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УДК 502.753, 57.022
Juan-Vicedo J., Casas-Martínez J. L.
MICROPROPAGATION OF ECONOMICALLY IMPORTANT, WILD AND ENDEMIC SPECIES OF LAPIEDRA AND NARCISSUS (AMARYLLIDACEAE) AS A BASES FOR THEIR CONSERVATION AND INDUSTRIAL PRODUCTION IN SPAIN
Jorge Juan-Vicedo, PhD, Research Collaborator, Laboratory of Plant Biotechnology, Research Institute CIBIO, University of Alicante, Spain: [email protected];
Jose Luis Casas-Martínez, PhD, Associate Professor, Laboratory of Plant Biotechnology, Research Institute CIBIO, University of Alicante, Spain
A protocol for bulb micropropagation has been obtainedfor 11 endemic, rare, threaten and/or economically important Amaryllidaceae species from Spain. The results show that this approach is suitable for propagation of these 11 species. However, some species .showed limitations in the multiplication phase and this protocol should be optimized in case of commercial-scale propagation.
Keywords: Amaryllidaceae, micropropagation, Lapiedra, Narcissus, in vitro cultures, germplasm conservation.
The Amaryllidaceae family has a high economical potential both for the chemical pharmaceutical industry as well as the ornamental plants market (Hanks, 2002). It is well represented in Spain, where both the genus Narcissus and Lapiedra find their center of origin and, in the case of Narcissus, its center of diversification too (Bolos and Vigo, 2001; Rivera et al., 2006). The genus Lapiedra Lag. has only one species described in the world: Lapiedra martinezii Lag. It is a small, late summer - autumn flowering geophyte that grows in thermophilous and semiarid plant communities dominated or co-dominated by the dwarf palm Chamaerops humilis L., Osyris lanceolata Hochst. & Steud. and Maytenus senegalensis (Lam.) Exell, in several types of Mediterranean macchias, and in woodlands with Pistacia lentiscus L. and Quercus coccifera L. Also, it is found in vertical rocky slopes, crevices and coastal cliffs ranging from the sea level up to 860 m.a.s.l. It is a Baetic-Moroccan endemism distributed in the South-Western Mediterranean, but most of its populations are placed in Spain, whereas in North Africa a punctual citation was done in Morocco (Bolos and Vigo, 2001). As far as Narcissus concern, it displays a huge diversity, estimated in more than 150 taxa only in Spain. These species use to grow in high mountains, wet grasslands, riverbeds and shady environments (Hanks, 2002; Rivera et al., 2006). The huge diversification in species, subspecies and wild forms and varieties makes Narcissus one of the most interesting mega-diverse genera in the world. From the economical point of view, Narcissus has an undoubted value as ornamental plant (Rivera et al., 2006). In addition, new varieties and wild species and forms are appreciated for fine collectors and breeders: this could be the reason why it is easy to find very rare materials, and even endemic or threaten species (including Lapiedra) over the internet in forums, gardening webpages and different online shops sold as seed or bulb materials at high prices in comparison to the conventional gardening varieties. However, the most promising economical interest lies on the richness in bioactive alkaloids that ca be exploited in the chemical industry under different preparations (reagents,
pesticides, etc.) and in the pharmaceutical industry, especially those materials rich in galanthamine: an appreciated alkaloid with acetylcholinesterase activity employed to treat Alzheimer's disease (Hanks, 2002). Finally, several Amaryllidaceae have been recognized as endemic, rare and/or threatened in the national checklist of threatened plants (Moreno et al., 2008) and in most of the regional ones not only in Spain, but also in Portugal, France and Italy. As a result, an international strategy its being managed by the International Union for the Conservation of Nature (IUCN) for the conservation of these species at a Mediterranean level.
In vitro culture techniques can be valuable tools for both the ex situ conservation of endemic, rare, and threatened species as well as an important tool for mass-propagation of selected genotypes to be used as novel industrial crops or cell factories producing certain target chemical compounds. Among these culture techniques, micropropagation is the true-to-type propagation of a selected genotype using in vitro techniques (Debergh and Read, 1991; Werbrouck and Debergh, 1996). The use of the available in vitro techniques (such as micropropagation) is a suitable strategy for both commercial mass-propagation (Winkelmann et al, 2006) and/or conservation of plant germplasm (Tasheva and Kosturkova, 2013). These techniques can be employed for the conservation of genetic resources such as threatened plants, species with recalcitrant seeds or with vegetative propagation, and of plant genotypes of outstanding interest such as their attractive structures or colours for gardening or the production of secondary metabolites (Tasheva and Kosturkova, 2013). For all these reasons, the development of usual biotechnological approaches (e.g. in vitro cell cultures) constitute an interesting tool in order to manage these plants fordifferent purposes.
However, in spite of the huge amount of scientific literature on application of biotechnological approaches for mass propagation of commercial cultivars, the information on wild (rare, endemic or threatened) species is almost lacking for all Amaryllidaceae in general. The huge diversity of this family sometimes implies a difficult management for ex-situ conservation
as the routines like germplasm collection, management and propagation are a very time consuming and expensive tasks. This is especially important when dealing with micropropagation procedures. Moreover, bulbous plants present some additional difficulties that collectively make quite challenging to obtain a reliable protocol even for one single species. Therefore, our aim was to obtain a micropropagation protocol that could be used for more than one single species.
Whole plants and seeds were collected form wild locations and maintained in garden culture conditions during one season before starting experiments. The experiments were performed during the vegetative period, just before blossoming. Three different types of explants were used for initiation of in vitro cultures: adult bulbs, seeds and seedlings. In Lapiedra martinezii we also tested young fruits and flower stalks as initiation explants. In vitro cultures were initiated sowing 150 replicates of explants of each species (previously sterilized) into test tubes containing 30 mL of initiation medium. The multiplication medium was prepared with basal MS+vitamins, supplied with 3% of sucrose and 5,5 g/L 'Plant Agar'. A standard combination of growth regulators was tested attending to the results of the previous works. The cultures were daily checked and the yield of the explants was recorded after two months of cultivation at 26°C in a 16-hours light/8-hours dark photoperiod (photosynthetically active radiation 42 l^mol m-2s-1).
Initiation
The effect of a cold pre-treatment on initiation of in vitro cultures was assessed by exposure of bulbs to 9°C during 4 weeks in a cold chamber in darkness. Afterwards, treated and non-treated bulbs were sterilized and cut into sections and dipped in liquid medium containing % Murashigue and Skoog medium and 300 ppm of sodium dichloroisocyanurate (NaDCC) for 48 hours. Finally, scales were dissected in pieces of approximately 7 x 7 mm containing about 2 mm of the bottom plate in order to obtain the tween scales. In all cases, 24 explants were transferred into test tubes containing full strenght MS salts supplemented with 5 g of activated charcoal and solidified with 5.5 g 'Plant Agar (Duchefa, The Netherlands) and cultured for four weeks. Cultures were incubated under fluorescent light of 50 ^mol m 2 s 1 for 16 h per day and at 25 ± 1°C. In all cases, 15 mL of medium were taken in test tubes.
Media were sterilized at 121°C and 1 atm pressure for 20 minutes. The experiment of the effect of a cold pre-treatment was carried out in N. rupicola, N. cantrabricus, N. hedraeanthus, N. alcaracensisand N. yepesii.
Regeneration of bulblets (Multiplication)
An experiment was conducted to test the effect of a multiplication medium on regeneration of bulblets of 11 species (Table 1). The medium employed was composed by MS containing 30 g of sucrose, supplemented with 10 ^M BA plus 5 ^M NAA and solidified with 5.5 g Plant Agar. Medium preparation and culture conditions were the same as explained above and culture period was extended up to 8 weeks and number of shoots produced per explant was registered.
Rooting and Acclimatization
Bulblets harvested from 8-week-old cultures that were more than 10 mm in diameter were transferred to different media in order to promote rooting in vitro before transplantation to ex vitro conditions. The rooting medium were composed by MS salts, 30 g/L sucrose and solidified with 5.5 g/L agar. Media were supplemented with 10 ^M BA plus 5 ^M NAA. Rooted bulblets were planted into potting mixture of peat moss and vermiculite (1:1) and placed in acclimatization chamber under 24±1°C and 100% of relative humidity (HR) in a 16-hours light/8-hours dark photoperiod (photosynthetically active radiation 42 ^mol m-2s-1). These conditions were maintained during one month. Afterwards, HR was reduced to 70% and the light intensity was increased up to 80 ^mol m-2s-1. The plants were maintained under these conditions one more month before transference to outdoor conditions.
Initiation of in vitro cultures
Among all the tested explants, only scales obtained from bulbs gave some positive results. However, it is remarkable the high contamination rates achieved during the first 3 weeks after inoculation in initiation media for all samples. Exposure to a 4-weeks of cold treatment had a highly significant positive effect (p<0.0001) on explant survival during the initiation phase for all the species tested in comparison to the non-treated explants. N. alcaracensis had the best initiation success with a survival rate of 75% explants. The other species showed lower survival rate of 33% (N. cantabricus and N. hedraeanthus) 16.70% (N. rupicola) and 12.5% (N. yepesii).
Table 1. Commercial interests (current or potential) and conservation interests (by means of the condition of endemic, rare and/or threaten plant in Spain) of the 11 studied species of Amaryllidaceae
Species Endemic to Spain Commercial interests Rare plant in Spain Threatened
Lapiedra martinezii NO YES (potential) YES Not
Narcissus alcaracensis S.Rios , D.Rivera , Alcaraz & Obon. YES YES (potential) YES EN
Narcissus bulbocodiumL. NO YES (current) NO Not
Narcissus cantabricus DC. YES YES (current) YES Not
Narcissus confusus Pugsley YES YES (current) YES Not
Narcissus eugeniae Fern.Casas YES YES (potential) YES VU
Narcissus hedraeanthus (Webb & Heldr.) Colmeiro YES YES (potential) YES Not
Narcissus jonquilla L. YES YES (current) NO Not
*Narcissus triandrus L. subsp. pallidulus (Graells) D.A. Webb YES YES (current) NO Not
Narcissus rupicolaDufour exSchult.f.YES YES (current) NO Not
Narcissus yepesii S.Rios , D.Rivera , Alcaraz & Obon. YES YES (potential) YES VU
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Regeneration of bulblets
The in vitro cultured expiants developed small, white, protuberances on the surface of scale sections within the first month of culture. The bulblets were clearly visible by the 5th of 7th weeks and they started developing green leaves after this period. The optimum shoot proliferation in vitro is normally related to a specific balance of cytokinin/auxin. The results here obtained showed that the response of the different species' explants (in terms of multiplication rate) was significantly affected by the concentration of growth regulators used. The multiplication medium tested induced variable response on regeneration of bulblets in the different species tested (Figure 1). For instance, the highest multiplication rates were achieved in N. bulbocdium and N. triandrus subsp. pallidulus that showed yield of 9 new bulbils per explant. This is in accordance with the previous report on wild N. bulbocodium in Portugal (Santos et al., 1998). The lowest rates corresponded to N. cantabricus and N. confusus. Although the yield is not high for commercial purposes is still acceptable in order to maintain a collection of in vitro plants to propagate for further reintroductions.
Number of shoots yielded per expiant
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4
5 2 1 0
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■Niunhcr of shoots yielded per expiant
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Figure 1. New bulbil production yielded per explant after two months of culture in multiplication medium supplemented with 10 ^M BA plus 5 ^M NAA and based on what Santos et al. (1998) Rooting of bulbils was spontaneous within the two months of multiplication for all species, and acclimatization was high (>70 %) during this period for all the species tested. All non-rooted explants were transferred to the rooting medium composed by 10 ^M BA plus 5 ^M NAA and they produced roots after three weeks.
To sum up, the most challenging phase in these species' in vitro cultivation was the sterilization and further culture initiation; once the culture are initiated and growth is restored, multiplication takes place in variable rates depending on the species but, in general, in quite acceptable nevels for all the tested plants. Rooting was spontaneous and subsequently
acclimatization was also performed without considerable loses.
These results provide a good strategy to initiate and maintain an in vitro collection of the above mentioned plants. The relative high propagation rates in most of species allow using this protocol as a common strategy for this plants' micropropagation in ecological restauration and ex situ conservation. However, the multiplication phase should be optimized for commercial purposes in those taxa that performed the lowest yields. In addition, the possibility maintain a stock of in vitro cell, tissue and bulb cultures can provide new material for phytochemical research orientated to the secondary metabolite's (alkaloid) research . In any case, the wide positive response obtained with this protocol makes us think that it can be perfectly applied not only for the studied plants, but also to other Amaryllidaceae.
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