Optimization of Plant Production by Seed Treatment in Two Wild Subspecies of Narcissus Pseudonarcissus Rich in Alkaloids
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molecules Article Optimization of Plant Production by Seed Treatment in Two Wild Subspecies of Narcissus pseudonarcissus Rich in Alkaloids Raquel Herranz 1, Miguel A. Copete 1,2,*, José M. Herranz 1,2, Elena Copete 1 and Pablo Ferrandis 1,2 1 ETSIAM, Department of Plant Production and Agricultural Technology, University of Castilla-La Mancha, University Campus s/n, 02071 Albacete, Spain; [email protected] (R.H.); [email protected] (J.M.H.); [email protected] (E.C.); [email protected] (P.F.) 2 Botanical Institute, University of Castilla-La Mancha, Avenida de la Mancha s/n, 02006 Albacete, Spain * Correspondence: [email protected]; Tel.: +34-967-599-204 Academic Editors: Jaume Bastida and Philippe Jeandet Received: 24 August 2020; Accepted: 25 September 2020; Published: 27 September 2020 Abstract: The daffodil Narcissus pseudonarcissus L. contains alkaloids of pharmaceutical interest. Wild daffodil populations have diverse genetic backgrounds and various genetic traits of possible importance. Developing protocols for plant production from seeds may ensure the availability of a large reservoir of individuals as well as being important for species with bulbs that are difficult to acquire. The closely related Narcissus pseudonarcissus subsp. munozii-garmendiae and subsp. nevadensis were investigated in this study because the alkaloids isolated from both are of high pharmacological interest. At the dispersal time, the seeds of both were dormant with underdeveloped embryos, i.e., morphophysiological dormancy (MPD). Experiments were conducted outdoors and under controlled laboratory conditions. Embryo growth and the percentages of radicle and seedling emergence were calculated under different temperature–light stratifications. In N. munozii-garmendiae, embryo growth occurred during warm stratification (28/14 ◦C or 25/10 ◦C) and the radicle then emerged when the temperature decreased, but the shoot was dormant. In N. nevadensis, the seeds germinated when cold stratified (5 ◦C) and then incubated at cool temperatures. Thus, N. munozii-garmendiae and N. nevadensis exhibit different levels of MPD, i.e., deep simple epicotyl and intermediate complex, respectively. Plant production protocols from seeds were established for both taxa in this study. Keywords: alkaloid; Amaryllidaceae; deep simple epicotyl morphophysiological dormancy; dormancy breakage; embryo growth; germination; intermediate complex morphophysiological dormancy; Narcissus pseudonarcissus L. 1. Introduction Many species of Narcissus (Amaryllidaceae) contain small chemical molecules with various medicinal properties. The pharmaceutical potential of Amaryllidaceae alkaloids including galanthamine, lycorine, haemanthamine, and pancratistatin, is well recognized, primarily as anticancer drugs [1]. Recently, 21 known Amaryllidaceae alkaloids with various structural types and one previously undescribed alkaloid named narcimatuline were isolated from the fresh bulbs of Narcissus pseudonarcissus L. and identified as potential drugs for use in the treatment of Alzheimer’s disease [2]. Many previous studies of alkaloids from the Amaryllidaceae were conducted with ornamental Narcissus cultivars e.g., [2–4] because of their large-scale cultivation for the ornamental plant industry. By contrast, the alkaloids present in wild populations of daffodils have been analyzed less frequently e.g., [5–8]. Wild plant resources have great potential for supplying raw materials to the pharmaceutical Molecules 2020, 25, 4439; doi:10.3390/molecules25194439 www.mdpi.com/journal/molecules Molecules 2020, 25, 4439 2 of 19 industryMolecules but 2020 wild, 25, x subspecies FOR PEER REVIEW are usually unsuitable for agricultural exploitation because of limitations2 of 19 in terms of their availability or a lack of previous analyses. The limitation due to low resource availabilityfrequently can e.g., be [ minimized5–8]. Wild plant by seed resource collections have andgreat sexual potential reproduction for supplying of da rawffodils, material ands althoughto the littlepharmaceutical is known about industry the seed but physiology wild subspecies in Narcissus are usually[9], several unsuitable studies for have agricultural been published exploitation on this subjectbecause in recent of limitations years [10 in– 14terms]. of their availability or a lack of previous analyses. The limitation due to low resource availability can be minimized by seed collection and sexual reproduction of daffodils, In a recent review of Narcissus systematics, Aedo [15] recognized the existence of 33 taxa (subspecies and although little is known about the seed physiology in Narcissus [9], several studies have been level) in the Iberian Peninsula, where 15 are endemic, thereby demonstrating the importance of published on this subject in recent years [10–14]. this geographic area for the evolution of the genus. In the present study, we focused on section In a recent review of Narcissus systematics, Aedo [15] recognized the existence of 33 taxa Pseudonarcissus(subspecies ,level) which in includesthe Iberian nine Peninsula, taxa with where seeds 15 thatare endemic, contain underdevelopedthereby demonstrating embryos the at theimportance dispersal time,of this and geographic thus it area is necessary for the evolution to promote of the embryo genus. In growth the present to facilitate study, we germination. focused Pseudonarcissuson section Pseudonarcissalso exhibitsus, physiologicalwhich includes dormancy nine taxa (PD), with whichseeds mustthat contain be overcome underdeveloped with several monthsembryos of warm at the and dispersal/or cold time stratification., and thus it This is necessary dormancy to classpromote is known embryo as growth morphophysiological to facilitate dormancygermination (MPD). Pseudonarcissus and it is considered also exhibits the most physiological difficult todormancy overcome (PD) [16, ].which Thus, must although be overc allo taxame in thewith section severalPseudonarcissus months ofhave warm a highand/or capacity cold forstratification. vegetative This propagation dormancy from class bulbs, is known germination as of theirmorphophysiological seeds is a complex dormancy problem (MPD) [17]. and Indeed, it is considered both Vandelook the most difficult et al. [10 to] andovercome Newton [16]. et Thus al., [ 11] highlightedalthough theall taxa diffi inculty the section of germinating PseudonarcissusNarcissus have pseudonarcissusa high capacity forseeds. vegetative Furthermore, propagation three from levels of MPDbulbs, havegermination been described of their seed ins section is a complexPseudonarcissus problem [17][12. Indeed–14],, and both thus Vandelook further et detailed al. [10] and study is necessary.Newton et al. [11] highlighted the difficulty of germinating Narcissus pseudonarcissus seeds. Furthermore, three levels of MPD have been described in section Pseudonarcissus [12–14], and thus In the present study, we investigated seed germination in N. pseudonarcissus subsp. further detailed study is necessary. munozii-garmendiae (Fern. Casas) Fern. Casas (referred to as N. munozii-garmendiae in the following) and In the present study, we investigated seed germination in N. pseudonarcissus subsp. munozii- N. pseudonarcissus subsp. nevadensis (Pugsley) A. Fern. (referred to as N. nevadensis in the following). garmendiae (Fern. Casas) Fern. Casas (referred to as N. munozii-garmendiae in the following) and N. Bothpseudonarcissus taxa have very subsp. limited nevadensis geographical (Pugsley) distributions A. Fern. (referred and smallto as N. populations nevadensis thatin the are following sensitive). to habitatBothperturbations. taxa have very limited Therefore, geographical they are considereddistributionsthreatened and small populations and are included that are in sensitive the Red to List of Spanishhabitat perturbations. Vascular Flora Therefore, [18]. Understanding they are considered their threatened germination and processesare included is essentialin the Red for Listex-situ of plantSpanish production Vascular for Flora medicinal [18]. Understanding applications of their their germination alkaloid contents, processes as is well essential as for for boosting ex-situ plant the wild plantproduction populations for medicinal and ornamental applications uses of (trumpet their alkaloid daffodils), contents, thereby as well avoiding as for boosting the damaging the wild e plantffects on wildpopulations populations and caused ornamental by the uses large-scale (trumpetextraction daffodils), ofthereby bulbs. avoiding In addition, the damaging maximizing effects their on wild genetic diversitypopulations via sexual caused reproduction by the large will-scale facilitate extracti geneticon of bulbs improvements. In addition, in themaximiz genusing to allowtheir genetic increased productiondiversity of via the sexual alkaloids reproduction required will by thefacilitate pharmaceutical genetic improvement industry.s In in addition, the genus the to allow long-term increas storageed of seedsproduction may beof possiblethe alkaloids under required low temperature by the pharmaceutical and moisture industry. conditions, In addition, thereby the providing long-term the advantagestorage of seeds storing may large be possible amounts under of live low genetic temperature plant materialand moisture in a conditions, very small thereby space. providing the advantage of storing large amounts of live genetic plant material in a very small space. According to Berkov et al. [7], both N. munozii-garmendiae