Seedling production in the industrially important agarophyte Gracilaria dura (Gracilariales, Rhodophyta) K. R. Saminathan, K. S. Ashok, V. Veeragurunathan & Vaibhav A. Mantri Journal of Applied Phycology ISSN 0921-8971 Volume 27 Number 4 J Appl Phycol (2015) 27:1541-1548 DOI 10.1007/s10811-014-0450-z
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Author's personal copy J Appl Phycol (2015) 27:1541–1548 DOI 10.1007/s10811-014-0450-z
Seedling production in the industrially important agarophyte Gracilaria dura (Gracilariales, Rhodophyta) K. R. Saminathan & K. S. Ashok & V. Veeragurunathan & Vaibhav A. Mantri
Received: 16 April 2014 / Revised and accepted: 26 October 2014 / Published online: 6 November 2014 # Springer Science+Business Media Dordrecht 2014
Abstract The commercial exploitation of seaweeds largely depends on success of consistent biomass production. The present study demonstrates technical feasibility for rapid production of viable seedlings clonally through selective propagation of apical fragments of Gracilaria dura. The highest regeneration was recorded at 25 psu salinity, 5 μmol photons m−2 s−1 irradiance and 25 °C where it ranged between 80.83± 3.95 and 28.77±2.03 %. The percentage growth in vertical polythene-tube-column culture was ranged between 40.298± 27.25 and 55.687±19.39 % and found to be effective in producing large numbers of seedlings. These seedlings showed fast acclimation to outdoor culture and recorded growth rates of 1.37 to 2.25 % day−1 after 1 month. The seedlings grew rapidly when outplanted in open sea with growth rates of 3.06–3.29 % day−1, comparable to commercial farming of Kappaphycus alvarezii along the southeast coast of India. The average length of harvested plants was found to be 7.71±0.89 cm, while fresh weight was 34.60±9.81 g. Further study to understand seasonal variation in regeneration and growth is imperative to scale-up the methodology for yearround production.
Keywords Aquaculture . Clonal propagation . Daily growth rate . Seedling production . Outplanting Electronic supplementary material The online version of this article (doi:10.1007/s10811-014-0450-z) contains supplementary material, which is available to authorized users. K. R. Saminathan : K. S. Ashok : V. Veeragurunathan : V. A. Mantri (*) CSIR-Central Salt & Marine Chemicals Research Institute, Marine Algal Research Station , Mandapam Camp 623 519, India e-mail:
[email protected] V. Veeragurunathan : V. A. Mantri Academy of Scientific and Innovative Research (AcSIR), CSIR, New Delhi 110001, India
Introduction A recent technical report of FAO (2013) reported a global increase in seaweed production from about 4 million wet tonnes in 1980 to over 20 million wet tonnes in 2010. Nevertheless, it also highlighted encouraging trend in biomass augmentation through aquaculture over wild harvest, of which red seaweeds account for ca. 47 %. The worldwide annual consumption of agarophytes stands at 72,300 dry tonnes to produce 9,600 t of agar with a market value of US$173 million (Bixler and Porse 2011). Gracilaria alone accounts for over 80 % of the harvest of commercial agarophytes, the bulk of which comes through artificial cultivation. The continuous supply of seed material is a determining step for successful mariculture program. The organogenetic capacity of red seaweeds has been successfully utilized to develop micropropagation techniques aimed at artificial seed stock production for aquaculture (Iima et al. 1995; Collantes et al. 2004; Titlyanov et al. 2006a, b). Furthermore, development of protocols for clonal propagation is crucial towards achieving large-scale production of seedlings. These methods are now available for several species of Gracilaria, namely Gracilaria papenfussii (Polne-Fuller and Gibor 1987), Gracilaria verrucosa (Kaczyna and Megnet 1993), Gracilaria vermiculophylla (Yokoya et al. 1993), Gracilaria chilensis (Collantes and Melo 1995), Gracilaria textorii, Gracilaria acuminata (Huang and Fujita 1997), Gracilaria corticata (Kumar et al. 2007), and Gracilaria changii (Young et al. 2014). Further, it is evident that seedling production is considerably influenced by several environmental factors. Hernández-González et al. (2007) have reported that the germling production of Gigartina skottsbergii is enhanced by manipulating temperature, irradiance, and nutrient supply. Therefore, standardizing key parameters promoting regeneration and growth is imperative.
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Commercial seaweed farming is being promoted in India as an alternative livelihood for coastal fisher folk (Krishnan and Narayanakumar 2013). This was achieved primarily due to the indigenous market available for various industrial products derived from Kappaphycus alvarezii through our research efforts such as liquid seaweed fertilizer (Eswaran et al. 2004), K-rich salt (Ghosh et al. 2007), and carrageenanbased biodegradable thin films (Ghosh et al. 2006). Similarly, an innovative, cost effective, and green method that has been developed to obtain high quality agarose from dry algal biomass of Gracilaria dura (Siddhanta et al. 2005; Meena et al. 2014) has attracted industrial attention. Despite its industrial utility, scanty biomass, restricted distribution, and short life-span are major impediments to achieve viable cultivation practice (Gupta et al. 2011). The further improvement in aquaculture would depend on obtaining a continuous and reliable supply of quality seed material along with backward integration of viable culture protocol giving impetus to commercial operations. The initial attempts to produce viable germlings from carpospores of this alga were successful but have limitations in commercial operations due to considerable spore mortality as well as temporal availability of reproductive fronds (Mantri et al. 2009). Thus, a new approach to develop a practical method for production of seedling through clonal propagation is desirable. The present study is based on the assumption that fragments obtained from apical tips are suitable to generate quick seed stock because of their fast growth and adoptability to outdoor culture and outplanting. The methods described herein are the first steps towards establishment of successful artificial cultivation technique for G. dura.
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readily produced buds from cut ends within a week, which were used for plantlet production.
Material and methods
Seedling production and standardization of culture conditions About 1 mm apical tips were cut from several fragments under dissecting microscope by sterile razor blade. The tips were washed several times by autoclaved seawater. The fragments of variable size were obtained after chopping these tips on sterilized glass slide. Each fragment contained enormous amount of growing cells. These fragments were then cultured initially in 20 mL sterile seawater (SSW) for 1 week and subsequently in same amount of PES medium in petri plates. The cultures were kept under above described conditions with occasional shaking twice a day, and periodic observations were made under dissecting microscope. The photographic documentation at the appropriate stage was made using digital camera. The medium was replenished once in a week. Standardization of the culture conditions was achieved by studying new shoots emerging under differential salinities (20, 25, and 30 psu); irradiances (5, 15, and 25 μmol photons m−2 s−1) and temperatures (15, 20, 25, and 30 °C) with a 12-h light and 12-h dark photoperiod. Each parameter was standardized separately; standardization of salinity was carried out first followed by light irradiance and finally temperature. The parameter where maximum regeneration was recorded was kept constant during the next experiment. The fragments were observed randomly from each petri plates under a stereo-zoom microscope to ascertain regeneration at the end of 2 weeks. Ten microscopic views were used to calculate regeneration rate for each plate, while the experiments were carried out for each treatment in triplicate (n=3). New shoot formation was recorded, and average values were expressed as percentage regeneration against total fragments in the microscopic view.
Collection and establishment of unialgal culture The adult thalli of G. dura were collected from the cultivation ground of the Marine Algal Research Station at Thonithurai (09° 16.92′ N 079° 11.40′E), Gulf of Mannar coast near Mandapam, India. The unialgal culture was established as described earlier (Mantri et al. 2009). Briefly, the thalli were brushed to remove the adhering dirt and then fragmented into small pieces. They were further treated with 0.5 % liquid detergent in sterilized seawater (SSW) for 60 s, 1 % povidone-iodine (5 %w/v iodine) for 30 s to reduce bacterial load, eliminate zooplankton and ciliates. They were washed repeatedly with SSW and cultured in 250-mL conical flasks using PES medium (Provasoli 1968) for 1 month. The incubation was carried out at 25±1 °C under daylight white fluorescent lamps at 15 μmol photons m−2 s−1 irradiance with a 12-h light and 12-h dark photoperiod in a Multi Thermo Incubator (MTI202, Eyela, Japan); 600 μL GeO2 [20 μg mL−1] was added to each culture flask to eliminate diatom growth. The fragments
Ve r t i c a l c o l u m n c u l t u re t o a c c e l e r a t e s e e d l i n g growth Seedlings of ca. 5 mm were cultured in vertically placed polythene-tube-columns (henceforth referred as vertical columns) using l L PES medium (25 °C, 15 μmol photons m−2 s−1, and 30 psu salinity) for 15 days. The light intensity was maintained at 15 μmol photons m−2 s−1 irradiance using day light cool white fluorescent lamps with 12-h light 12-h dark photoperiod. In all 10–15 seedlings were cultured in each vertical column. These seedlings were constantly kept under motion using aeration provided from the bottom of the tube. The baggage tie was used in invert position to hold the culture tubes vertically, which enabled repeated use of baggage tie in a costeffective manner (Supplementary material, Figs. 1, 2, 3, and 4). The medium was replenished at weekly interval, and the specific as well as relative growth rate and percent growth in terms of biomass growth was calculated at the end 15 days culture. The specific growth rate (SGR) % day -1 was calculated using the formula ln W2 −ln W1/t×100; while relative growth rate (RGR)
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g day-1 was calculted using the formula ln W2 −ln W1/; where W2 is the final fresh weight in grams, W1 is the initial fresh weight in grams, t is the number of culture days, and ln is the Napierian logarithm. Based on the SGR, the prediction in weight gain after 90 days has been computed using geometric progression theory as described by Yong et al. (2013). Formula=W1 ×(RGR/100+1)t where W1 is the initial fresh weight in gram and t is the number of culture days, and RGR is relative growth rate. The vertical column culture was achieved at 54 tubes scale. However, growth data was obtained from 10 tubes (n=10) of three independent experiments. Outdoor seedling culture and outplanting The seedlings after reaching about 1–1.2 cm length were transferred to outdoor tank culture. The plastic tanks (35 L capacity) having white internal surface were used to cultivate seedlings for 4 weeks (Supplementary material 5, 6, and 7). Continuous aeration was provided, and 2/3 seawater was replenished daily with no external addition of nutrients. The aeration tube was installed centrally, which enable the seedlings to tumble by upward water currents. Two separate tanks were used during these experiments (n=2), and about 150–200 fragments were inoculated. Each cycle of cultivation was carried out for 30 days. Ten individual fragments from each tank were measured to record increase in fragment length and daily growth rate based on fresh weight gain in the biomass. The experiment was performed for three subsequent batches in series. The abiotic factors such as irradiance, salinity, temperature, and pH were recorded every alternate day throughout the experiment, and the range was provided in Table 1. About 200–300 seedlings grown in the outdoor culture tanks were transported under cool conditions to the cultivation ground of the Marine Algal Research Station at Thonithurai, near Mandapam to evaluate their growth performance in open sea. The seedlings were enclosed in perforated polythene bags and covered in nylon net bags to avoid loss by grazing and wave action. The cleaning of net bags was carried out periodically to avoid siltation and epiphyte infestation. After 2 weeks of initial culture in perforated bags, they were grown in open sea on polythene ropes for 2 more weeks before raft culture was undertaken.
Table 1 Abiotic factors registered during outdoor cultivation of Gracilaria dura Parameter
Value
Photon flux density inside the tank Salinity Air temperature Seawater temperature pH
4–63 μmol photons m−2 s−1 32–36 psu 26.73–30.63 °C 25.9–29.23 °C 8.0–8.32
Cultivation of seedlings on floating rafts The open sea cultivation was carried out using square bamboo raft (2×2 m) as described by Ganesan et al. (2011). Briefly, the outplanted seedlings were grown about 4–5 cm in height—comprising of 730 g total biomass—were used as initial seed material. Four to five seedlings were tied together with nylon rope and inserted in 3 mm polypropylene rope, 25 such bunches were seeded and 20 ropes were tied per raft (Fig. 3b, c). The raft was covered with fishnet and agronet to avoid grazing and drifting of plants. The whole assembly was anchored in deep waters by a pair of stones using 6 mm polypropylene rope. The harvest was made after 8 weeks (57 days). The daily growth rate (DGR) was calculated using the formula given by Ganesan et al. (2011). DGR % day-1 =ln (W2/W1)/t×100, where W2 is the final fresh weight in gram, W1 is the initial fresh weight in gram, t is the number of culture days, and ln is the Napierian logarithm. After harvesting, 50 untagged plants were randomly selected to determine the average length and fresh weight of the cultivated plants.
Results Establishment of unialgal culture, seedling production, and standardization of culture conditions Incubation in SSW rather than PES along with various treatments given prior to establishment of algal culture was effective in obtaining clean cultures. The treatment with liquid detergent (0.5 % for 10 min) and iodine (1 % for 30 s) stopped degradation of algal tissue as a result of bacterial growth and reduced zooplankton as well as ciliates. The GeO2 was detrimental for diatom growth. Buds were produced from the apical end of cut fragments within 3 to 4 weeks of culture. The apical fragments obtained by chopping the buds varied in shape and size (Fig. 1a). In static culture, fragments tended to settle at the bottom of the petri plates and gentle shaking further accelerated their growth. The pretreatment of algal tissue also considerably reduced the contamination by fouling organisms, which is one of the major constrains in seedling production. The fast growth was observed in all fragments after 4 weeks of culture in PES medium (Fig. 1b). Differences in regeneration were recorded under varying salinity (20– 30 psu), temperature (15–30 °C), and irradiance (5–25 μmol photons m−2 s−1) [Fig. 2a–c]. The standardization of culture conditions for effective regeneration revealed that 25 psu salinity (75.67±7.47 %), 5 μmol photons m−2 s−1 irradiance (79.52±3.15 %), and 25 °C temperature (80.83±3.95 %) at a 12:12 h light/dark photoperiod was optimal to obtain the maximum regeneration of apical fragments under in vitro culture.
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Fig. 1 a Clonal propagation from apical tissue (scale bar=500 μm), b Regeneration of new thallus (scale bar=300 μm)
a
b
after 30 days of culture in outdoor tanks in all the three batches, while non-aerated outdoor cultures showed mortality as well as retarded growth (data not shown). The average increase in seedling length of 20 individuals ranged from 45 to 53 % during outdoor culture, while the daily growth rate varied between 1.37 and 2.25 % day−1. The highest growth was observed in the second batch of seedlings transferred. The perforated polythene bags together with use of agronet provided protection to seedlings from herbivores, ensured adequate exchange of surrounding water for sustainable growth, and restricted drifting of seedlings (Supplementary material Fig. 7). The seedlings produced several new proliferations upon outplanting in the open sea within the first week. Then, more robust growth followed. The biomass of 730 g fresh weight was produced after 6 weeks of culture in perforated polythene bags which corresponds to 3.29 % day −1 DGR. These seedling after initial culture on polythene ropes reached about 2–4 cm in length.
Vertical polythene-tube-column culture Seedlings produced from apical tips have specific temperature, salinity, and light requirements. The larger seedlings tend to grow well between 25–32 psu salinity and 10–15 μmol photons m−2 s−1 (separate experiments were conducted, data not shown). The vertical columns were suitable for cultivation of seedlings suspended in liquid phase (Fig. 3a, Supplementary material, Fig. 4; Table 2). The bubbling at the bottom allowed seedlings to be distributed throughout the column. After 2 weeks of culture, the seedlings reached the size of about 10–15 mm. Among the three sets of experiments, average specific growth rate varied from 1.079±0.586 to 1.453±0.408 % day−1, while relative growth rate was found to be between 0.011±0.006 and 0.015±0.004 %. The percentage growth ranged betwee 40.298 ± 27.25 and 55.687 ± 19.39 %. The average predicted weight gain of the fragments in vertical column culture after 90 days ranged from 0.164± 0.101 to 0.211±0.139 g, which was about 183 to 282 % higher.
Cultivation of seedlings on floating rafts
Outdoor seedling culture and outplanting
The seedlings planted on the monocline into the raft (Fig. 3b) reached harvestable size in 8 weeks and assumed morphology to that of naturally occurring thalli. The thalli were mature, darkly pigmented, and branched while they did not produce any reproductive structures. The use of agronet reduced incidences of epiphytic infestation. The biomass increase was sustainable throughout the raft cultivation, and 4192 g fresh wt was harvested after about 8 weeks of culture (57 days),
The seedlings transfered in the outdoor culture tanks (Supplementary material Fig. 6; Table 3) quickly increased in size and weight. The continuous aeration and replanishing of 2/3 seawater daily had positive effects on growth and acclimatization of G. dura seedlings in outdoor conditions. All the laboratory-generated seedlings were able to survive
b
c 90
90 80 70 60 50 40 30 20 10 0
80 70
% Regeneration
% Regeneration
% Regeneration
a
60 50 40 30 20 10 0
20
25
Salinity (psu)
30
5
15
25
Irradiance (µmol photons m–2 s–1)
Fig. 2 Standardization of culture conditions for regeneration of apical fragment (n=3). a Differential response to various salinities. b Differential response to various light irradiances (salinity kept constant at 25 psu).
90 80 70 60 50 40 30 20 10 0
15
20
25
30
Temperature (0C)
c Differential response to various temperatures (salinity and irradiance kept constant at 25 psu and 5 μmol photons m−2 s−1, respectively)
Author's personal copy J Appl Phycol (2015) 27:1541–1548 Fig 3 a Vertical polythene-tubecolumn culture (scale bar= 15 cm). b Initial seeding by tie-tie method on polythene ropes (scale bar=1 cm). c Floating bamboo raft method adopted for open sea cultivation (scale bar=25 cm). d Fully grown plants after 45 days of culture cycle (scale bar= 1.5 cm)
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a
b
c
d
with a DGR corresponding to 3.06 % day−1 day (Fig. 3c). The average length of harvested plants was found to be 7.71± 0.89 cm, while fresh weight was 34.60±9.81 g. The individual seedlings grew over 50 g in some cases (Fig. 3d, Supplementary material, Fig. 7). The drift accumulation over the raft surface was also avoided due to the agronet covering.
Discussion The year-round supply of adequate and viable seedlings is pivotal in establishing commercial cultivation. However, restricted distribution of G. dura along the Saurashtra and Kanyakumari coasts coupled with seasonality has limited exploitation for industrial utilization (Mantri 2009). To the best of our knowledge, this is the first report of clonal seedling production in G. dura. Elimination of contamination is a persistent issue at nursery level (Holdt et al. 2014). However, use of locally available chemicals such as liquid detergent, povidone-iodine, and GeO2 alone ensured production of clean unialgal cultures in the present investigation. Alternatively, treatment with sodium hypochlorite (0.2 g L−1) for 2 min also has been suggested (Holdt et al. 2014). The use of antibiotics has been reported to slow down regeneration and, therefore, was not used in the present study. The chemical treatment greatly helped in reducing contamination in initial culture thus resulting in achieving higher regeneration. The higher regeneration of 80.83±3.95 % was recorded at 25 °C, 25 psu salinity, and 5 μmol photons m−2 s−1 irradiance. This was more than regeneration from protoplasts of Gracilaria gracilis (Huddy et al. 2013) and carpospores of
G. dura (Mantri et al. 2009) under similar culture conditions. The higher regeneration reported here could be attributed to higher revival capacity of vegetative cells in apical region of the thallus. The totipotency of cells from the apical region could be utilized for obtaining maximum number of propagules per donor plant, which has been hitherto explored only in Gelidium sp., Palmaria palmata (Titlyanov et al. 2006a, b), and G. chilensis (Collantes et al. 2004). The regeneration recorded in this study was more than G. changii (47.62± 26.51 %), while in the latter, the medium was supplemented with 10 mM L−1 K2SO4 along with 10 mg L−1 2,4-D and 0.001 mg L−1 kinetin (Yong et al. 2013). Kumar et al. (2007)) reported 40–60 % regeneration in G. corticata at 30 μmol photons m−2 s−1 irradiance; however, plants at 5 μmol photon m−1 s−1 did not show any regeneration. This study also suggested that dim light promoted quick healing and better survival in this species. It corroborated well with observations made in previous studies (Hernández-González et al. 2007; Holdt et al. 2014). It would further help in nursery management for this alga where maintenance of stock can be carried out in dim light while high light can be provided for promoting growth. The use of unenriched seawater for initial culture also allowed rapid healing as reported for G. skottsbergii (Hernández-González et al. 2007), while addition of nutrients after 1 week enhanced vegetative growth. It is clear that asexual propagation in red algae can lead to reduced genetic diversity; consequently, repeated use of single clone might drastically retard growth in commercial farms (Oliveira et al. 2000; Guillemin et al. 2008). However, selecting fast growing, disease resistant strains from chimeric populations (Santelices 2001) among different life cycle stages (Gupta et al. 2011) can lead to improvement in the cultivar. The method described here can achieve mass
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Table 2 Vertical polythene-tube-column culture Date
Initial weight (g)
Final weight (g)
Growth (%)
Relative growth rate (g day−1)
Specific growth rate (% day−1)
Predicted weight after 90 days (g)
1st setup 17.9.13 20.9.13 21.9.13 22.9.13 23.9.13 24.9.13 25.9.13 26.9.13
0.094 0.083 0.063 0.059 0.049 0.057 0.058 0.045
0.108 0.114 0.087 0.084 0.102 0.075 0.076 0.059
14.403 37.576 36.759 42.797 107.143 31.179 30.472 29.834
0.004 0.011 0.010 0.012 0.024 0.009 0.009 0.009
0.449 1.063 1.043 1.188 2.427 0.905 0.887 0.870
0.141 0.214 0.161 0.171 0.424 0.129 0.129 0.099
0.042 0.028 0.058±0.019
0.047 0.044 0.080±0.024
11.905 60.909 40.298±27.25
0.004 0.016 0.011±0.006
0.375 1.586 1.079±0.586
0.059 0.113 0.164±0.101
0.107 0.104 0.084 0.087 0.057 0.072 0.057 0.036 0.054 0.036 0.069±0.026
0.116 0.141 0.099 0.092 0.072 0.08 0.121 0.077 0.073 0.07 0.094±0.024
7.925 35.337 18.263 5.747 26.316 10.519 112.335 113.889 35.514 94.444 46.029±43.540
0.003 0.010 0.006 0.002 0.008 0.003 0.025 0.025 0.010 0.022 0.011±0.009
0.254 1.009 0.559 0.186 0.779 0.333 2.510 2.534 1.013 2.217 1.139±0.933
0.135 0.257 0.138 0.103 0.115 0.097 0.528 0.342 0.133 0.259 0.211±0.139
0.048 0.052
0.072 0.071
51.681 34.890
0.014 0.010
1.389 0.998
0.165 0.128
0.060 0.053 0.045 0.052 0.055 0.045 0.057 0.054 0.052±0.005
0.079 0.090 0.087 0.073 0.091 0.075 0.086 0.081 0.081±0.008
30.000 71.780 93.548 39.924 67.093 67.634 51.096 49.221 55.687±19.39
0.009 0.018 0.022 0.011 0.017 0.017 0.014 0.013 0.015±0.004
0.875 1.803 2.201 1.120 1.711 1.722 1.376 1.334 1.453±0.408
0.132 0.264 0.319 0.143 0.252 0.208 0.195 0.180 0.199±0.063
27.9.13 28.9.13 Average±SD 2nd setup 3.10.13 5.10.13 7.10.13 9.10.13 10.10.13 11.10.13 12.10.13 14.10.13 15.10.13 17.10.13 Average±SD 3rd setup 10.1.14 11.1.14 12.1.14 13.1.14 14.1.14 15.1.14 16.1.14 17.1.14 18.1.14 19.1.14 Average±SD
production of elite germplasm of desired trait and consistent ploidy to cater for the needs of commercial aquaculture. Table 3 Outdoor tank culture Month
Average length increase (%)
DGR (% day-1)
First batch Second batch Third batch
45 (±0.41) 39 (±0.46) 53 (±0.39)
1.37 (±0.19) 2.25 (±0.14) 1.96 (±0.70)
Furthermore, the growth reported in the vertical column culture was similar to that of Chondrus crispus cultured in a bubble column photobioreactor (Holdt et al. 2014). The higher growth rate encountered can be attributed to the fact that clonal propagation allows only vegetate development, inhibiting reproductive development. Despite seedling production being achieved in Gelidium sp. and Palmaria palmata from meristematic tissue (Titlyanov et al. 2006a, b), involvement of a freeze–thawing protocol makes this technique cumbersome. The use of an airlift
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photobioreactor for explants as well as tissue culture of G. changii was recommended for mass production of seedlings (Yong et al. 2013). Alternatively, isolation of protoplasts from G. dura as a seed stock for aquaculture was described recently (Gupta et al. 2011). Nevertheless, cost and technical skills involved in such techniques for commercial production of seedlings are yet to be validated. Furthermore, these studies did not attempt outplanting to establish overall reliability for field cultivation. The spore-based seedling production was proved successful in several species of Gracilaria (Oliveira et al. 2000). However, spore culture methods are more relevant to strain selection, breeding, and restoring vigor in event of senescence than year-round seedling production due to inherent seasonality barrier in life cycle phases, high mortality of microscopic stages, and slow growth. Further, reproductive methods need extra energy cost for production and maintenance of gametes and spores (Halling et al. 2005). The outdoor culture of G. dura was useful in acclimatizing seedlings prior to their transplantation in the open sea. The growth rates (1.37 to 2.25 %) recorded in this study were comparable with outdoor culture of Gracilaria edulis (1.6 – 4.3 %) and Gracilaria arcuata (2.71 %), where in the former inorganic fertilizer (Kaliaperumal et al. 2003), and in the latter fish effluent, was used (Al-Hafedh et al. 2012). Cirik et al. (2010) reported 1.21±0.34 to 4.03±1.63 % daily growth rate for G. verrucosa in Johnson-modified medium in outdoor tanks. Although unenriched seawater was used in the present study for outdoor cultivation, nutrient formulation and flux, frequency, and time of application are matters of future investigation (Hernández-González et al. 2007). The use of perforated polythene bags for outplanting avoided drifting and grazing. Furthermore, we propose use of these seedlings for densifying the natural stock to augment biomass which is currently dismal for industrial utilization. The commercial cultivation of G. chilensis is being undertaken by suspended mono-lines and bottom planting (Buschmann et al. 1995). The bottom planting methods recorded a decrease in yield after a few harvests presumably due to repeated removal of apical meristem and associated factors (Halling et al. 2005). However, floating rafts have ease in seeding, maintenance, and harvesting and was proven successful in G. edulis (Ganesan et al. 2011). The raft culture accompanied with net convening also avoids problems such as siltation, grazing, accumulation of drift, etc. Further, the seedlings grown in the floating rafts have access to higher nutrients and dissolved oxygen due to continuous surf action at the sea surface (Mantri et al. 2009). The management of accumulated drift, higher cost, and exposure of seedlings to higher light and elevated temperature are some of the bottlenecks experienced in the raft culture of G. edulis (Ganesan et al. 2011). Moreover, the raft culture is not advisable in sites where wave action is intense. The daily growth rate of outplanted seedlings in open sea conditions (3.06–3.29 %)
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was similar to our earlier study where laboratory-generated tetrasporophytes were outplanted in the sea (Mantri et al. 2009). Bezerra and Marinho-Soriano (2010) reported comparable relative growth rates (4.3–4.4 %) in Gracilaria birdiae, whereas, daily growth rate was 3.6–5.9 % in G. edulis in open sea (Ganesan et al. 2011). The growth rates reported in our previous as well as this study corroborated with those reported in commercial cultivation of K. alvarezii (Periyasami et al. 2014). From the foregoing, it is evident that rapid and needbased mass production of seedling via clonal propagation of apical cells could be achieved in G. dura. These seedlings could be subsequently cultured in outdoor tanks (nursery culture) and outplanted (open sea cultivation). Thus, this alga can be farmed along with K. alvarezii by the raft method along the southeast coast as an additional aquaculture species. This will bring employment opportunities and improve the livelihood of the coastal rural population. We consider that further study to understand seasonal variation in regeneration, growth, and cultivation would help to scale-up the methodology for year-round production. Further, we intend to test different cultivation methods such as single rope floating raft technique, net culture, long line method, etc. to test their efficacy for large-scale commercial farming. The immediate research priorities include optimization of cultivation parameters along with harvesting strategies. The improvement in raft design to withstand rough-sea conditions would bring more area under cultivation as it can be undertaken in subtidal and offshore waters also. Acknowledgments The financial assistance from Council of Scientific and Industrial Research, New Delhi is greatly acknowledged. The initial funding for clonal propagation was received from the Department of Biotechnology, New Delhi (BT/PR/6266/AAQ/03/251/2005). This work has been undertaken as a part of an in-house project entitled “Scaled-up farming for technology demonstration of promising seaweeds.” We would like to thank anonymous referees and Prof. Borowitzka for constructive comments. Thanks are due to Dr. K. Eswaran and Dr. C. R. K. Reddy for discussion on cultivation and clonal propagation, respectively. This contribution has CSIR-CSMCRI PRIS Registration No.: 062/2014.
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