IN VITRO PROPAGATION AND EX SITU CONSERVATION OF THE WESTERN-MEDITERRANEAN ENDEMIC SPECIES LAPIEDRA MARTINEZII LAG. (AMARYLLIDACEAE)
Jorge Juan Vicedo
INSTITUT UNIVERSITARI D’INVESTIGACIÓ CIBIO
FACULTAT DE CIÈNCIES
TÍTOL: IN VITRO PROPAGATION AND EX SITU CONSERVATION OF THE
WESTERN-MEDITERRANEAN ENDEMIC SPECIES LAPIEDRA MARTINEZII
LAG. (AMARYLLIDACEAE)
AUTOR: JORGE JUAN VICEDO
Tesi presentada per a optar al grau de DOCTOR PER LA UNIVERSITAT D’ALACANT,
MENCIÓ DE DOCTOR INTERNACIONAL
PROGRAMA DE DOCTORAT EN BIODIVERSITAT I CONSERVACIÓ
Dirigida per:
SEGUNDO RÍOS RUIZ (Professor titular de Botànica)
JOSÉ LUIS CASAS MARTÍNEZ (Professor titular de Fisiologia Vegetal)
Per a la realització d’aquest treball d’investigació he gaudit d’una beca-contracte per a la
Formació de Doctors del Vicerrectorat d’Investigació, Desenvolupament i Innovació de la
Universitat d’Alacant (Referència FPU-UA: 2010-21685934). Tanmateix, i dins del programa de recerca de l’esmentada Universitat, he gaudit de finançament per a tres estades d’investigació a altres centres, dos d’ells estrangers, i poder optar així a la menció de Doctor Internacional.
Resultats recollits en la present memòria han generat les següents publicacions en revistes indexades:
- Juan-Vicedo, J.; Fernández-Pereira, J.; Ríos, S.; Casas J-L. and Martín, I. (2016). Seed
germination and storage behaviour of Lapiedra martinezii (Amaryllidaceae). Seed Science
and Technology, 44(1), 1-8.
- Juan–Vicedo, J.; Marchev, A.; Ríos, S.; Casas J-L. and Pavlov A. (2016). Callus induction
in Lapiedra martinezii Lag. (Amaryllidaceae): a South-West Mediterranean endemic
medicinal plant. Journal of Medicinal Plants Research (en premsa).
Durant els anys 2011-2014 vaig realitzar tres estades en diferents centres d’investigació
(un nacional i dos estrangers) de les quals, els seus resultats han estan inclosos en la present memòria: part dels experiments dels Capítols 3 i 5 es van dur a terme al Centro de Recursos
Fitogenéticos (Instituto de Investigaciones Agrarias y Tecnología Agroalimentaria) d’Alcalá de
Henares, Madrid. Els experiments corresponents al cultiu in vitro dels calls (Capítol 4) es van desenvolupar íntegrament al Laboratory of Applied Biotechnologies del Stephan Angeloff
Institute of Microbiology, Bulgarian Academy of Sciences (Plovdiv, Bulgaria). Part dels resultats obtinguts al Capítol 4, es van obtindre a la Faculty of Pharmacognosy de la
University of Vienna (Viena, Àustria).
Per últim, m’agradaria agrair a totes les persones que m’han ajudat durant tots aquests anys al laboratori, al camp, amb l’estadística, la redacció, les correccions de l’anglès i els tràmits administratius, etc. a poder materialitzar el treball realitzat al voltant de Lapiedra martinezii en la present memòria. També, agrair a la meva família, companys i amics per la seva paciència, consell i suport: moltes gràcies ☺.
0.- SUMMARY...... 1
1.- GENERAL INTRODUCTION...... 25
1.1.- The genus Lapiedra in the Mediterranean Region: L. martinezii Lag...... 27
1.2.- The ex-situ conservation strategies...... 33
1.2.1.- Seed germination physiology research and its importance in plant propagation
and ex situ conservation...... 33
1.2.2.- The in vitro culture techniques as a tool for plant propagation and
conservation...... 35
2.- OBJECTIVES………………………..……………………………………………….37
3.- SEED DORMANCY AND GERMINATION OF Lapiedra martinezii
Lag.……………………………………………………………….………………………….41
3.1.- Introduction…………………………..………………………………………………...43
3.1.1.- Anatomy of seeds………………………………………………………...……43
3.1.1.1.- Seeds of Amaryllidaceae: anatomy and types……………………….43
3.1.1.2.- Structure of Lapiedra seeds…………………………………………44
3.1.2.- Seed germination physiology………………………………………………….44
3.1.2.1.- Seed dormancy and germination…………………………………….44
3.1.2.2.- Seed dormancy of Amaryllidaceae species……………………...…..48
3.1.2.3.- Seed germination of Amaryllidaceae species……………………….50 3.1.2.4.- The role of cold, warm and gibberelline pre-treatments to overcome
dormancy and promote seed germination…………………………………..…51
3.2.- Objectives…………...………………………………………………………………….53
3.3.- Material and methods………………..………………………………………………...53
3.3.1.- Seed collection and preparation……………………………………………….53
3.3.2.- Seed germination of Lapiedra martinezii……………………………………..55
3.3.2.1.- The effect of temperature on seed germination……………………..55
3.3.2.2.- The effect of light regime on seed germination……………………..55
3.3.2.3.- The effect of a cold pre-treatment on seed germination……………55
3.3.2.4.- The effect of a warm pre-treatment on seed germination…………..55
3.3.2.5.- The effect of gibberellic acid on seed germination…….……………56
3.3.2.6.- Statistical analyses…………………………………………………..56
3.4.- Results and Discussion…………………..…………………………………………….58
3.4.1.- Seed germination of Lapiedra martinezii…………………………………...... 58
3.4.1.1.- The effect of temperature on seed germination……………………..58
3.4.1.2.- The effect of light regime on seed germination……………………..58
3.4.1.3.- The effect of a cold pre-treatment on seed germination……………61
3.4.1.4.- The effect of a warm pre-treatment on seed germination………….63
3.4.1.5.- The effect of gibberellines on seed germination…………………..65 3.5.- Conclusions……………………..………………………………………………………67
4.- BIOTECHNOLOGICAL APPROACHES TO THE MANAGEMENT
OF L. MARTINEZII
GERMPLASM…...………………………………………………………………………69
4.1.- Introduction………………..…………………………...………………………………71
4.1.1.- Micropropagation techniques and process…………………………………….71
4.1.1.1.- The basis of micropropagation……………………………………....72
4.1.1.2.- Stages in the micropropagation process……………………………..73
4.1.2.- Application of in vitro techniques in bulbous plant propagation…………...…74
4.1.3.- Callus induction as a source of new germplasm……………………………....75
4.2.- Objectives…………………………...………………………………………………….76
4.3.- Material and methods……………..…………………………………………………...77
4.3.1.- Bulb collection and preparation…………………………………………….…77
4.3.2.- In vitro seedling cultures………………………………………………………77
4.3.2.1.- Seed collection and preparation……………………………………..77
4.3.2.2.- Seed surface sterilization………………………………………….....78
4.3.2.3.- Initiation of seedlings cultures………………………………………78
4.3.2.4.- Stock maintenance of seedlings cultures…………………………….81
4.3.2.5- Rooting and Acclimatization of microplants………………………...81 4.3.3.- Tissue culture of bulb scales………………………………………………..…82
4.3.3.1.- Bulb sterilization…………………………………………………….82
4.3.3.2- Initiation of in vitro cultures…………………………………………82
4.3.3.3.- Multiplication………………………………………………………..83
4.3.3.4- Rooting and Acclimatization………………………………………...84
4.3.4.- Callus induction as a source of new germplasm………………………………85
4.3.4.1.- Obtaining in vitro callus cultures……………………………………85
4.3.5.- Statistical analyses…………………………………………………………….87
4.4.- Results and Discussion………………..……………………………………………….88
4.4.1.- In vitro seedling cultures……………………………………………………....88
4.4.1.1.- Initiation of seedlings cultures………………………………………88
4.4.1.2.- Stock maintenance of seedlings cultures and rooting……………….91
4.4.1.3- Acclimatization of microplants……………………………………....92
4.4.2.- Tissue culture of bulb scales…………………………………………………..93
4.4.2.1.- Bulb sterilization and initiation of in vitro cultures…………………93
4.4.2.2.- Multiplication of bulb scales……………………………………...…96
4.4.2.3- Rooting and Acclimatization…………………………………..…....104
4.4.3.- Callus induction as a source of new germplasm……………………………..105
4.4.3.1.- Obtaining in vitro callus cultures…………………………………..106 4.5.- Conclusions…………………...…………………………………………………..…...115
5.- STORAGE BEHAVIOUR AND LONG-TERM PRESERVATION OF
L. martinezii SEEDS…………………………………………………………………...119
5.1.- Introduction………………..………………………………………………………….121
5.1.1.- Seed Storage behavior and desiccation tolerance……………………………121
5.1.2.- Long-term seed storage as ex situ conservation strategy: storage below 0ºC and
cryopreservation……………………………………………………….…………….122
5.1.3.- The Seed Moisture Content (SMC) and its role on long term seed
storage…………………………………………………………………………...…..123
5.2.- Objectives………………...…………………………………………………………...128
5.3.- Material and Methods……………..…………………………………………………128
5.3.1.- Determination of the Seed Moisture Content (SMC) and desiccation
tolerance…………………………………………………………………………..…128
5.3.2.- Seed storage behavior and long-term conservation of L. martinezii………...130
5.3.2.1.- Seed germination trials…………………………………………..…131
5.3.3.- Statistical analyses…………………………………………………………...132
5.4.- Results and Discussion………..……………………………………………………...132
5.4.1- Long term conservation of seeds…………………………………………..…132
5.4.1.1.- Determination of the Seed Moisture Content (SMC)…………...…132
5.4.1.2.- Seed desiccation tolerance………………………………………....134 5.4.1.3.- Long term seed storage…………………………………………….134
5.5.- Conclusions………………………..…………………………...……………………..137
6.- GENERAL CONCLUSONS……………………………………………………..139
7.- PROTOCOLS...... 144
8.- REFERENCES…………………………………...…………………………………153
0.- SUMMARY
~ 1 ~
~ 2 ~
SUMMARY
1.- Introducció general
1.1.- Lapiedra martinezii Lag.
Lapiedra martinezii Lag. (Amaryllidaceae) és un geòfit que creix en comunitats vegetals termòfiles i semiàrides dominades pel margalló, Chamaerops humilis L., i Osyris lanceolata Hochst. & Steud. i en diversos tipus de de màquies mediterrànies i matollars.
Tanmateix es troba freqüentment en rocalles, escletxes, esquerdes en indrets pedregosos i penya-segats costaners, des del nivell del mar fins als 650 m.s.n.m. a la Península Ibèrica i el
Nord d’Àfrica (Aedo, 2010; Bolòs i Vigo, 2001; Ríos et al., 2013).
L. martinezii té un elevat potencial econòmic degut a la seva riquesa en alcaloides amb gran interès per a la indústria farmacèutica (Larsen et al., 2010; Ríos et al., 2013). A banda, és una planta amb potencial interès per a la indústria de plantes bulboses ornamentals i també té un cert interès en conservació degut a la relativa baixa abundància que mostra al llarg de la seva àrea de distribució, i a les amenaces que s’han detectat a les seves poblacions (Ríos et al., 2013).
1.2.- Les estratègies de conservació ex-situ: la investigació en fisiologia de llavors i l’ús de tècniques de cultiu in vitro com a eina per a la propagació sostenible i la conservació
La germinació té una importància capital al cicle biològic de les espècies vegetals i el seu èxit reproductiu, i es pot considerar l’etapa més vulnerable ja que representa l’entrada de les plantes als ecosistemes (Harper, 1977; Navarro i Guititán, 2002; Rajjou et al., 2012;
Weitbrecht et al., 2011). Per a moltes espècies, el moment de l’any on la germinació és possible pot ser prou limitat (per exemple, només a la tardor, o la primavera, coincidint amb l’estació humida). No obstant això, l’època favorable per a la germinació d’altres espècies es
~ 3 ~ prou llarga, per exemple, durant tot el període de creixement (Baskin i Baskin, 2001). Les plàntules de la majoria de les espècies vegetals emergeixen tan prompte com les llavors han germinat al sòl. Aleshores, el rellotge biològic de l’emergència de les plàntules està principalment regulat pel trencament de la letargia i la presència dels requeriments adequats per a la germinació de les llavors. Per a algunes altres espècies, un considerable lapse de temps hi existeix entre el moment de l’eixida de la radícula al camp, i l’emergència de la plàntula (Vandelook i Van Assche, 2008).
Les llavors són la via més important de reproducció i dispersió en plantes (Nikolaeva,
2001). La informació sobre la germinació té, potencialment, un gran valor econòmic, per exemple per a la propagació d’arbres autòctons amb important valor econòmic, mates, lianes, herbes i gespes, per a restauració d’ecosistemes degradats, i planificació per un eficaç control de les males herbes (Baskin i Baskin, 2001). Junt amb açò, el desconeixement de la dormició en llavors d’espècies econòmicament importants causen importants problemes (Nikolaeva,
2001).
Per tant, la investigació en la biologia de les llavors i, particularment, en les condicions òptimes per al trencament de la dormició i promoure la germinació són objectius essencials per plantes amb importància econòmica. Amés, la informació relativa a la fisiologia de la germinació és necessària per a establir procediments de conservació ex situ apropiats i rutines de treball adequades als bancs de germoplasma, així com la planificació de programes de restauració de la coberta vegetal amb espècies autòctones (Baskin i Baskin,
2001; Copete et al., 2011; Walters, 2015).
Tenint en compte que tant la propagació vegetativa, com la recol·lecció directa de material vegetal de les poblacions naturals no podria satisfer la demanda de bulbs que podria ser requerida per a propòsits industrials, el cultiu mitjançant tècniques biotecnològiques, com
~ 4 ~ ara les tècniques de cultiu in vitro (George i Debergh, 2008) semblen ser la via més adequada per a dur a terme un ús sostenible d’aquest recurs, així com per a previndre la sobreexplotació i extinció de les poblacions naturals, com ha passat en altres ocasions.
2.- Objectius
El treball ací presentat s’ha dut a terme amb l’objectiu de desenvolupar un mètode apropiat per a la propagació in vitro de germoplasma de L. martinezii (llavors, plàntules, escales de bulbs i bases foliars) que podrien ser emprades per a propòsits industrials, així com estudiar el comportament de les llavors front a l’emmagatzematge amb la intenció de preservar-les ex situ als bancs de germoplasma. Tanmateix, els resultats obtinguts podrien ajudar a establir un procediment general per a la producció i conservació de plantes endèmiques i amenaçades de la família de les Amaril·lidàcies a Espanya. Concretament, els objectius específics són:
1.- Estudiar les condicions per al venciment de la dormició i la fisiologia de la
germinació de les llavors de L. martinezii.
2.- Establir diferents cultius in vitro partint de material vegetal divers (llavors, bulbs i
fulles) i provar diferents vies morfogèniques (organogènesi directa i indirecta) de L.
martinezii, així com desenvolupar un protocol de micropropagació adequat per als
bulbs de L. martinezii.
3.- Avaluar el comportament de les llavors de L. martinezii front a l’emmagatzematge,
amb la intenció de desenvolupar un protocol apropiat per a la seva conservació a llarg
termini.
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3.- Dormició i germinació de les llavors de Lapiedra martinezii Lag.
3.1.- Introducció
Les llavors de L. martinezii són ovades i negres, amb un estrofíol gran i discolor al principi, però que finalment ennegreix. La mida de les llavors seques és d’uns 2-3 mm, i per a llavors fresques d’uns 4-5 mm (Ríos et al., 2013). El principals objectius en aquesta primera secció del treball van ser determinar la temperatura òptima de germinació de llavors de L. martinezii, provar l’efecte de diferents tractaments per a promoure la germinació: tractaments de il·luminació i foscor junt amb diferents temperatures, pretractaments de fred i calor, així com diverses concentracions d’àcid giberèlic.
3.2.- Materials i mètodes
Les llavors van ser collides de càpsules madures al moment de la dispersió natural
(Octubre de 2011 i 2013 en Santa Pola, Alacant). Les llavors de 2013 van ser emprades per a provar l’efecte de l’àcid giberèlic, mentre que els altres experiments van ser duts a terme amb llavors recol·lectades l’any 2011. En tots els casos, la mitja embrió-endosperm (E:S) de les llavors va ser mesurada, una vegada collides i emprant una lupa binocular equipada amb micròmetre (Vandelook i Van Assche, 2008). Els experiments van començar una setmana després de la recol·lecció de les llavors.
Per a tots els assajos de germinació, quatre rèpliques amb 50 llavors sanes van ser emprades. Les llavors es van posar sota un paper de filtre (518G Filter-Lab) banyat amb 5 mL d’aigua destil·lada en plaques Petri de 90 mm de diàmetre. Les plaques Petri van ser incubades en càmeres de cultiu (Ibercex, model F-4, Madrid, Spain) baix un fotoperíode de 8- hores llum/16-hores obscuritat (radiació fotosintèticament activa 25 µmol m-2s-1). La
~ 6 ~ germinació va ser avaluada en tres temperatures alternes i (30/20ºC, 25/16ºC i 17/10ºC) i tres constants (20ºC, 17ºC i 4ºC) als fotoperíodes de llum/obscuritat (d’ara endavant, llum) i obscuritat. Les condicions d’obscuritat van ser imposades al envoltar les plaques Petri en paper d’alumini.
Per a determinar si hi havia dormició del tipus fisiològic, es va provar l’efecte d’un pretractament de fred al incubar llavors humectades a 4ºC durant 15 i 30 dies. Per a comprovar si hi havia dormició morfològica es va aplicar també un pretractament de calor sec mitjançant l’empaquetament de les llavors mantingudes a humitat ambiental (condicions de laboratori) en una bossa d’alumini amb doble tancament, i posterior escalfament en bany termostàtic a 40ºC durant 20 dies. Després dels pretractaments, les llavors van ser incubades a les temperatures de 30/20º, 25/16º, 17/10º i 20ºC en condicions de llum. Per a estudiar l’efecte de l’àcid giberèlic en la germinació, una mostra de llavors va ser submergida en concentracions de GA3 de 0.0, 0.01, 0.125, 0.25, 0.5, 0.75, 1 i 2 g/L durant 48 hores i després incubades a 20ºC en condicions de llum.
La germinació de les llavors va ser monitoritzada regularment per un període de 100 dies. La majoria de la germinació va ocórrer dins els primers 30-40 dies d’incubació i cap germinació va ser registrada després dels 50-60 dies de cultiu. Els següents paràmetres de germinació van ser mesurats:
- Percentatges de germinació final: es va considerar que una llavor havia germinat quan el coleòptil havia aconseguit una longitud de 0.5 cm després de trencar la testa.
- Temps Umbral: el dia on es registrava la primera germinació.
- T50: el dia on es registrava el 50% de la germinació d’una rèplica concreta.
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Per a l’anàlisi estadístic de les dades, es van dur a terme diversos anàlisi de la variança
(ANOVA) d’un o dos factors, i les diferències significatives es van determinar amb el
Fisher’s Least Significant Differences test (Fisher’s LSD) al 5%. Els percentatges de
germinació final es van transformar amb la funció arcsinus. Per als altres paràmetres
(temps Umbral i T50) es van utilitzar els valors sense transformar. Totes les dades van
ser analitzades amb el paquet estadístic Infostat 2008.
3.3.- Resultats i Discussió
Els embrions de L. martinezii han de créixer dins de la llavor fins que es produeix la germinació. Per tant, considerem que les llavors d’aquesta espècie tenen dormició morfològica (Baskin i Baskin, 2001; 2014; Nikolaeva, 1999). Després de la imbibició, la germinació ocorre en diferents règims de temperatura i condicions de llum i obscuritat.
S’aconsegueixen percentatges de germinació propers als 100% a 20ºC (tant en llum com en obscuritat). Elevats percentatges de germinació (>60%) s’han obtingut amb els règims de
17ºC, 30/20ºC i 25/16ºC, mentre que les llavors rarament van germinar a 17/10ºC. En general, les condicions de llum van influir positivament en la velocitat de germinació (temps Umbral i
T50) però no en els percentatges de germinació final. Resultats semblants s’han documentat en algunes altres espècies d’ambients semiàrids (González-Benito et al., 2006; Marchioni-
Ortu i Bocchieri, 1984; Schütz i Milberg, 1997) i altres bulboses de floració tardorenca
(Keren i Evenari, 1974; Marques i Draper, 2012; Nikopoulos et al., 2008), mentre que altres bulboses de floració primaveral, o be bulboses de floració tardorenca però amb una distribució continental o de muntanya, mostren diferents nivells de dormició morfofisiològica i el procés de germinació s’allarga fins sis mesos o més (Copete et al., 2011; 2014; Herranz et al., 2013; 2015).
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El pretractament de calor aplicat va millorar la germinació al augmentar el percentatges de germinació final, disminuir el temps Umbral i el T50 significativament en la majoria dels casos. Per contra, el pretractament de fred va minvar tots els paràmetres de germinació a totes les temperatures assajades i les llavors, molt probablement, van entrar en dormició secundària. L’aplicació de GA3 va afectar a la germinació a les concentracions de 0-
0.75 g/L, mentre que la proporció de llavors germinades va disminuir significativament a les concentracions més elevades de 1 i 2 g/L.
4.- Ús d’eines biotecnològiques per a la gestió de germoplasma de L. martinezii
4.1.- Introducció
La biotecnologia vegetal es defineix com el conjunt d’activitats científiques de cultius cel·lulars i manipulació genètica integrades per a emprar els recursos vegetals existents i obtindre productes derivats de les plantes amb interès comercial. La biotecnologia vegetal inclou (Ziv, 1997):
- El cultiu de teixits vegetals: micropropagació, cultius a gran escala, regeneració de
plantes i eliminació de patògens.
- Manipulació genètica (modificació de certes característiques hortícoles): pol·linització
in vitro, rescat d’embrions i fertilització in vitro, producció de plantes haploides, cultiu
de calls, fusió de protoplasts i cultiu d’híbrids somàtics, transformació i manipulació
genètica, empremta molecular d’ADN i mapeig de genomes.
Les tècniques de cultiu de teixits (tant en medis sòlids com líquids) han sigut amplament emprades per a la producció comercial de planta ornamental, incloent la propagació de quasi
~ 9 ~ totes les espècies i varietats comercials de geòfits (de Klerk, 2012; George i Debergh, 2008;
Hvoslev-Eide i Preil, 2005; Santos et al. 1998). La micropropagació és la forma genuïna de propagació de genotips selectes emprant les tècniques de cultiu in vitro. La regeneració de plantes amb aquestes tècniques es basa en la totipotència de les cèl·lules vegetals; és a dir, la habilitat que tenen cèl·lules vegetals aïllades i mantingudes en cultiu en (des-) diferenciar-se, re-diferenciar-se i regenerar noves plantes.
Tanmateix, aquestes tècniques de cultiu in vitro s’utilitzen per a obtindre genotips estables amb una elevada producció de metabòlits secundaris amb interès per a la indústria alimentària i farmacèutica (Avato et al., 2005; Georgiev et al., 2010; Hanks, 2002) així com per a la conservació ex situ de plantes rares, endèmiques o amenaçades (González-Benito and Martin,
2011; Laguna, 1998; Marco, 2010; Nikopoulos et al., 2008).
Per totes aquestes raons, s’hi provarà l’eficiència de diferents cultius in vitro (tant en medis sòlids com en líquids) en la micropropagació de L. martinezii.
4.2.- Materials i mètodes
Es van seleccionar plantes sanes d’una població del Terme de Santa Pola (Santa Pola,
Alacant). Aquesta població no està inclosa en cap àrea protegida i es localitza en una vessant de muntanya a 3 m.s.n.m. (coordenades 38º13’30.70’’N/ 0º30’45.11’’W). Es va col·lectar una mostra de bulbs al període de fructificació, durant la primera setmana d’octubre de 2013. Es van collir plantes senceres i es van tractar de diferents maneres per a obtindré els distints explants (llavors, bulbs o bases foliars) tal i com s’explica en les següents seccions.
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4.2.1.- Cultiu in vitro de plàntules.
Iniciació i manteniment d’un stock de plàntules in vitro.
Les llavors es van esterilitzar en superfície mitjançant una curta immersió (30 s) en etanol al 70%, seguit d’una immersió en HgCl2 al 0.1% durant 3 min. Aleshores, les llavors es van rentar tres vegades en aigua destil·lada estèril i es van dur a germinar. Les llavors esterilitzades van ser sembrades en plaques Petri de 90 mm de diàmetre amb un disc de germinació de paper (518 Filter-Lab) humectat amb 5 mL d’aigua destil·lada estèril. Les condicions ambientals aplicades pr a la germinació van ser la temperatura i il·luminació
òptimes obtingudes al capítol anterior. Una vegada les llavors havien germinat (coleòptil emergit i radícula, bulbet i primera fulla diferenciats) les plàntules es van esterilitzar amb immersió en etanol al 70% durant 30 s, seguit d’una immersió en hipoclorit càlcic al 7% durant 20 min i rentades 3 vegades en aigua destil·lada estèril. Posteriorment, les arrels i fulles van ser parcialment eliminades, netejant els teixits necròtics o danyats.
Finalment, els bulbets es van sembrar en diferents medis d’iniciació formulats en base als resultats obtinguts en Georgieva et al. (2010) i es van incubar en càmera de cultiu a 24ºC baix fotoperíode de 16 hores llum/8 hores obscuritat. Els subcultius es van dur a terme cada
28 dies i el percentatge d’explants supervivents i contaminats es van registrar al primer i tercer mes.
Les plàntules es van sembrar en medi nutritiu IN1, composat de sals Gamborg B5 suplementat amb 500 mg/L de caseïna, 2 mg/L d’adenina, 10 mg/L de glutation, 10 mg/L de sacarosa i 5.5 g/L de ‘Plant Agar’. Els cultius van ser revisats setmanalment durant un període de 2 mesos i els tubs contaminats o amb plantes mortes es van retirar de l’experiment.
Posteriorment, les plantes in vitro van ser transferides a pots plens amb 250 mL de medi nutritiu sòlid, sense hormones, formulat amb salts MS+vitamines, 30 g/L de sacarosa i ~ 11 ~
5.5 g/L de ‘Plant Agar’. Aquests cultius es van mantindre 10 mesos en càmeres de cultiu a
17±1ºC i fotoperíode de 16 hores llum/8 hores obscuritat (radicació fotosintèticament activa de 42 µmol m-2s-1) i la supervivència de les plàntules en el temps es va avaluar.
Arrelament i aclimatació.
Les plantes obtingudes es van transferir a la fase d’aclimatació en condicions ex vitro.
Es van plantar tres rèpliques dels bulbets en una mescla de turba i vermiculita (5:4) prèviament autoclavada (durant 30 minuts a 121ºC i 1 atm de pressió) i disposat en càmera de cultiu amb control de llum i humitat. Es van dur a terme dos fases d’aclimatació en càmera de cultiu, prèvies a la transferència a condicions ambientals. La primera fase d’aclimatació es va desenvolupar durant un mes, amb una temperatura de 24ºC, humitat relativa (HR) de 100% i un fotoperíode de 16 hores llum/8 hores obscuritat (radicació fotosintèticament activa de 42
µmol m-2s-1). En la segona fase, també amb una durada d’un mes a 24ºC, la HR es va reduir fins al 70%, mentre que la intensitat lumínica es va incrementar fins als 80 µmol m-2s-1. En qualsevol cas, es van regar les plants setmanalment i després de la segona fase d’aclimatació les plantes es van transferir a condicions ambientals exteriors en el pati de l’Institut d’Investigació CIBIO de la Universitat d’Alacant.
4.2.3.- Cultiu de teixits de bulbs
Esterilització i inici de cultiu in vitro
En aquest cas, els bulbs es van separar de les plantes senceres i es van netejar amb un detergent comercial. Set procediments d’esterilització de bulbs, combinant diferents tractaments tèrmics i químics, es van provar. De cada bulb esterilitzat, es va retirar dos terços de la part apical, així com els dos catafil·les externs i les parts brunenques del disc basal, corresponents a teixit necròtic. La resta del bulb, es va seccionar longitudinalment per a obtindre 3-4 explants que posteriorment es van sembrar en medi d’inici IN1 (descrit ~ 12 ~ anteriorment) atès els bons resultats obtinguts al cultiu de plàntules. De forma complemetària, es van fer sembres en medi IN1 suplementat amb 2 g/L de carbó actiu.
Les condicions de cultiu per a la iniciació van ser 24ºC de temperatura, al principi en règim d’obscuritat durant 10 dies (imposades al envoltar els carrils de tubs d’assaig amb làmines de paper d’alumini) i posteriorment amb el fotoperíode convencional per a cultiu in vitro descrit en apartats anteriors, durant 30 dies més. Cada explant es va sembrar en tubs d’assaig amb 15 mL de medi de cultiu. Quatre lots de 25 tubs d’assaig van ser emprats per a cada medi d’inici (un total de 100 explants per medi).
Multiplicació in vitro
Una vegada iniciats els cultius in vitro es van anar multiplicant els bulbs amb el medi de multiplicació MUL1 (BAP=2 mg/L i NAA=0.12 g/L; basat en les fórmules emprades per a
Narcissus bulbocdium L. en Santos et al., 1998) fins a obtindre un stock de plantes prou nombrós com per a poder dur a terme un assaig de multiplicació complet amb diversos medis.
La resta de medis (14 en conjunt) també es van formular atenent als resultats obtinguts en altres treballs de micropropagació de bulboses (especialment de la família Amaryllidaceae).
Independentment de la combinació de reguladors del creixement emprada en el medi, tots els medis estaven composats per sals MS+vitamines, 30 g/L de sacarosa i solidificats amb 5.5 g/L de ‘Plant Agar’. La durada del període de multiplicació va ser de 8 setmanes, amb les condicions de cultiu descrites per a la fase d’iniciació (temperatura i fotoperíode amb llum).
Després d’aquest període de cultiu, es van mesurar els següents paràmetres:
- Nombre de brots per explant: formació de nou bulbets per cada explant sembrat.
- Calibre: ample i llarg (mm) d’aquests nou bulbets formats in vitro.
- Arrelament: el percentatge d’arrelament va ser visualment determinat.
~ 13 ~
- Formació de calls: presencia o absència, i estimació visual del grau de proliferació de
call o d’estructures no-clorofíliques semblants al call.
Un total de 30 rèpliques van ser emprades per a les mesures en cada medi de cultiu
(medi de multiplicació). Després del període de multiplicació, l’arrelament va ser espontani en pràcticament tots els medis provats i, per tant, no va ser necessari dissenyar una fase específica d’arrelament in vitro. L’aclimatació es va dur a terme seguint el mateix procediment descrit per a la secció ‘cultiu in vitro de plàntules’.
4.2.4.- Inducció i formació de call com a font de germoplasma nou.
Per a aquests experiments es van emprar les fulles retirades dels bulbs emprats en la secció anterior. Les fulles es van netejar amb sabó comercial ‘Domestos’ i posteriorment esterilitzat amb una dissolució d’etanol al 70% durant 30 s, seguida d’immersió en hipoclorit càlcic al 7% durant 20 min. Finalment, els explants van ser rentats sis vegades en aigua destil·lada estèril, eixugats en paper de filtre estèril en condicions de flux laminar, i finalment seccionats en explants d’un cm de llarg, aproximadament, prèvia eliminació de les parts necròtiques.
La iniciació dels cultius in vitro de calls es va dur a terme al sembrar dos rèpliques de
3-4 explants cadascuna en plaques Petri per a condicions de llum i obscuritat. Els explants de fulles es van cultivar amb les superfícies abaxials en contacte amb el medi de cultiu, i prou submergides com per a assegurar que tota l’àrea ferida pels talls quedava en contacte amb el medi. El medi de cultiu emprat va ser MS+vitamines, suplementat amb 30 g/L de sacarosa, i solidificat amb 5.5 g/L de ‘Plant Agar’ (Duchefa). A aquesta fórmula general, es va afegir un total de 50 combinacions de reguladors del creixement
(benzilaminopurina, kinetina i 2,4-D) per a provar la inducció i formació de call. Per a tots els medis de cultius descrits al present treball, el pH va ser ajustat a 5.8 i posteriorment
~ 14 ~ autoclavat durant 15 min a 121ºC i 1 atm de pressió. Una quantitat aproximada de 40 mL de medi va ser dispensat a cada placa Petri de 9 cm de diàmetre, en condicions de flux laminar.
El cultiu es va dur a terme a 26ºC de temperatura, tant en condicions de llum
(fotoperíode de 16 hores llum/8 obscuritat i radiació fotosintèticament activa de 42 µmol m-2s-1) com de obscuritat completa (càmera de cultiu sense il·luminació). Els cultius van ser revisats diàriament durant els primers tres mesos d’incubació, i els explants contaminats van ser retirats de l’experiment. Posteriorment, les revisions es van fer setmanalment. Sis períodes de subcultiu diferents (28 dies cadascun) es van dur a terme en les condicions anteriorment descrites, abastint per tant el temps total d’experimentació de
7 mesos. L’avaluació del desenvolupament del call es va fer mesurant els següents paràmetres:
- Temps Umbral en la inducció del call: el dia on es registra el primer signe de
desdiferenciació.
- Eficiència en la inducció del call (nivell de desdiferenciació dels teixits): mitjançant
una estimació del volum de teixit original que s’ha desdiferenciat en cada període de
subcultiu de mesos.
- Característiques del call: textura (compact, friable o semi-friable), color i
supervivència del call durant el període de cultiu.
4.2.5.- Anàlisi estadístic
Per a l’anàlisi de les dades, els percentatges d’arrelament, formació de call, així
com l’eficiència en la inducció de call es van transformar mitjançant la funció
arcsinus. La resta de paràmetres (número de bulbets produïts per explant, calibre, etc.)
~ 15 ~
van ser analitzats sense cap transformació. Diversos anàlisi de la variança (ANOVA)
d’un factor es van dur a terme i les diferències significatives es van determinar amb el
Fisher’s Least Significant Differences test (Fisher’s LSD) al 5% emprant el programa
estadístic Infostat 2008.
4.3.- Resultats i Discussió
4.3.1.- Cultiu in vitro de plàntules.
Durant el primer mes després de la imbibició de les llavors, totes les plàntules es van desenvolupar i els bulbets podien ser distingits. Un total de 9 medis d’inici es van formular atenent els resultats positius en especies semblants (Georgieva et al., 2010). Aquests medis es van formular amb sals MS+vitamines suplementats amb diferents concentracions de sacarosa and Fe (sequestrene). Altres medis amb aproximadament la meitat de salts, com ara ½ MS o
Gamborg B5, es van provar. Els millors resultats es van obtindre en medis Gamborg B5, suplementats amb 10 g/L de sacarosa (sense carbó actiu), mentre que aquells medis més concentrats en sals (MS) o sacarosa (30 g/L) van mostrar els pitjors resultats.
Degut a la continua aparició d’infeccions als cultius, es va estudiar si es tractava de microbis endògens (bacteris o fongs), que pogueren comprometre els subseqüents experiments. Per a això, es van submergir algunes porcions d’explants en medi líquid i es van tindre en cultiu durant 3 setmanes en condicions d’agitació, a 25ºC i fotoperíode llum.
Posteriorment, es van seleccionar al atzar 3 rèpliques del cultiu, i una mostra del líquid que contenien es va inocular a medis selectius de bacteris o fongs (tres rèpliques cadascun, un total de nou mostres) i es van incubar a 37.2ºC en obscuritat durant 7 dies. Els resultats van mostrar la presència del mateix tipus de colònia bacteriana en 7 de les nou mostres avaluades, mentre que les altres dos no van mostrar signes de creixement microbià. Cap signe de creixement de fongs es va registrar a les rèpliques sembrades en Malt-Agar. Aquests resultats
~ 16 ~ demostren que els cultius in vitro no tenen fongs endògens, mentre que una part significativa d’ells tenen bactèries que, potencialment poden florir al medi i fer malbé els cultius. Per això, vam decidir afegir una mescla d’antimicrobians durant tres setmanes, per controlar la proliferació bacteriana als cultius, i aquesta es va aconseguir reduir durant el perdiode de cultiu.
Per a avaluar si un stock de plantes pot ser preservada in vitro sense cap subcultiu durant un període més extens, una prova sobre la supervivència i l’increment en la mida dels bulbs mantinguts en medi d’iniciació, a una temperatura més baixa (17ºC) durant deu mesos.
Després d’aquest període de cultiu, es va observar una supervivència del 100% dels explants, junt amb un increment considerable en el calibre dels mateixos. Aquest procediment proporciona una aproximació intermèdia a la conservació ex situ de germoplasma a temperatures més baixes i proporciona un material genèticament divers, apropiat per a estratègies de reintroducció i restauració poblacional. Després d’aquest període de cultiu, 113 bulbs van ser transferits a la fase d’aclimatació, on un 86% dels explants van sobreviure després del primer més. D’aquestes, un 100% van superar la segona fase d’aclimatació i la transferència al exterior (condicions ambientals del campus Universitat d’Alacant).
4.2.3.- Cultiu de teixits de bulbs
L’esterilització d’aquest material va ser especialment problemàtica. Només el tractament tèrmic aplicat a 54ºC durant 60 min va ser efectiu per als explants, posteriorment submergits en una dissolució comercial de lleixiu al 30% (5% de clor actiu) amb tres gotes de tween 20 durant 10 minuts. Aquest procediment està basat en els resultats obtinguts per Hol i
Van Der Linde. (1992), Langen-Gernts et al. (1998) i Sochacki i Orlikowska (2005). El nivell de desinfestació va ser prou elevat, permetent l’inici de cultius asèptics en un 21.59% i un
15.46% dels explants per als medis d’inici sense i amb carbó actiu, respectivament. La resta
~ 17 ~ de protocols d’esterilització emprats no van donar cap resultat positiu, amb taxes de contaminació d’entre el 94 i el 100% després de dos setmanes de cultiu.
En la fase de multiplicació, el medi emprat com a control (sense reguladors del creixement) va aportar els resultats més baixos de productivitat (amb 0.10 bulbets per explant). Això mostra que certa propagació es pot obtindre sense l’aplicació exògena de reguladors del creixement. Aquest fet també ha sigut reportat en corms de gladiol ‘Yamit’
(Steinitz and Lilien-Kipinis, 1989). No obstant això, la producció per explant obtinguda en L. martinezii és molt baixa encara i descartem aquest medi com a possible medi de multiplicació. La combinació de BA=4 i NAA=0.12 g/L va mostrar la millor resposta morfogenètica en termes de producció per explant (tassa de 5.77±0.31) i de calibre (4.07±0.48 mm de llarg i 6.10±0.65 mm d’ample). Aquest medi també va generar un arrelament molt elevat (98.18%), i la proliferació d’estructures no-clorofíliques i/o semblants al call va ser relativament baixa (24.84%) tenint en compte els resultats obtinguts a la resta de medis de multiplicació avaluats.
Amb una formula de medi de cultiu molt semblant, Santos et al. (1998) van obtindre també resultats òptims amb N. bulbocodium. També, Hussey et al., (1982) va trobar molt efectives, en la propagació de Narcissus, les fórmules amb concentracions elevades de citocinines (des de 3-4 mg/L fins 10 mg/L de BAP/BA) respecte d’auxines. Treballs duts a terme en altres amaril·lidàcies apunten també en aquesta direcció, tant en organogènesi directa, com en organogènesi indirecta (regeneració) independentment de la presencia, el tipus i la concentració d’auxina (Berkov et al., 2010; Chen et al., 2005; Georgiev et al., 2012;
Hussey, 1982; Ivanov et al., 2011; Nikopoulos i Alexopoulos, 2008; Pavlov et al., 2007;
Santos et al., 1998; Seabrook et al., 1976; Seabrook i Cumming, 1982; Sellés et al., 1999;
Squires i Langton, 1990). Aquest fet pot estar relacionat, molt probablement, amb el paper
~ 18 ~ que les citocinines juguen en la supressió de la dominància apical i en la promoció directa del creixement dels brots (Cline, 1994; Ongaro i Leyser, 2008; Van Staden et al., 2008).
Resultats sub òptims s’han obtingut amb el medi formulat amb BA=4 i IBA=0.12 mg/L en termes de formació de nous bulbets (3.40±0.32), calibre (3.77±0.48 mm d’alçada i
5.67±0.63 mm d’amplària) i arrelament (82.80%). Malgrat això, la proliferació d’estructures semblant al call va ser de les més elevades (36.94%), fet que ens fa descartar aquest medi com a alternativa per a la micropropagació, especialment si l’objectiu és conservació i restauració de les poblacions naturals. Els altres medis de cultiu van mostrar una baixa producció de nous bulbets (més baixa de 2.6 bulbets per explant), mentre que el calibre va romandre majoritàriament sense diferències significatives respecte dels resultats òptims. L’arrelament va ser, en general, major del 75% en totes aquestes combinacions, mentre que la proliferació d’estructures semblants al call va ser variable, mostrant valors situats entre 7.1 i 37.76%.
Un experiment complementari dut a terme en sistemes d’immersió permanent en medi líquid va mostrar que el medi amb BA=2 i NAA=0.12 g/L va resultar en una molt menor producció (0.7 nou bulbets per explant) que el sòlid (2.10) i les fulles van aparèixer massa desenvolupades i vitrificades (aquesta última condició es va corregir al cultivar en medi sòlid durant 2-4 setmanes). El resultat ací exposat no suporta la idea d’una millor producció utilitzant medis líquids en la micropropagació de L. martinezii.
L’arrelament i aclimatació dels bulbs produïts in vitro amb explant de bulb va ser espontani als medis de multiplicació en el transcurs del primer subcultiu (2 mesos) en la majoria de les mostres. Els explants que no van mostrar un arrelament espontani, van desenvolupar arrels durant el mes posterior a la transferència a un medi composat de MS suplementat amb 30 g/L de sacarosa i 5.5 g/L de ‘Plant Agar’ sense reguladors del creixement. Durant la fase d’aclimatació, els bulbs van desenvolupar túnica i, després de la
~ 19 ~ transferència a condicions ambientals exteriors, el 89% dels bulbs van sobreviure, mostrant uns percentatges d’èxit semblants als de l’aclimatació de plàntules in vitro, i també a l’èxit obtingut a altres amaril·lidàcies cultivades in vitro (Santos et al., 1998).
4.3.3..- Inducció i formació de call com a font de germoplasma nou.
En general, els calls obtinguts mostraren una textura compacta, i un tipus de desenvolupament agregat, amb color groc. Es va arribar al creixement complet durant el segon i tercer mes de cultiu baix condicions d’obscuritat, mentre que aquelles rèpliques disposades en condicions d’il·luminació gairebé van mostrar cap signe de diferenciació durant el període sencer de cultiu (7 mesos). Els calls no es van obtindre mai en presència de citocinines a soles i la formació del call va ser òptima a elevades proporcions d’auxines respecte de citocinines.
5.- Comportament de les llavors de L. martinezii front a l’emmagatzematge i conservació a llarg termini
5.1.- Introducció
Les estratègies de conservació ex situ constitueixen una reserva a llarg termini per als recursos genètics silvestres i faciliten el seu ús per a diferents propòsits. L’emmagatzematge de llavors és, actualment, una estratègia amplament emprada no només per a espècies agrícoles amb importància econòmica i els seus parents silvestres, sinó que també per a plantes amenaçades i flora silvestre endèmica en general (Hay i Probert, 1993; Walters,
2015). Les llavors poden ser classificades depenent del seu comportament front a l’emmagatzematge en tres categories principals (Hong i Ellis, 1996): ortodoxes (poden ser dessecades, sense danys, fins a nivells baixos en contingut d’humitat), recalcitrants (poden ser
~ 20 ~ dessecades fins a un límit que en qualsevol cas no és molt ample) i intermèdies (mostren un comportament intermedi).
El punt crític per a conservar llavors per a un temps prolongat és determinar la resposta front a diferents nivells de dessecació, així com al emmagatzematge a baixes temperatures (Hong i Ellis, 1996; ISTA; 1993; Rao et al., 2007). Les condicions de conservació ex situ normalment impliquen emprar temperatures per baix dels 0ºC, normalment entre -18º i -20ºC, que poden variar depenent del nivell d’humitat de les llavors.
La criopreservació és el manteniment de qualsevol estructura biològica (llavors en aquest cas) a temperatures del nitrogen líquid: és a dir, aproximadament entre -196º i -150ºC
(Benson, 1999). La criopreservació constitueix un dels mètodes més eficaços de conservació a llarg termini de llavors ja que, en aquestes condicions, l’activitat metabòlica i els processos bioquímics dins les cèl·lules es veuen aturats i, per tant, permeten la conservació per períodes de temps, a priori, il·limitats (Benson, 1999; Panis, 2001).
5.2.- Materials i mètodes
El comportament de les llavors de L. martinezii front a l’emmagatzematge va ser avaluat al comparar la germinació de llavors fresques amb aquella de llavors dessecades i emmagatzemades a -20ºC (llavors recollides el 2011) i -80ºC durant 90 dies, dessecades i submergides en nitrogen líquid per cinc dies (llavors recollides el 2013) seguint les indicacions de Hong i Ellis (1996). La dessecació es va dur a terme a 20ºC, en caixes hermètiques amb gel de sílice (proporció gel de sílice:llavors de 10:1, wt/wt), on la HR es va equilibrar al 7% (mesurat amb un sensor portàtil, EL-USB-2). Els test de germinació es van dur a terme a 20º abans i després de l’emmagatzematge en fred.
~ 21 ~
5.3.- Resultats i discussió
La dessecació de llavors fins a nivells d’humitat del 4.10% no va tindre cap efecte significatiu en la germinació. Aquest fet podria estar relacionat amb les condicions ambientals que escauen a les poblacions naturals de L. martinezii al moment de la dispersió de les llavors i, per tant, la tolerància a la dessecació seria un caràcter adaptatiu per a L. martinezii, front a la tolerància limitada que s’ha descrit en altres espècies d’amaril·lidàcies a Europa (Newton et al., 2013).
L’emmagatzematge a -20º, -80ºC i –amb nitrogen líquid no va tindre tampoc cap efecte significatiu en els paràmetres de germinació estudiats. La immersió en nitrogen líquid va minvar lleugerament els percentatges de germinació final, encara que la proporció de llavors germinades va continuar sent molt elevada. Finalment considerat, tots els resultats obtinguts en aquesta tesi sobre fisiologia de llavors de L. martinezii demostren el comportament ortodox d’aquestes front a la dessecació. Per tant, els mètodes convencionals de conservació de llavors als bancs de germoplasma es poden emprar sense cap risc de pèrdua de qualitat de les col·leccions d’aquesta espècie.
6.- Conclusions generals
- Les llavors de L. martinezii tenen dormició morfològica ja que els embrions han de
créixer dins de la llavor, abans que es produixca l’emergència del coleòptil.
- La germinació òptima es produeix a 20ºC amb percentatges de quasi un 100% de
germinació final en llum i obscuritat.
- Les condicions de llum van influir a la velocitat de germinació, però els percentatges
de germinació final van romandre sense diferències significatives.
- El pretractament de fred va minvar tots els paràmetres de germinació a totes les
temperatures assajades, mentre que el pretractament de calor els va incrementar.
~ 22 ~
- L’aplicació exògena de giberelines no va afectar en cap paràmetre a la germinació a
les concentracions d’entre 0-0.75 g/L, mentre que les proporcions de llavors
germinades va decréixer a les concentracions majors d’1 i 2 g/L.
- L’establiment de cultius in vitro de L. martinezii és una tasca prou difícil per les
elevades proporcions de contaminació que s’obté durant el procés d’inici de cultiu,
degut a la presència de bactèries endògenes. No obstant això, una mescla d’agents
antimicrobians formulada amb 500 mg/L de cefotaxima, 5 mg/L d’ampicilina i 15
mg/L de kanamicina durant tres setmanes va ajudar a mantindré els cultius amb uns
nivells de proliferació bacteriana inferiors al 30%.
- El medi d’inici de cultiu dissenyat amb Gamborg B5 i suplementat amb 10 g/L de
sacarosa, va ser el més apropiat per a l’inici de cultius in vitro tant de plàntules com de
bulbs.
- Els cultius de plàntules en medi MS, suplementat amb 30 g/L de sacarosa, són una
bona alternativa per a desenvolupar un stock de material in vitro, que pot mantenir-se
sense subcultivar durant 10 mesos a temperatures de cultiu més baixes (17ºC), sense
suspendre el creixement.
- La millor resposta morfogenètica a la multiplicació es va obtindre amb el medi sòlid
MS suplementat amb 30 g/L de sacarosa, BA=4 i NAA=0.12 mg/L en termes de
producció de bulbets (5.77 per explant) i calibre (4.07x6.10 mm), com en l’arrelament
(96.18%). Amés, la proliferació d’estructures tipus call va ser relativament baixa
(24.84%) en comparació amb altres medis de multiplicació provats.
- L’arrelament va ser espontani en tots els medis de multiplicació testats i en una ampla
proporció de les rèpliques durant el primer període de subcultiu, amb uns valors
variables des de 76.43% fins al 100% (excepcionalment 49.68%). Per tant, no va ser
necessari dissenyar una fase específica d’arrelament amb medis específics.
~ 23 ~
- El protocol general d’aclimatació ací provat va tindre un elevat percentatge de plantes
supervivents per a ambdós tipus d’explants: plàntules (amb una supervivència del 86%
per a les plàntules produïdes in vitro) i bulbs (supervivència del 89% per als clons
procedents d’explants de bulbs) una vegada transferides a condicions ambientals
exteriors.
- Les condicions d’obscuritat a 26ºC van afavorir clarament la inducció de call, el seu
creixement i desenvolupament, mentre que el fotoperíode testat amb llum va tindre un
efecte marginal en la producció de calls.
- L’aplicació de 2,4-D és necessària per a la formació del call, independentment de la
citocinina emprada al medi (BAP o KIN).
- Proporcions elevades d’auxines front a citocinines (al menys 2 vegades) van ser
necessàries per a la inducció del call i per a arribar a una òptima eficiència en la seva
inducció, que ocorre de forma completa als dos-tres mesos de cultiu.
- Els calls amb un nivell de desenvolupament òptim van mostrar un color groc i quasi
tots van ser compactes o lleugerament friables.
- Les llavors de L. martinezii poden ser dessecades fins nivells de contingut en humitat
del 4.10%, i emmagatzemades a -20º, -80ºC o mitjançant immersió en nitrogen líquid,
sense cap efecte significatiu en la germinació.
- Pel que fa al comportament de les llavors de L. martinezii front a l’emmagatzematge,
les dades ací obtingudes demostren que són ortodoxes.
~ 24 ~
1.- GENERAL INTRODUCTION
~ 25 ~
~ 26 ~
1.- GENERAL INTRODUCTION
1.1.- The genus Lapiedra Lag. in the Mediterranean Region: Lapiedra martinezii Lag.
The genus Lapiedra Lag. has only one species described in the world: Lapiedra martinezii Lag. It is a small, late summer - autumn flowering geophyte belonging to the
Amaryllidaceae family. The flowers are white, actinomorphic, with 6 persistent tepals and grupped in apical pseudoumbella infloresecences. Flowers and leaves do not normally show contemporaneous development, and the latter appearing during the fruit-ripening stage in most populations (Aedo, 2010; Ríos et al., 2013).
The fruits are dehiscent capsules, although dispersion mechanism is not explosive.
After the opening of fruit, many seeds remain joined to the placenta. The bulb is more or less rounded and tunicated, covered with blackish scales, extended as a sheat up to the lower part of the leaves (Ríos et al., 2013). The bulb caliber ranges from 2,6-6,5 x 2,4-6,7 cm (Aedo,
2010). Each bulb produces (1)2-4(5) narrow linear leaves of dark-green colour with glossy aspect and a typical central witish band. Vegetative propagation frequently takes place by subequal division in 2-3 or more, so that the new bulbs appeared in tightly grouped colonies
(Ríos et al., 2013).
L. martinezii grows in thermophilous and semiarid plant communities dominated or co-dominated by the dwarf palm Chamaerops humilis, Osyris lanceolata Hochst. & Steud. and Maytenus senegalensis, in several types of Mediterranean macchias, and in woodlands with Pistacia lentiscus and Quercus coccifera. Also, it is commonly found in vertical rocky slopes, crevices and coastal cliffs ranging from the sea level up to 860 m.a.s.l. (Aedo, 2015;
Bolòs and Vigo, 2001; Ríos et al., 2013).
~ 27 ~
Figure 1.1.- Lapiedra martinezii Lag. habitat and plant material (whole plant and detail of different parts): a) Thermophilous and coastal plant communities with Chamaerops humilis, Rhamnus lycioides, Stipa tenacissima and Asparagus albus in the proximities of Santa Pola’s Cape (Santa Pola, Spain); b) L. martinezii individuals in rock crevices in Santa Pola; c) Population of L. martinezii in coastal cliffs of Penyal d’Ifach (Calp, Spain); d) Flowering colony of L. martinezii; e) Detail of flowering individual of L. martinezii with contemporaneous leaves; f) L. martinezii roots, bulbs and leaves in adult post-flowering individuals; g) detail of bulbs, a bulblet produced by vegetative propagation and roots from adult plants; h) detail of ripen fruits; i) detail of seeds.
It is a Baetic-Moroccan endemism distributed in the South-Western Mediterranean, but most of its populations are placed in the Iberian Peninsula. In Spain, it grows from Malaga to Valencia (with doubious presence in Castellón and Córdoba Provinces) and in North Africa
~ 28 ~ a punctual citation was done in Morocco (Aedo, 2015; Bolòs and Vigo, 2001; Maire, 1959;
Ríos et al., 2013; http://www.floravascular.com). Local names found are cebolla marranera, hierba de la estrella, and narciset valencià for Andalusia, Murcia and Valencia regions respectively (Ríos et al., 2013).
a
b
Figure 1.2.- Potential distribution of Lapiedra martinezii Lag.: a) in the Mediterranean Basin (redrawn from Bolòs and Vigo, 2001); and b) in Spain redrawn from http://www.floravascular.com (b). The green-colored areas show the Provinces were the plant has been found and the grey-colored areas show the Provinces with doubted presence according to Aedo (2010).
~ 29 ~
The Amaryllidaceae are economically important as ornamentals in the horticultural industry (Bishop et al., 2001; Hanks, 2002; Rivera et al., 2003; Santos et al., 1998). Also, this family plays an important role in the pharmaceutical industry (Bastida et al., 2001; Berkov et al., 2014; Hanks, 2002). Survival of populations of species of Amaryllidaceae in the wild is becoming increasingly threatened by bulb collection from the natural habitat for the horticultural bulb trade (Davis, 1999; Rivera et al., 2003), the pharmaceutical industry, and the habitat loss due to anthropic pressure (Demur et al., 2010; Marques et al., 2007;
Nikopoulos and Alexopoulos, 2008; Nikopoulos et al., 2008; Roselló-Graell et al., 2003;
Serra, 2007). As a consequence, several species of Amaryllidaceae are being considered and listed on the International Union for the Conservation of Nature -IUCN Red Lists of
Threatened Species at the Regional, National, European, Mediterranean and Global Levels
(IUCN, 2015; Moreno et al., 2008).
As far as L. martinezii concerns, the most promising economical interest undoubtedly relies on its content in biologically active alkaloids. The most common alkaloid found in this species is lycorine (Ríos et al., 2013). This alkaloid has a potential use in the chemical industry and, particularly, in the pharmaceutical industry could be used for the treatment of diseases such as Leukemia or Parkinson, among others (Bastida et al., 2011; McNulty, 2009;
2010; Larsen, 2010; Ríos et al., 2013; Suau et al., 1988; Suau et al., 1990). Although the chemical synthesis of lycorine-type, as well as galanthamine-type alkaloids, has been successfully performed (Liu et al., 2014; Magnus et al., 2009; Sellés et al., 1999; Yamada et al., 2009), plants remain an important source of these compounds for the pharmaceutical industry (Georgieva et al., 2007; Berkov et al., 2011).
Due to the interest of these chemical compounds, the number of scientific works carried out on members of the Amaryllidaceae familiy where lycorine- and galanthamine- related compounds were isolated has increased during the last decade (Berkov et al., 2010; ~ 30 ~
Berkov et al., 2014; Georgieva et al., 2007; Georgiev et al., 2012; Hanks; Pavlov et al., 2007;
Pigni et al. 2012; 2013; Berkov et al. 2014; Sellés et al., 1999) including the recent findings on L. martinezii (Larsen, 2010; Ríos et al., 2013).
L. martinezii Lag. has also certain interest in conservation biology. According to the available information on this topic, the main threats for these species can be summarized as follows:
1) Overgrazing could be considered as a relevant factor of threat in several populations
throughout the Iberian Peninsula (Ríos et al., 2013). This was also described for other
Amaryllidaceae such as several Narcissus species and, in some cases, it might
compromise and decrease the competence conditions of the plants (Ríos et al., 2010).
2) The massive and uncontrolled harvesting for the pharmaceutical industry is an
important risk to bear in mind for this species, since plants remain an important source
of alkaloids (Georgieva et al., 2007; Berkov et al., 2011).
3) The most of its populations are included in, or close to current or potential touristy
areas along its distribution area. Although the touristic pressure seems to be
decreasing in last years in coastal and sub-littoral areas of Spain, the deep
transformation and subsequent fragmentation of their natural environments does not
allow to easy recovering of its populations, even leading to the complete extinction of
big populations as it was reported for other littoral distributed plants such as
Pancratium maritimum (Balestri & Cinelli, 2004; Costa, 1999; Demur et al., 2010;
Grassi et al., 2005; Nikopoulos and Alexopoulos, 2008; Nikopoulos et al., 2008;
Serra, 2007).
4) The uncontrolled harvesting of germplasm (mainly bulbs) for horticultural purposes is
a possible risk to bear in mind for this species as happened for other bulbous plants in
~ 31 ~
the past such as daffodils (Miller 1754; Parkinson 1629; Rivera et al. 2003; Santos et
al., 1998).
Ríos et al. (2013) found L. martinezii in 22 habitats of European Community Interest according to D92/43/CEE, and is present in Spain in at least 22 natural protected areas.
Actually, is under evaluation by the IUCN (preliminary assessed as Least Concern for the
Mediterranean Region, Catherine Numa personal comment). It is considered as Least
Concern in Andalusia Region (Blanca et al., 2011). However, it is not found in big quantities along its narrow distribution area in Spain (Ríos, personal comment). It is considered to show average abundance in the Valencian Region (Mateo and Crespo, 2009); locally abundant in some thermic points of Murcia (http://www.regmurcia.com) and Almeria
(http://www.almerinatura.com). Finally, scarce information related to the very few African populations is available (Ríos et al., 2013).
In spite of the increasing interest on this species, there is a complete lack of scientific information on its reproductive biology, propagation and tissue cultures, and response to conventional ex situ seed conservation actions. Therefore, to study the germination biology of this species, as well as to find a protocol for the in vitro propagation of L. martinezii, may be useful for the industrial production of this species, and its conservation, thus respecting its natural populations.
The reproductive strategy of L. martinezii depends on both seed production and natural vegetative propagation (Aedo, 2010; Rios et al., 2013). However, the populations within their natural habitats are quite sparse and appear not to be very abundant with a remarkable threat due to the overgrazing (Ríos et al., 2013). Moreover, the time required to achieve the adult stage comprises usually some years which makes not operative to grow bulbs from seeds.
Although vegetative propagation is faster for growing bulbs, the natural rates of vegetative
~ 32 ~ propagation in vivo of wild bulbs are generally low, much lower than the in vitro propagation rates (de Klerk, 2010; Kim and De Hertogh, 1996; Lilien-Kipnis and Ziv, 1992; Rees, 1969;
Van Rossum et al., 1997).
Since vegetative propagation, or collection from natural habitats, do not make up for the bulbs that could be demanded for industrial purposes, cultivation seems to be the way to obtain a sustainable use of this resource, as well as to prevent overexploitation or extinction of natural populations.
Our work was undertaken with the aim of developing a method for the in vitro propagation of L. martinezii germplasm (seeds, seedlings, bulb scales and leaf bases) that can be used for industrial purposes, as well as to study the seed storage behaviour in order to preserve it ex situ in seedbanks. Also, it may help to establish a general procedure for the in vitro production of bulbs and ex situ storage of seeds of endangered wild species of
Amaryllidaceae in Spain.
1.2.- The ex situ conservation strategies
1.2.1.- Seed germination physiology research and its importance in plant propagation and ex situ conservation.
Germination has a crucial importance in the biological cycle of plant species and in their reproductive success being the most vulnerable stage as it represents the entry of the plants in the ecosystem (Harper, 1977; Navarro and Guititán, 2002; Rajjou et al., 2012;
Weitbrecht et al., 2011). Species have a characteristic germination season (or seasons). For many species, the time of year when germination is possible is quite limited, e.g., only in the autumn, spring or the wet season. In contrast, the germination season for other species is long, e.g. throughout the growing season (Baskin and Baskin, 2001). Seedlings of most species
~ 33 ~ emerge shortly after the seed has germinated in the soil. Thus, timing of seedling emergence is mainly regulated by dormancy breaking and germination requirements of the seed. In some other species, a considerable time lag exists between the moment of radicle protrusion in the field and emergence of the seedling (Vandelook and Van Assche, 2008).
The most favourable period for seed germination and seedling establishment can vary according to several factors such as:
- Geographical distribution of plants (Vandelook and Van Assche, 2008).
- Climatic conditions (Vandelook and Van Assche, 2008).
- Habitat preference (Nikolaeva, 2001).
- Life cycle (Nikolaeva, 2001).
Seeds are the most important way of reproduction and dissemination of plants
(Nikolaeva, 2001). Information on seed germination potentially has great monetary value, e.g., for propagation of native economically important trees, shrubs, vines, forbs and grasses, restoration of damaged ecosystems, and planning for the effective control of weeds (Baskin and Baskin, 2001). Along with this, dormancy in seeds of many economically important plants causes great problems (Nikolaeva, 2001). Therefore, research on seed biology and particularly on germination and dormancy-breaking conditions, are essential goals for economically important plants. Moreover, the information on seed germination physiology is necessary to stablish suitable procedures for ex situ conservation and germination routines in genebanks and in restoration programs of wild species (Baskin and Baskin, 2001; Copete et al., 2011; Walters, 2015).
~ 34 ~
1.2.2.- The in vitro culture techniques as a tool for plant propagation and conservation.
The use of the available in vitro techniques (such as micropropagation) is a suitable strategy for both the propagation and/or conservation of plant germplasm (González-Benito and Martin, 2011; Tasheva and Kosturkova, 2013). These techniques can be driven to the conservation of genetic resources such as endemic, rare and threatened plants, species with recalcitrant seeds or with vegetative propagation, and also of plant genotypes of outstanding interest such as their attractive structures or colors for gardening or the production of secondary metabolites (Engelmann, 2009; Tasheva and Kosturkova, 2013).
The micropropagation industry has expanded from 130 million plantlets produced worldwide in 1986 to some 1-1.5 billion produced in last decade (Prakash, 2009). In the specific case of geophytes, the culture and production of corm and bulb plants would not have attained their full potential without the achievements of plant biotechnology during the last decades (de Klerk, 2010; Ziv, 1997). Particularly, tissue culture is applied commercially (and thus constitutes a cost-effective method of production) in lily and zantedeschia (de Klerk,
2012) and several elite materials of Narcissus cultivars (Squires and Langton, 1990).
Plant micropropagation is also a powerful tool in germplasm conservation that has been applied to a wide variety of rare, endemic and threatened plants (Chandra et al., 2006;
González-Benito and Martin, 2011; Laguna, 1998; Piovan et al., 2010; Demeter et al., 2010;
Sarasan et al., 2006; Pence, 2011) with successful results in some wild Amaryllidaceae species such as Narcissus bulbocodium (Santos et al., 1998), Pancratium maritimum
(Panayatova et al., 2008), Leucojum aestivum (Georgieva et al., 2010) and N. cavanillesii
A.Barra & G.López (David Draper, personal comment.)
~ 35 ~
~ 36 ~
2.- OBJECTIVES
~ 37 ~
~ 38 ~
2.- OBJECTIVES
The main object of this research was to develop a specific strategy for the sutainable propagation and ex situ conservation of Lapiedra martinezii Lag., which can provide the bases for the exploitation of this species with different purposes, as well as to provide new insights for the ex situ conservation of its germplasm.
This strategy might be the first point to establish the scientific and technological bases for the development of the genebank at the University of Alicante. To achieve this goal, the work was structured throughout the following specific objectives:
1.- To study the dormancy-breaking conditions and seed germination physiology of L.
martinezii seeds.
2.- To establish in vitro cultures from different explants (seeds, bulbs and leaves) and
different morphogenetic pathways (direct and indirect organogenesis) of L. martinezii,
as well as to develop an appropriate protocol for L. martinezii bulb micropropagation
3.- To evaluate the seed storage behavior of L. martinezii in order to develop an
appropriate protocol for their long-term conservation.
~ 39 ~
~ 40 ~
3.- SEED DORMANCY AND GERMINATION OF
Lapiedra martinezii
~ 41 ~
~ 42 ~
3.- SEED DORMANCY AND GERMINATION OF L. martinezii
3.1.- Introduction
3.1.1.- Anatomy of seeds
Martin (1946) distinguished 12 types of seeds based on embryo morphology, relative amount of endosperm, and position of the embryo in relation to the endosperm. This classification is still widely used to describe seed types because, sometimes, these seed features are closely related to the seed dormancy type and the germination process (Baskin and Baskin, 2001). With respect to seeds with linear embryos, some interesting differences are found between monocots and dicots. Linear embryos in most dicots are surrounded completely by endosperm, but in some species the radicular end of the embryo touches the base of the seed. The radicular end of the embryo also touches the base of the seed in large number of monocots, including the Amaryllidaceae. The linear embryo extends to both ends of the seed in some other monocot families such as Ponteridaceae and Cannaceae (Baskin and
Baskin, 2001).
3.1.1.1.- Seeds of Amaryllidaceae: anatomy and types
The Amaryllidaceae are extremely variable in fruit and seed characters, particularly among the American, African and Eurasian clades (Meerow et al., 1999, Newton et al.,
2013). Dry, hard, wedge-shaped or irregularly round seeds are characteristic of most of the
Eurasian clade (except Lycoridae), frequently with an elaiosome at the chalazal end (Meerow et al., 1999). Among all genera of the family, Pancratium is the most polymorphic for seed type (Werker and Fahn, 1975). According to Martin (1946) the Amaryllidaceae have underdeveloped linear embryos embedded in the endosperm.
~ 43 ~
3.1.1.2.- Structure of Lapiedra seeds.
Seeds of L. martinezii are ovate and black, with large, with a large strophiole discolorous at first, but finally also blackish. The size of dried seeds is 2-3 mm, and for fresh seeds, it is 4-5 mm (Ríos et al., 2013).
3.1.2.- Seed germination physiology
3.1.2.1.- Seed dormancy and germination
According to Baskin and Baskin (2004) a dormant seed, or other germination unit, is one that does not have the capacity to germinate in a specified period of time under any combination of normal physical environmental factors (temperature, light/dark, etc.) that otherwise is favourable for its germination. A non-dormant seed, on the other hand, is one that has the capacity to germinate over the widest range of normal physical environmental factors possible for the genotype. This inability to germinate under favourable conditions is related either to the properties of the seeds themselves (organic dormancy) or to lack of favourable environmental conditions (imposed dormancy). The ability of seeds to be in dormancy prevents premature seedling emergence on the one hand, and on the other hand it promotes formation of a seed reserve in the soil, which provides conservation of the plant’s genetic viability (Nikolaeva, 2001).
Various systems for classifying seed dormancy have been purposed during the last decades. An overview of them is presented in Baskin and Baskin (2014). However, Nikolaeva was the first seed biologist to develop a comprehensive classification scheme for the various types of seed dormancy and used for the first time names and formulas to refer to the different dormancy types and their interrelations (Nikolaeva, 1969, 1977, 2001). The last version of her scheme is still the best general classification for seed dormancy types available today and it is widely used in studies of seed germination physiology and ecology (Baskin and Baskin, 2001, ~ 44 ~
2004; 2014). Nikolaeva (1969, 1977) distinguished two broad types of organic seed dormancy: endogenous and exogenous (Table 3.1). In endogenous dormancy, some characteristic of the embryo prevents germination, whereas in exogenous dormancy, some characteristic of the structures, including endosperm (sometimes perisperm), seed coats, or fruit walls, covering the embryo prevents germination. Before seeds with either endogenous or exogenous dormancy can germinate, changes must occur in seeds that remove the block or blocks to germination (Baskin and Baskin, 2001).
According to the scheme and classification on seed dormancy purposed by Nikolaeva
(1969, 1977) and explained in Baskin and Baskin (2001; 2014) the types of seed dormancy can be summarized as follows:
1.- Endogenous Dormancy
1.1.- Physiological Dormancy (PD): is caused by a physiological inhibiting mechanism of
the embryo that prevents radicle emergence. However, structures that cover the embryo,
including endosperm, seed coats, and indehiscent fruit walls, may play a role in
preventing germination. Most seeds with PD are permeable to water (Baskin and Baskin,
2001; 2014). This type of dormancy is widely distributed in nature (Nikolaeva, 2001).
Occurs in representatives of all 12 types of seeds described by Martin (1976) and several
families of gymnosperms, as well as all major clades of angiosperms (Baskin and Baskin,
2001; 2014). PD has three different levels of depht, also subdivided into some sublevels.
Duration of cold stratification and response of seeds to dry storage and to treatment with
gibberellic acid are used as indicators of depth of dormancy (Nikolaeva, 2001).
1.2- Chemical Dormancy: seeds do not germinate due to the presence of inhibitors in the
pericarp, either produced in or translocated to the seed, where they block embryo growth. ~ 45 ~
Further, chemical dormancy is broken by removal of the pericarp and leaching of the fruits. Germination inhibitors have been found in the embryo, endosperm, and seed coats of seed and in structures that sometimes are dispersed along with the seeds. Traditionally this dormancy was included as a category of Exogenous Dormancy (Baskin and Baskin,
2001; 2004; Nikolaeva, 1977). However, following the newest scheme of classification
(Baskin and Baskin, 2014) we consider as a component within the PD (Finch-Savage and
Leubner-Metzger, 2006; Baskin and Baskin, 2014).
1.3.- Mechanical Dormancy: is due to the presence of a hard, woody, fruit wall (usually endocarp, or mesocarp). Stony endocarps are found in many families of angiosperms such as Anacardiaceae, Rosaceae, Juglandaceae and Oleaceae (Baskin and Baskin, 2014).
Traditionally this dormancy was included as a category of exogenous dormancy (Baskin and Baskin, 2001; 2004; Nikolaeva, 1977). However, following the newest scheme of classification (Baskin and Baskin, 2014) we consider as a component within the PD
(Finch-Savage and Leubner-Metzger, 2006, Baskin and Baskin, 2014).
1.4.- Epycotil Physiological Dormancy: is the term to describe a delay of about 3-4 weeks
(or longer) in emergence of shoot after the radicle has emerged in seeds with fully developed embryos (Baskin and Baskin, 2014). Traditionally this category of dormancy was included as a subcategory within Morphophysiological Dormancy, named as deep simple epicotyl MD (Baskin and Baskin, 2001; 2004; Finch-Savage and Leubner-
Metzger, 2006; Nikolaeva, 1977; 2001). However, following the newest scheme of classification (Baskin and Baskin, 2014) we consider as a separate category because it refers only to seeds with fully developed embryos whose also displays PD (Baskin and
~ 46 ~
Baskin, 2014). Some species of ‘white oaks’ (genus Quercus, subgenus Lepidobalanus) are examples of Epicotyl PD seeds.
1.5.- Morphological Dormancy (MD): the germination is prevented at the time of dispersal. The embryo, although could be fully differentiated into radicle and cotyledon(s), is not fully grown (underdeveloped) at the moment of dispersal. Thus, embryo growth is required before germination occurs. In seeds of other species, the embryo is just a mass of cells at the time of dispersal and germination does not take place until both differentiation and growth occur (Baskin and Baskin, 2001; 2014).
1.6.- Morphophysiological Dormancy (MPD): occurs in seeds with rudimentary or linear
(underdeveloped) embryos, and is combined with physiological dormancy. Two general kinds of events (described in two or three different stages) must happen before seeds with
MPD can germinate: 1) the embryo must grow to a species-specific critical size and 2) physiological dormancy of the embryo must be broken (Finch-Savage and Leubner-
Metzger, 2006; Baskin and Baskin, 2001; 2004; 2014; Nikolaeva, 2001). These seeds require a dormancy-breaking treatment, e.g. a defined combination of warm (normally for the embryo growth) and/or cold stratification (to overcome the physiological component of the dormancy), which in some cases can be replaced by gibberellic acid application
(Finch-Savage and Leubner-Metzger, 2006). The nature of germination of seeds with
MPD is one of the most complicated and still little investigated problems (Nikolaeva,
2001). Traditionally, eight levels of MPD, based on the protocol for seed dormancy break and germination, were considered (Baskin and Baskin, 2001; 2004; Finch-Savage and
~ 47 ~
Leubner-Metzger, 2006; Nikolaeva, 2001). However, the newest scheme of classification
(Baskin and Baskin, 2014) distinguish nine different levels.
2.- Exogenous Dormancy.
2.1.- Physical Dormancy (PY): the primary reason for the lack of germination is the
impermeability of seed (or fruit) coats to water. PY is present in at least 15 families of
angiosperms. Based on embryo morphology, seven types of seeds are found among the
15 families known (Baskin and Baskin, 2001; 2014).
3.1.2.2.- Seed dormancy and germination of Amaryllidaceae species
So far, the main type of seed dormancy described in Amaryllidaceae species is the endogenous dormancy. Seeds of the Amaryllidaceae species would undoubtedly display MD since their representatives have underdeveloped embryos at the time of seed dispersal
(Martin, 1946). However, Marques and Draper (2012) observed that embryos were fully grown at the time of seed dispersal in N. cavanillesii and N. serotinus and concluded that seeds of these species were non dormant. In other Amaryllidaceae, it is quite common to find seeds with PD associated with MD (Copete et al., 2011; 2014; Herranz et al., 2013a; 2013b;
2015). Thus, these seeds have MPD (Baskin and Baskin, 2001; 2014). As it is shown in
Vandelook and Van Assche (2008) the level of embryo growth required before germination is variable among species. In terms of dormancy, this means that a gradient can be observed in the extent of morphological dormancy.
~ 48 ~
Seed Dormancy Types Type Cause Broken by Endogenous dormancy 1.- Physiological Physiological inhibiting mechanism (PIM) of germination Warm and/or cold stratification 2.- Chemical Germination inhibitors Leaching 3.- Mechanical Woody structures restrict growth Warm and/or cold stratification 4.- Epycotil Physiological Dormancy PIM of shoot emergence Warm and/or cold stratification 5.- Morphological Underdeveloped embryo Appropriate conditions for embryo growth/germination 6.- Morphophisiological PIM of germination and underdeveloped embryo Warm and/or cold stratification
Exogenous dormancy 1.- Physical Seed (fruit) coats impermeable to water Opening of specialized structure
Table 3.1.- Types and causes of seed dormancy and the different treatments to broke it (scheme adapted from Baskin and Baskin, 2014 as a simplified version of Nikolaeva’s, 1977, Classification Scheme).
~ 49 ~
3.1.2.3.- Seed germination of Amaryllidaceae species.
According to the general scheme for germination of close related species Allium cepa
(Fahn, 1990) after a more or less prolonged dormancy, if any, the germination takes place.
Germination is essentially a resumption of embryo growth after uptake water, or imbibition.
During imbibition, the water content of the seed rises, usually fast at first then slower, and the quiescent tissue becomes metabolically active, associated with several ultrastructural changes.
Enzymes already present are activated and new proteins with specific enzymatic activities are synthesized for the digestion and utilization of the different kinds of stored materials. Cell extension and cell division are initiated and proceed according to a programmed pattern. This growth requires a continuous supply of water and nutrients. Before the embryo becomes self- supporting seedling, it utilizes the food stored in the endosperm and embryo itself.
After the mobilization of food reserves takes place, the cotyledon elongates at its base and pushes the embryo axis from the seed, forcing the coleoptile or cotyledonary sheet to emerge through the seed coat at the micropyle. Furthemore, the elongation occurs in the root and the shoot. The shoot (corresponding to the first leave) breaks through the cotyledonary sheet and raises above the ground. The haustorial tip of the cotyledon remains embedded in the endosperm, probably, to draw upon the food reserves (Fahn, 1990; Vandelook and Van
Assche, 2008). The part emerging above the ground becomes green and photosynthetically active. Afterwards, the part of the cotyledon above the bend dries out from the tip down.
Finally, adventitious roots emerge (Vandelook and Van Assche, 2008).
~ 50 ~
3.1.2.4.- The role of cold, warm and gibberellic acid pre-treatments to overcome dormancy and promote seed germination.
Some treatments are commonly used to break dormancy and/or enhance germination in wild plants. Among these, cold stratification, warm temperatures and application of exogenous gibberellins are widely used (Baskin and Baskin, 2001). The first publication on seed germination of Mediterranean Amaryllidaceae was done by Keren and Evenary (1974) in
Pancratium maritimum from Israel, and high germination percentages were obtained after incubation in a wide range of temperatures. Similar results were obtained in Nikopoulos et al.,
(2008) and Baslestri and Cinelli (2004) for this species, in Greece and Italy respectively.
However, the latter suggested the presence of dormancy due to the small delay on complete seed germination, but the authors did not discuss this topic. Thompson (1977) studied seed germination of Narcissus bulbocodium and obtained high germination percentages after a warm stratification followed by a cooler seed exposure. Blanca et al. (1999) obtained similar results for the Spanish N. bugei after chilling pre-treatment. This suggests that these species have some level of Physiological Dormancy and, therefore, stratification is needed in order to overcome it and induce seed germination. Also, studies carried out in N. longispathus (Blanca et al., 1999) and N. nevadensis (Blanca et al., 1999; Lorite et al., 2007) showed a lack of germination under conventional thermoperiods, suggesting the need of pre-treatments to promote seed germination and thus, the presence of seed dormancy (Baskin and Baskin,
2014). However, data on temperature or light requirements for embryo growth, dormancy- break and germination of these species were lacking.
The first systematic study published on this topic for Eurasian Amaryllidaceae species was done for N. pseudonarcissus (and two other temperate forest spring geophytes) growing in Belgium (Vandelook and Van Assche, 2008). The authors determined that seeds have
Morphophysiological Dormancy (MPD) and germination took place after warm pre-treatment ~ 51 ~ followed by low temperatures (<10ºC). In this line, several studies have been carried out on
Amaryllidaceae in the Mediterranean Basin during the last years, showing different levels of
MPD: Narcissus hispanicus (Copete et al., 2011), N. eugeniae (Copete et al., 2014) and N. radinganorum (Herranz et al., 2015) both showing deep simple epicotyl MPD that is overcome after an alternate cycle of warm and cold stratification; N. alcaracensis with intermediate complex MPD that requires only cold stratification to break seed dormancy
(Herranz et al., 2013a) and N. longispathus non-deep complex MPD (Herranz et al., 2013b) that requires warm stratification followed by cold one to germinate. In contrast, N. serotinus germinated under a wide range of temperatures, while N. cavanillesii only did it at 15ºC, but seeds of both two species were considered non-dormant (Marques and Draper, 2012).
Regarding growth regulators, it is generally accepted that gibberellic acid induces dormancy-break and promotes seed germination. Gibberellic acid promotes seed germination in some species with different levels of PD. Also, gibberellic acid enhances germination in some other non-dormant species (Baskin and Baskin, 2004). There are some examples of enhancement of seed germination by means of exogenous gibberellic acid application in cultivated plants (Baskin and Baskin, 2001). In wild plants, it has been reported that gibberellic acid promoted seed germination in Narcissus nevadensis and N. tortifolius (Blanca et al., 1999), Pancratium maritimum at lower concentrations than 0.5 g/L (Balestri & Cinelli,
2004). Higher gibberellic acid concentrations than 1 g/L promoted germination in other wild taxa as it was shown for Ferula gummosa Boiss and Teucrium polium L. (Nadjafi et al.,
2005), Carpinus betulus L. and C. orientalis Mill. (Pipinis et al., 2012), Melastoma dodecandrum Desr. (Tang et al., 2012) and Butia capitata Becc. (Dias et al., 2013).
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3.2.- Objectives
The main objectives in this chapter were to determine the optimum temperature regime for seed germination of L. martinezii, to test the effect of different treatments on promoting germination. Concretely, we wanted to test:
- The effect of different temperatures on seed germination.
- The effect of light on seed germination.
- The effect of a cold pre-treatment on seed germination.
- The effect of a warm-pretreatment on seed germination.
- The effect of gibberellines on seed germination.
3.3.- Material and Methods
3.3.1.- Seed collection and preparation
Seeds were harvested from mature capsules at the time of natural dispersal in October
2011 and 2013 in Santa Pola, (Spain). The seeds from 2013 were used to test the effect of gibberellic acid treatment, whereas the other experiments were performed with the seeds collected in 2011. In all cases, mean embryo-endosperm (E:S) of L. martinezii seeds was measured once collected using a dissecting microscope equipped with a micrometer
(Vandelook and Van Assche, 2008). Seeds were kept in the laboratory at room conditions
(22-25ºC and 50% HR) until experiments were initiated one week after seed collection.
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Figure 3.1.- Location of the plant material source area in the proximities of Santa Pola’s Cape (Santa Pola, Spain).
Figure 3.2.- Seed samples kept in the laboratory at room conditions (22-25ºC and 50% HR) for the experiments.
~ 54 ~
3.3.2.- Seed germination of L. martinezii
3.3.2.1.- The effect of temperature on seed germination
Seed germination was assayed at three alternate (30/20, 25/16 and 17/10ºC) and three constant (20, 17 and 4ºC) temperatures. In all germination assays, four replicates of 50 healthy seeds were used. Seeds were previously sterilized by immersion in 20% commercial bleach solution for 15 minutes and rinsed six times in distilled water. Afterwards, the seeds were placed on a filter paper disk (518G Filter-Lab) moistened with 5 mL distilled water in a
90 mm-diameter Petri dish. Petri dishes were incubated in a growth chamber (Ibercex, model
F-4, Madrid, Spain).
3.3.2.2.- The effect of light regime on seed germination
Two light regimes or photoperiods were used along with the temperature regimes above mentioned: 1) an 8-hour light / 16-hour dark photoperiod (photosynthetically active radiation 25 µmol m-2 s-1) under the light/dark photoperiod (light hereafter) and 2) a complete darkness photoperiod. Dark conditions were imposed by wrapping plates with aluminium foil.
3.3.2.3.- The effect of a cold pre-treatment on seed germination.
To determine if physiological dormancy is present, a cold stratification pre–treatment was performed by incubating seeds at 4ºC for 15 and 30 days under wet conditions.
3.3.2.4.- The effect of a warm pre-treatment on seed germination.
A warm pre-treatment was also applied by packaging dry seeds in double-sealed laminated aluminium foil bags and heating them in a thermostatic bath at 40ºC for 20 days.
After pre-treatments of cold and warm seeds were incubated at 30/20, 25/16 and 17/10ºC and
20ºC in light conditions.
~ 55 ~
3.3.2.5.- The effect of gibberellic acid on seed germination.
To study the effect of gibberellic acid on seed germination, a sample of seeds was immersed in 0.0, 0.01, 0.125, 0.25, 0.5, 0.75, 1 and 2 g/L concentrations of GA3 for 48 hours and then incubated at 20ºC in light.
For this and subsequent experiments, germination was monitored regularly for over
100 days. Most germination occurred within the first 30-40 days and no germination was registered after 50-60 days of culture. The following germination parameters were measured:
- Final seed germination percentage: a seed was considered to have germinated when the coleoptile had protruded by 5 mm.
- Germination onset: day when the first seed germinated.
- T50: day when 50% germination was achieved.
3.3.2.6.- Statistical analyses
All analyses were carried out by means of ANOVA and significant differences between means identified with Fisher’s Least Significant Differences test (Fisher’s LSD) at the 5% level. Final germination percentages were arcsine square root transformed. For the other parameters (onset and T50), non-transformed data were used. All data were analysed using the Infostat 2008 package.
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a b
c d
e f
Figure 3.3.- Preparation and process of the Lapiedra martinezii Lag. seed germination experiments : a) sowing seeds for the temperature and light regimes experiments; b) replicates incubated at 17/10ºC after cold pre-treatment; c) application of dry-warm pre-treatment to seeds in double-sealed laminated aluminium foil bags in thermostatic bath; d) immersion of seeds in different concentrations of GA3; e) Growth chambers employed to incubate seeds; f) regular seed germination counting in laminar flow cabinet.
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3.4.- Results and Discussion
3.4.1.- Seed germination of Lapiedra martinezii seeds.
3.4.1.1.- The effect of temperature on seed germination.
At the time of seed dispersal, L. martinezii seeds showed a mean embryo-endosperm ratio (E:S) of 0.78 ± 0.09 and 0.76 ± 0.10 (2011 and 2013 seed samples, respectively). After germination, the E:S ratio was 0.92 ± 0.02 and 0.91 ± 0.05 respectively. This shows that the embryo had to grow inside the mature seed before they could germinate. According to this, the embryo was not fully developed at the time of seed dispersal and seeds of L. martinezii have morphological dormancy (Baskin and Baskin, 2001; Nikolaeva, 2001). Seeds reached the highest and fastest germination rates at 20ºC, and over 90% of the seeds germinated in both light and darkness. At this temperature, germination onset occurred after one week and
50% germination was reached after around 15 days (figure 1). Mean final germination
(between light and dark conditions) of 85.2, 83.5 and 70.2% were obtained at 17, 25/16 and
30/20ºC, respectively. Mean (between light and dark) germination onset and T50 ranged from
13 to 16 days and from 21 to 26 days, respectively, under these temperature regimes.
Germination was nil at 4ºC, and germination percentages of less than 5% and very low germination speed were obtained at 17/10ºC (figure 1). In all cases, once germination occurred (emergence of coleoptile), all seedling structures (radicle, bulbil and leaf) developed in less than two weeks.
3.4.1.2.- The effect of light regime on seed germination.
Light conditions did not have a significant effect on germination at any temperature, although germination was generally slower in light. Germination onset and germination speed
~ 58 ~ were shorter in darkness at all temperatures, except at the optimal temperature of 20ºC (figure
1). A seed germination optimum at 20ºC has also been documented for other plant species from the same environments in the Mediterranean region, such as Chamaerops humilis L.
(González–Benito et al., 2006), Launaea arborescens Murb. (Schütz and Milberg, 1997),
Crithmum maritimum L. (Marchioni-Ortu and Bocchieri, 1984) and Pancratium maritimum
L. (Keren and Evenari, 1974, Nikopoulos et al., 2008). This temperature regime also seems to be common for the seed germination of plants from other semiarid regions around the world
(Rojas-Aréchiga and Vázquez-Yánez, 2000).
In a study on the seed germination behaviour of five autumn flowering geophytes growing in Mediterranean grasslands, Marques and Draper (2012) found that seed germination was optimum at 20ºC for four of five autumn-flowering species studied. These authors also found that light had a marginal effect on final germination, although darkness increased germination speed in all cases. In our study, we also found a similar pattern where germination was generally faster in dark conditions. Navarro and Guitian (2003) suggested that the good germination rates obtained with seeds of Petrocoptis grandiflora and P. viscosa in dark conditions could benefit seeds that fall into cracks or crevices in the rock face. This strategy would help L. martinezii seeds to germinate in rocky outcrops, where the species has commonly been reported to occur (Aedo, 2015; Bolòs and Vigo, 2001; Ríos et al., 2013).
L. martinezii seeds are normally dispersed in October. This dispersal phenology allows seeds to avoid the extremely dry conditions during the summer in the seed source area and to concentrate the germination during October, when remarkably higher rainfall is registered
(Table 3.2) and thus, continuous moisture in soil might be guaranteed. The highest germination rates were obtained at constant temperatures of 20ºC (figure 1). This temperature is very close to the mean temperatures in October in the seed source area. Also, the suboptimal regime of 25/16ºC is in agreement with the cycle of maximum/minimum ~ 59 ~ temperatures registered in the seed source area in this month. The temperatures in November are relatively close to the optimal (17ºC) and suboptimal (25/16ºC) temperatures obtained in laboratory experiments. Therefore, a proportion of seeds would be also able to germinate during November in natural habitats.
Fast seed germination has been documented for some other late summer-autumn flowering geophytes with more or less underdeveloped embryos in Mediterranean ecosystems
(Baskin and Baskin, 2001; Balestri and Cinelli, 2004; Nikopoulos et al., 2008; Marques and
Draper, 2012; Juan-Vicedo et al., 2013). An explanation to this rapid germination during the short rainfall period would be that seeds have limited desiccation tolerance at dispersal as shown for other Amaryllidaceae (Berjak and Pammenter, 2004; Sershen et al., 2008; Newton et al., 2013). In our opinion, a more plausible explanation for L. martinezii would be that rapid germination and seedling establishment ensures the high production of bulbils during the short rainfall period before the winter starts, regardless of the amount of moisture at the time of dispersal. This quick germination and bulb formation (as a structure to store water) is a convenient strategy for plants growing in semiarid regions (Baskin and Baskin, 2001).
Moreover, the results obtained in this study on seed storage behaviour do not support the hypothesis of limited desiccation tolerance of L. martinezii seeds at dispersal (discussed in
Chapter 5). In contrast to this strategy, other Mediterranean bulbs with spring–flowering phenology have morphophysiological dormancy (Copete et al., 2011; 2014; Herranz et al.,
2013; 2015) and is needed more than six months to complete germination.
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Figure 3.4.- Germination progress curves for seeds of Lapiedra martinezii Lag. at different temperatures with a 8-hour photoperiod (light) or in the dark. Different letters on final germination points indicate significant differences (P < 0.05).
3.4.1.3.- The effect of a cold pre-treatment on seed germination.
To determine if physiological dormancy was present, a cold stratification pre– treatment was performed by incubating seeds at 4ºC for 15 and 30 days under wet conditions.
After the pre-treatments, seeds were incubated at 30/20, 25/16 and 17/10ºC and 20ºC in light conditions. The results obtained after the cold pre-treatment showed an inverse relationship between pre–treatment duration and seed germination percentages and speed at all the assayed ~ 61 ~ temperatures (Figure 3.6). When extended up to 30 days, the cold pre-treatment had a significant negative effect on germination as shown by the delay in germination onset, increase in T50 and decrease in germination percentages. These results and the low germination rates obtained under the colder regimes (17/10 and 4ºC) suggest that L. martinezii seed germination is highly cold-sensitive. Considering this limitation at cool temperatures, it seems very unlikely that this species is able to germinate and colonise successfully in areas with colder autumns such as continental and mountain areas in the
South-West Mediterranean. This would explain why L. martinezii displays a littoral and sub- littoral pattern of distribution. The limited germination at cold temperatures could be related to the similarities of the current environmental conditions within its distribution area, and the warm conditions where this species originated as suggested by Ríos et al. (2013). These authors purposed three different scenarios for the origin and biogeographic distribution of L. martinezii. In all scenarios, the origin is around the Alboran Sea, and then spreading along the warmest and most arid lands of the Iberian Peninsula (reaching North Africa) after the desiccation of the Tethys Sea.
Thus, the lower rates of seed germination under the coldest temperature regimes assayed and after the cold pre-treatment may be explained as an adaptation to avoid massive germination in an unfavorable period. Furthermore, cold exposure seems to induce secondary dormancy in a considerable proportion of seeds which would restrict germination after winter even though environmental conditions are more appropriate. This view is supported by the
0% germination obtained during 3 months after seeds had been maintained at 4ºC for 100 days and then transferred to 20º. However, most of the non-germinated seeds were stained by the tetrazolium test showing that they were still alive (data not shown).
In nature, seeds which fail to germinate in autumn would probably remain torpid in winter and could germinate in the following spring when temperatures rise. At colder ~ 62 ~ exposures, they would enter a period of secondary dormancy that could be overcome with a more complex temperature cycle (for instance, those comprised between spring and the next autumn). The induction of secondary dormancy by cold exposure has been reported in other
Amaryllidaceae such as Narcissus radinganorum (Herranz et al., 2015).
3.4.1.4.- The effect of a warm pre-treatment on seed germination.
A warm pre-treatment was also applied by packaging dry seeds in double-sealed laminated aluminium foil bags and heating them in a thermostatic bath at 40ºC for 20 days.
The exposure of seeds to a warm-dry pre-treatment had a positive effect on seed germination capacity and germination speed (Figure 3.6). This effect was especially prominent at the warmest temperature, where the germination percentage increased from 63 to 87%. In general, germination onset and T50 were significantly shorter in seeds treated under the
30/20, 25/16 and 17/10ºC regimes, although the germination percentage was still very low under the latter.
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Figure 3.5.- Onset, T50 and final seed germination of Lapiedra martinezii after cold (left) and warm (right) pre-treatments under different temperature regimes in light. Different treatment durations (days) are represented by different bar patterns. For each pre-treatment and parameter, different letters indicate significant differences (P < 0.05).
In nature, this response would increase the germination capacity of seeds ripened during the period of harsh environmental conditions (high temperatures and drought) in late summer before the start of the rainfall period. Once the rainfall period has started, seeds would be able to germinate under a relatively wide range of conditions (temperature and light). A warm pre-treatment is normally required to break MD and induce embryo growth in ~ 64 ~ seeds with underdeveloped embryos (Baskin and Baskin, 2001). In L. martinezii seeds, the overall time required for embryo growth and seed germination is between seven days at 20ºC and approximately 16 days at 30/20ºC. If a warm-dry pre-treatment is applied, a significant reduction in the time required for germination is observed (figure 2). This fact suggests that embryos grow during this warm pre-treatment and thus, seeds can germinate faster once transferred to the different laboratory temperatures. Therefore, this confirms that seeds of L. martinezii have morphological dormancy.
3.4.1.5.- The effect of gibberellines on seed germination.
To study the effect of gibberellic acid on seed germination, a sample of seeds was immersed in 0.0, 0.01, 0.125, 0.25, 0.5, 0.75, 1 and 2 g/L concentrations of gibberellic acid
(GA3) for 48 hours and then incubated at 20ºC in light. These gibberellic acid pre-treatments did not significantly influence final germination (99.6, 99.0, 100, 100, 9.5 and 100% with 0.0,
0.01, 0.125, 0.25, 0.5 and 0.75 g/L GA3, respectively) or germination speed (Table X). The
higher concentrations of 1 and 2 g/L GA3 had a significant inhibitory effect on seed germination (P < 0.05), whereas the germination speed remained the same as with the lower concentrations (Table 3.3). The proportion of germinated seeds in 1 and 2 g/L pre-treatments was 84.5 and 88.0%, respectively. There is no general agreement concerning the possible inhibitory effect of this growth regulator. However, some studies have demonstrated a decrease in seed germination in wild plants, as we report here, at high concentrations (Rojas-
Aréchiga et al., 2011).
~ 65 ~
Month January February March April May June July August September October November December T (mean) 11.0 12.0 14.2 16.2 19.2 23.0 25.6 26.2 23.7 19.6 15.5 12.4 T (max) 16.0 17.3 19.7 21.5 24.7 28.6 31.4 31.8 29.3 24.8 20.6 17.3 T (min) 6.1 6.7 8.7 10.9 13.7 17.4 19.9 20.6 18.2 14.4 10.4 7.6 Rainfall 20.0 23.0 22.0 29.0 26.0 16.0 4.0 8.0 35.0 61.0 41.0 32.0
Table 3.2.- Mean, maximum, minimum monthly temperatures (ºC) and monthly rainfall (liters) in Santa Pola, Alicante (Spain). Information obtained from the electronic resource (http://es.climate-data.org/).
Concentration of gibberellic acid (g l-1) 0 0.01 0.125 0.25 0.5 0.75 1 2 Onset (days) 11.5c 10.75bc 10.75bc 10ab 10ab 10ab 9a 9.25a T50 (days) 21ab 20.25ab 21.5ab 22bc 19.75a 21ab 24c 23.75c Final germination (%) 99.6bc 99bc 100c 100c 99.5bc 100c 84.5a 88a
Table 3.3.- Effect of gibberellic acid on seed germination of Lapiedra martinezii Lag. The experiment was carried out at 20ºC with an 8-hour photoperiod. For each parameter, different letters indicate significant differences (P < 0.05).
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3.5.- Conclusions.
- Embryos of Lapiedra martinezii Lag. seeds had to grow within the seed before the
coleoptile emergence took place and thus this species’ seeds show morphological
dormancy.
- Optimal seed germination was achieved at 20ºC with germination percentages of
almost 100% both in light and darkness.
- Suboptimal germination percentages (>60%) were obtained at 17ºC, 30/20ºC and
25/16ºC.
- Light conditions influenced germination speed, but did not affect germination
percentages.
- Cold stratification decreased germination parameters at all the assayed temperatures.
- Warm pre-treatment generally increased germination rates.
- The application of GA3 did not affect germination at concentrations between 0-0.75
g/L, while the proportion of germinated seeds decreased at higher concentrations
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~ 68 ~
4.- BIOTECHNOLOGICAL APPROACHES TO
THE MANAGEMENT OF Lapiedra martinezii
GERMPLASM
~ 69 ~
~ 70 ~
4.- BIOTECHNOLOGICAL APPROACHES TO THE MANAGEMENT
OF L. martinezii GERMPLASM.
4.1.- Introduction.
Plant biotechnology is defined as the integrated scientific activity of cell culture and gene manipulation for tailoring plant resources to generate commercial plant products. It amalgamates two disciplines: tissue culture and molecular biology. The plant biotechnology includes (Ziv, 1997):
- Plant tissue culture: micropropagation, large-scale cultures, plant regeneration and
pathogen elimination.
- Genetic manipulation (modification of horticultural traits): in vitro pollination,
fertilization and embryo rescue, production of haploid plants, callus culture, protoplast
culture and somatic hybrids, transformation and genetic manipulation, DNA
fingerprinting and genome mapping.
Plant tissue culture includes a collection of in vitro techniques used to maintain, grow or regenerate plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition under strictly controlled environmental conditions (Larkin and
Scowcroft, 1981). Tissue culture techniques have been used extensively for the micropropagation of almost all the commercially grown geophytes (Ziv, 1997).
4.1.1.- Micropropagation techniques and process.
Micropropagation is the true-to-type propagation of a selected genotype using in vitro techniques (Debergh and Read, 1991; Werbrouck and Debergh, 1996). Micropropagation is achieved by enhancing axillary bud development, organogenesis and adventitious bud formation or by somatic embryogenesis. Regeneration of plants under these techniques is ~ 71 ~ based on plant totipotency: the ability of isolated plant cells in culture to dedifferentiate, redifferentiate and regenerate new plants (George and Debergh, 2008; Ziv, 1997).
Some of the advantages of these techniques with respect to classical vegetative propagation include (George and Debergh, 2008):
- The amount of plant material (explants) needed to start the micropropagation
process is smaller than in vegetative propagation by classical methods.
- The micropropagated material obtained is pathogen free.
- Factors controlling vegetative regeneration (temperature, growth regulators and
light regime) can be controlled. Thus, the multiplication rate can be optimized for
each plant.
- Allows the clonal multiplication of plants with difficulties to be propagated by
classical methods.
- Plant production is not submitted to seasonal changes.
- The produced plants can be stored for extended periods.
- It requires less space and time for storing plant stocks.
4.1.1.1.- The basis of Micropropagation
According to George and Debergh (2008) two different ways are, basically,
employed to carry out in vitro micropropagtion in plants (Figure 4.1).
1.- Direct Organogenesis: adventitious shoot formation from meristems or other
tissues.
2.- Indirect Organogenesis: regeneration of plants from an undifferentiated cell mass
(callus).
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Figure 4.1.- Scheme of the different strategies used to propagate plants in vitro from axillary buds and from adventitious buds according to George and Debergh (2008).
4.1.1.2.- Stages in the micropropagation process.
Based on Werbrouck and Debergh (1996) and George and Debergh (2008) the micropropagation process generally involves five stages:
1.- Stage 0: the preparative stage. The plant material for in vitro culture is prepared;
the aim is to obtain hygienic and physiologically better adapted starting material.
2.- Stage 1: initiation of cultures. The aim is to obtain a reliable starting
contamination-free material. Multiplication is not important.
3.- Stage 2: multiplication. The main objective of this stage is to increase and maintain
the shoot stock; meristematic centers are induced and developed into buds and/or
shoots. ~ 73 ~
4.- Stage 3: Shoots are usually elongating and roots appear.
5.- Stage 4: acclimatization. Transfer to greenhouse conditions in order to ensure their
survival ex vitro.
4.1.2.- Application of in vitro techniques in bulbous plants propagation.
Micropropagation via tissue culture might solve the problem of slow natural propagation rates of bulbous plants. Principally, in tissue culture, every type of tissue can be used as a starting material, but for in vitro culture of geophytes such as tulips or daffodils generally two types of tissue are of interest: bulb scales and stalks (George and Delbergh,
2008; Santos et al., 1998; van Rossum et al., 1997). The bulb scale explants consist in cuttings of the sterilized bulb into several parts containing meristematic tissue that constitutes the explant (George and Debergh, 2008).
The use of liquid media sometimes optimizes the plant multiplication by means of increasing micropropagation rates and reducing the level of plant stress in culture by diluting the potentially toxic compounds that may be released (de Klerk, 2012; George and Debergh,
2008; Hvoslev-Eide and Preil, 2005; Marchev et al., 2011). Some works searching biomass production have been successfully done with some bulbous plants using liquid media under culture conditions of permanent immersion. Some examples are Charybdis numidica (Jord. &
Fourr.) Speta (Wawrosch et al., 2005), Hippeastrum x chmielii Chm. (Ilczuk et al., 2005),
Lilium L. sp., Fritillaria thunbergii Miq., Hippeastrum hybridum Hort., Gladiolus L.,
Hyacinthus orientalis L. (Takayama and Akita, 2005), Ixia L. hybrids (Ruffoni et al., 2005),
Narcissus L. sp. (Chen and Ziv, 2001) and Nerine Herb. (Lilien-Kipnis and Ziv, 1992).
Other applications of in vitro techniques include to supply plant material for DNA analyses (Sarasan et al., 2006). Finally, the increasing demand on secondary metabolites for the pharmaceutical industry or food technologies has increased the amount of ~ 74 ~ biotechnological tools used to produce high-efficiency plants under controlled growth conditions in order to obtain stable genotypes (Avato et al., 2005).
For all these reasons we will test the efficacy of different in vitro cultures (in solid and liquid systems) on L. martinezii micropropagation. The results obtained could also be applied to other rare, endemic or threatened Amaryllidaceae species.
4.1.3.- Callus induction as a source of new germplasm.
Underground organs (bulbs, corms, tubercles, etc) are often used as a source of explants for in vitro direct organogenesis in geophytes (George and Delbergh, 2008; Santos et al., 1998; van Rossum et al., 1997). These organs are in contact with different types of microbes that suppose a high risk of infections and contaminations in vitro. In fact, one of the biggest problems concerning in vitro cultures is the sterilization of plants (Sochacki and
Orlikowska, 2005; Ziv, 1997). The contamination rate found in in vitro cultures is mostly influenced by growth conditions, especially by the soil type and the weather (Sochacki and
Orlikowska, 2005). Therefore, the use of alternative pathways (for instance, callus culture based on leaf, root or flower explants) to obtain axenic and less labor in vitro cultures is of particular interest (George and Delbergh, 2008).
Calli cultures can be regarded as a tool for genetic manipulation throughout the modification of certain horticultural traits (Ziv, 1997). In addition, callus induction is a key point both for micropropagation throughout indirect organogenesis (George and Delbergh,
2008; Pavlov et al., 2007), thus obtaining germplasm for ecosystem restoration and conservation (Irvani et al., 2010; Piovan et al., 2010) and also as a more efficient system for in vitro alkaloid production (Pavlov et al., 2007).
~ 75 ~
L. martinezii is a high valuable species, with economic potential and certain interest in conservation. Thus, it seems important to determine the conditions for the establishment of different in vitro calli cultures as a new germplasm to avoid damages in natural populations and support further research on biotechnology, phytochemistry or crop plant production for the ornamental and/or pharmaceutical industries, as well as conservation-restoration purposes.
Therefore, we present here a study of different methods for the L. martinezii in vitro callus induction and establishment.
4.2.- Objectives
The main goal of this chapter was to establish different in vitro cultures starting from bulbs, seeds or leaf explant and exploring different morphogenetic pathways (direct and indirect organogenesis) of L. martinezii in order to obtain an appropriate protocol for bulb micropropagation. The specific objectives were:
- To optimize the sterilization protocol for L. martinezii bulb explants.
- To optimize the initiation phase in seedling and bulb-scale cultures.
- To study the morphogenetic response of bulb explants in 14 different
multiplication media.
- To assess the rooting and acclimatization capacity of produced bulblets.
- To check the plant survival in stock seedling cultures after a long culture
period at 17ºC.
- To establish the optimal conditions and growth regulator concentrations for
callus induction as a source of new germplasm.
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4.3.- Material and methods
4.3.1.- Bulb collection and preparation
Healthy plants showing typical morphological features were selected from the population of Santa Pola Cape (Santa Pola, Alicante, Spain). This population is not included in any protected area and it is located in a mountain through at 3 m.a.s.l. (coordinates:
38º13’30.70’’N/ 0º30’45.11’’W).
A sample of bulbs was harvested from this population in the fruiting period during the first week of October, 2013. The bulbs were sown in a peat-moss mixture and placed at room conditions (T= 22-25ºC and HR=40-55%), being watered every week until the leaves appeared. At this time, whole plants were taken and treated as is explained in the following sections in order to obtain the different explants (bulbs or leaf bases) for the in vitro culture
(Figure 4.2.
4.3.2.- In vitro seedling culture
4.3.2.1.- Seed collection and preparation
Seeds were collected from mature capsules were harvested on October, 2011 and 2013 in the above-mentioned population (Santa Pola, Alicante). The capsules were naturally dried at room conditions in the laboratory (T= 22-25ºC and HR=40-55%) during one week.
Afterwards, healthy seeds were used for laboratory trials:
~ 77 ~
4.3.2.2.- Seed surface sterilization
Seeds were surface-sterilized by a short dip (30 s) in 70% ethanol followed by immersion in 0,1% HgCl2 for 3 min. Seeds were then rinsed three times in sterile distilled water and placed to germinate.
4.3.2.3.- Initiation of seedling cultures
Sterilized seeds were sown in a 90 mm-diameter Petri dish with a single disc of moistened sterilized filter paper (518G Filter-Lab) at the bottom. The environmental conditions applied for seed germination were those optimal temperature and light regimes obtained in Chapter 3: 20°C constant temperature and a 16 h L/8 h D photoperiod.
Once germinated, the seedlings were sterilized as follows: 70% ethanol for 30 s followed by immersion in 7% calcium hypochlorite for 20 min and 3 rinses in sterile distilled water.
Afterwards, rootlets and leaflets were partially removed, cleaning the damaged tissues.
Finally, the bulblets were sown in different initiation media (Table 4.1) and placed into a culture room at 24° C under a 16 h L/8 h D photoperiod. Subcultures were conducted every
28 days and the percentage of contaminated and surviving explants recorded after one and three months.
~ 78 ~
a b c
d e f
g h i
Figure 4.2.- Harvesting and preparation of Lapiedra martinezii Lag. plant material for in vitro culture experiments: a) selection and harvesting of healthy material in autumn from natural habitats; b) preparative stage and plant preconditioning in laboratory; c) general cleaning of plants; d) root and necrotic parts; e) preparation of bulb scales for sterilization; f) preparation of leaf explants for sterilization; g) chemical bulb explant sterilization; h) chemical leaf explant sterilization; i) germinated seed as a source of explants for in vitro cultures.
~ 79 ~
Phase/Composition IN1 IN2 IN3 IN4 IN5 IN6 IN7 IN8 IN9 Medium MS+vit (g/L) - 4.4 4.4 2.2 2.2 4.4 Medium G5 3.2 3.2 3.2 3.2 - - - - - Caseine (mg/L) 500 500 500 500 - - - - - Adenine (mg/L) 2 2 2 2 - - - - - Glutation(mg(L) 10 10 10 10 - - - - - Sucrose (g/L) 10 30 60 90 30 10 30 10 10 Activated charcoal (g/L) - 2 2 2 - - - - - Sequestren (mg/L) ------10 Plant Agar (g/L) 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5
Table 4.1.- Different initiation media tested for L. martinezii bulblet explants based on standard protocols and in those which gave good results in Leucojum aestivum L. (Georgieva et al., 2010).
~ 80 ~
4.3.2.4.-Stock maintenance of seedlings cultures
The seedlings were sown in nutritive medium IN1 composed by salts Gamborg B5 supplemented with 500 mg/L of caseine, 2 mg/L of adenine, 10 mg/L of glutation, 10 mg/L of sucrose and 5.5 g/L of Plant Agar. Cultures were weekly checked throughout a 2-months period and those contaminated or died plantlets were discarded. Afterwards, the microplants were transferred into jars filled with 250 mL of hormone-free, solid nutrient medium composed by basal MS medium+vitamins, 30 g/L of sucrose and ith 5.5 g/L of Plant Agar.
These cultures were maintained for 10 months in a growth chamber at 17±1ºC and a 16 h L:8 h D photoperiod (photosynthetically active radiation 42 µmol m-2s-1) and bulblet survival evaluated.
4.3.2.5.- Rooting and acclimatization of microplants
The obtained microplants were transferred into the acclimatization phase in ex vitro conditions once calibers reached ≥ 1 x 1,5 cm after 8 weeks of culture in MUL5 medium.
Acclimatization to ex vitro conditions was carried out by planting three replicates of the bulblets in a mixture of peat moss and vermiculite (5:4) previously autoclaved (during 30 minutes at 121ºC and 1 atm of pressure) and placed into a growth chamber with light and humidity control. We carried out this phase following steps:
- Material preparation: the first step was to take out plantlets (bulblets with roots and
leaves) out of the jars and wash the rest of agar in distilled water in order to avoid
microbial proliferation and death tissues were removed.
- Acclimatization I. Transfer to initial ex vitro conditions: the initial conditions for
acclimatization were constant temperature of 24±1°C and 100% of relative humidity
(HR) in a 16 h L:8 h D photoperiod (photosynthetically active radiation 42 µmol m-2s-
1). These conditions were maintained during one month.
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- Acclimatization II. Hardening: HR was reduced to 70% and the light intensity was
increased up to 80 µmol m-2s-1. The plants were maintained under these conditions
one more month and pots were watered weekly.
- Transference of plants to outdoor conditions: the plants were finally soaked in pots
with the mixture above described under outdoor conditions at the courtyard of the
CIBIO. The plants were watered every two weeks and after six months, the survival
percentage was measured.
4.3.3- Tissue culture of bulb scales
4.3.3.1.- Bulb sterilization
The whole plants were washed in tap water with commercial detergent. Afterwards, the old brown bulb scales and death tissues, as well as roots, were removed and the bulbs were cleaned. Seven procedures of sterilization combining different chemical and thermic treatments were tested (Table 4.4). After disinfestation treatment, the bulbs were transferred to the laminar flow cabinet, and then cut into 2 cm-segments with at least one axillary bud.
These scales were used as explants in the initiation cultures.
4.3.3.2- Initiation of in vitro cultures
From each sterilized bulb, the two-third of the apical portion was removed as well as the two external scales of the remaining portion. A thin portion of brown tissue in the bottom plate was also discarded. The remaining portion of the bulb was longitudinally sectioned to obtain 3-4 explants by scaling bulb segments. These explants were sown in the initiation medium IN1 as it gave good results of seedling survival in initiation media as is reported in
Georgieva et al. (2010). Also, a replicate of this medium with 2 g/L activated charcoal was employed. Initiation cultures were carried out in a growth chamber at 24±1ºC in dark
~ 82 ~ conditions imposed by wrapping the test tubes with aluminum foil. The cultures were maintained in the dark during 10 days in order to avoid the oxidation of phenolic compounds.
Afterwards, a 16 h L:8 h D photoperiod (photosynthetically active radiation 42 µmol m-2s-1) was applied during around 30 more days. Each explant was sown in one test tube with
15 mL of culture medium. Four batches of 25 test tubes each were employed for each culture medium (overall 100 explants for each initiation medium).
4.3.3.3.- Multiplication
Once the in vitro cultures were initiated and established, the multiplication phase took place. A first proof of multiplication was done with a single subcultivation cycle of 2 months in M1 and M2. As we had positive response on the initiated cultures in M1, four subsequent cultures of 2 months each in M1 were carried out in order to obtain more bulblets for the multiplication phase. The objective was to optimize the amount of new bulblets from each explant. A total number of fourteen culture media were used to test the morphogenic response of these explants. All the media were based on standard MS+vitamins, 30 g/L sucrose and 5.5 g/L Plant Agar (Duchefa). The pH was adjusted at 5.75 with NaOH 0.1 N or KOH 0.1 N prior to autoclaving during 20 minutes at 121ºC. As in the previous initiation stage, the explants were sown in test tubes containing 15 mL of multiplication medium.
After an 8-weeks period of culture the following parameters were measured:
- Number of shoots per explant: new bulblet formation per each explant.
- Caliber: width and length (mm) of the new bulblets formed in vitro.
- Rooting: rooting was visually scored following the criteria of Sáez et al. (1994): 0 =
no roots; 1= roots <0.5 cm; 2= 1-2 roots >= 1 cm or any number of roots 0.5-1 cm
long; 3= 3 or more roots >= 1 cm.
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- Callus formation: following a similar procedure, the presence and degree of
development of callus or callus-like structures was determined: 0 = absence of callus;
1 = callus development that not affected the tissue of the new bulblet formation (for
instance, the old protective leaves from the previous explant); 2 = callus developed in
the tissue of the new bulblet formed (regardless the presence/absence in other old
structures) that affected less than 50% of the explant; 3 = callus developed in the tissue
of the new bulblet formed that occupies more than 50% of the explant or less than
50% but with remarkable abnormalities in leaf, root and/or bulb appearance.
A total number of 30 replicates were taken for the measurements in each culture medium. The different multiplication media were selected attending to the good results in close related species. Their composition is shown in Table 4.5.
4.3.3.4- Rooting and Acclimatization
Rooting of multiplied bulblets was spontaneous in multiplication medium within the first subculture (2 months) in most samples. The non-rooted explants developed roots within a month after transference to MS medium supplemented with 30 g/L sucrose and 5.5 g/L of
‘Plant Agar’ (Duchefa, The Nederlands) without (growth regulators free. For this reason it was not necessary to optimize the rooting phase protocol. Prior to the transference to the acclimatization chambers, the bulblets were cleaned.
Acclimatization was done at ex vitro conditions by planting the bulblets in an autoclaved (during 30 minutes at 121ºC) soil mixture of peat moss and vermiculite (5:4) placed into a growth chamber with light and humidity control. The conditions for acclimatization were the same as explained previously: constant temperature of 24±1°C and
100% of relative humidity (HR) in a 16 h L:8 h D photoperiod (photosynthetically active radiation 42 µmol m-2s-1) during one month. After this period, the HR was decreased to 70%
~ 84 ~ values for one more month and plants were watered weekly. Finally, the pots were placed under moderate illumination conditions outside the laboratory. The plants were watered every two weeks and after six months, the percentage of survivors was measured.
4.3.4.- Callus induction as a source of new germplasm
For callus induction experiments young leaf bases were used as a source of explants.
This plant material was first cleaned with ‘Domestos’ commercial bleach-soap and then surface sterilized with 70% ethanol for 30 seconds, followed by immersion in 7% calcium hypochlorite during 20 minutes. Explants were finally rinsed six times in distilled water, dried on sterile filter paper under laminar flow cabinet and cut into explants of around 1 cm in length, after removal of necrotic parts.
4.3.4.1.- Obtaining in vitro cultures
The initiation of callus in vitro cultures was done by sowing two replicates of 3-4 explants each one into petri dishes for both light and dark conditions. Leaf explants were cultured with their abaxial surfaces in contact with the medium and placed deep enough to get almost all the wounded area produced by the cuts also in contact with medium. The basal medium employed was MS+vitamins, supplied with 3% of sucrose, and solidified with 5.5 g/L of ‘Plant Agar’ (Duchefa, The Nederlands), 50 different combinations of growth regulators: benzylaminopurine, kinetine and 2,4-D presented the in Table 4.2. For all media, the pH was adjusted to 5.8 prior to autoclaving for 15 minutes at 121º C and 1 atm pressure.
An approximate amount of 40 mL of media was dispensed into 9 cm petri plates in laminar box conditions.
The cultivation was carried out at 26ºC, in darkness or under a 16 h L:8 h D
(photosynthetically active radiation 42 µmol m-2s-1) photoperiod. Cultures were checked daily during the first three months of cultivation, and contaminated explants were removed from the ~ 85 ~ experiment. Afterwards, the explants were weekly checked. Six subcultures were done in the same conditions as above every 28 days, embracing therefore the overall experimentation time 7 months. Assessment on callus development was made by measuring:
a) The onset of callus induction: the day where the first macroscopic sign of
dedifferentiation is recorded.
b) Callus induction efficiency (level of tissue dedifferentiation): an estimation of the
volume of the original tissue that is dedifferentiated in each 2-months period of
culture.
c) Callus characteristics: texture (compact, soft, friable or semi-friable), color and
survival of callus within a 6-months period of cultivation.
Benzylaminopurine Kinetine 2,4-D (mg/L) (mg/L) (mg/L) 0.1 - 0.5;1.0;2.0;4.0 0.5 - 0.5;1.0;2.0;4.0 1.0 - 0.5;1.0;2.0;4.0 - 0.1;0.2;0.5;1.0;2.0;4.0 - 0.1;0.2;0.5;1.0;2.0;4.0 - - - - 0.1;0.2;0.5;1.0;2.0;4.0;5.0;6.0;7.0;8.0 - 0.1 0.5;1.0;2.0;4.0 - 0.5 0.5;1.0;2.0;4.0 - 1.0 0.5;1.0;2.0;4.0
Table 4.2.- Different combinations of growth regulators tested for callus induction in leaf explants of L. martinezii. An overall of 50 different concentrations made up with benzylaminopurine, kinetin and 2,4-D were tested in basal medium MS+vitamins, with 30g/L of sucrose and 5,5 g/L of Plant Agar.
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4.3.5.- Statistical Analyses
Percentages of rooting and callus formation, obtained from rooting and callus scores respectively, as well as percentages of callus induction efficiency were arcsine square root transformed. The remaining parameters (number of shoots per explant, width, leng, etc.) were analyzed without any transformation. One-way or two-way analyses of variance (ANOVA) were carried out and significant differences between means were revealed with the Fisher’s
Least Significant Differences (for calli culture experiments) and Tukey’s multiple range tests
(for bulb explant multiplication) at the 5% level. All data were analyzed using the Infostat
2008 package.
The ANOVA were carried out on callus induction efficiency percentages, excluding data corresponding to all combinations with null response to improve variance homogeneity and the accuracy of the analysis.
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4.4.- Results and discussion
4.4.1.- In vitro seedling cultures
4.4.1.1.- Initiation of seedling cultures
In order to obtain a stock of in vitro plants developed from seedlings, a sample of seeds was sown for further initiation of in vitro cultures. In a frame of 1 month after seed imbibition all seedlings of L. martinezii were developed and bulblets could be distinguished.
A total amount of 9 initiation media were tested for seedling in vitro cultures. These media were based on standard MS+vitamins (IN5, IN6 and IN9) supplemented with different combinations of sucrose and Fe (sequestrene). Other media such as half-strenght
MS+vitamins (IN7-8) and Gamborg B5 (IN1-4) were also evaluated. The latter were based on the initiation medium employed by Georgieva et al. (2010) with different concentrations of sucrose. The best results on survival of bulblets in vitro were achieved in Gamborg B5 medium supplemented with 10 g/L sucrose and without activated charcoal, while those with the higher concentrations or/with standard concentrations of MS salts were the lowest (Table
4.3).
Medium Survival of bulblets (%) 15 days 1 month 3 months IN1 41 40 26,25 IN2 n=240 39,58 29,58 4,45 IN3 42,91 31,25 5,42 IN4 42,5 39,17 11,7 IN5 40,32 35,48 2,42 IN6 43,55 40,32 8,06 IN7 n=124 44,35 42,74 12,9 IN8 39,52 44,35 15,32 IN9 45,16 41,13 3,22
Table 4.3- Number of surviving Lapiedra martinezii seedlings (bulblets) after 1 and 3 months of in vitro culture as a function of the different initiation media tested. ~ 88 ~
a b
1
2
Figure 4.3.- Contamination of Lapiedra martinezii Lag. in vitro cultures: a) fungal contamination (1) and bacterial contamination in bulb explants (2); b) different bacterial colonies in seedling explants.
Due to the continuous appearance of microbial infections in our cultures (Figure 4.3) we wanted to test (after a period of quarantine and in apparently microbes-free samples) if there were endogenous bacteria or fungi that may compromise the subsequent experiments.
Thus, we submerged some portions of the explants in liquid media MS with sucrose and kept them for 3 weeks under light conditions (25°C). Afterwards, 3 random flasks were taken and a sample of the liquid was inoculated by cross-hatched grooves in two different types of solid nutrient media (three replicates each): Malt-Agar (selective for fungi) and standard nutritive agar (selective for bacteria) at 37.2°C in the dark during 7 days.
The results showed the presence of the same kind of colony of bacteria in the 7 of the
9 samples sown in nutritive agar, while the other 2 remained without bacterial colonies
(Figure 4.4). Any sign of fungal growth was recorded in any of the replicates sown in Malt-
Agar. These results demonstrate that the in vitro cultures are fungi-free.
The results obtained in the medium selective for bacteria showed that a certain portion of our explants would have endogenous bacteria (Figure 4.4). Therefore, bacterial colonies would appear and spread after cutting and subculturing even if the in vitro stocks look sane.
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Then, we decided to add the mixture of antibiotics used to control bacterial proliferation in seedling cultures during 3 weeks before starting experiments.
a
b
Figure 4.4.- Microbial assay to determine the presence of endogenous fungi and bacteria: a) batch of petri dishes showing endogenous infection with bacteria; b) a detail of bacterial colonies, which have the same features to those found on in vitro cultures, regarding colour, shape, viscosity, etc..
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This mixture was based on the formula employed in Urginea maritima (L.) Baker
(Wawrosch personal comment) but modified (without fungicides as we did not find fungi in our cultures) and is as follows: 500 mg/L cefotaxime, 5 mg/L ampiciline and 15 mg/L kanamycin.
A second batch of initiation was carried out with IN1 medium supplemented with the antimicrobial solution explained above during three weeks. An assessment of save plantlets was done after a 2-months period of culture. In these conditions, contamination varied from
85% to 25% in seedling cultures.
It was very difficult to obtain microplants growing in aseptic conditions, especially without bacterial growth during the three first months of culture. However, the use of the antimicrobial solution helped to control the proportion of infected cultures. Finally, the good results in this kind of culture allo in vitro plantlets production from seeds, which is recommendable in plant restoration programs due to its higher genetic diversity.
4.4.1.2.- Stock maintenance of seedling cultures and rooting.
In order to test whether a stock of plants can be preserved at in vitro conditions without any subculture, a test of survival rate and measurements on calibers were carried out using in vitro plants maintained in initiation medium at 17ºC during 10 months without any subculture. The plantlets were initially sown with a portion of the main rootlet and the leaflet.
About ten months later, the bulblets produced an average of 4-5 well developed roots, 2-4 leaves and calibers raised from a maximum initial width of 2.05±0.2 mm and height of
5.97±2.1 mm (n=42) up to 16.42±3.7 mm width and 53±12.9 height (including leaves, n=51).
The preservation of micro-plants in standard MS-medium for 10 months was possible without any subculture. This procedure provides an intermediary approach to ex situ germplasm storage (Pence, 2011) in which bulb cultures can be maintained at relatively low temperatures
~ 91 ~ for at least 10 months without any subculture as it has been described for some other species
(Withers, 1991). The main inconvenient of in vitro cultures applied to conservation is that vegetative propagation produces clones. Thus, a subsequent risk arises in case of reintroduction of reinforcing in natural populations (Laguna, 1998), especially for threatened plants or popoulations with scarce amount of individuals. Therefore, this stock of in vitro cultures provides a
4.4.1.3.- Acclimatization of microplants.
The acclimatization phase is extremely important. Since the plants are not totally autothrophic at the time of transfer to ex vitro conditions, they are most likely unable to produce the required amount of carbohydrates for their growth. Also, high humidity and low light intensity are common handicaps in the transference into the ex vitro phase (George and
Debergh, 2008). After culture, 113 bulbs were transferred to the acclimatization phase and,
86% of this material (98 plants) survived after the first month of acclimatization (with
HR=100% and photosynthetically active radiation 42 µmol m-2s-1).
The 100% of these plants survived after the second stage of acclimatization
(hardening) and transference into open field conditions in a one-year period of observation.
These results show that this procedure of in vitro cultures provides a complementary source of explants that can be employed as a source of starting material for further studies. Also, the acclimatization procedure used was suitable because a high percentage of plants were able to grow in open field conditions.
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4.4.2- Tissue culture of bulb scales:
4.4.2.1.- Bulb sterilization and initiation of in vitro cultures
The main objective in this section was to obtain an aseptic plant tissue culture. Thus, the explants should be contamination-free and show a certain rate of growth prior to their transference to the multiplication stage. Obtaining an aseptic culture able to start an initiation culture was a very challenging task in L. martinezii bulb scale cultures. Seven procedures for sterilization combining chemical and temperature treatments were tested (Table 4.4).
The bulbs were surface-sterilized by combining different immersion times in 70 %
EtOH followed by 15 min in 30% of commercial bleach (NaOCl, around 5 % active chlorine) containing 0.05 % Tween-20, based on the general procedures that also gave good result in other bulbous plants (Sage et al., 1999). Also, a treatment of 0.1% mercuric chloride (alone or in combination with bleach) was tested according to the good results on bulb decontamination obtained by Stanilova et al. (1994) and Zagorska et al. (1997). As contamination rate still remained high, we also applied a hot water treatment based in Hol and Van Der Linde (1992)
Langen-Gernts et al. (1998) and Sochacki and Orlikowska (2005). This procedure allows intensive bulb disinfestation and is especially effective against endogenous microbs. The treatment comprised bulb immersion in two different temperatures (54ºC and 60ºC) for one hour, combining some of the chemical treatments above described.
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Time in Immersion in Immersion in Hot water Initiation Contamination Death Growth Protocol 70% EtOH 0.1% HgCl bleach solution 2 (60 min) medium (%) (%) rate (%) (s) (3 min) (20 min) IN1 43,14% (n=102) 56,86% 0% 1 60 Yes - - IN4 39,80% (n=98) 60% 0% IN1 49,34 % (n=77) 45,35% 5,20% 2 30 Yes - - IN4 55 % (n= 80) 45% 0% IN1 100% (n=98) 0% 0% 3 30 - Yes - IN4 100% (n=95) 0% 0% IN1 36,84% (n=57) 63,16% 0% 4 30 Yes Yes - IN4 35,41% (n= 48) 64,59% 0% IN1 29,41% (n=102) 48,99% 21,59% 5 - - Yes 54ºC IN4 31,96% (n=97) 37,42% 15,46% IN1 40% (n=100) 60% 0% 6 - - Yes 60ºC IN4 35,35% (n=99) 64,65% 0% IN1 31,82% (n=44) 68,18% 0% 7 30 Yes - 54ºC IN4 27,09% (n=48) 72,91% 0%
Table 4.4.- Different protocols for the bulb sterilization of Lapiedra martinezii Lag. Evaluation on explant contamination (both due to fungi and bacteria), death of the explants and symptoms of growth after 28 days of culture were recorded for the two initiation media tested.
~ 94 ~
Sterilization of plant material from this species was found to be extremely difficult.
The general procedure consisting in 30 seconds of immersion in 70% EtOH followed by an immersion of 20 min in 30% commercial bleach solution supplemented with three drops of
Tween-20 was completely unsuccessful, with 100% contamination after two weeks. Higher concentrations of bleach and/or EtOH and longer exposures for both were also tested but oxidized the explant in all cases (data not shown). As a result, the death of the tissues during the first month of culture was achieved in all cases. Only reducing the immersion time in
EtOH down to 30 seconds followed by a 2-min immersion in HgCl2 gave some growth
(5,20% of explants) in the initiation medium IN1, but this was still a low rate of survival
(Table 4.4).
The application of a 60 min bath in water at 54ºC to explants previously immersed in a
30% solution of commercial bleach (5% active chlorine) with three drops of tween 20 during
10 minutes, notably increased the amount of surviving explants that displayed growth in both initiation media tested (21, 59% and 15,46% for IN1 and IN2 respectively). The application of hot water sterilization treatments at this same temperature (Hol and Van Der Linde, 1992) or slightly lower Sochacki and Orlikowska, 2005) improved the micropropagation protocols in Narcissus and Lilium speciosum Thunb. ‘Rubrum No. 10’ (Langen-Gerrits et al., 1998).
The success of a hot water pre-treatment depends on the differential heat sensibility of pathogen and host (Langen-Gerrits et al., 1998). Increasing the temperature of the bath up to
60º resulted in a massive death of explants, although the contamination was indeed reduced.
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4.4.2.2.- Multiplication of bulb scales
The culture media for the multiplication phase were selected attending to the good results in close related species (Table 4.5). For instance, MUL1 and MUL5 gave high bulblet yield in N. bulbocodium (Santos et al., 1998) and MUL5 was also very successful for some
Narcissus species (Hussey, 1982). Also, combinations including IBA with BAP gave the best results in Santos et al. (1998). Thus, we tested the effect of these growth regulators in
MUL12-13. MUL2 yielded a considerable number of bulblets for Leucojum aestivum in
Georgieva et al., (2010) and was often used as a regeneration and multiplication medium from calli cultures of some Amaryllidaceae such as Leucojum aestivum (Pavlov et al., 2007). A similar composition of the medium was also optimum for the multiplication of Peloponesse
(Greece) populations of Pancratium maritimum (Nikopoulos and Alexopoulos, 2008;
Nikopoulos et al., 2008).
For MUL3 we added 5 µM of paclobutrazol (PAC) to our previous MUL1 medium.
For MUL4 we also used the previous base of the MUL1 medium and added 9 µM of ancymidol (ANC). The growth retardants paclobutrazol and ancymidol, are inhibitors of gibberellin biosynthesis. They have been shown to promote shoot proliferation, inhibition of leaf elongation and induction of meristematic bud clusters for several geophytes with economic importance (Hvoslev-Eide and Preil, 2005) including Narcissus tazzeta cv ´Ziva´
(Chen and Ziv, 2001), Charybdis numidica (Kongbangkerd and Wawrosch, 2003), Crinum macowanii (Slabbert et al., 1993) and Hosta ‘Blue vision’ (Maki et al., 2005) and corm development in Gladiolus (Steinitz and Lilien-Kipinis, 1989). Also, the same auxin/cytokinin ratios tested with an alternative auxin (IBA) or cytokinin (KIN and 2iP) were tested in
MUL6-11. After two months of subcultivation, the morphogenetic response of explants to the different culture media was recorded.
~ 96 ~
Compound Multiplication medium composition MUL0 MUL1 MUL2 MUL3 MUL4 MUL5 MUL6 MUL7 MUL8 MUL9 MUL10 MUL11 MUL12 MUL13 MS+vit (g/L) 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 NAA (mg/L) - 0.12 1.15 0.12 0.12 0.12 0.12 0.12 0.12 0.12 IBA (mg/L) 0.12 0.12 0.12 0.12 2iP (mg/L) - 2 4 KIN (mg/L) 2 4 2 4 BA (mg/L) - 2 2 2 2 4 - - 2 4 PAC µM - - - 5 ------ANC µM - - - - 9 ------Sucrose (g/L) 30 30 30 30 30 30 30 30 30 30 30 30 30 30 PlantAgar (g/L) 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5
Table 4.5.- Different multiplication media for bulb micropropagation of L. martinezii Lag. An overall of 13 different combinations of naphtalenacetic acid (NAA), 6-(γ,γ-dimethylallylamino) purine (2iP), 6-benzylaminopurine (BA) paclobutrazol (PAC), ancymidol (ANC) and indole-3-butyric acid (IBA) were tested in basal MS medium + vitamins, supplemented with 30 g/L sucrose and 5,5 g/L of Plant Agar. A growth regulator-free medium (M0) was included as a control.
~ 97 ~
The medium employed as a control, MUL0 (growth regulators free) gave the lowest multiplication rates (0,10 shoots per explant). These results show that a certain propagation rate can be achieved without the application of exogenous growth regulators (Table 4.6). This was also described in ‘Yamit’ Gladiolus’ corms (Steinitz and Lilien-Kipinis, 1989). The nutritive media tested here as MUL0 has a considerable amount of sucrose (30 g/L). Taking into consideration the beneficial effect of sucrose for bulbling (Mielke et al., 1989; Santos et al., 1998; Sellés et al., 1999) an explanation to this response could be that some bulblets were formed as a result of this sucrose content. On the other hand, this phenomenon could be also explained as spontaneous shoot proliferation related to the normal levels of endogenous growth regulators that are responsible of vegetative propagation in nature. However, the yield here reported is still very low and we discard it as a plausible multiplication medium.
MUL5 was the medium that showed better production, not only in terms of new bulblets formed per explant (multiplication rate) but also in terms of caliber (width and length, Table 4.6). This medium performed a rooting of 96.18% and a proliferation of callus- like structures of 24.84% which is not too much taking into consideration the results for all media tested. Therefore, seems that MUL5 is the best multiplication medium in terms of the general morphogenic response. The medium which performed suboptimal yield was MUL13, however the shoot proliferation is almost the half and the proliferation of callus or callus-like structures was the highest of the tested media. Therefore we would not employ it as an alternative to MUL5.
Intermediate results were obtained with MUL1, MUL3-4, MUL 7 and MUL12 with a yield between 2.10-2.50 shoots produced per explant (Table 4.6). The caliber for all these media also showed intermediate values, with only slight variations in MUL12 and MUL4.
Rooting and callus proliferation appeared to be more variable among these media.
~ 98 ~
Morphogenic response of Lapiedra martinezii Lag. explants in the presence of different combinations of growth regulators Combination of growth Number of Rooting Callus Callus regulators shoots Width (mm) Lenght (mm) Rooting (%) score (%) score MUL0 0.10±0.06 a 3.33±0.09 ab 6.00±0.26 bc 100 c 3 0.00 a 0 MUL1 2.10±0.26 c 2.97±0.49 ab 4.76±0.69 ab 100 c 3 16.56 abc 0.4 MUL2 0.80±0.00 ab 2.07±0.25 a 3.1±0.44 a 49.68 a 1.5 35.67 d 0.9 MUL3 2.23±0.29 cd 3.2±0.44 ab 5.13±0.63 abc 100 c 3 15.92 abc 0.4 MUL4 2.57±0.27 cd 3.43±0.50 ab 5.53±0.67 abc 100 c 3 15.92 abc 0.4 MUL5 5.77±0.31 e 4.07±0.48 ab 6.10±0.65 bc 96,18 bc 2.9 24.84 bc 0.63 MUL6 1.63±0.26 bc 4.83±0.53 b 7.73±0.80 c 100 c 3 12.74 ab 0.4 MUL7 2.27±0.33 cd 2.87±0.34 ab 4.67±0.42 ab 92.36 bc 2.8 7.01 a 0.2 MUL8 1.50±0.18 bc 3.6±0.56 ab 4.60±0.61 ab 89.81 bc 2.7 28.66 bc 0.77 MUL9 1.83±0.35 bc 2.8±0.47 ab 3.70±0.62 ab 76.43 b 2.4 27.39 bc 0.73 MUL10 1.70±0.31 bc 2.83±0.32 ab 4.17±0.49 ab 100 c 3 30.57 c 0.87 MUL11 1.87±0.27 bc 3.77±0.54 ab 5.57±0.73 abc 89.81 bc 2.7 31.21 c 0.87 MUL12 2.50±0.25 cd 2.33±0.31 a 3.80±0.53 ab 82.80 bc 2.5 37.76 d 0.97 MUL13 3.40±0.32 d 3.77±0.48 ab 5.67±0.63 abc 82,80 bc 2.5 36.94 d 1.03
Table 4.6.- Multiplication of Lapiedra martinezii Lag. Morphogenic response of L. martinezii Lag. bulb-scale explants in the presence of different combinations of growth regulators after eight weeks of culture: formation of new bulblets per explant; caliber determination (width and length) as well as observations on rooting and callus and/or callus-like structures formation was studied. Different letters indicate statistical differences at 5% level.
~ 99 ~
The lowest callus score recorded among these media corresponded to MUL1 and
MUL4. Taking into consideration the general morphogenic response of these media we would advice MUL1 and MUL4 as suboptimal media for multiplication. Low shoot proliferation was achieved with MUL6 and MUL8-11 as they regenerated less than 2 bulblets per explant.
Calibers showed measurements not significantly different to the media that performed optimal and intermediate multiplication values. Although the rooting in these media was variable, all of them produced high percentage of rooting (higher than 75%). However, the percentage of formation of callus and callus-like structures is higher than 25% and along with results of shoot proliferation we would discard these media as multiplication media.
a b c
d e f
g h i
Figure 4.6.- Morphogenetic response of bulb explants sown in different multiplication media: a) – c) shoots produced and caliber measurements; d) – e) proliferation of callus and callus-like structures; f) – i) root development in multiplication media.
~ 100 ~
Finally, MUL2 gave a limited response and appeared not to be effective in producing new bulbils, good calibers as well as rooting percentages (Table 4.6). Moreover the formation of callus and callus-like structures is quite high. Therefore we would not employ this medium for multiplication. The results obtained showed that the response of the explants was affected by the concentration of auxin (NAA) used, since the same levels of BAP (2 mg/L) gave a better yield with low NAA levels (0.12 mg/L) (Table 4.6). These results contrast with those obtained in Leucojum aestivum (Georgieva et al., 2010) where the optimum multiplication rate resulted at higher concentrations of NAA in respect to the BAP (our MUL2) and was the preferred medium for tissue culture (bulb micropropagation and plant regeneration from callus) on some other Amaryllidaceae (Berkov et al., 2010; Georgiev et al., 2012; Ivanov et al., 2011; Pavlov et al., 2007; Squires and Langton, 1990). Nikopoulos and Alexopoulos
(2008) also had very good results with the micropropagation of Greek populations of
Pancratium maritimum with this medium (MUL2). However, Panayatova et al. (2008) obtained a very low response for the Bulgarian populations of P. maritimum at similar high auxin concentrations, in agreement with the results reported here. Sochacki and Orlikowska
(2005) obtained the highest yield for Narcissus ‘Bursztynek’ with 0,5 mg/L IAA with 13.1 shoots per explant after 9 weeks of culture. Santos et al. (1998) obtained the lowest multiplication rate with medium with 2 mg/L BAP plus 1 mg/L NAA. However, the yield is higher than those obtained in our work.
Therefore, the concentration of growth regulators in general, and auxins in this particular case, seems not to play the same role in close related species or even among different populations of the same species. This fact was also pointed out in other plants belonging to other botanical families and is probably due to the interaction of specific genotypes with the culture medium (Marco, 2010).
~ 101 ~
The addition of both anti-gibberellin agents 5 µM of PAC and 9 µM of ANC to MUL3 and MUL4 media had a slight better yield than their counterpart without anti-gibberellin agent
(MUL1) after an 8-weeks culture period, but is still low and not statistically different at the
5% level. This strongly contrasts with the good results obtained with PAC and ANC on shoot proliferation in several geophytes (Chen and Ziv, 2001; Hvoslev-Eide, and Preil, 2005;
Kongbangkerd and Wawrosch, 2003; Maki et al., 2005; Slabbert et al., 1993; Steinitz and
Lilien-Kipinis, 1989). However, further experiments with a wider concentration range of these compounds, as well as longer culture periods would be desirable in order to finally determine the effect of PAC and ANC in L. martinezii in vitro cultures.
In our experiments, medium M5 had almost a multiplication rate three times higher than M1, with double concentration of BA. Both media yielded with comparable rates to the similar formulas of the media used for the micropropagation of N. bulbocodium (Santos et al.,
1998) with an average bulblet production of 4,5 (after 12 weeks) for M1 and 7.1 (after 6 weeks of culture) for M5. Also Hussey (1982) (cited in Santos et al., 1998) found very effective the combination of M5 in shoot formation for some Narcissus species. In fact, concentrations of cytoquinins (from 3-4 mg/L up to 10 mg/L of BAP and BA) have been reported to give very good results on shoot production both in direct and in indirect organogenesis for several Amaryllidaceae species, regardless of the presence, the type and concentration of auxin (Berkov et al., 2010; Chen et al., 2005; Georgiev et al., 2012; Hussey,
1982; Ivanov et al., 2011; Nikopoulos and Alexopoulos, 2008; Pavlov et al., 2007; Santos et al., 1998; Seabrook et al., 1976; Seabrook and Cumming, 1982; Sellés et al., 1999; Squires and Langton, 1990). This fact could be related most likely to the role of this growth regulator in apical dominance suppression and direct promotion of bud growth (Cline, 1994; Ongaro and Leyser, 2008; Van Staden et al., 2008).
~ 102 ~
Therefore, concentrations of BAP ranging from 3-4 mg/L, sometimes related with low concentrations of auxin (normally NAA), seem to induce the highest bulblet formation in the most of the studied Amaryllidaceae as we reported here.
A complementary experiment carried out in liquid medium showed that the production from bulb explant in M1 resulted in a lower yield (0.7 bulbs per explant) than in the solid one
(2.10) and leaves appeared overdeveloped and hyperhydrated (Figure 4.7). After 3 weeks of culture in solid MS medium supplemented with 30 g/L sucrose and growth regulators free, the leaves become non-hyperhidrated. However, the leaf overdevelopment was concomitant with a low bulb development. These results do not support the idea of better production using in vitro conditions of permanent immersion in liquid cultures at the conditions described.
a b c
d e f
Figure 4.7.- In vitro cultures of Lapiedra martinezii for permanent immersion in liquid medium experiments: a) stock of plants grown in solid medium; b) rotatory shakers; c) growth achieved in liquid medium: flask during the period of culture; d) bulblets formed in liquid medium showing typical hyperdhydric features; e) first day of subculture in solid medium (MS), showing typical hyperhydricity features; f) cultures recovered the non-hyperydric state after 3 weeks of culture in solid (MS growth regulators free).
~ 103 ~
4.4.2.3- Rooting and Acclimatization
There was no need of a specific rooting phase with different rooting media for this species. Rooting of bulblets in multiplication medium was 100% in MUL0-1; MUL3-4;
MUL6 and MUL10 and higher than 80% in MUL5; MUL7-8 and MUL11-13. Only MUL2 showed low spontaneous rooting in multiplication medium within the first subculture
(49.68%). However, the non-rooted explants developed roots within a month after transference to MS medium supplemented with 30 g/L sucrose and 5.5 g/L of ‘Plant Agar’
(Duchefa, The Nederlands) without growth regulators. This fact constitutes a clear advantage for the micropropagation of L. martinezii especially for industrial purposes since the in vitro rooting phase almost doubles the price of microcuttings (de Klerk, 2002). Afterwards, 113 in vitro plants were soaked in propagators (three different replicates) filled with peat moss- vermiculite mixture and placed in the growth chamber. During the acclimatization phase, the bulbs developed a tunic, and after the transference to outdoor conditions, 89% of the bulbs survived in accordance with the obtained results in the acclimatization of seedling cultures.
Transplantation success was also high in N. bulbocodium after one month of acclimatization in culture chamber, with percentages of survival from 92% up to 95% in Santos et al. (1998).
~ 104 ~
a b
c d
Figure 4.8.- Acclimatization phase in Lapiedra martinezii micropropagation: a) rooted bulblets were cleaned before acclimatization; b) transplantation of in vitro bulblets in soil mixture; c) ex vitro acclimatization in growth chamber; d) bulbs produced in vitro transferred to outdoor conditions.
4.3.3.- Callus induction as a source of new germplasm.
The callus induction for plant regeneration provides a complementary conservation option (Irvani et al., 2010; Piovan et al., 2010) and is a key point for micropropagation and for obtaining in vitro systems for alkaloid production (Pavlov et al., 2007). In this section we wanted to determine the conditions for the in vitro establishment of callus cultures as a strategy to obtain complementary in vitro cultures with multiple purposes. Thus, leaf bases
~ 105 ~ were sterilized and sown in solid medium in order to test the effect of 50 different combinations of growth regulators and 2 photoperiods (light/dark and dark) at 26ºC.
4.3.3.1.- Obtaining in vitro callus cultures.
Leaf explants were incubated on different media to promote the induction of callus.
The first symptom of callus development was observed after 8 days of culture in darkness. A proliferation of spheroid tissue structures on the cut edge of the explants was first identified in medium number 1. The same structures were also identified within the first subculture period in media number 2, 3, 5, 9, 11, 13, 39 and 40 in the dark (Figure 4.12).
a b
c d
Figure 4.9.- Different stages of Lapiedra martinezii Lag. callus development: a) callus induction onset: detail of spheroidal tissue structures in the cut edge in medium 1 (darkness) after 8 days of culture; b) callus development after two months of culture in medium 47 (darkness); c) callus completely formed in medium 41 after four months of culture at 26ºC in darkness; d) detail of granular, aggregated structures in compact-soft friable callus (medium 39 in darkness).
. ~ 106 ~
The complete growth was reached between the second and third month under dark conditions, whereas scarcely signs of dedifferentiation during the whole period of culture were observed in light (Table 4.7, Figure 4.10). Only medium 3 showed signs of dedifferentiation within the first month of culture (the only one which reached the 100% of growth after the whole culture period) and two more media (8 and 50) showed signs of dedifferentiation but without formation a stable callus at the end of the 6-months culture period.
Dark provoked higher callus induction efficiency, onset and growth than light in
Pancratium maritimum (Georgiev et al., 2010) and Crinum x powellii ‘Album’ (Niño et al.,
2005). Dark also gave better results for callus induction in another Amaryllidaceae such as
Hymenocallis littoralis (Sundarasekar et al., 2012), Leucojum aestivum (Pavlov et al., 2007),
Pancratium maritimum (Berkov et al., 2010) and Narcissus tazetta L. var. chinensis Roem
(Chen et al., 2005), although in these last four studies, no comparisons with light regimes were done. In contrast, the experiments on Narcissus confusus callus formation were carried under a 16 h L:8 h D photoperiod (at 25º C) and authors obtained normal good results (Sellés et al., 1999). Also, Wang et al. (2011) developed callus in Clivia miniata under a light photoperiod although at a different photoperiod: 12 h L:12 h D. We have no a clear explanation for the results obtained in light/dark and in the presence of different combinations of growth regulators. Perhaps, the different constitution and different levels of endogenous growth regulators in the explants may explain this.
~ 107 ~
Table 4.7.- Effects of photoperiod and combination of growth regulators on Lapiedra martinezii callus onset, callus induction efficiency and callus characteristics. The degree of development was measured as a percentage of the explant initial volume dedifferentiated during thre measurements done during the observation period (six months). Different letters following the percentage data indicate significant differences at the 5 % level according to the Fisher’s LSD test.
~ 108 ~
Figure 4.10.- Callus induction efficiency of Lapiedra martinezii measured as a percentage of callus formed in relation to the initial tissue volume, in the 25 combinations of growth regulators (overall 50 initial variants) were some response were recorded. Values followed by different letters indicate significant differences at the 5% level. Bars indicate SE.
~ 109 ~
Among the well-established calli, the fastest dedifferentiation rates and growth were reached between the second and the third month of culture under dark conditions in media number 39 and 40 in the dark. The variants 1, 9, 11, 13, also displayed a good callus formation rate
(Table 4.7, Figure 4.9).
The onset of callus formation for these well-established calli ranged from 8 days to 20 days (14.6 days on average) in darkness and 28 days in light conditions (Table 4.7). Similar results were obtained by Wang et al. (2011) in Clivia miniata. This is relatively faster than other Amaryllidaceae such as Hymenocallis littoralis that ranged from 15 up to 40 days
(Sundarasekar et al., 2012), or 35 days in Leucojum aestivum (Pavlov et al., 2007) or
Pancratium maritimum (Berkov et al., 2010; Georgiev et al., 2010) or between 15 and 21 days in Narcissus tazetta L. var. chinensis Roem (Chen et al., 2005), between 28 and 46 in
Narcissus pseudonarcissus cv. Golden Harvest and cv. St. Keverne (Sage et al., 1999) or even
46 in Narcissus confusus with 42 days until the first signs of dedifferentiation (Sellés et al.,
1999). However, in all cases, the final necessary time to complete the dedifferentiation process and get established in L. martinezii as well as in the other studied Amaryllidaceae was around three months.
~ 110 ~
High auxin/cytokinin ratios favoured callus initiation, variants 1, 2, 3, 4, 6, 7, 8, 11,
12, 39, 40, 41, 42, 44, 45, 46, 49, 50 in the dark and 3, 8 and 50 in light and callus establishment, media 1, 11, 39, 40 and 41 in dark and 3 in light. However, this balance appeared not to be exclusive since an equilibrated ratio between growth regulators also promoted the callus development (variants 5, 43 and 48) (Table 4.7, Figure 4.10) and one of them (5) developed a well-established callus. Also, a low auxin/cytokinin ratio promoted callus induction in four of the media (9, 13, 14 and 47) with two well-established callus
(media 9 and 13).
It is generally accepted that high auxin and low cytokinin in the medium promotes cell proliferation with the consequent formation of callus (Perianez-Rodriguez et al., 2014;
Skoog and Miller, 1957). In Amaryllidaceae species such as Hymenocallis littoralis
(Sundarasekar et al., 2012) the highest percentages of callus induction and onset were obtained when the amount of 2,4-D was 3-, 4-fold that of BAP. With lower 2,4-D/BAP ratios, but always high auxin in relation to cytokinin (3-4 mg/L 2,4-D and 2 mg/L BAP), Pavlov et al. (2007) obtained the best results in Leucojum aestivum callus formation. In the same manner, double amount of 2,4-D (4 mg/L) with respect to BAP (2 mg/L) was the optimum for
Pancratium maritimum callus development (Berkov et al., 2010; Georgiev et al., 2010).
However, Berkov et al. (2010) also found that the opposite ratio (1 mg/L 2,4-D and 2 mg/L
BAP) gave optimum results in Pancratium maritimum callus induction pointing out that some variation can be achieved in the response to growth regulators even within the same plant material. In Narcissus tazetta L. var. chinensis Roem. (Chen et al., 2005) also a double amount of 2,4-D in relation to BA (1 mg/L 2,4-D and 0,5 mg/L BA) whereas the worst results were obtained when 0,5 mg/L 2,4-D in absence of cytokinin was applied. However. For
Narcissus pseudonarcissus ‘Golden Harvest’ and ‘St Keverne’ (Sage et al., 1999) 2,4-D also gave the best results in combination with BAP when applied at equal ratios with BAP or at a ~ 111 ~
10:1 ratio (5µM 2,4-D and 0.5 or 5 µM BAP). In this case, 2,4-D also gave some positive response, coinciding with our results. This rule also works for Clivia miniata (Wang et al.,
2011) since the best media for both kind of explants had greater amounts of auxins (single
2,4-D, or in combination with NAA) than cytoquinins (BA).
Figure 4.10.- Efficiency of growth regulators (single or combined) on Lapiedra martinezii callus induction efficiency (CIE). For each variant, the CIE in relation to the number of variants with this combination, is showed.
It seems that callus formation in L. martinezii is dependent on 2,4-D application
(Figure 4.10), coinciding with the results from Sage et al. (1999) who found no response
(somatic embryo development) without 2,4-D on Narcissus pseudonarcissus ‘Golden
Harvest’ and ‘St. Kaverne’. ~ 112 ~
Figure 4.11.- Calli cultures of Lapiedra martinezii Lag. Some of the best callus induction efficiency was found in medium 39 (left) and 40 (right).
Most of the well-established callus obtained in our experiments (variants 1, 5, 11, 39,
40 and 41) showed a compact-soft friable, clump-type development (Figure 4.11) whereas those classified strictly as compact appeared only in variants 9, 13, L3. The callus was stable in culture conditions and was not completely friable although those compact-soft friable showed some granular, aggregated structures. Anyway all of them were compact. Only one of the well-established callus displayed white colour, whereas the vast majority of them where completely yellow.
The studies on callus development in Amaryllidaceae species are still scarce and therefore no general conclusions may be still withdrawn. However, some works conducted on this same family agree with our results: callus are generally yellow (rarely white) and compact with normally granular structure. For instance, in Hymenocallis littoralis
(Sundarasekar et al., 2012), Pancratium maritimum (Berkov et al., 2010; Georgiev et al.
(2010), Leucojum aestivum (Pavlov et al., 2007) or Narcissus tazetta L. var. chinensis Roem
(Chen et al., 2005). This feature of callus would be also extended to other close related genus
~ 113 ~ and families with bulbous plants within the Monocotyledonous such as Croccus (Chichiriccò,
1989; Demeter, 2010).
Although the texture of callus was dependent on the type of auxin used, all callus obtained in Narcissus confusus cultures by Sellés et al., (1999) were also yellowish and compact. Those developed in the presence of 2,4-D were granular and non-embryogenic, whereas those cultured with picloram were friable with many globular structures
(embryogenic). Also, under a certain balance of growth regulators, some calluses appeared friable instead of compact in Hymenocallis littoralis (Sundarasekar et al., 2012) or Narcissus tazetta L. var. chinensis Roem (Chen et al., 2005). However, we cannot observe this pattern of dependence between texture and growth regulator balance in our study.
The type of explant also seemed to influence the nature of callus generated in Clivia miniata Wang et al. (2011). Calluses derived from the base of the petal became yellow and friable and those derived from young ovaries became compact yellow-green. In spite of the difference on textures, both types were optimum. Only in Crinum x powelli ‘Album’ Niño et al. (2005) obtained different color of calluses, such as cream color, for both types of textures, friable and nodular.
In our study all non-yellowish callus (white-hyaline, white-yellowish and brownish- colored callus) died or stopped their growth within six months of culture. All these callus were obtained always with 2,4-D alone or combined with cytokinin, but never with a single cytoquinin, either benzylaminopurine or kinetin (Table 4.7, Figure 4.10). When an auxin (2,4-
D) was applied alone, a high concentration of it (2 or 4 mg/L) was required to promote callus induction but any of them was able to induce a well-established callus after 6 months of culture. It seems that callus which do not form compact clumps (such as watery, hyaline
~ 114 ~ calluses) do not dedifferentiate completely. Similar results were obtained by Sellés et al.,
(1999) from Narcissus confusus watery explants that did not increase in biomass and did not get stable callus. Finally, further experiments using other plant organs (roots, flower buds, fruits and stems) would increase the range of genetic material that can be obtained in these in vitro conditions.
Young leaves provided a good source of explants for callus development in Lapiedra martinezii. However, in other studies conducted in related plants such as in Croccus heuffelianus, leaves did not respond in the same way (Demeter et al., 2010). Our results indicate that young leaves are an interesting plant material for callus induction in L. martinezii. and does support that endogenous levels of growth regulators or they response might vary between organs, as it was purposed by Piovan et al. (2010=.
4.5.- Conclusions
- The in vitro culture of Lapiedra martinezii is a quite challenging task due to the high
levels of microbial contamination, especially due to the endogenous bacteria, in
seedling and bulb scale cultures.
- A mixture of antimicrobials consisting in; 500 mg/L cefotaxime, 5 mg/L ampicillin
and 15 mg/L kanamycin for three weeks of culture was found suitable to reduce
contamination to a acceptable level.
- An initiation media with low concentration of salts and sucrose such as Gamborg B5
supplemented with 10 g/L of sucrose, suited better for seedling cultures than more
concentrated ones. This medium also worked well as an initiation medium for bulb
scales cultures.
~ 115 ~
- Seedling cultures developed well as a stock for 10 months at low temperature (17ºC)
in MS medium supplemented with 30 g/L sucrose and withoug growth regulators.
- The inhibitors PAC and ANC did not provoke any response in our cultures at all
concentrations tested.
- The best morphogenic response was obtained in medium MS supplemented with 30
g/L sucrose, BA= 4 mg/L and NAA = 0,12 mg/L in terms of shoot production (5.77
bulblets per explant) and caliber (4.07±0.48 x 6.10±0.65). Also, the rooting was very
high in this medium (96.18%). Moreover, the proliferation of callus-like structures
was relatively low (24.84%) in comparison to the other media tested.
- Rooting was spontaneous in all multiplication media during the first subculture with a
variable percentage of 76.43% to 100% (exceptionally 49.68%). Thus, there was not
necessary to include a rooting stage with specific media in the micropropagation
process, although transference to MS solid medium supplemented with 30 g/L and
without growth regulators increased rooting up to 100% in all the multiplication media
tested that gave rooting under 100%.
- The acclimatization procedure tested here had a very high success, with 86% and 89%
of survival for microplants obtained from seedlings and bulblets, respectively, after
transference to ex vitro conditions.
- Complete darkness clearly favored callus induction, growth and development, while
light had a marginal effect on it.
- High auxin/cytokinin ratios (of at least 2) were necessary for callus initiation and to
achieve the highest callus efficiency in most cases.
- 2,4-D is necessary for the callus formation, regardless the citokinin present in the
medium.
- Calli were initiated after 14.5 days of culture and between 2 and 3 months on average
were necessary to obtain well-stablished cultures. ~ 116 ~
- The well-established calli were yellow and almost all of them showed compact-soft
friable textures.
~ 117 ~
~ 118 ~
5.- STORAGE BEHAVIOUR AND LONG-TERM
PRESERVATION OF Lapiedra martinezii SEEDS
~ 119 ~
~ 120 ~
5.- STORAGE BEHAVIOUR AND LONG-TERM PRESERVATION OF
Lapiedra martinezii SEEDS
5.1.- Introduction
5.1.1.- Seed storage behavior and desiccation tolerance
Ex situ conservation strategies constitute a long term safeguard for wild genetic resources and facilitate their use for different purposes (Engels et al., 2008; Perez-García et al., 2007). Seed banking is now widely used, not only for economically important crop plants and crop wild relatives, but also for endangered plants and native flora in general (Hay and
Probert, 2013). Seed collections of wild species are intrinsically more complex to manage than collections of cultivated plants as greater a priori knowledge of seed biology characteristics is needed (Walters, 2015), including essential aspects such as germination requirements and seed storage behavior.
The seeds can be classified depending on their storage behavior into three main categories: orthodox, recalcitrant and intermediate. The main point is to determine their response to different degrees of desiccation as well as to cold storage at different temperatures
(Hong and Ellis, 1996; ISTA, 1993; Rao et al., 2007). The main features of these three kinds of seeds related to the seed storage behaviour are exposed in the following definitions based on Hong and Ellis (1996).
- Orthodox: those seeds that can be dried, without damage, to low levels of moisture
content and, over a wide range of environments, their longevity increases with a
decrease in seed moisture content and temperature in a predictable way (Hong and
Ellis, 1996; Roberts, 1973). Orthodox seeds do not necessary tolerate desiccation at all
stages of their development and maturation (Hong and Ellis, 1996). Ideally, orthodox
~ 121 ~
seeds should be collected when, for the majority of seeds in the targeted population,
germinability, desiccation tolerance and longevity are at their maximum (Newton et
al., 2013).
- Recalcitrant: seeds that cannot be dried without damage. When fresh recalcitrant seeds
begin to dry, viability is first slightly reduced as moisture is lost, but then begins to be
reduced considerably at a certain moisture content termed the ‘critical moisture
content’ even until the viability is completely lost. Maturation drying to low moisture
contents on the mother plant does not occur (Hong and Ellis, 1996).
- Intermediate: show a mixed behavior. The essential feature is that the negative relation
between seed longevity in air-dry storage and moisture content is reversed at values
below those in equilibrium (at 20ºC) with about 40-50% RH. Sometimes this feature is
associated with damage immediately after desiccation (Hong and Ellis, 1996).
Orthodox and recalcitrant seeds were first defined by Roberts (1973). However, these two categories did not account satisfactorily for all observations on seed storage behavior (see examples in Hong and Ellis, 1996). Then, the intermediate category was introduced by Ellis et al (1990).
5.1.2.- Long-term seed storage as ex situ conservation strategy: storage below 0ºC and crypreservation
The long-term preservation consists in keeping the seeds under environmental controlled conditions in order to ensure their viability for extended periods of time, normally in the so-called ‘base collections’. The preservation conditions usually imply temperatures
~ 122 ~ below 0ºC, between -18º and -20ºC than can vary depending on the seeds moisture content
(Engels and Visser, 2003; Hong and Ellis, 1996; Rao et al., 2007).
Cryopreservation is the maintenance of any biological structures (seeds in this case) at the temperatures of liquid nitrogen, approximately -196 to -150ºC (Benson, 1999; González-
Benito, 1998). It is one of the most effective methods for the long-term conservation of seeds
(Engelmann, 2009; Hirano et al., 2009) because when done properly it allows stopping all metabolic activity and biochemical processes within cells, and therefore allowing conservation for unlimited periods (Benson, 1999; Stanwood, 1985; Pritchard, 1984; Walters,
2015; Panis, 2001; Panis and Lambardi, 2005). Also, cryopreservation could be cheaper and safer than other ex situ conservation strategies (Engelmann, 1991). However, in some species has been documented a decreasing in germination capacity and seed viability after cryopreservation (Pritchard, 2004). For this reason, we will try three different temperatures of storage between -20º and -196ºC in order to determine the optimum strategy for a simple long-term ex situ conservation of Lapiedra martinezii seeds.
5.1.3.- The seed moisture content (SMC) and its role on long-term seed storage
Seed moisture content (SMC) is a critical factor to bear in mind when designing long- term preservation strategies, especially when the target biological structures have high levels of moisture content such as intermediate or recalcitrant seeds. Also, SMC is the most important factor to determine the speed of seed deterioration (Hong and Ellis, 1996). This has an important impact on the seed longevity in genebanking even at slight changes on seed moisture content (Rao et al., 2007). Therefore is very important to determine seed moisture content prior to deal with seed storage strategies. The internationally recommended standards are that seeds should to be dried to 5 % ±2 % moisture content prior to sub-zero storage
(FAO/IPGRI, 1994). Rao et al. (2007) also recommend seed moisture content between 3%
~ 123 ~ and 7% for long term storage. However, seeds with good storage features (like cereals) can be stored with seed moisture contents up to 7-11 % for medium term storage. On the other hand
Ellis et al. (1993; 2006), Hong and Ellis (1996) and Gomez-Campo (2006) pointed out the importance of reaching high levels of desiccation, known as ultra-dry seed storage, which values of SMC lower than 3% in long-term seed storage. The long-term storage of orthodox seeds is the most widely used method for ex situ preservation of plant genetic resources. It is the most efficient, economic and safe for the preservation of seeds from temperate regions with orthodox seeds (Pérez-García et al., 2007).
Other ex situ methods based on in vitro technology are the suitable option for recalcitrant seeds, clonal propagation, or species that do not produce seeds regularly
(González-Benito and Martín, 2011; Panis et al., 2001; Panis and Lambardi, 2005).
The goal of genebanks is essentially the maintenance of seed viability of a much wider range of species for long-term periods, for instance 100 years or more (Hong and Ellis, 1996).
According to Harrington (1972) this goal can be achieved in orthodox seeds by decreasing both the seed moisture content and the storage temperature (the seed longevity is doubled every decreasing of 5º in storage temperature and 1% of seed moisture content).
In spite of the increasing works on seed storage behavior and seed cryopreservation of wild species in the last years (Caetano et al., 2009; Hay and Probert, 2013; Panis and
Lambardi, 2005; Probert et al., 2009; Walters, 2015) there is a considerable lack of information on seed storage behaviour of wild Amaryllidaceae from the Eurasian species
(Newton et al., 2013). Seed storage behavior ranges from predominantly orthodox seeds in the American clade (Newton et al., 2013) to non-orthodox or recalcitrant seed storage behavior in the African one (Berjak and Pammenter, 2004; Sershen et al., 2008). As far as we know, seeds of this family are normally dried and stored following the conventional protocols
~ 124 ~ for orthodox seed collections (FAO/IPGRI, 1994; Newton et al., 2013; Rao et al., 2007) in
European Genebanks. In this geographical context, we find works on seed storage behaviour of some Amaryllidaceae species: Nikopoulos et al. (2008) and Juan-Vicedo et al. (2013) pointed out the good desiccation tolerance and thus, the orthodox storage behavior of
Pancratium maritimum seesd. Also, the results of Herranz et al. (2015) in Narcissus radinganorum suggest the orthodox behavior of seeds of this species. However, limited desiccation tolerance at seed dispersal was obtained by Newton et al. (2013) in Narcissus pseudonarcissus and Galanthus nivalis. In the latter study, as most seeds were unable to germinate following conventional desiccation procedures at the point of natural dispersal, conventional collections and drying protocols may not be suitable for seeds collections of these species.
Information regarding seed physiology related to the storage behavior of L. martinezii is still lacking. An approach to predict seed storage behaviour is discussed in Hong and Ellis
(1996). According to this, presumably, we are dealing with orthodox seed attending to some of the fruit and features (small seeds from dehiscent capsules) and the ecology of natural populations (semiarid environments in the Mediterranean Region). However, the degree of moisture and frost tolerance has to be determined in order to finally determine the seed storage behaviour of this species.
Therefore, discrimination among the orthodox, recalcitrant or intermediate categories is essential to determine whether this species can be maintained successfully over the long- term by conventional methods in case of orthodox seeds. On the contrary, specific procedures to improve desiccation tolerance and/or storability, as wel as alternative methods based on other forms of the germplasm to short-, medium- and long-term storage would be applied in case that we find non-orthodox behaviour of seeds (FAO/IPGRI, 1994; Hong and Ellis, 1996;
Newton et al., 2013,; Rao et al., 2007; Sershen et al., 2008). ~ 125 ~
Following the protocol of Hong and Ellis (1996) the first step to determine seed storage behavior considers desiccation tolerance to low moisture contents (for instance, up to
5%). The second is to confirm it after at least 90 days of cold storage at different environments. After each step, a germination test to assess the seed viability has to be carried out. For orthodox seeds, the use of ultra-dry levels and, particularly, the use of silica gel within the seed containers is highly recommendable. The advantages of using silica-gel are multiple: a) It dry seeds to ultra-dry levels, b) it efficiently maintains those moisture levels within waterproof containers, c) it warns by changing its color if anything goes wrong and d) it provides a second important preserving mechanism by absorbing toxic gases produced during the seed aging (Gómez-Campo, 2006).
~ 126 ~
Figure 5.1.- Simplified outline of a protocol to determine seed storage behaviour. Based in Hong and Ellis (1996).
In order to establish optimal protocols for the long-term seed conservation of L. martinezii it is necessary to know the desiccation tolerance of these seeds as well as the germination after preservation under different low temperatures.
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5.2.- Objectives
The main objective in this chaper was to evaluate the seed storage behavior of
Lapiedra martinezii in order to develop an appropriate long-term seed conservation protocol.
This goal was achieved by means of developing the following concrete objectives:
- To determine the seed moisture content of fresh seeds and desiccated seeds
after two different drying treatments.
- To study the response of seeds to desiccation throughout a germination test.
- To assess the response of seeds to desiccation and cold storage at different
temperatures under zero by means of a germination test.
5.3.- Material and Methods
5.3.1.- Determination of the Seed Moisture Content (SMC) and seed desiccation tolerance
The most accurate procedure to determine the seed moisture content is the oven desiccation method. Initially it was described by the International Seed Testing Association, ISTA (2005).
Based on the chemical composition of the seeds, there are two methods:
1) Low constant temperature method for oily seeds,
2) High constant temperature for the other type of seeds.
In our experiments, SMC was expressed in terms of the weight by water loss after dehydration. This was determined following the low constant temperature oven method for
~ 128 ~ oily seeds described in ISTA (2005) and summarized in Rao et al. (2007). As we could not find any information related to the biochemical composition of L. martinezii we used the method for oily seeds because it is the most conservative and can be applied for both kind of seeds (oily and non-oily seeds). To calculate SMC we followed this procedure (Rao et al.,
2007):
- Desiccation of the glass containers at 130º during one hour and cooling in a desiccator.
- Mark and weight each container with and without the tapes.
- Two randomly choosen seed subsamples from the accession were weighted in the
containers with and without the tapes.
- Put the containers without the tapes in the oven kept at 103±2ºC.
- Dry the seeds during 17±1 hours.
- Put the tapes on the containers and cool them in a desiccation chamber for 45 minutes.
- Register the weight of the containers of both samples.
- Calculate SMC according to the following formula:
SMC= (P2-P3/P2-P1) x 100
Where:
P1= Weight of the container with the tape
P2= Weight of the container with the seed sample and tape, before drying
P3= Weight of the container with the seed sample and tape, after drying.
~ 129 ~
Once the seed moisture content was determined, we obtained the fresh seed weight of
L. martinezii samples. To study the loss of water content with the two drying methods (drying chamber and silica gel) we measured in alternate days the weight of the seed lots and determine SMC as above.
Figure 5.2.- Seed desiccation methods used for Lapiedra martinezii Lag. seeds: desiccation chamber (left) and airtight boxes with silica gel (right).
5.3.2.- Seed storage behavior and long-term conservation of L. martinezii
The storage behaviour of L. martinezii seeds was evaluated by comparing the germination of fresh seeds with that of seeds desiccated (in chamber and silica-gel) and stored at -20 (sample of 2011), and desiccated in silica-gel and stored at -80ºC (samples of 2013) for
90 days. Additionally, an immersion in liquid nitrogen (-196ºC) of seeds desiccated in silica- gel (sample of 2013) for five days was done. This procedure was designed following the general protocol to determine the seed storage behaviour reported in Hong and Ellis (1996).
Seed desiccation took place at 20ºC, in airtight boxes with silica gel (ratio of silica gel:seeds
10:1, by weight) in which relative humidity was 7% (measured with a portable sensor, EL-
USB-2) and in dehydration chamber in which relative humidity was 13%.
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Figure 5.3.- Immersion of Lapiedra martinezii seeds in liquid nitrogen for five days: a) cryogenic tank; b) cryovials with seed samples.
Silica gel-desiccated seeds of 2013 were placed inside polypropylene cryovials and submitted to three different cryopreservation temperatures: 1) Normal freezing temperatures of -20ºC during 90 days; 2) Ultra freezing temperatures (-80ºC) and 3) immersion in liquid nitrogen (- 196ºC) for 5 days. After cryopreservation, the cryovials were unfrozen in a water bath at 40ºC and seeds were taken to equilibrate humidity content at room conditions during one day before sowing in the germination trials at the conditions mentioned above. A control of fresh seeds was also incubated and germination parameters were studied.
5.3.2.1.- Seed germination trials
The seed germination tests were carried out attending to the optimal results obtained in
Chapter 2, before and after dehydration and storage. Laboratory conditions for seed germination were: constant temperature of 20ºC under a 8 hours light /16 hours dark photoperiod (photosynthetically active radiation: 42 µmol m-2s-1).
~ 131 ~
We measured the following parameters:
a) Final seed germination percentage: a seed was considered to be germinated when the
coleoptile reached at least 0,5 cm after seed protrusion.
b) Onset: the day of the first germination.
c) T50: the day when 50% of germination is achieved.
5.2.4.- Statistical analyses
Final germination percentages were arcsine square root transformed, and the normality and homoscedasticity of the data were checked. For the other parameters (onset and T50), non-transformed data were used. One-way analyses of variance (ANOVA) were carried out and significant differences between means were revealed with the Fisher’s Least Significant
Differences (Fisher’s LSD) test at the 5% level. All data were analysed using the Infostat
2008 package.
5.4.- Results and Discussion
5.4.1- Long-term conservation of seeds
5.4.1.1- Determination of the SMC
As we did not know the biochemical composition of L. martinezii seeds, we proceeded with the the low constant temperature oven method (ISTA, 2005; Rao et al., 2007) proposed for oily seeds: SMC of fresh seeds was 9.19. Seed desiccation of L. martinezii took approximately 40 days. However, most of the water loss in both samples occurred within the
~ 132 ~ first two weeks of desiccation (Figure 5.4). Afterwards, the water loss was lower and thus desiccation took place at slower speed until certain values. SMC of L.martinezii desiccated seeds was 4.44 (in desiccation chamber) and 4.10 (in desiccator with silica gel). These values are around the recommended international values for seed storage at subfreezing temperatures, which are included within a range of 3-4% and 6-7% of SMC (FAO/IPGRI,
1994; Gómez-Campo, 2006; Hay & Probert, 2013; Hong and Ellis, 1996; Rao et al., 2007).
Figure 5.4.- Seed desiccation evolution in samples of Lapiedra martinezii (seeds of samples of 2011) for both desiccation methods tested (dehydration chamber and silica gel desiccation) measured as a loss of weight.
~ 133 ~
5.4.1.2- Seed desiccation tolerance
The final germination percentages of control and desiccated seeds of L. martinezii did not significantly differ between the two desiccation treatments applied to the seed samples, where the lowest values of SMC were achieved (Figure 5.4). Desiccation only affected to the germination onset, but this was a very slight effect that did not have any consequence in the germination speed (T50) and final germination percentages. This behavior was also obtained in the 2013 seed sample where the onset was slightly longer in all the pre-treated seeds in comparison to the control (Figure 5.5).
5.4.1.3- Long-term seed storage.
The final germination of the 2011 samples with respect to controls did not show any significant differences after the two levels of desiccation and the desiccation along with 90 days of cold storage at -20ºC. In the samples of 2013 we wanted to test whether lower storage temperatures of -80ºC and -196ºC had any effect on the seed germination and viability for seeds (Figure 5.5). In this case, only a slight inhibitory effect, but not significant, on final seed germination was obtained after the immersion in LN. In any case, percentages of seed germination still remained high. An explanation to this might be that the 2013 sample could have achieved a slight decrease in the storage capacity of seeds by means of crystal formation and tissue damage of seeds. However, the results obtained at temperatures of -80ºC did not support this idea, as this temperature is also very low and the same kind of problems could have been arosen in seeds. Also, it is generally considered that SMC comprised between 5-
18% is safe for liquid nitrogen storage (Hong and Ellis, 1996; Stanwood and Roos, 1979).
This suggests that seeds of L. martinezii could have suffered some slight alterations due to the immersion. However, these alterations can not be considered significant as the decrease on
~ 134 ~ final seed germination and onset is not significantly different in respect to the control (Figure
5.5). A significant reduction in onset was obtained for both samples after desiccation and cold storage at all subzero temperatures tested. However, the most of these treatments did not affect T50 and therefore we can assume that these procedures did not affect significantly the germination speed. Only, in some cases, T50 was significantly affected by the desiccation and cold storage. However these differences are not so big in comparison to the control and we can not say that the desiccation and storage procedures here applied are harmful for L. martinezii seeds.
The ability of seeds to survive desiccation is an important functional trait and is an integral part of the regeneration ecology that is still relatively under-studied (Tweedle et al.,
2003). The wide desiccation tolerance found in this study for L. martinezii is probably necessary in these species in situ as they may experience conditions in their natural habitat that would result in rapid drying to low moisture content at, or after the time of seed dispersal, before optimal conditions for germination take place. These conditions would be, according to the results obtained in Chapter 2, the start of the short rainfall period in the autumn. As most seeds were able to germinate following these desiccation procedures at the point of natural dispersal, conventional collection and drying protocols (FAO/IPGRI, 1994; Hong and Ellis,
1996; Rao et al., 2007; Walters, 2015) are reasonably suitable for producing high quality seed collections of these species. These findings contrasts with the results obtained by Newton et al. (2013) for Narcissus pseudonarcissus and Galanthus nivalis where limited desiccation tolerance of seeds at dispersal as was found. Orthodox seeds are those that can be stored in these ‘conventional’ seed banks. Such seeds tolerate drying to very low moisture contents,
≤3–7% fresh weight (Roberts, 1973). Therefore, the obtained results suggest the orthodox storage behaviour of L. martinezii seeds.
~ 135 ~
Figure 5.5.- Onset, T50 and final seed germination of Lapiedra martinezii seed samples of 2011 (left) and 2013 (right). The germination tests were carried out at 20ºC and under an irradiance of 25 µmol m-2s-1 and an 8 hours light/16 hours dark photoperiod. For each pre-treatment and parameter, different letters indicate significant differences (p<0.05). For the samples of 2011 (left) treatments are as follow: 1, control; 2, seeds dehydrated under 13% R.H.; 3, seeds dehydrated under 7% R.H.; 4, seeds dehydrated under 13% R.H. and stored at -20ºC for 90 days; 5, Seeds dehydrated under 7% R.H. and stored at -20ºC for 90 days. For the samples of 2013 (right) treatments are as follow: 1, control; 2, seeds dehydrated under 7% R.H. and stored at -20ºC for 90 days; 3, seeds dehydrated under 7% R.H. and stored at -80ºC for 90 days; 4, seeds dehydrated under 7% R.H. and submerged in liquid nitrogen (-196ºC) for 48 hours. The bars above the columns indicate standard deviation.
~ 136 ~
L. martinezii 2011 L. martinezii 2013 Final Germination (%) Onset (days) T50 (days) Final Germination (%) Onset (days) T50 (days) Control 98,5±1,91 7,0±0 15,5±0 97±3,46 6,25±1,50 15,5±1 Desiccation Chamber 99,5±1 10,5±1 14,5±1,91 - - - Silica Gel Desiccator 98,0±2,83 11,0±0 16,0±0 - - - Desiccation Chamber + Storage -20ºC 96,5±2,52 10,25±0,50 16,5±1 - - - Silica Gel Desiccator + Storage -20ºC 97,5±2,52 10,25±0,50 18,25±2,36 98,5±1,91 10,25±1,50 19,0±1,15 Silica Gel Desiccator + Storage -80ºC - - - 99,0±1,15 11,0±0 18,0±0 Silica Gel Desiccator + Immersion -196ºC - - - 92,5±1 10,25±1,50 16,25±2,06
Table 5.6. Summary of the diffrenet treatments done on seeds of the two samples studied of Lapiedra martinezii (2011 and 2013). The final percentage of seed germination, onset and T50 were determined. Values are acompanied with ± the standard error. Different letters among columns indicate non significant differences at the 5% level according to the Fisher’s LSD.
5.5.- Conclusions
- Seeds of L. martinezii can be dried until SMC levels of 4.10 without any significant
effect on final germination percentages.
- Also, seeds from this species can be cold stored at temperature of -20ºC and final seed
germination remains the same as the control, suggesting an orthodox behavior.
- L. martinezii seeds can be preserved up to temperatures of -80ºC without any
reduction on final seed germination.
- Immersion in liquid nitrogen did not decrease significantly the final germination
percentage in L. martinezii seeds.
- Seed germination is not significantly affected by desiccation and storage treatments
and thus conventional techniques of seed conservation in genebanks are appropriate
alternatives for long-term germplasm conservation.
~ 137 ~
~ 138 ~
6.- GENERAL CONCLUSIONS
~ 139 ~
~ 140 ~
6.- GENERAL CONCLUSIONS
- Seeds of Lapiedra martinezii have morphological dormancy as embryos had to grow
within the seed before the coleoptile emergence took place.
- Optimal seed germination was achieved at 20ºC with germination percentages of
almost 100% both in light and darkness, although suboptimal germination percentages
(>60%) were also obtained at 17ºC, 30/20ºC and 25/16ºC.
- Light conditions influenced germination speed, but did not affect germination
percentages.
- Cold stratification decreased germination parameters at all the assayed temperatures,
whereas warm pre-treatment generally increased germination rates.
- The application of GA3 did not affect germination at concentrations between 0-0.75
g/L, while the proportion of germinated seeds decreased at higher concentrations
- .The in vitro culture of Lapiedra martinezii is a quite challenging task, especially due
to the presence of endogenous bacteria. A mixture of antimicrobials consisting in; 500
mg/L cefotaxime, 5 mg/L ampicillin and 15 mg/L kanamycin for three weeks of
culture was found suitable to reduce contamination to an acceptable level.
- Culture medium based in Gamborg B5 salts and supplemented with 10 g/L of sucrose,
suited better for initiation of seedling and bulb scale cultures.
- Seedling cultures developed well as a stock for 10 months at low temperature (17ºC)
in MS medium supplemented with 30 g/L sucrose and without growth regulators.
- The best morphogenic response was obtained in medium MS supplemented with 30
g/L sucrose, BA= 4 mg/L and NAA = 0.12 mg/L in terms of shoot production (5.77
bulblets per explant) and caliber (4.07±0.48 x 6.10±0.65). Also, the rooting was very
high in this medium (96.18%). Moreover, the proliferation of callus-like structures
was relatively low (24.84%) in comparison to the other media tested. ~ 141 ~
- Rooting was spontaneous in all multiplication media during the first subculture with a
variable percentage of 76.43% to 100% (exceptionally 49.68%). Thus, there was not
necessary to include a rooting stage with specific media in the micropropagation
process, although transference to MS solid medium supplemented with 30 g/L and
without growth regulators increased rooting up to 100% in all the multiplication media
tested that gave rooting under 100%.
- The acclimatization procedure tested here had a very high success, with almost 90% of
survival for both types of microplants (seedlings and bulblets) after transference to ex
vitro conditions.
- Complete darkness clearly favored callus induction, growth and development, while
light had a marginal effect on it.
- 2,4-D is necessary for the callus formation, regardless the citokinin (BAP or KIN)
present in the medium.
- High auxin/cytokinin ratios (of at least 2) were necessary for callus initiation and to
achieve the highest callus efficiency in most cases in a frame of 2 and 3 months.
- The well-established calli were yellow and almost all of them showed compact-soft
friable textures.
- Seeds of L. martinezii can be dried until SMC levels of 4.10, and stored at -20, -80ºC
and by immersion in liquid nitrogen without any significant effect on final
germination percentages suggesting their orthodox behaviour.
~ 142 ~
~ 143 ~
7.- PROTOCOLS
~ 144 ~
~ 145 ~
7.- PROTOCOLS.
Com a resultat d’aquest treball d’investigació, s’han desenvolupat els següents protocols de treball per a la conservació i/o propagació sostenible de L. martinezii que poden satisfer les necessitats de les rutines de treball amb diferents finalitats.
PROTOCOL 1.- RUTINA DE CONSERVACIÓ PER A BANCS DE GERMOPLASMA
1.- Recolecció de llavors durant la tardor.
2.- Germinació d’una mostra a 20ºC i fotoperíode 16 hores llum i 8 obscuritat.
3.- Dessecació recomanada entre el 4.1-8% d’humitat seminal.
4.- Conservació a -20ºC.
5.- Regeneració i estudi periodic de viabilitat per germinació de mostres a 20ºC i fotoperíode 16 hores llum i 8 obscuritat.
~ 146 ~
PROTOCOL 2.- ESTERILITZACIÓ DE MATERIAL I INICI DE CULTIU IN VITRO
D’ESCALES DE BULBS
1.- Recolecció de bulbs adults en la tardor.
2.- Neteja dels bulbs amb detergent commercial, i retirada de les arrels, catàfils i parts necrosades dels bulbs.
3.- Tractament tèrmic (termoteràpia): submergir bulbs en aigua escalfada a temperatura constant de 54ºC durant 60 min.
4.- Retirada del terç apical dels bulbs i secció de la part remanent en tres-quatre escales.
5.- Immersió de les escales en una dissolució de lleixiu comercial al 30% durant 20 min.
6.- Rentar els explants 3 vegades en aigua destil·lada estèril.
7.- Inoculació en medi d’inici Gamborg G5+10 g/L sacarosa+500 mg/L de caseïna, 2 mg/L d’adenina, 10 mg/L de glutation i 5.5 g/L Plant Agar (manteniment 10 dies en obscuritat).
8.- Manteniment durant 4-8 semanes en medi d’inici en condicions de llum.
9.- Acondicionament preventiu dels explants (previ a multiplicació o creació de stocks) mitjançant la transferència a MS+vit+500 mg/L de cefotaxima, 5 mg/L d’ampicilina i 15 mg/L de kanamicina durant tres setmanes.
~ 147 ~
PROTOCOL 3.- PROPAGACIÓ DE BULBS DES DE STOCKS INICIATS IN VITRO
1.- .- Retirada del terç apical dels bulbs, arrels, fulles i catàfils externs; secció en 2-4 escales.
2.- Transferència a medi de multiplicació: MS+vit+30 g/L sacarosa+5.5 g/L Plant
Agar+BA=4 mg/L+NAA=0.12 mg/L (cultiu durant 8 setmanes en fotoperíodes llum/obscuritat a 24ºC).
3.- Si durant el període de cultiu la proliferació de bactèries > 40- 50%:...... 3.1
3b.- Si durant el període de cultiu la proliferació de bactèries < 40-50%...... 4
3.1.- Retirada del cultiu dels explants més afectats i esterilització d’aquells
que mostren un nivell de contaminació més baix mitjançant la immersió en
etanol al 70% durant 30 seg i rentar amb aigua destil·lada estèril.
3.2.- Sembra dels explants esterilitzats, els explants no danyats i nous explants
extrets dels stocks iniciats (pas 1) en medi MS+vit+500 mg/L de cefotaxima,
5 mg/L d’ampicilina+15 mg/L de kanamicina+5.5 g/L Plant Agar durant
tres setmanes. Després, passem al punt 4.
4.- Durant el període de cultiu, es produixen 3 o més arrels ≥ 1 cm...... 5
4b.- Durant el període de cultiu, es produixen arrels < 1 cm o no es produixen...... 4.1
~ 148 ~
4.1.- Transferència a medi MS+vit+30 g/L sacarosa+5.5 g/L Plant Agar
durant 3-4 setmanes. Posteriorment, una vegada l’arrelament és adequat, es
passa al punt 5.
5.- Neteja dels bulbets (retirada de l’agar, parts necrosades, etc.) i plantació en una mescla de turba i vermiculita ex vitro (5:4) prèviament autoclavada.
6.- Aclimatació preliminar: cultiu en càmera a 24ºC, HR de 100% i un fotoperíode de 16 hores llum/8 hores obscuritat (radicació fotosintèticament activa de 42 µmol m-2s-1) durant 4 setmanes.
7.- Enduriment: cultiu en càmera a 24ºC, HR reduïda fins al 70%, i intensitat lumínica incrementada fins als 80 µmol m-2s-1 durant 4 setmanes.
8.- Transferència a condicions ambientals d’exterior.
~ 149 ~
PROTOCOL 4.- MANTENIMENT DE STOCK DE PLÀNTULES IN VITRO
1.- Esterilització superficial de llavors: immersió en etanol al 70% (30 s), seguit d’una immersió en HgCl2 al 0.1% (3 min) i rentar tres vegades en aigua destil·lada estèril.
2.- Germinació a 20ºC i fotoperíode 16 hores llum i 8 obscuritat durant aproximadament 5 setmanes.
3.- Esterilització de les plàntules: immersió en etanol al 70% (30 s), seguit d’una immersió en hipoclorit càlcic al 7% (20 min) i rentar tres vegades en aigua destil·lada estèril.
4.- Eliminació parcial de fulles i radícules, així com de restes de la llavor (coleòptil) i sembra en medi d’inici: Gamborg B5+500 mg/L de caseïna+2 mg/L d’adenina+10 mg/L de glutation+10 mg/L de sacarosa+5.5 g/L de Plant Agar durant 8 setmanes.
5.- Si durant el període d’inici la proliferació de bactèries > 40- 50%:...... 5.1
5b.- Si durant el període de cultiu la proliferació de bactèries < 40-50%...... 6
5.1.- Retirada del cultiu dels explants més afectats i esterilització d’aquells
que mostren un nivell de contaminació més baix mitjançant la immersió en
etanol al 70% durant 30 seg i rentar amb aigua destil·lada estèril.
5.2.- Sembra dels explants esterilitzats, els explants no danyats i nous explants
extrets de les accessions de llavors (pas 1) en medi MS+vit+500 mg/L de
cefotaxima, 5 mg/L d’ampicilina+15 mg/L de kanamicina+5.5 g/L Plant
Agar durant tres setmanes. Després, passem al punt 6. ~ 150 ~
6.- Sembra en medi d’inici: Gamborg B5+500 mg/L de caseïna+2 mg/L d’adenina+10 mg/L de glutation+10 mg/L de sacarosa+5.5 g/L de Plant Agar i cultiu durant 8 setmanes.
7.- Obtenció de stocks: transferència dels bulbs a creixement a 17ºC en pots de 250 mL de medi sense hormones, formulat amb salts MS+vitamines+30 g/L de sacarosa+5.5 g/L de
Plant Agar.
~ 151 ~
PROTOCOL 5.- OBTENCIÓ DE CALLS IN VITRO
1.- Recolecció de bulbs adults en la tardor, en el moment de la foliació.
2.- Separació i neteja de les fulles amb detergent comercial. Retirar la part basal de la fulla
(en contacte amb el bulbs) i la part superior (verda).
3.- Esterilització superficial amb etanol al 70% durant (30 s) i immersió en hipoclorit càlcic al 7% (20 min). Finalment, rentar les fulles sis voltes en aigua destil·lada estèril.
4.- Seccionar el material esterilitzat en explants d’un cm de llarg, aproximadament, prèvia eliminació de les parts necròtiques.
5.- Sembra de les fulles en medis MS+vit+30 g/L sacarosa+5.5 g/L Plant Agar amb les següents combinacions de reguladors del creixement (mg/L): 1) BAP=0.1+2,4-D=0.5; 2)
BAP=1+2,4-D=0.5 ;3) BAP=1+2,4-D=2; 4) KIN=0.1+2,4-D=0.5; 5) KIN=0.1+2,4-D=1; 6)
KIN=0.1+2,4-D=2.
6.- Mantindre en càmera de cultiu a 26ºC i obscuritat, i subcultivar cada 4 setmanes.
~ 152 ~
9.- REFERENCES
~ 153 ~
~ 154 ~
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