Micropropagation and in Vitro Flowering of an Ornamental Aquarium Plant Lindernia antipoda L. (Alston) 
,
Sheeja George 
,
Anjara Raj ,
Sree Lakshmi S. ,
Aneykutty Joseph Author Correspondence author
International Journal of Aquaculture, 2016, Vol. 6, No. 8 doi: 10.5376/ija.2016.06.0008
Received: 29 Feb., 2016 Accepted: 12 Apr., 2016 Published: 23 Sep., 2016
Jabir T., Sheeja George., Anjana Raj., Sree Lakshmi S., Aneykutty Joseph., 2016, Micropropagation and in vitro flowering of an ornamental aquarium plant Lindernia antipoda (L.) Alston, 6(8): 1-10 (doi: 10.5376/ija.2016.06.0008)
1 Introduction
Lindernea antipoda (L.) Alston is an evergreen amphibian herbaceous plant belongs to the family Linderniaceae, found in Tropical Asia. It is a large and taxonomically difficult genus; most species are found in swamps and also found in temporarily submerged areas. They are mainly used for filtration and cleaning of water in canals and lakes. Some of the species belonging to the genus Lindernia are used as ornamental aquarium plants. L. antipoda (L.) produces small white flowers and bright green leaves and is widely used as an aquarium plant. Native range of Lindernea sp. is tropical and subtropical Asia from India and Sri Lanka, throughout Malaysia and eastward to northern Australia; apparently adventives further eastward (Smith, 1992). The systematic position of L. antipoda (L.) Alston is as follows.
Kingdom: Plantae
Division: Tracheophyta
Class: Magnoliopsida
Order: Lamiales
Family: Linderniaceae
Natural propagation of aquatic plant species is limited due to the production of small number of plants with a long cultivation period, disease and requirement of large space for propagation (Sulaiman, 2004). In vitro propagation of aquarium plants can solve many of these problems such as unreliable supply, over collection, variable plant quality and frequent losses from poorly characterized water transmitted diseases and destruction of endangered species (Kane et al., 1999; Sarasan et al., 2006). Hence, conservation and commercial production of Lindernia sp. in order to meet the market demand can be achieved via the development of efficient and economically viable micropropagation protocols.
Moreover, the collection of plant materials from the wild resources can be reduced or prevented with the production of clonal propagated plants. Previous studies have reported the in vitro propagation protocols for some aquarium plants namely Aponogeton sp. (Kukulczanka et al., 1980), Cryptocoryne lucens (Kane et al., 1990), Anubias barteri (Huang et al., 1994), Cryptocorine wendtii (Kane et al., 1999; Mo and Jiang, 2003), Ludwigia repens (Ozturk et al., 2004), Cryptocorine becketti, Cryptocorin lutea and Rotala rotundifolia (Micheli et al., 2006). However, there has been no report on the effect of medium on the shoot proliferation of Lindernea sp. The objective of the present study is to establish an efficient micropropagation protocol for the mass production of L. antipoda (L.) plantlets.
2 Materials and Methods
2.1 Explants collection and preparation
Disease free, young and healthy nodal explants were collected from Malabar Botanical Garden, Olavanna, Kozhikkode, Kerala state, India. Terminal shoots bearing 4 to 5 nodes were cut off from plants growing in the field. They were washed under running tap water for 10 minutes followed by washing with few drops of Cleansol solution. Finally, thorough washing was done for several times using sterile distilled water.
2.2 Explants sterilization
The leaves were removed from the explants and washed under running tap water for 30 minutes in order to wash off the external dust/contaminants. After this treatment, the explants were surface disinfected using a strong surface sterilant, sodium hypochlorite (4% Chlorine) of different concentrations for various time intervals (Table 1). Then the explants were removed from the sterilizing solution and rinsed thoroughly twice with sterile double distilled water. In the next step, explants were soaked in an aqueous solution containing 150 mg/l bavistin (BASF, India Limited) and 0.03 % Ampicillin for 10 minutes in a laminar flow hood. Then the explants were thoroughly washed twice with sterile double distilled water for 5 minutes.
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Table 1 Response of L. antipoda (L.) for surface sterilization with various concentration of NaOCl |
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2.3 Initiation of cultures
Sterilized explants were transferred aseptically to sterilized glass plate and the material was cut in to both basal as well as the top portion to remove undesirable/dead portions after surface sterilization. For shoot initiation from explants mainly two different strength of MS media were used, Full strength MS (Full MS) and half strength MS (Half MS). Each nodal explant was aseptically placed in an erect position in the test tube containing medium. Tubes were kept in the growth room with temperature conditions 25±2°C, with a photoperiod of 16 hours daylight and 8 hrs night break under the cool white fluorescent light of average 2500 lux (cool white fluorescent tube light 40 W GE).
2.4 Establishment of cultures
After approximately 9-10 days of inoculation, the axillary bud break was seen in some explants. When the explants attained bud proliferation, these cultures were transferred to jars containing fresh medium. After 21- 25 days of incubation, the initiated plants were taken out of the test tube using clean and sterilized forceps, medium adhered to the plants was removed, undesirable/brownish leaves were removed from the plants and were put into the culture bottles containing autoclaved semi-solid media having the same combinations as that for the culture initiation. The bottles were then placed in culture room under the standard conditions of temperature (25 ± 2°C) for 16/8 hrs of day/night break under the cool white fluorescent light of average 2500 lux (cool white fluorescent tube light 40 W GE).
2.5 Axillary shoot proliferation
Multiple shoots/cluster were transferred from the culture bottle to a sterile glass plate; brown leaves were removed from the primary shoots and sectioned into one node piece after removing the leaves. These nodal segments were transferred to the multiplication media (Table 2). These culture bottles were then incubated in the growth room. These steps were repeated every 25-30 days for the next sub-culturing.
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Table 2 Description of various media for the shoot multiplication of L. antipoda (L.) |
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2.6 Transplantation and acclimatization of the plantlets
After 10 – 14 days of culture on rooting media, the rooted plantlets were transplanted to pots or trays for hardening prior to their final transfer to aquarium. Rooted plantlets were taken out of the culture bottles with the help of forceps and washed thoroughly with water to remove any remaining of the medium. Bavistin (0.1%) treatment was given to the plants in order to protect them from fungal attack in the near future. After this, the plants were carefully planted in poly bags containing different ratio of Neopete (Sterlings Farm Research Services Pvt. Ltd.) & soil mixtures in 1:1 ratio. After planting, plantlets are thoroughly watered and kept in poly house under humidity range of approximately 80%. These plantlets were sprinkled with water time to time as per the requirement and after two weeks and transferred to shade house having humidity range of approximately 60%. The plantlets are then transferred to open area after 9-10 days and kept there for ten days prior transferring them to the aquarium.
3 Discussion
3.1 Surface sterilization of explant
Sodium hypochlorite solution (NaOCl, 4% available chlorine, Fisher Scientific Prod. No. 27908) was used for surface sterilization of explant. After treatment with sodium hypochlorite over 40% of the cultures were contaminated by fungi and or bacteria. Response of L. antipoda (L.) explants to surface sterilisation with various concentration of NaOCl is provided in the Table 2. From the table, it was clear that 10% NaOCl for 10 minutes was the best surface sterilization procedure for L. antipoda (L.) explants. In this, 60.2% explants survived and 39.8% were contaminated. Use of 10% NaOCl for 20 min caused the death of explants (80%). The survival rate was only 10.4% but had lower contamination rate 9.6%. Hence, 60.2% survival rate on treated with 10% NaOCl for 10 min, the contamination rate was 35.5%, to avoid this systemic contamination bavistin was effectively used. By pre-treatment of the explants using 0.15% of bavistin for 3 minutes ceased the rate of contamination to 15.5% and sprouting percentage of shoots raised to 84.5%. But, when bavistin with 0.3% concentration was used, contamination rate was reduced to 6% while rate of sprouting also ceased to 48%. The variable response of Lindernia sp. explants to bavistin treatment is provided in Table 3. It is proved that various concentration of bavistin was tried and 0.15% was optimum for initiating a better response in L. antipoda (L.)
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Table 3 Effect of fungicide (bavistin) treatment on culture establishment of L. antipoda (L.) nodal explants |
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Murashige and Skoog (1962), one of the widely used plant tissue culture media, was used for present study. For shoot initiation from explants two different ionic strength of MS media were used, Full strength MS (Full MS) and half strength MS (Half MS) (Figure 1). In full MS medium, the growth rate of axillary buds was about 55.6% while in ½ MS media it was 95.6% (Figure 2). Here, half of the concentration of MS medium was taken, because it was observed that, the half strength MS medium favored better growth response and axillary shoot proliferation than full strength MS medium.
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Figure 1 Culture initiation of L. antipoda L. on ½ MS media basal media |
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Figure 2 Culture establishment of L. antipoda L. on ½ MS media + 1mg/l BAP after 4 weeks of culture initiation |
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After culture establishment, plants multiplied in different culture media. The semisolid and liquid culture media of MS and ½ MS media was used. Highest multiplication was obtained on half strength MS liquid media (Figure 3). The response of L. antipoda (L.) on solid and liquid culture media having different ionic strength is explained in Table 4.
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Figure 3 Multiple shoot proliferation of L. antipoda L. on BM3 media after 2 weeks of culture initiation |
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Table 4 Effect of culture medium types and condition on in vitro shoot proliferation of L. antipoda culture initiation after 28 days |
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Although the response of explants in ½ MS basal medium was not at all satisfactory, excellent responses were obtained in half strength MS medium supplemented with various concentrations of growth regulators (Table 5). Results revealed that ½ MS medium supplemented with BAP, Kn & NAA showed significant difference in terms of shoot proliferation effect. The best response of culture initiation was obtained in half strength MS medium supplemented with 1 mg/l BAP. BAP concentration 0.1-2 mg/l had highest culture establishment capability (40.3 - 85%) and also culture initiation was very fast within 10 days. But in Kinetin and NAA, culture establishment was below 40% (MS control 35%) and shoot initiation took more than 10 days. Further studies were focused to find out the most appropriate medium for maximum shoot proliferation. MS media with Cytokinin proved to be most effective, for multiple shoot proliferation. After 25-30 days of first subculture, established cultures were transferred to culture jars having respective media combinations. Multiplication of shoot cultures was carried out by culturing nodal segments/clusters excised from in vitro raised plants.
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Table 5 Effect of various concentrations of growth regulators on L. antipoda L., in BM1 - BM5 Media |
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Effect of various concentration of growth regulators on shoot proliferation are provided in the Table 6. Observations were taken for evaluating the growth of explants by taking parameters like inter nodal distance, average shoot length and number of nodes. The experiment was carried out in seven media having different concentrations of growth regulators each with 3 replications; only results of best media are given. The media that showed best results were BM3 and BM5 with highest average shoot length of 5.56 and 6.3 respectively with 100% cluster formation. Sub culturing was carried out after 25-30 days using the same medium combinations as for initiation and establishment stages. Shoot clusters obtained in each subculture was divided in appox 1 cm2 size with approx 4-5 small shoots in each cluster and inoculated in each bottle.
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Table 6 The effect BAP and IAA supplemented into MS medium on the shoot proliferation of L. antipoda L after 4 weeks of culture |
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It was observed that the highest number of shoots (>3 cm) originated from the basal node and average number of shoots formed in case of BM3 media (MS+1mg/l BAP+0.2mg/l NAA) is 16.6 ; 5.19 in BM1 (MS+0.1mg/l BAP+0.1mg/l NAA); 10.62 in BM4 (MS+2mg/l BAP+0.4mg/l NAA) and lowest number of shoot regenerated in case of MS(Control) was 3.24. In term of number of nodes/explants, same order followed i.e. (BM3 MS+1mg/l BAP+0.2mg/l NAA) having 6.5, BM1 (MS+0.1mg/l BAP+0.1mg/l NAA) having 4.09, 6.20 in BM4 (MS+2mg/l BAP+0.4mg/l NAA) and in case of MS (Control) was 3.13.
3.2 In vitro flowering
In vitro conditions including media components, concentration and ratio of plant growth regulators and culture conditions affected in vitro flowering. Flower initiated in MS media supplemented with 1 mg/l BAP & 0.2 mg/l NAA (BM3) produces multiple shoots with flowering after 4–6 weeks of cultures (Figure 4). The production of flowering shoots continued for many subcultures spanning a period one year.
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Figure 4 in vitro flowering of L. antipoda (L.) on BM3 media 4 weeks after culture initiation |
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In the present study, incubation of flowering cultures on BAP supplemented with NAA in the medium was necessary for flowering after 6 weeks. Photoperiod was found to be important for in vitro flowering. Maximum in vitro flowers were found at 16 hrs ± 2 light periods; it was observed that plants incubated under 12 hrs or shorter photoperiods (8 hrs) were negatively affected for floral bud development. Optimum temperature for efficient in vitro flowering was 25°C ± 2°C.
3.3 In vitro rooting and hardening
Rooting was also observed along the medium. Highest number of root observed on the BM4 media is 12.5 due to 0.4 mg/l NAA. Small amount of rooting was observed on BM1, BM2 & BM3 is respectively. i.e., NAA along with BAP also stimulates rooting in the medium.
It was observed that during each passage, the number of leaves/shoots has increased substantially along with the height of shoots. The leaf size was approximately 0.5-0.8 cm. It was observed that morphogenic responses exhibited in the form of shoots or roots are correlative to a specific auxin/cytokinin ratio. In comparison to MS basal media, for BM3, there is a significant increase in number of shoots (5.28 times) and average shoot length (3.14 times).
After sufficient rooting, plantlets were transferred to the pots having Neopeat (Sterlings Farm Research Services Pvt. Ltd.) for acclimatization. Agar was removed from the rooted plantlets and washed with tap water. The roots of each plant were dipped in 0.1% Bavistin solution to avoid future fungal attack and then roots of explants were inserted carefully into the Neopete. Then potted plants were transferred to the tanks with 2-3 inches water level. The system is completely covered with polythene and humidity maintained is approx 80% and temperature was 28–30°C and kept there for 15 days. 90% plant survived in the polythene propagator system.
4 Discussion
In vitro establishment of L. antipoda (L.), through vegetative explants presented complications due to excessive contamination, as has been recorded for other aquatic plants (Balestri et al., 1998; Jenks et al., 2000). The establishment of aseptic shoot explants of L. antipoda (L.) was proved difficult due to the bacterial contamination which was found to retard the shoot growth and rooting and eventually led to the death of the young plantlets. Difficulties in the establishment of axenic aquatic plant cultures have been reported (Dore Swamy and Mohan Ram, 1969; Madsen, 1985; Godmaire and Nalewajko, 1986). Difficulty in obtaining aseptic cultures has also been reported for another aquatic plant, Nympiodes indica (Jenks et al., 2000).
For surface sterilization of aquatic plants sodium hypoclorite (NaOCl) was effectively used than HgCl2. NaOCl was successfully used in the surface sterilization of Bacopa monniera (Tiwari et al., 1998). Our results indicate 10% NaOCl for 10 min as the most effective method for surface sterilization of Lindernia sp. Fungal contamination was a major problem in the study, so that a systemic fungicide, bavistin was used for inhibiting the growth of fungus on explants. Pre-soaking the explants in fungicide solution for reducing fungal contamination has been suggested by Broome and Zimmerman (1972) in Black berry, Sharma et al. (2010) in Bacopa monneri and Thomas et al. (2006) in Papaya (Carica papaya). 0.2% bavistin is effectively used to kill endophytic fungus. The results obtained with these studies agree with these reports in the case of bavistin concentration as a fungicide.
MS media with half ionic strength was the suitable media for culture initiation of Lindernia sp. Half strength MS medium has been successfully reported to be the most suitable media for Ipomea (Priya et al., 2005), Myriophyllum (Sumitha et al., 2005) and Gymnema (Anu et al., 1994). The media tried to initiate the axillary bud development including growth regulator free medium and medium supplemented with various concentrations of BAP, Kinetin and IAA. Thus, BAP was found to be very effective medium for culture initiation and establishment when compared to Kinetin and IAA. BAP was reported to be the effective medium in the culture establishment in Myriophyllum sp. (Kane and Gilman, 1991) and Spilanthes acmella (Haw and Keng, 2003). Shrivastva and Rajani (1999) reported that out of two cytokinins, BAP was found to be more suitable than Kn. As BAP resulted in quicker and better response then the latter while, addition of NAA (0.2 mg/l) proved synergistic (Srivastava et al., 2002). Hence, different combination of BAP and NAA was selected for the multiplication of L. antipoda L explants. Among the different combinations of BAP and NAA was tried, the maximum shoot proliferation and shoot length was obtained on BM3 Media (MS+1mg/l BAP+0.2 mg/l NAA).
The stimulatory effects of BAP on bud break multiple shoot formation reported earlier in Ocimum sanctum (Patnaik and Chand, 1996). However, further increase in concentration of BAP above the optimum level reduced the rate of shoot multiplication. According to Hongrat et al. (2005), maximum number of shoots of Cryptocoryne cordata was regenerated in MS medium supplemented with BAP at 1 mg/l.
In BM3 media 1mg/l BAP & 0.2 mg/l NAA stimulates high multiple shoot proliferation and shoot length. In Paederia foetida and Centella asiatica multiple shoots were obtained in MS medium supplemented with BAP 1.0 mg/l (Singh et al., 1999), and for Rauwolfia serpentina MS medium supplemented with BAP and NAA (Sehrawat et al., 2001) was the best. In Aponogeton madagascariensis 2 mg/l BAP and 0.5 mg/l NAA was the optimum formula that could increase the number of plantlet (Van Bruggen, 1987).
Liquid shake cultures are proven extremely successful in plant tissue culture propagation (Tewary and Oka, 1999; Han et al., 2004; Kadota and Niimi, 2004). According to Piatczak et al. (2005), the fast proliferation of shoots in liquid medium is due to the fact that the shoots were totally immersed in the liquid medium presenting a large surface area for the uptake of nutrients and plant growth regulators. Higher shoot proliferation in liquid medium was also reported for Dioscorea japonica (Kadota and Nimii, 2004).
Flowers were initiated on MS medium with all combinations of cytokinin (BAP) and auxin (NAA), however the flowering shoots were found on combination of 1 mg/l BAP with 0.2 mg/l NAA.(Table 6 and Figure 4). Mostly BAP has been used to induce in vitro flowering (Wang et al., 2002; Saritha and Naidu, 2007; Jana and Shekhawat, 2011). Phytohormones play a major role in the process of flowering by bringing about changes such as initiation of mitosis and regeneration of cell division and organ formation by cytokinin (Tylor et al., 2005; Rathore et al., 2014). Synergic combination of both auxin and cytokinin was found finest for flowering. Combination of auxin and cytokinin was alone used to induce flowering in Withania somnifera (Saritha and Naidu, 2007) and Cleome viscosa (Rathore N.S. et al., 2014). All concentrations of NAA possess rooting after flowering. L. antipoda (L.) was amphibious types of aquatic plants; hence polythene propagator system was recommended (Yapabandara et al., 2006). 100% survival rate was obtained in polythene propagator system. Then the plants were successfully transplanted in to aquarium.
In conclusion, in the present work we successfully developed a suitable micropropagation protocol for large scale multiplication of aquarium plants L. antiopoda (L.) Alston. The best medium for shoots multiplication and root proliferation was MS media supplemented 1mg/l BAP and 0.2mg/l NAA. Shoot multiplication and in vitro flowering was established in MS media supplemented with 1mg/l BAP & 0.2 mg/l NAA (BM3). The results of the study delineate successful in vitro proliferation of aquarium ornamental plant L. antiopoda (L.) Alston.
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