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Seed dormancy and germination of the medicinal holoparasitic plant Cistanche deserticola from the cold desert of northwest China
Accepted Manuscript Seed dormancy and germination of the medicinal holoparasitic plant Cistanche deserticola from the cold desert of northwest China Jia Wang, Jerry M. Baskin, Carol C. Baskin, Guofang Liu, Xuejun Yang, Zhenying Huang PII:
S0981-9428(17)30130-4
DOI:
10.1016/j.plaphy.2017.04.010
Reference:
PLAPHY 4856
To appear in:
Plant Physiology and Biochemistry
Received Date: 13 February 2017 Revised Date:
7 April 2017
Accepted Date: 7 April 2017
Please cite this article as: J. Wang, J.M. Baskin, C.C. Baskin, G. Liu, X. Yang, Z. Huang, Seed dormancy and germination of the medicinal holoparasitic plant Cistanche deserticola from the cold desert of northwest China, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.04.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Seed dormancy and germination of the medicinal holoparasitic plant
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Cistanche deserticola from the cold desert of Northwest China
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Jia Wang1,4, Jerry M. Baskin2, Carol C. Baskin2,3, Guofang Liu1, Xuejun Yang1*,
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Zhenying Huang1
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Chinese Academy of Sciences, Beijing 100093, P.R. China
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Department of Biology, University of Kentucky, Lexington, KY 40506, USA
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Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY
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40546, USA
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University of Chinese Academy of Sciences, Beijing 100039, P.R. China
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State Key Laboratory of Vegetation and Environmental Change, Institute of Botany,
Corresponding author
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Fax: +86-10-62836276
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E-mail address: [email protected]; [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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Cistanche deserticola is a holoparasitic plant with high medicinal value that
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reproduces only by seeds. However, the requirements for seed dormancy break and
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germination of this species remain unclear. The freshly matured dust-like seeds
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consist of a water-permeable seed coat and an undifferentiated oval-shaped embryo
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embedded in endosperm. No fresh seeds germinated in water or a 10-5 M fluridone
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solution at any incubation temperature within 60 days. Length of embryos in seeds
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incubated in warm- and cold-started stratification sequences had increased 10.4 and
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11.7 % after 50 and 40 weeks, respectively. After 6 months, length of embryos in
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seeds stratified at 5 ºC had increased by 12 %. Germination of fresh seeds and of
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seeds stratified at 5 ºC for 6 months and then incubated in mixed fluridone/gibberellic
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acid 3 (GA3) solutions at 30/20 ºC germinated to only 2.6 and 11.7 %, respectively.
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Embryos of fresh seeds and of cold-stratified seeds had increased 29.4 and 15.8 % in
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length, respectively, at the time of germination, but they never differentiated into
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organs. The highest germination (54.4 %) was for seeds incubated in a 10-5 M solution
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of fluridone in darkness in spring that had overwinter on the soil surface in the natural
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habitat. Our study indicates that breaking of physiological dormancy (PD) occurs first
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and then the embryo grows to a critical length (0.44 mm) without differentiation into
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organs prior to seed germination. Seeds for which PD had been broken were induced
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to germinate by fluridone and GA3 at high temperature. Taken together, these results
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suggest that C. deserticola seeds have a specialized kind of morphophysiological
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dormancy. This study reveals possible ways to release seed dormancy that will be
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useful in propagating this medicinal species.
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Keywords: Cistanche deserticola; holoparasite; seed germination; specialized
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morphophysiological dormancy; undifferentiated embryo
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1. Freshly matured seeds are dust-like, with an undifferentiated oval-shaped embryo
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embedded in endosperm.
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2. C. deserticola seeds have a specialized morphophysiological dormancy (MPD).
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3. The physiological (PD) component of dormancy is broken before the embryo
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grows.
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4. Embryos grew to a critical length prior to germination but did not differentiate into
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organs.
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5. After PD is broken, the seeds could be induced to germinate by fluridone and by a
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combination of fluridone and GA3 at 30/20 ºC.
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Seed germination is a critical stage in the life history of plants and especially for those
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that grow in deserts (Gutterman, 1993). Seeds may be nondormant at maturity and
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thus germinate soon after dispersal if environmental conditions are favorable for them
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to do so. However, favorable conditions may not persist long enough for the resulting
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plant to become established. Seed dormancy prevents seeds from germinating in such
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conditions, thus reducing the chances of seedling mortality and thereby contributing
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to the success of population regeneration (Baskin and Baskin, 2014).
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Based on the seed dormancy classification system developed by the Russian seed
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physiologist Mariana G. Nikolaeva between 1967 and 2001 (Baskin and Baskin, 2008)
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and expanded and modified by Baskin and Baskin (2014), there are five classes of
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dormancy. Seeds with physiological dormancy (PD) are water-permeable and have a
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fully developed embryo with a physiological inhibiting mechanism that prevents
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radicle emergence, seeds with morphological dormancy (MD) an underdeveloped
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(small) embryo that needs to grow before the seed germinates, seeds with
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morphophysiological dormancy (MPD) an underdeveloped embryo that is
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physiologically dormant, seeds with physical dormancy (PY) a fully developed
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nondormant embryo but germination is prevented due to water-impermeability of the
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seed (or fruit) coat and seeds with combinational dormancy a fully developed embryo
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that is physiologically dormant and the seed (or fruit) coat is water impermeable
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(PY+PD). In addition to these kinds of dormancy, Baskin and Baskin (2014)
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recognized specialized kinds of morphological and morphophysiological dormancy
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maturity that never differentiate into a root-shoot axis (Baskin and Baskin, 2014).
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These specialized kinds of dormancy are the ones that occur in “dust seeds” of
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holoparasites such as Orobanchaceae species and in mycoheterotrophs such as orchids
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(Baskin and Baskin, 2014).
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Cistanche deserticola Ma (Orobanchaceae) is a perennial holoparasite herb that
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parasitizes the roots of the cold desert shrub Haloxylon ammodendron (C. A. Mey.)
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Bunge (Amaranthaceae, subfamily Chenopodioideae). It occurs in the temperate arid
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cold deserts of northwest China and the Republic of Mongolia. Plants bloom in May
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to June, and fruits mature from June to August. C. deserticola has been used as a
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traditional medicinal plant in China for hundreds of years. However, the species has
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become endangered due to over-collection and the difficulty of regenerating it from
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seeds, which is the only way it reproduces. That is, C. deserticola does not reproduce
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vegetatively (Fu, 1992; Xu et al., 2009).
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In general, results of studies on the seed germination of C. deserticola are
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inconsistent. In a study by Qiao et al. (2007) fluridone induced 53 % of the seeds to
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germinate, but Fen (2012) obtained only 2.9 % germination of seeds treated with this
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ABA biosynthesis inhibitor. Zhang et al. (2008) reported that seeds treated with two
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cycles of 50 ºC for 1 hour and 4 ºC for 1 week germinated to 6.7 %. Chen et al. (2009)
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found that the germination percentage of seeds cold-stratified at 5 ºC for 120 days
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was >70 % in GA3 solution but that GA3 had no effect on fresh (non-cold stratified)
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seeds. However, Zhang et al. (2009) reported that GA3 induced germination of fresh
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seeds. The sensitivity of seeds to exogenous GA3 might vary with the physiological
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status of the seeds, which could explain the inconsistent results obtained by different
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authors. Although several studies have been conducted on seed germination of C.
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deserticola, we still do not have an efficient way to produce plants from seeds, i.e. the
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number of plants produced per number of seeds sown in very low (Sun et al., 2008).
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In the field, farmers dig holes adjacent to the host plant and put approximately 200
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seeds in each hole. However, this practice of propagating plants leads to very low
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numbers of plants due to dormancy of the seeds. According to information provided
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by local farmers in our study site, less than three seeds can develop into plants in each
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hole. Xu et al. (2009) showed that fresh stem yield of C. deserticola was about 0.2-2.2
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t ha-1. However, this high crop yield was based on a huge number of seeds, most of
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which were wasted. Thus, a more efficient method is needed for propagating plants of
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this species from seeds.
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Thus, we suggest that learning more about the morphology and physiology of
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germination of the seeds under natural and simulated natural conditions may help in
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this endeavor. Since mature seeds of C. deserticola contain a globular embryo that is
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undifferentiated (Li et al., 1989; Ma et al. 1997) and fresh seeds are dormant (see
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references cited above), we assumed that seeds have a specialized kind of
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morphophysiological dormancy (sensu Baskin and Baskin, 2014). Thus, we
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hypothesized that (1) the small embryo needs to grow or differentiate prior to
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germination, (2) physiological dormancy needs to be released before the germ-tube
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needs a chemical stimulant (which in nature would be produced by the host) to
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germinate. To test these hypotheses, we asked following questions. (1) Do fresh seeds
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of C. deserticola have a specialized kind of MD or MPD? (2) How do embryos in
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intact seeds respond to different temperatures? (3) Do embryos need to grow before
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the seeds germinate? (4) Does seed dormancy/germination respond to seasonal
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changes of soil temperatures in the natural habitat?
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2. Materials and methods
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2.1. Seed collection and measurement of plant height
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Infructescences of C. deserticola with freshly matured seeds were harvested from
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plants in the Tengger Desert, Inner Mongolia, China, (38°79′N, 105°54′E, 1379 m
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a.s.l.) on 10 August 2013 and 30 August 2014. This area has a typical continental arid
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climate with a mean annual precipitation of 182 mm, about 70 % of which occurs
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from July to September. Mean annual temperature is 8.5 °C, and mean temperature of
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the coldest (January) and hottest (July) months are -15.7° and 30 °C, respectively
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(data from WorldClim database, http://www.worldclim.org).
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Seeds were extracted from the fruits (capsules) and separated from other plant
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material using a soil sieve (mesh size 0.6 mm). The seeds were air-dried for 3 days at
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ambient laboratory conditions (20-25 ºC, 17–32 % relative humidity) before initiation
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of experiments.
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Ten adult plants of C. deserticola were haphazardly selected in the natural habitat, 8
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and height of each plant from the sand surface to the top of inflorescence was
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measured.
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2.2. Seed morphology and water imbibition
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Embryos were dissected from seeds using forceps and a dissecting needle and
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measured using a Nikon SMZ1000 dissecting microscope (Tokyo, Japan) equipped
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with a calibrated micrometer. Length of 25 embryos and length and width of 25 fresh
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seed were measured. The 1000-seed mass was determined by weighing five replicates
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of 1000 seeds.
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Rate of water uptake (imbibition) was monitored in freshly mature seeds. Initial
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seed mass was determined by weighting four replicates of 1000 air-dried seeds. Four
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replications of 1000 seeds were placed on filter paper in a 5.5 cm Petri dishes
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moistened with 3 ml distilled water and incubated at room temperature (20-25 °C).
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Seed weight was monitored at 1-minute intervals for 10 minutes. Percentage of
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increase in seed mass based on initial seed mass was calculated.
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Seed structure was studied by fixing fresh seeds in FAA solution (70% ethanol:
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acetic acid: formaldehyde, 18:1:1, by vol.) and then dehydrating them in a graded
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ethanol series. Then, the seeds were embedded in paraffin and cut into 8 µm sections
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with a rotary microtome (Leica RM2235, Germany). Sections were stained either with
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safranin or fast green and photographed under a light microscope (Nikon 80i, Japan).
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Germinated seeds were observed and photographed under a Nikon SMZ1500
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dissecting microscope (Tokyo, Japan).
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2.3. Initial germination tests
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Freshly mature seeds were placed in 5.5 cm Petri dishes on two layers of Whatman
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No. 1 filter paper moistened with 3 ml of 10-5 M fluridone (Sigma, USA) (Qiao et al.,
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2007; Chen et al., 2012) and incubated in both light (12 h photoperiod, 100 µmol m-2
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s-1, cool white fluorescent light) and continuous darkness (seeds placed in a light-tight
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bag) at 15/5, 20/10, 25/15 and 30/20 ºC (12/12 h) for 12 weeks. Four replicates of 100
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seeds were used for each treatment. The high temperature coincided with the 12 h
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light period and the low temperature with the 12 h dark period. The alternating
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temperature regimes represent approximations of mean daily maximum and minimum
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air temperatures for each month during the growing season in the natural habitat:
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April and October, 15/5 ºC; May and September, 20/10 ºC; June and August 25/15 ºC;
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and July, 30/20 ºC. Germination of seeds incubated in light was monitored every 3
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days, and those incubated in darkness were checked every 6 days under a green safe
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light (Emitting diode power supply, YP-TH0301, YIPU, China) in a dark room. Final
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germination percentages were determined after 3 months. Emergence of the germ tube
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through the seed coat was the criterion for germination.
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2.4. Effect of warm- and cold-started stratification on embryo growth
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Our purpose here was to monitor embryo growth in seeds in a warm-started and in a
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cold-started temperature sequence that simulated seasonal temperature regimes in the
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field. The sequence began with either warm (25/15 ºC) or cold (5 ºC) stratification. 10
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10 weeks of warm → 3
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cycles of (12 weeks of cold → 16 weeks of warm) and the cold -started stratification
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procedure of 3 cycles of (12 weeks of cold → 16 weeks of warm) → 12 weeks of
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cold.
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Each warm stratification treatment in a sequence represented high temperature
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months (average temperature above 15 ºC) and each cold stratification the low
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temperature months (average temperature between 0 and 10 ºC). Seeds of C.
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deserticola are dispersed from late July to late August, and they are exposed to warm
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stratification until October (ca. 10 weeks). Then, they are exposed to cold
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stratification (about 12 weeks) in October, November and March (soil frozen in
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December to February), to warm stratification (about 16 weeks) again from June to
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September, etc. Seeds in both warm-started and cold-started temperature sequences
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were kept moist (seeds checked every two days and distilled water added if necessary)
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during treatment. Embryo length was determined for 25 haphazardly-chosen seeds at
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time zero and at the end of each warm and cold treatment in the two sequences.
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2.5. Effect of cold stratification and germination stimulants on germination
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The purpose of this experiment was to test the effect of cold stratification alone and in
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combination with the germination stimulants fluridone, an inhibitor of ABA
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biosynthesis, GA3, a plant hormone and GR24 (Sigma, USA), a synthetic
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strigolactone, on promoting germination of C. deserticola seeds. Intact seeds that had
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been cold-stratified at 5 ºC in darkness for 6 months and freshly matured seeds 11
ACCEPTED MANUSCRIPT (control) were used in the experiment. Four replicates of 100 seeds for each treatment
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were soaked in distilled water (control) or in solutions of fluridone (10-5 M), GA3
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(10-5 M), fluridone (10-5 M)/GA3 (10-5 M) or GR24 (10-6 M) and incubated in
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darkness at 30/20 and at 5 ºC for 6 months. In our initial study (unpublished data), no
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fresh seeds germinated either in 10-3, 10-5 or 10-7 M GA3 or in 10-6, 10-8 or 10-10 M
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GR24. Furthermore, treatment of seeds with 10-4, 10-5 and 10-6 M fluridone resulted in
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no seed germination. Germination was monitored weekly using the green safe light
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described above. Length of embryo in each of 20 seeds in each treatment was
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measured monthly during the cold stratification period. For germinated seeds, a small
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protuberance (germ tube) emerged through the seed coat. Length of embryos that had
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grown was measured when the seed coat had split, but the germ tube had not emerged
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from the seed.
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2.6. Embryo growth and release of physiological dormancy in the field
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Two thousands freshly matured seeds were placed in each of 18 (12 × 7 cm) nylon
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mesh bags. Six bags of seeds each were buried at depths of 0 (surface), 2 and 5 cm in
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sand in the natural habitat in which seeds were collected. Sand temperatures at all
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burial depths were recorded at 2-hour intervals by an iButton DS1992L temperature
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data logger (Maxim Integrated Device, California, USA). Three bags were exhumed
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at 2-month intervals from September 2014 to September 2015. For seeds from each
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burial period and depth, embryo lengths of 25 seeds were measured prior to the
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germination test. Germination was tested by incubating seeds in distilled water and in
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a 10-5 M fluridone solution in darkness at 30/20 ºC for 12 weeks. Germination was
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monitored every 3 days using the green safe light. Emergence of the germ tube was
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the criterion for germination.
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2.7. Statistical analysis
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Means and standard errors were calculated for germination percentages and embryo
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lengths. One-way ANOVA was used to analyze the results of warm- and of
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cold-started stratification treatments on embryo growth and of the effects of cold
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stratification and chemical stimulants on seed germination. Fisher’s LSD test was
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used to test for significant differences in multiple comparisons. Data were arcsine
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square root transformed when necessary to meet assumptions of ANOVA for
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normality and homogeneity of variance. Statistical analyses were performed using
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SPSS Version 21.0 for Windows 8.0 (SPSS, Chicago, IL, USA).
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3. Results
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3.1. Plant height, and seed morphology and water imbibition
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Average height of C. deserticola plants was 0.48 ± 0.02 m, and they were parasitic on
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the roots of H. ammodendron (Fig. 1A). Mean seed length and width were 1.21 ± 0.02
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mm and 0.84 ± 0.02 mm, respectively (Fig. 1B). Mass of 1000 seeds was 122.18 ±
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1.27 mg. After 4 minutes, seeds were fully imbibed, and seed mass had increased to
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163.2 ± 5.1 % of the original mass.
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Fresh seeds had a dark alveolate seed coat, a perisperm (nucellar material) and a 13
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was located between the perisperm and the seed coat (Fig. 1C). During germination,
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neither radicle nor cotyledons was formed. The radicular pole of the embryo grew and
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developed into a radicle-like germ tube (Fig. 1D).
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3.2 Initial germination test
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None of the freshly matured seeds had germinated after 3 months in the 10-5 M
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fluridone solution in either light or darkness at any of the temperature regimes tested.
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3.3. Effect of warm- and cold-started stratification on embryo growth
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Embryos grew very slowly in both warm- and cold-started stratification treatments
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(Fig. 2A, 2B). Mean embryo length of freshly matured seeds was 0.29 ± 0.01 mm.
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The lengths of embryos of seeds subjected to warm- (94 weeks) and cold- (96 weeks)
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started stratification were 0.35 ± 0.01 mm and 0.36 ± 0.01 mm, respectively. In both
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warm- and cold-started stratification treatments, embryo length had increased
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significantly (P < 0.05) after 50 and 40 weeks, respectively.
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3.4. Effect of cold stratification and germination stimulants on germination
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Neither fresh nor cold-stratified (5 ºC for 6 months) seeds germinated at 5 ºC in any of
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the stimulant solutions. At 30/20 ºC, no cold-stratified seeds germinated in the GA3 or
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GR24 solutions, but they germinated to 11.7 ± 3.4 % in the fluridone/GA3 solution,
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which was significantly higher than that of fresh seeds (P < 0.05; Fig. 3A). 14
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0.01 mm in length. During incubation at 30/20 ºC for another 6 months, embryos of
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cold-stratified seeds continued to grow, to 0.44 mm in length, before protrusion of the
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germ tube from the seed occurred. Length of embryos in fresh seeds (CK) germinated
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at 30/20 ºC in fluridone/GA3 solution grew from 0.34 ± 0.01 to 0.44 ± 0.01 mm within
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6 months (Fig. 3B).
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3.5. Embryo growth and seed dormancy break in the field
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During sand burial, temperatures at 0, 2 and 5 cm were -12.4 to 47.6 ºC, -7.0 to 34.6
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ºC and -5.2 to 32.0 ºC, respectively (Fig. 4A). No obvious embryo growth occurred in
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seeds buried at depths of 0, 2 or 5 cm from September 2014 to September 2015 (Fig.
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4B). Of the 6 retrieval times, no exhumed seeds germinated in distilled water, but
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germination of exhumed seeds in fluridone solution occurred in November, January
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and March for seeds from all three burial depths. In general, seeds exposed at the soil
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surface germinated better than those buried at 2 cm and 5 cm. The peak of
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germination (54.4 ± 6.4 %) occurred in March of 2015, for seed retrieved from the
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soil surface. For seeds burial in soil at 2 and 5 cm, the peak of germination (44.5 ±
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3.5 % and 19.9 ± 3.9 %, respectively) was in January 2015. Germinability of seeds
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buried at all three depths decreased to 0% by May, and it remained at 0% through
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September, when the study ended (Fig. 4C). The non-germinated seeds were intact
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and firm and thus likely viable.
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Seeds of C. deserticola are water-permeable and have an undifferentiated embryo that
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must grow to a length of 0.44 mm inside the seed prior to germination (emergence of
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germ tube), showing that they have a morphological (or growth requirement)
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component of dormancy. Further, they also have a physiological component of
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dormancy. Thus, up to 54% of the seeds kept on the sand surface (0 cm) or buried in
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the natural habitat at depths of 2 and 5 cm for 2 to 5 months germinated when treated
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with 10-5 M fluridone, whereas no seeds in the water controls did so. Fluridone, GA3
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and GR24 failed to induce germination of fresh seeds. Taken together, these results
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show
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morphophysiological dormancy as described by Baskin and Baskin (2014).
that
fresh
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C.
deserticola
have
a
specialized
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of
According to our study, the following three steps are needed for seed germination
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in nature. (1) Following dispersal in late summer, seeds need to be exposed to a period
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of low temperatures during winter to release physiological dormancy. (2) Then, the
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embryo needs to grow to a critical length (0.44 mm) before the seeds can germinate.
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(3) Finally, the seeds need to be stimulated to germinate by chemicals (strigolactones?)
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from host roots. We propose the following morphological stages in germination of C.
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deserticola: Seed (stage 0) → growth (c. 0.1 mm) of embryo inside seed via cell
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elongation (Stage 1) → further elongation of embryo and splitting of seed coat (Stage
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2) → protrusion of germ tube from seed and seedling formation (Stage 3). Based on
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our observations in the field, after they germinate the dust-like seeds of C. deserticola
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grow into flowering plants about 0.5 m in height, being parasitic on host roots of H.
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ammodendron. This is consistent with the description of C. deserticola reported by Xu
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et al. (2009). Re-entrance of viable, buried seeds into dormancy (i.e. secondary dormancy) by
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May, after being capable of germinating to >50 % in March, probably indicates that
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they have the capacity to cycle between dormancy and nondormancy (i.e. an annual
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dormancy cycle), which has been demonstrated in buried seeds of Orobanche crenata
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(van Hewijk et al., 1994; Lopez-Granados and Garcia-Torres, 1999). Seeds of the cold
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desert autotrophic herbaceous halophyte Suaeda corniculata subsp. mongolica (Cao et
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al., 2012) and of the autotrophic cold desert shrub Kalidium gracile (Cao et al., 2014)
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also exhibit annual cycling between dormancy and nondormancy when buried in soil
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under natural or near-natural environmental conditions. Baskin and Baskin (2014)
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regard dormancy cycling as an adaptation that regulates the timing of germination so
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that it occurs at the time of the year when seedlings can become established. We
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suggest that the seeds of C. deserticola cycle out of and into dormancy until they
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either lose viability or come in contact with the roots of their host H. ammodendron
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during the nondormant phase of the dormancy cycle.
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Fen et al. (2012) stated that incubating C. deserticola seeds at 25 ºC for 60 days
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was a suitable condition for germination. However, we found that fresh seeds C.
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deserticola treated with fluridone did not germinate within 60 days at any temperature
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regime tested. Like seeds of other holoparasitic Orobanchaceae species (Xie et al.
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2010; Fernández-Aparicio et al. 2011), those of C. deserticola will not germinate in
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nature without a chemical signal from a host plant. Thus, various compounds have 17
ACCEPTED MANUSCRIPT been tested for their effects of germination of C. deserticola seeds. For example, Chen
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et al. (2012) reported that norflurazon treatment induced 65 % of the seeds to
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germinate. Fluridone is another compound that has received considerable research
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attention. As a carotenoid biosynthesis inhibitor (Yoshika et al., 1998; Saito et al.,
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2006), fluridone is effective in releasing seed dormancy and promoting germination
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by decreasing the concentration of ABA in many species, such as the autotrophic
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herbaceous species Nicotiana plumbaginifolia and Arabidopsis thaliana (Grappin et
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al., 2000; Ali-Rachedi et al., 2004) and presumably also in C. deserticola.
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Furthermore, Chen et al. (2009) showed that the critical period for induction of
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germination by GA3 was 4 months of cold stratification. Zhang et al. (2009) reported
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that approximately 4.7 % of C. deserticola seeds germinated with GA3 treatment.
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However, our unpublished data showed that seeds that had been cold stratified for 4
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months did not respond to exogenous GA3. Sensitivity to exogenous GA3 for seeds
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might be limited to a specific physiological status of the seeds, which could explain
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the inconsistent results between our study and those other researchers. Our study
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indicates that an effective way to promote germination of C. deserticola is to place
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fresh seeds on the sand surface in their natural habitat during winter, and then treat
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them with fluridone in spring. When we took overwintered seeds to the laboratory in
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March and incubated them in darkness at 30/20 ºC on filter paper moistened with a
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10-5 M solution of fluridone 55 % of them germinated. Some seeds cold stratified in
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the laboratory gained the ability to germinate in fluridone and fluridone/GA3 solution
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but not in GA3 solution at 30/20 ºC.
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seeds by GA3, Chen et al. (2009) reported that cold stratification at 5 °C for 4-5
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months increased the sensitivity of seeds to exogenous GA3 and that >70 % of the
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seeds germinated. However, Niu et al. (2006) stated that soaking seeds in water at
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24-25 °C for 30 days rather than cold-stratification at 4 °C for 60 days increased their
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sensitivity to exogenous GA3, and according to Zhang et al. (2009) approximately
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4.7 % of C. deserticola seeds germinated with a GA3 treatment only. We suggest that
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sensitivity of C. deserticola seeds to exogenous GA3 may be determined by the
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physiological status of the seeds, which could explain the inconsistent results between
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our study and those other researchers.
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The synthetic strigolactone analogue GR24 has been used to promote germination
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of Orobanche seeds (Baskin and Baskin, 2014). However, it did not promote
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germination of fresh or cold-stratified seeds of C. deserticola in our study. Thus, our
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results do not agree with those of Chen et al. (2012), who reported that 10-6 M GR24
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induced 46 % of the seeds of C. deserticola to germinate at 25 °C. It should be noted
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that a long period of incubation may decrease the sensitivity of seeds to GR24. For
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example, O. ramosa seeds lost their sensitivity to GR24 after incubation for more than
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60 days at 5 and 30 ºC (Gibot-Leclerc et al., 2004). Furthermore, Fen et al. (2012)
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stated that the effect of temperature on seed germination differed in various GR24
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concentrations.
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In conclusion, mature seeds of C. deserticola contain an undifferentiated and
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physiologically dormant embryo that never differentiates into organs such as 19
ACCEPTED MANUSCRIPT cotyledons, hypocotyl and radicle, i.e. seeds have a specialized kind of MPD. PD was
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broken by cold stratification, after which seeds could be induced to germinate by
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fluridone at 30/20 °C. This is the first documentation that seeds of Cistanche
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deserticola have a specialized kind of MPD and that they most likely undergo an
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annual dormancy cycle with regard to PD, being able to respond to a germination
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stimulant only after PD is broken. Our results on the dormancy breaking and
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germination requirements of C. deserticola seeds increase our understanding of the
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dynamics of the establishment phase of the life cycle of this holoparasitic flowering
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plant that will be useful in propagating it for medicinal purposes.
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ACKNOWLEDGEMENTS
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This research was supported by the Key Basic Research and Development Plan of P.
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R. China (2016YFC050080502) and the National Natural Science Foundation of P. R.
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China (31370705, 31570416).
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release and maintenance of mature seeds: studies with the Cape Verde Islands
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Cao, D.C., Baskin, C.C., Baskin, J.M., Yang, F., Huang, Z.Y., 2012. Comparison of
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Cao, D.C., Baskin, C.C., Baskin, J.M., Yang, F., Huang, Z.Y., 2014. Dormancy
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Garcia-Torres, L., Saxena, M.C., Verkleij, J.A.C., Pieterse, A.H., 1994. Seasonal
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changes in germination response of buried seeds of Orobanche crenata Forsk.
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Xu, R., Chen, J., Chen, S.L., Liu, T.N., Zhu, W.C., Xu, J., 2009. Cistanche deserticola
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Yoshioka, T., Endo, T., Satoh, S., 1998. Restoration of seed germination at
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supraoptimal temperatures by fluridone, an inhibitor of abscisic acid biosynthesis.
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Plant Cell Physiol. 39, 307-312.
Zhang, R.M., Bai, J., Lü, C.L., Chen, H.W., Gao, Y., 2008. Fluctuating temperature
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stratification induced seed germination of Cistanche deserticola. Sci. Silva. Sin.
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44, 170-173. (in Chinese with English abstract)
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Zhang, R.M., Chen, H.W., Zhang, D., Bai, J., Gao, Y., 2009. Chemical induction on
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the seed germination and haustoriam formation of Cistanche deserticola. Sci.
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Silva. Sin. 45, 39-44. (in Chinese with English abstract)
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Figure 1
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Figure 1. Plant and seed of C. deserticola. A, Two flowering plants parasitic on the roots of its host Haloxylon ammodendron. B, Whole seed. C, Longitudinal section of a freshly matured seed. D, Germinated seed.
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Figure 2. Embryo growth (mean length ± SE) of C. deserticola seeds subjected to two sequences of simulated seasonal temperature regimes. (A) Warm (25/15 ºC)-cold (5 ºC)-warm- stratification in darkness for 94 weeks. (B) Cold-warm-coldstratification in darkness for 96 weeks. Different letters indicate significant differences (P < 0.05).
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Figure 3. (A) Effects of distilled water, fluridone (Flu), GA3, Flu/GA3 and GR24 on germination (mean ± SE) of fresh seeds (CK) and of seeds cold stratified (5 ºC) for 6 months. Different lowercase letters indicate significant differences (P < 0.05) between treatments for fresh seeds and seeds stratified at 5 ºC and different uppercase letters significant differences between fresh and seeds stratified at 5 ºC in the same solution. (B) Embryo growth (mean length ± SE) in seeds during cold stratification and incubation at 30/20 ºC.
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ACCEPTED MANUSCRIPT Figure 4. (A) Mean monthly maximum and minimum temperatures on sand surface (0 cm) and at depths of 2 and 5 cm. Tmax, mean monthly maximum soil temperatures; Tmin, mean monthly minimum soil temperatures. (B) Embryo length (mean ± SE) from September 2014 to September 2015 in C. deserticola seeds on the sand surface and buried at the two sand depths. (C) Germination percentage (mean ± SE) of seeds retrieved from the field at bimonthly intervals from September 2014 to September 2015 and incubated in a 10-5 M fluridone solution in darkness at 30/20 ºC. No seeds in the water controls germinated.
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ACCEPTED MANUSCRIPT Contributions J.W., J.M.B., C.C.B., X.J.Y. and Z.Y.H. conceived and designed the experiments. J.W. and X.J.Y. conducted the experiments. J.W., X.J.Y., G.F.L. and Z.Y.H. performed data
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analyses. J.W., J.M.B., C.C.B., X.J.Y. and Z.Y.H. wrote the manuscript.
S0981-9428(17)30130-4
DOI:
10.1016/j.plaphy.2017.04.010
Reference:
PLAPHY 4856
To appear in:
Plant Physiology and Biochemistry
Received Date: 13 February 2017 Revised Date:
7 April 2017
Accepted Date: 7 April 2017
Please cite this article as: J. Wang, J.M. Baskin, C.C. Baskin, G. Liu, X. Yang, Z. Huang, Seed dormancy and germination of the medicinal holoparasitic plant Cistanche deserticola from the cold desert of northwest China, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.04.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Seed dormancy and germination of the medicinal holoparasitic plant
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Cistanche deserticola from the cold desert of Northwest China
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Jia Wang1,4, Jerry M. Baskin2, Carol C. Baskin2,3, Guofang Liu1, Xuejun Yang1*,
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Zhenying Huang1
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*
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1
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Chinese Academy of Sciences, Beijing 100093, P.R. China
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2
Department of Biology, University of Kentucky, Lexington, KY 40506, USA
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Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY
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40546, USA
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University of Chinese Academy of Sciences, Beijing 100039, P.R. China
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State Key Laboratory of Vegetation and Environmental Change, Institute of Botany,
Corresponding author
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Fax: +86-10-62836276
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E-mail address: [email protected]; [email protected]
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Cistanche deserticola is a holoparasitic plant with high medicinal value that
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reproduces only by seeds. However, the requirements for seed dormancy break and
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germination of this species remain unclear. The freshly matured dust-like seeds
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consist of a water-permeable seed coat and an undifferentiated oval-shaped embryo
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embedded in endosperm. No fresh seeds germinated in water or a 10-5 M fluridone
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solution at any incubation temperature within 60 days. Length of embryos in seeds
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incubated in warm- and cold-started stratification sequences had increased 10.4 and
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11.7 % after 50 and 40 weeks, respectively. After 6 months, length of embryos in
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seeds stratified at 5 ºC had increased by 12 %. Germination of fresh seeds and of
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seeds stratified at 5 ºC for 6 months and then incubated in mixed fluridone/gibberellic
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acid 3 (GA3) solutions at 30/20 ºC germinated to only 2.6 and 11.7 %, respectively.
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Embryos of fresh seeds and of cold-stratified seeds had increased 29.4 and 15.8 % in
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length, respectively, at the time of germination, but they never differentiated into
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organs. The highest germination (54.4 %) was for seeds incubated in a 10-5 M solution
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of fluridone in darkness in spring that had overwinter on the soil surface in the natural
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habitat. Our study indicates that breaking of physiological dormancy (PD) occurs first
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and then the embryo grows to a critical length (0.44 mm) without differentiation into
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organs prior to seed germination. Seeds for which PD had been broken were induced
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to germinate by fluridone and GA3 at high temperature. Taken together, these results
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suggest that C. deserticola seeds have a specialized kind of morphophysiological
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dormancy. This study reveals possible ways to release seed dormancy that will be
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useful in propagating this medicinal species.
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Keywords: Cistanche deserticola; holoparasite; seed germination; specialized
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morphophysiological dormancy; undifferentiated embryo
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1. Freshly matured seeds are dust-like, with an undifferentiated oval-shaped embryo
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embedded in endosperm.
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2. C. deserticola seeds have a specialized morphophysiological dormancy (MPD).
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3. The physiological (PD) component of dormancy is broken before the embryo
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grows.
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4. Embryos grew to a critical length prior to germination but did not differentiate into
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organs.
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5. After PD is broken, the seeds could be induced to germinate by fluridone and by a
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combination of fluridone and GA3 at 30/20 ºC.
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Seed germination is a critical stage in the life history of plants and especially for those
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that grow in deserts (Gutterman, 1993). Seeds may be nondormant at maturity and
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thus germinate soon after dispersal if environmental conditions are favorable for them
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to do so. However, favorable conditions may not persist long enough for the resulting
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plant to become established. Seed dormancy prevents seeds from germinating in such
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conditions, thus reducing the chances of seedling mortality and thereby contributing
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to the success of population regeneration (Baskin and Baskin, 2014).
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Based on the seed dormancy classification system developed by the Russian seed
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physiologist Mariana G. Nikolaeva between 1967 and 2001 (Baskin and Baskin, 2008)
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and expanded and modified by Baskin and Baskin (2014), there are five classes of
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dormancy. Seeds with physiological dormancy (PD) are water-permeable and have a
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fully developed embryo with a physiological inhibiting mechanism that prevents
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radicle emergence, seeds with morphological dormancy (MD) an underdeveloped
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(small) embryo that needs to grow before the seed germinates, seeds with
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morphophysiological dormancy (MPD) an underdeveloped embryo that is
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physiologically dormant, seeds with physical dormancy (PY) a fully developed
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nondormant embryo but germination is prevented due to water-impermeability of the
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seed (or fruit) coat and seeds with combinational dormancy a fully developed embryo
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that is physiologically dormant and the seed (or fruit) coat is water impermeable
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(PY+PD). In addition to these kinds of dormancy, Baskin and Baskin (2014)
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recognized specialized kinds of morphological and morphophysiological dormancy
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maturity that never differentiate into a root-shoot axis (Baskin and Baskin, 2014).
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These specialized kinds of dormancy are the ones that occur in “dust seeds” of
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holoparasites such as Orobanchaceae species and in mycoheterotrophs such as orchids
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(Baskin and Baskin, 2014).
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Cistanche deserticola Ma (Orobanchaceae) is a perennial holoparasite herb that
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parasitizes the roots of the cold desert shrub Haloxylon ammodendron (C. A. Mey.)
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Bunge (Amaranthaceae, subfamily Chenopodioideae). It occurs in the temperate arid
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cold deserts of northwest China and the Republic of Mongolia. Plants bloom in May
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to June, and fruits mature from June to August. C. deserticola has been used as a
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traditional medicinal plant in China for hundreds of years. However, the species has
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become endangered due to over-collection and the difficulty of regenerating it from
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seeds, which is the only way it reproduces. That is, C. deserticola does not reproduce
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vegetatively (Fu, 1992; Xu et al., 2009).
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In general, results of studies on the seed germination of C. deserticola are
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inconsistent. In a study by Qiao et al. (2007) fluridone induced 53 % of the seeds to
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germinate, but Fen (2012) obtained only 2.9 % germination of seeds treated with this
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ABA biosynthesis inhibitor. Zhang et al. (2008) reported that seeds treated with two
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cycles of 50 ºC for 1 hour and 4 ºC for 1 week germinated to 6.7 %. Chen et al. (2009)
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found that the germination percentage of seeds cold-stratified at 5 ºC for 120 days
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was >70 % in GA3 solution but that GA3 had no effect on fresh (non-cold stratified)
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seeds. However, Zhang et al. (2009) reported that GA3 induced germination of fresh
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seeds. The sensitivity of seeds to exogenous GA3 might vary with the physiological
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status of the seeds, which could explain the inconsistent results obtained by different
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authors. Although several studies have been conducted on seed germination of C.
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deserticola, we still do not have an efficient way to produce plants from seeds, i.e. the
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number of plants produced per number of seeds sown in very low (Sun et al., 2008).
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In the field, farmers dig holes adjacent to the host plant and put approximately 200
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seeds in each hole. However, this practice of propagating plants leads to very low
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numbers of plants due to dormancy of the seeds. According to information provided
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by local farmers in our study site, less than three seeds can develop into plants in each
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hole. Xu et al. (2009) showed that fresh stem yield of C. deserticola was about 0.2-2.2
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t ha-1. However, this high crop yield was based on a huge number of seeds, most of
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which were wasted. Thus, a more efficient method is needed for propagating plants of
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this species from seeds.
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Thus, we suggest that learning more about the morphology and physiology of
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germination of the seeds under natural and simulated natural conditions may help in
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this endeavor. Since mature seeds of C. deserticola contain a globular embryo that is
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undifferentiated (Li et al., 1989; Ma et al. 1997) and fresh seeds are dormant (see
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references cited above), we assumed that seeds have a specialized kind of
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morphophysiological dormancy (sensu Baskin and Baskin, 2014). Thus, we
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hypothesized that (1) the small embryo needs to grow or differentiate prior to
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germination, (2) physiological dormancy needs to be released before the germ-tube
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needs a chemical stimulant (which in nature would be produced by the host) to
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germinate. To test these hypotheses, we asked following questions. (1) Do fresh seeds
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of C. deserticola have a specialized kind of MD or MPD? (2) How do embryos in
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intact seeds respond to different temperatures? (3) Do embryos need to grow before
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the seeds germinate? (4) Does seed dormancy/germination respond to seasonal
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changes of soil temperatures in the natural habitat?
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2. Materials and methods
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2.1. Seed collection and measurement of plant height
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Infructescences of C. deserticola with freshly matured seeds were harvested from
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plants in the Tengger Desert, Inner Mongolia, China, (38°79′N, 105°54′E, 1379 m
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a.s.l.) on 10 August 2013 and 30 August 2014. This area has a typical continental arid
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climate with a mean annual precipitation of 182 mm, about 70 % of which occurs
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from July to September. Mean annual temperature is 8.5 °C, and mean temperature of
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the coldest (January) and hottest (July) months are -15.7° and 30 °C, respectively
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(data from WorldClim database, http://www.worldclim.org).
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Seeds were extracted from the fruits (capsules) and separated from other plant
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material using a soil sieve (mesh size 0.6 mm). The seeds were air-dried for 3 days at
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ambient laboratory conditions (20-25 ºC, 17–32 % relative humidity) before initiation
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of experiments.
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Ten adult plants of C. deserticola were haphazardly selected in the natural habitat, 8
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and height of each plant from the sand surface to the top of inflorescence was
146
measured.
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2.2. Seed morphology and water imbibition
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Embryos were dissected from seeds using forceps and a dissecting needle and
150
measured using a Nikon SMZ1000 dissecting microscope (Tokyo, Japan) equipped
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with a calibrated micrometer. Length of 25 embryos and length and width of 25 fresh
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seed were measured. The 1000-seed mass was determined by weighing five replicates
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of 1000 seeds.
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Rate of water uptake (imbibition) was monitored in freshly mature seeds. Initial
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seed mass was determined by weighting four replicates of 1000 air-dried seeds. Four
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replications of 1000 seeds were placed on filter paper in a 5.5 cm Petri dishes
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moistened with 3 ml distilled water and incubated at room temperature (20-25 °C).
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Seed weight was monitored at 1-minute intervals for 10 minutes. Percentage of
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increase in seed mass based on initial seed mass was calculated.
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Seed structure was studied by fixing fresh seeds in FAA solution (70% ethanol:
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acetic acid: formaldehyde, 18:1:1, by vol.) and then dehydrating them in a graded
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ethanol series. Then, the seeds were embedded in paraffin and cut into 8 µm sections
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with a rotary microtome (Leica RM2235, Germany). Sections were stained either with
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safranin or fast green and photographed under a light microscope (Nikon 80i, Japan).
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Germinated seeds were observed and photographed under a Nikon SMZ1500
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dissecting microscope (Tokyo, Japan).
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2.3. Initial germination tests
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Freshly mature seeds were placed in 5.5 cm Petri dishes on two layers of Whatman
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No. 1 filter paper moistened with 3 ml of 10-5 M fluridone (Sigma, USA) (Qiao et al.,
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2007; Chen et al., 2012) and incubated in both light (12 h photoperiod, 100 µmol m-2
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s-1, cool white fluorescent light) and continuous darkness (seeds placed in a light-tight
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bag) at 15/5, 20/10, 25/15 and 30/20 ºC (12/12 h) for 12 weeks. Four replicates of 100
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seeds were used for each treatment. The high temperature coincided with the 12 h
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light period and the low temperature with the 12 h dark period. The alternating
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temperature regimes represent approximations of mean daily maximum and minimum
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air temperatures for each month during the growing season in the natural habitat:
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April and October, 15/5 ºC; May and September, 20/10 ºC; June and August 25/15 ºC;
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and July, 30/20 ºC. Germination of seeds incubated in light was monitored every 3
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days, and those incubated in darkness were checked every 6 days under a green safe
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light (Emitting diode power supply, YP-TH0301, YIPU, China) in a dark room. Final
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germination percentages were determined after 3 months. Emergence of the germ tube
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through the seed coat was the criterion for germination.
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2.4. Effect of warm- and cold-started stratification on embryo growth
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Our purpose here was to monitor embryo growth in seeds in a warm-started and in a
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cold-started temperature sequence that simulated seasonal temperature regimes in the
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field. The sequence began with either warm (25/15 ºC) or cold (5 ºC) stratification. 10
ACCEPTED MANUSCRIPT The warm-started stratification procedure consisted of
10 weeks of warm → 3
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cycles of (12 weeks of cold → 16 weeks of warm) and the cold -started stratification
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procedure of 3 cycles of (12 weeks of cold → 16 weeks of warm) → 12 weeks of
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cold.
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Each warm stratification treatment in a sequence represented high temperature
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months (average temperature above 15 ºC) and each cold stratification the low
195
temperature months (average temperature between 0 and 10 ºC). Seeds of C.
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deserticola are dispersed from late July to late August, and they are exposed to warm
197
stratification until October (ca. 10 weeks). Then, they are exposed to cold
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stratification (about 12 weeks) in October, November and March (soil frozen in
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December to February), to warm stratification (about 16 weeks) again from June to
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September, etc. Seeds in both warm-started and cold-started temperature sequences
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were kept moist (seeds checked every two days and distilled water added if necessary)
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during treatment. Embryo length was determined for 25 haphazardly-chosen seeds at
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time zero and at the end of each warm and cold treatment in the two sequences.
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2.5. Effect of cold stratification and germination stimulants on germination
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The purpose of this experiment was to test the effect of cold stratification alone and in
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combination with the germination stimulants fluridone, an inhibitor of ABA
208
biosynthesis, GA3, a plant hormone and GR24 (Sigma, USA), a synthetic
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strigolactone, on promoting germination of C. deserticola seeds. Intact seeds that had
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been cold-stratified at 5 ºC in darkness for 6 months and freshly matured seeds 11
ACCEPTED MANUSCRIPT (control) were used in the experiment. Four replicates of 100 seeds for each treatment
212
were soaked in distilled water (control) or in solutions of fluridone (10-5 M), GA3
213
(10-5 M), fluridone (10-5 M)/GA3 (10-5 M) or GR24 (10-6 M) and incubated in
214
darkness at 30/20 and at 5 ºC for 6 months. In our initial study (unpublished data), no
215
fresh seeds germinated either in 10-3, 10-5 or 10-7 M GA3 or in 10-6, 10-8 or 10-10 M
216
GR24. Furthermore, treatment of seeds with 10-4, 10-5 and 10-6 M fluridone resulted in
217
no seed germination. Germination was monitored weekly using the green safe light
218
described above. Length of embryo in each of 20 seeds in each treatment was
219
measured monthly during the cold stratification period. For germinated seeds, a small
220
protuberance (germ tube) emerged through the seed coat. Length of embryos that had
221
grown was measured when the seed coat had split, but the germ tube had not emerged
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from the seed.
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2.6. Embryo growth and release of physiological dormancy in the field
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Two thousands freshly matured seeds were placed in each of 18 (12 × 7 cm) nylon
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mesh bags. Six bags of seeds each were buried at depths of 0 (surface), 2 and 5 cm in
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sand in the natural habitat in which seeds were collected. Sand temperatures at all
228
burial depths were recorded at 2-hour intervals by an iButton DS1992L temperature
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data logger (Maxim Integrated Device, California, USA). Three bags were exhumed
230
at 2-month intervals from September 2014 to September 2015. For seeds from each
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burial period and depth, embryo lengths of 25 seeds were measured prior to the
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germination test. Germination was tested by incubating seeds in distilled water and in
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a 10-5 M fluridone solution in darkness at 30/20 ºC for 12 weeks. Germination was
234
monitored every 3 days using the green safe light. Emergence of the germ tube was
235
the criterion for germination.
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2.7. Statistical analysis
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Means and standard errors were calculated for germination percentages and embryo
239
lengths. One-way ANOVA was used to analyze the results of warm- and of
240
cold-started stratification treatments on embryo growth and of the effects of cold
241
stratification and chemical stimulants on seed germination. Fisher’s LSD test was
242
used to test for significant differences in multiple comparisons. Data were arcsine
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square root transformed when necessary to meet assumptions of ANOVA for
244
normality and homogeneity of variance. Statistical analyses were performed using
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SPSS Version 21.0 for Windows 8.0 (SPSS, Chicago, IL, USA).
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3. Results
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3.1. Plant height, and seed morphology and water imbibition
249
Average height of C. deserticola plants was 0.48 ± 0.02 m, and they were parasitic on
250
the roots of H. ammodendron (Fig. 1A). Mean seed length and width were 1.21 ± 0.02
251
mm and 0.84 ± 0.02 mm, respectively (Fig. 1B). Mass of 1000 seeds was 122.18 ±
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1.27 mg. After 4 minutes, seeds were fully imbibed, and seed mass had increased to
253
163.2 ± 5.1 % of the original mass.
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Fresh seeds had a dark alveolate seed coat, a perisperm (nucellar material) and a 13
ACCEPTED MANUSCRIPT small undifferentiated (organless) embryo surrounded by endosperm. An air cavity
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was located between the perisperm and the seed coat (Fig. 1C). During germination,
257
neither radicle nor cotyledons was formed. The radicular pole of the embryo grew and
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developed into a radicle-like germ tube (Fig. 1D).
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3.2 Initial germination test
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None of the freshly matured seeds had germinated after 3 months in the 10-5 M
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fluridone solution in either light or darkness at any of the temperature regimes tested.
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3.3. Effect of warm- and cold-started stratification on embryo growth
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Embryos grew very slowly in both warm- and cold-started stratification treatments
266
(Fig. 2A, 2B). Mean embryo length of freshly matured seeds was 0.29 ± 0.01 mm.
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The lengths of embryos of seeds subjected to warm- (94 weeks) and cold- (96 weeks)
268
started stratification were 0.35 ± 0.01 mm and 0.36 ± 0.01 mm, respectively. In both
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warm- and cold-started stratification treatments, embryo length had increased
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significantly (P < 0.05) after 50 and 40 weeks, respectively.
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3.4. Effect of cold stratification and germination stimulants on germination
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Neither fresh nor cold-stratified (5 ºC for 6 months) seeds germinated at 5 ºC in any of
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the stimulant solutions. At 30/20 ºC, no cold-stratified seeds germinated in the GA3 or
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GR24 solutions, but they germinated to 11.7 ± 3.4 % in the fluridone/GA3 solution,
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which was significantly higher than that of fresh seeds (P < 0.05; Fig. 3A). 14
ACCEPTED MANUSCRIPT Embryos of seeds stratified at 5 ºC for 6 months grew from 0.34 ± 0.01 to 0.38 ±
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0.01 mm in length. During incubation at 30/20 ºC for another 6 months, embryos of
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cold-stratified seeds continued to grow, to 0.44 mm in length, before protrusion of the
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germ tube from the seed occurred. Length of embryos in fresh seeds (CK) germinated
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at 30/20 ºC in fluridone/GA3 solution grew from 0.34 ± 0.01 to 0.44 ± 0.01 mm within
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6 months (Fig. 3B).
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3.5. Embryo growth and seed dormancy break in the field
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During sand burial, temperatures at 0, 2 and 5 cm were -12.4 to 47.6 ºC, -7.0 to 34.6
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ºC and -5.2 to 32.0 ºC, respectively (Fig. 4A). No obvious embryo growth occurred in
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seeds buried at depths of 0, 2 or 5 cm from September 2014 to September 2015 (Fig.
288
4B). Of the 6 retrieval times, no exhumed seeds germinated in distilled water, but
289
germination of exhumed seeds in fluridone solution occurred in November, January
290
and March for seeds from all three burial depths. In general, seeds exposed at the soil
291
surface germinated better than those buried at 2 cm and 5 cm. The peak of
292
germination (54.4 ± 6.4 %) occurred in March of 2015, for seed retrieved from the
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soil surface. For seeds burial in soil at 2 and 5 cm, the peak of germination (44.5 ±
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3.5 % and 19.9 ± 3.9 %, respectively) was in January 2015. Germinability of seeds
295
buried at all three depths decreased to 0% by May, and it remained at 0% through
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September, when the study ended (Fig. 4C). The non-germinated seeds were intact
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and firm and thus likely viable.
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Seeds of C. deserticola are water-permeable and have an undifferentiated embryo that
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must grow to a length of 0.44 mm inside the seed prior to germination (emergence of
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germ tube), showing that they have a morphological (or growth requirement)
303
component of dormancy. Further, they also have a physiological component of
304
dormancy. Thus, up to 54% of the seeds kept on the sand surface (0 cm) or buried in
305
the natural habitat at depths of 2 and 5 cm for 2 to 5 months germinated when treated
306
with 10-5 M fluridone, whereas no seeds in the water controls did so. Fluridone, GA3
307
and GR24 failed to induce germination of fresh seeds. Taken together, these results
308
show
309
morphophysiological dormancy as described by Baskin and Baskin (2014).
that
fresh
seeds
of
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C.
deserticola
have
a
specialized
kind
of
According to our study, the following three steps are needed for seed germination
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in nature. (1) Following dispersal in late summer, seeds need to be exposed to a period
312
of low temperatures during winter to release physiological dormancy. (2) Then, the
313
embryo needs to grow to a critical length (0.44 mm) before the seeds can germinate.
314
(3) Finally, the seeds need to be stimulated to germinate by chemicals (strigolactones?)
315
from host roots. We propose the following morphological stages in germination of C.
316
deserticola: Seed (stage 0) → growth (c. 0.1 mm) of embryo inside seed via cell
317
elongation (Stage 1) → further elongation of embryo and splitting of seed coat (Stage
318
2) → protrusion of germ tube from seed and seedling formation (Stage 3). Based on
319
our observations in the field, after they germinate the dust-like seeds of C. deserticola
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grow into flowering plants about 0.5 m in height, being parasitic on host roots of H.
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ammodendron. This is consistent with the description of C. deserticola reported by Xu
322
et al. (2009). Re-entrance of viable, buried seeds into dormancy (i.e. secondary dormancy) by
324
May, after being capable of germinating to >50 % in March, probably indicates that
325
they have the capacity to cycle between dormancy and nondormancy (i.e. an annual
326
dormancy cycle), which has been demonstrated in buried seeds of Orobanche crenata
327
(van Hewijk et al., 1994; Lopez-Granados and Garcia-Torres, 1999). Seeds of the cold
328
desert autotrophic herbaceous halophyte Suaeda corniculata subsp. mongolica (Cao et
329
al., 2012) and of the autotrophic cold desert shrub Kalidium gracile (Cao et al., 2014)
330
also exhibit annual cycling between dormancy and nondormancy when buried in soil
331
under natural or near-natural environmental conditions. Baskin and Baskin (2014)
332
regard dormancy cycling as an adaptation that regulates the timing of germination so
333
that it occurs at the time of the year when seedlings can become established. We
334
suggest that the seeds of C. deserticola cycle out of and into dormancy until they
335
either lose viability or come in contact with the roots of their host H. ammodendron
336
during the nondormant phase of the dormancy cycle.
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Fen et al. (2012) stated that incubating C. deserticola seeds at 25 ºC for 60 days
338
was a suitable condition for germination. However, we found that fresh seeds C.
339
deserticola treated with fluridone did not germinate within 60 days at any temperature
340
regime tested. Like seeds of other holoparasitic Orobanchaceae species (Xie et al.
341
2010; Fernández-Aparicio et al. 2011), those of C. deserticola will not germinate in
342
nature without a chemical signal from a host plant. Thus, various compounds have 17
ACCEPTED MANUSCRIPT been tested for their effects of germination of C. deserticola seeds. For example, Chen
344
et al. (2012) reported that norflurazon treatment induced 65 % of the seeds to
345
germinate. Fluridone is another compound that has received considerable research
346
attention. As a carotenoid biosynthesis inhibitor (Yoshika et al., 1998; Saito et al.,
347
2006), fluridone is effective in releasing seed dormancy and promoting germination
348
by decreasing the concentration of ABA in many species, such as the autotrophic
349
herbaceous species Nicotiana plumbaginifolia and Arabidopsis thaliana (Grappin et
350
al., 2000; Ali-Rachedi et al., 2004) and presumably also in C. deserticola.
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Furthermore, Chen et al. (2009) showed that the critical period for induction of
352
germination by GA3 was 4 months of cold stratification. Zhang et al. (2009) reported
353
that approximately 4.7 % of C. deserticola seeds germinated with GA3 treatment.
354
However, our unpublished data showed that seeds that had been cold stratified for 4
355
months did not respond to exogenous GA3. Sensitivity to exogenous GA3 for seeds
356
might be limited to a specific physiological status of the seeds, which could explain
357
the inconsistent results between our study and those other researchers. Our study
358
indicates that an effective way to promote germination of C. deserticola is to place
359
fresh seeds on the sand surface in their natural habitat during winter, and then treat
360
them with fluridone in spring. When we took overwintered seeds to the laboratory in
361
March and incubated them in darkness at 30/20 ºC on filter paper moistened with a
362
10-5 M solution of fluridone 55 % of them germinated. Some seeds cold stratified in
363
the laboratory gained the ability to germinate in fluridone and fluridone/GA3 solution
364
but not in GA3 solution at 30/20 ºC.
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366
seeds by GA3, Chen et al. (2009) reported that cold stratification at 5 °C for 4-5
367
months increased the sensitivity of seeds to exogenous GA3 and that >70 % of the
368
seeds germinated. However, Niu et al. (2006) stated that soaking seeds in water at
369
24-25 °C for 30 days rather than cold-stratification at 4 °C for 60 days increased their
370
sensitivity to exogenous GA3, and according to Zhang et al. (2009) approximately
371
4.7 % of C. deserticola seeds germinated with a GA3 treatment only. We suggest that
372
sensitivity of C. deserticola seeds to exogenous GA3 may be determined by the
373
physiological status of the seeds, which could explain the inconsistent results between
374
our study and those other researchers.
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The synthetic strigolactone analogue GR24 has been used to promote germination
376
of Orobanche seeds (Baskin and Baskin, 2014). However, it did not promote
377
germination of fresh or cold-stratified seeds of C. deserticola in our study. Thus, our
378
results do not agree with those of Chen et al. (2012), who reported that 10-6 M GR24
379
induced 46 % of the seeds of C. deserticola to germinate at 25 °C. It should be noted
380
that a long period of incubation may decrease the sensitivity of seeds to GR24. For
381
example, O. ramosa seeds lost their sensitivity to GR24 after incubation for more than
382
60 days at 5 and 30 ºC (Gibot-Leclerc et al., 2004). Furthermore, Fen et al. (2012)
383
stated that the effect of temperature on seed germination differed in various GR24
384
concentrations.
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In conclusion, mature seeds of C. deserticola contain an undifferentiated and
386
physiologically dormant embryo that never differentiates into organs such as 19
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388
broken by cold stratification, after which seeds could be induced to germinate by
389
fluridone at 30/20 °C. This is the first documentation that seeds of Cistanche
390
deserticola have a specialized kind of MPD and that they most likely undergo an
391
annual dormancy cycle with regard to PD, being able to respond to a germination
392
stimulant only after PD is broken. Our results on the dormancy breaking and
393
germination requirements of C. deserticola seeds increase our understanding of the
394
dynamics of the establishment phase of the life cycle of this holoparasitic flowering
395
plant that will be useful in propagating it for medicinal purposes.
396
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ACKNOWLEDGEMENTS
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This research was supported by the Key Basic Research and Development Plan of P.
399
R. China (2016YFC050080502) and the National Natural Science Foundation of P. R.
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China (31370705, 31570416).
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Yoshioka, T., Endo, T., Satoh, S., 1998. Restoration of seed germination at
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supraoptimal temperatures by fluridone, an inhibitor of abscisic acid biosynthesis.
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Plant Cell Physiol. 39, 307-312.
Zhang, R.M., Bai, J., Lü, C.L., Chen, H.W., Gao, Y., 2008. Fluctuating temperature
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stratification induced seed germination of Cistanche deserticola. Sci. Silva. Sin.
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44, 170-173. (in Chinese with English abstract)
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Zhang, R.M., Chen, H.W., Zhang, D., Bai, J., Gao, Y., 2009. Chemical induction on
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the seed germination and haustoriam formation of Cistanche deserticola. Sci.
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Figure 1
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Figure 1. Plant and seed of C. deserticola. A, Two flowering plants parasitic on the roots of its host Haloxylon ammodendron. B, Whole seed. C, Longitudinal section of a freshly matured seed. D, Germinated seed.
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Figure 2
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Figure 2. Embryo growth (mean length ± SE) of C. deserticola seeds subjected to two sequences of simulated seasonal temperature regimes. (A) Warm (25/15 ºC)-cold (5 ºC)-warm- stratification in darkness for 94 weeks. (B) Cold-warm-coldstratification in darkness for 96 weeks. Different letters indicate significant differences (P < 0.05).
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Figure 3
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Figure 3. (A) Effects of distilled water, fluridone (Flu), GA3, Flu/GA3 and GR24 on germination (mean ± SE) of fresh seeds (CK) and of seeds cold stratified (5 ºC) for 6 months. Different lowercase letters indicate significant differences (P < 0.05) between treatments for fresh seeds and seeds stratified at 5 ºC and different uppercase letters significant differences between fresh and seeds stratified at 5 ºC in the same solution. (B) Embryo growth (mean length ± SE) in seeds during cold stratification and incubation at 30/20 ºC.
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ACCEPTED MANUSCRIPT Figure 4. (A) Mean monthly maximum and minimum temperatures on sand surface (0 cm) and at depths of 2 and 5 cm. Tmax, mean monthly maximum soil temperatures; Tmin, mean monthly minimum soil temperatures. (B) Embryo length (mean ± SE) from September 2014 to September 2015 in C. deserticola seeds on the sand surface and buried at the two sand depths. (C) Germination percentage (mean ± SE) of seeds retrieved from the field at bimonthly intervals from September 2014 to September 2015 and incubated in a 10-5 M fluridone solution in darkness at 30/20 ºC. No seeds in the water controls germinated.
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ACCEPTED MANUSCRIPT Contributions J.W., J.M.B., C.C.B., X.J.Y. and Z.Y.H. conceived and designed the experiments. J.W. and X.J.Y. conducted the experiments. J.W., X.J.Y., G.F.L. and Z.Y.H. performed data
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analyses. J.W., J.M.B., C.C.B., X.J.Y. and Z.Y.H. wrote the manuscript.