
Fig. 2. Effect of subcultures on shoot multiplication of Lawsonia inermis cultured on MS basal medium supplemented with 0.25 mg/l BA, 0.25 mg/l Kn, 0.5 mg/l ascorbic acid and 3 % sucrose. Subcultures took place every 4 weeks. Means of 20 cultures/subculture; repeated thrice.
Discussion
The present study showed that it was possible to explore the morphogenetic potential of L. inermis (Lal by application of growth regulators and light condition. With the use of different combinations of cytokinins for the induction and multiplication of shoots derived from apical and axillary meristems. The regulatory action of cytokinin and apical dominance helped the in vitro shoot induction and multiplication was well documented (Wickson and Thimann 1958). The maximum shoot induction and multiplication was observed both in apical and axillary meristems cultured on MS medium supplemented with 0.25 mg/l BA, 0.25 mg/l Kn and 0.5 mg/l ascorbic acid within 4 weeks of culture under a 14hr photoperiod. At higher concentrations of BA or Kn, the rate of shoot proliferation declined. The axillary meristems produced more number of shoots than the apical meristems. Similar results were reported in Psoralea corylifolia (Saxena et al. 1998), Picrorhiza kurroa (Lal et al. 1988, Upadhyay et al. 1989) and Plumbago zeylanica (Rout et al. 1999). Mao et al (1995) reported that BA proved superior to other cytokinins for multiple shoot induction of Clerodendrum colebrookianum. Our results demonstrated that the inclusion of either IAA or IBA in the culture medium did not help in shoot multiplication. Prolonged culture on the proliferation and multiplication media containing IAA or IBA resulted in the blackening of the basal ends of the developing shoots. The results are consistent with earlier reports indicating cytokinins and auxins affect shoot multiplication in other plants using shoot tip or axillary bud explants (Mathew and Hariharan 1990, Rout et al. 1992, Rout and Das 1993, Rout et al 1999) The results showed that the number of shoots per culture was increased in continuous light both in apical and axillary meristems than 14h photoperiod. The interaction of photoperiod and plant growth regulators have significant effect on shoot morphogenesis as reported earlier (Baraldi et al. 1988, Samantaray et al. 1995). With the increase in the concentration of either BA or Kn, the percentage of shoot multiplication declined. The results also implies that there were differences among the treatments for both the percentage of culture developing multiple shoots and the mean numbers of shoots per culture. The variation of response resulted due to the varying concentrations of growth regulators used in the medium and light condition as reported earlier in Lavandula latifolia (Calvo and Segura 1989) and Zingiber officinale (Rout and Das 1997). The rate of multiplication was high and stable up to the 5th subculture and declined in subsequent subcultures. This might be due to the balancing of the endogenous and exogenous growth regulators and the ionic concentration of nutrient salts as reported earlier in other plants (Zimmerman 1985, Rout and Das 1997). The elongated shoots were rooted the maximum in MS basal salts supplemented with 0.25 mg/l IBA with 2% sucrose. The rooting ability was reduced with the increase in the concentration of IAA or IBA in the medium. The percentage of shoots forming roots and days to rooting significantly varied with different concentrations of IAA or IBA. Similar observations were made in Plumbago rosea (Satheesh Kumar and Bhavanandan 1988) and Psoralea corylifolia (Saxena et al. 1998). The rooted plantlets were established in the field and grew normally. ]]>
In conclusion, an attempt was made to develop an in vitro protocol for mass multiplication of L. inermis by manipulating the nutrient salts, growth regulators and culture conditions. The pattern of morphogenesis on various phytohormonal regimes largely confirm to those reported in other plant species (Koblitz et al. 1983, Ideada et al. 1988, Lal et al. 1988). This investigation may be useful for conservation of economic plant species.
Acknowledgements
The authors acknowledge the Department of Forest and Environment, Government of Orissa for necessary facilities.
Resumen
Se desarrolo un protocolo exitoso para la propagación en masa de Lawsonia inermis Linn., una planta medicinal importante. Se indujeron múltiples tallos en meristamos apicales y auxiliares derivados de explantes maduros de L. inermis en medio de Murashige y Skoog (1962) suplementado con 0.25 mg/l 6-benzylaminopurina (BA), 0.25 mg/l quinetina (Kn), 0.5 mg/l ácido ascórbico y 3 % (w/v) sucrosa. La tasa de multiplicación fue más alta cuando los cultivos fueron incubados bajo luz continua que bajo el fotoperíodo de 14hr. El enraizamiento se logró al tranferir los microtallos al medio basal, semi-sólido MS suplementado con 0.25 mg/l de ácido indol-3-butiríco (IBA) luego de 10 días de cultivo. Las plántulas micropropagadas fueron aclimatizadas y cultivadas con éxito en el suelo.
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