Morphophysiology and cryopreservation of seeds of Dendrobium nobile Lindl. (Orchidaceae) at different stages of development

The immature seeds of Dendrobium nobile acquire resistance to freezing and long-term storage in a cryobank at the age of 6 months after pollination. Seed maturation of Dendrobium nobile Lindl to establish the optimal stage for long-term storage of immature seeds in liquid nitrogen was studied. Immature seeds can germinate in vitro starting from 3 months after pollination (MAP) but only develop up to the stage of embryo swelling or protocorm without rhizoids. The maximum staining of embryos with vital dyes FDA and TTC occurs at 4 and 5 MAP that corresponds to the release of suspensor beyond the embryo sac. Embryo staining does not correlate with germination capacity and seed viability in cryogenic storage. The immature seeds acquire resistance to drying in airflow and cryostorage at the age of 6 MAP. 11% of seeds germinated after cryogenic storage at 6 MAP, and 81–94% at 7–10 MAP.


Introduction
The ongoing interest in orchid cultivation is explained not only by the ornamental features of the flowers but also by diverse biologically active compounds, produced in their stems and leaves. Dendrobium nobile Lindl. is widely used in traditional Eastern and modern Western medicine. The stems and leaves of this plant contain valuable biologically active substances with antimutagenic and anticancerogenic properties. Dendrobin, mosquatiline, gigantol, denbinobin, nobilin, dendrophenol and many other substances isolated from D. nobile can be used to treat a variety of ageingrelated diseases (Bhattacharyya et al. 2013(Bhattacharyya et al. , 2014Cakova et al. 2017;Xu et al. 2017).
One of the measures for the protection of orchids is their cultivation ex situ (Pritchard and Seaton 1993). Seed reproduction contributes to the genetic variability of plants, which ensures the survival of species in different environmental conditions. Rapid loss of germination and mycosymbiotrophy of orchid seeds make it necessary to use special biotechnological methods for industrial use. Thus, the vital staining method (van Waes and Debergh 1986) is used to test the viability of orchid seeds, cryopreservation methods are developed for long-term storage in liquid nitrogen (LN) (Pritchard et al. 1999;Seaton and Pritchard 2003;Merritt (Alam et al. 2002;Faria et al. 2004;Kananont et al. 2010;Soares et al. 2012).
One of the ways to optimize the germination and longterm storage of orchid seeds may be the immature seeds culture (Lo et al. 2004;Vijayakumar et al. 2012;Udomdee et al. 2014). However, the requirements for seed germination in vitro and cryogenic storage are different. Immature seeds that contain a lot of water in cells are better for in vitro germination, and mature dried seeds are better for cryogenic storage.
This work aims to determine the time of morphophysiological maturity of Dendrobium nobile seeds for in vitro cultivation and long-term storage in LN.

Materials and methods
Plants of Dendrobium nobile Lindl. (Orchidaceae Juss.) ( Fig. 1A) were cultivated in the greenhouse with a moderately warm temperature regime (day 18-26 °C, night 14-18 °C) at a relative humidity of 60-75% and natural light. The flowering of different clones continued from November 2019 to March 2020. The fruits were obtained by artificial allogamous pollination. The age of the seeds and ovules was calculated in months after pollination (MAP).
Germination ability was evaluated before and after cryogenic storage of seeds. The fruits were surface sterilized with 70% ethanol. The seeds were extracted with a sterile tool. To determine in vitro germination ability, seed samples were removed from disinfected fruits on the day of collection and placed on half-strength MS (1/2 MS) hormone-free media (Murashige and Skoog 1962) without drying.
The initial humidity of seeds on harvest day was determined by the weight loss method after drying to constant weight at 95 °C. For cryogenic storage, the samples were partially dehydrated in a laminar airflow chamber at room temperature and 40-60% relative humidity for 2-6 h (Makeen et al. 2005). The dehydrated seeds in sterile cryovials (Nunc, USA) were immersed in LN for 1 month.
After cryogenic storage, immature seeds (3-6 MAP) were germinated in vitro without additional sterilization. The more mature seeds (7-12 MAP) were disinfected in 5% calcium hypochlorite for 15 min and washed with sterilized distilled water. Samples were germinated for 2 months in Petri dishes on a hormone-free 1/2 MS basic medium in the dark at 25 °C. The percentage of protocorms was calculated in three replications of 100-200 seeds in each. Bacteriological Agar (Oxoid Ltd, Bastingstoke, England) (0.7%, w/v) was used to solidify the media dispersed in Petri dishes. The pH of the media was adjusted to 5.7 and media were sterilized by autoclaving at 121 °C (0.1 MPa) for 25 min. All samples were germinated for 2 months in Petri dishes in the dark before transferring them to a 16/8 h (light/dark) photoperiod at a light intensity of 50 µmol m −2 s −1 provided by fluorescent lamps (OSRAM, L 18 W/77 Fluora, Germany) at 25 ± 2 °C.
The viability of seeds was determined by vital staining with 0.005% fluorescein diacetate (FDA, Sigma-Aldrich, USA) or 1% 2,3,5-triphenyltetrazolium chloride (TTC, SERVA, Germany). The samples were stained on a glass slide in the dark wet chamber for 2 h, washed several times with distilled water, and mounted in 50% glycerol. Samples were investigated with an FV1000D confocal microscope (Olympus, Japan) and Axioplan (Carl Zeiss, Germany). The fluorescence was excited with violet (405 nm) and blue (473 nm) lasers at 50% power. The signal was recorded in blue (425-460 nm), green (485-530 nm) and red (560-660 nm) channels. Standard errors of mean values were calculated and are reported in Table 1.

Results and discussion
D. nobile fruit is a dehiscent capsule (Fig. 1B). The time of seed ripening is 12-13 MAP.
The archesporial cell, the inner integument initials and megasporogenesis were noted at the age of 2 MAP. Maturation of the embryo sac and fertilization occurred at 2.5-3 MAP. A few-celled (2-4 cell) embryo formed at 3-3.5 MAP. The suspensor at this stage was still within the embryo sac (Fig. 1C). The developmental stages from zygote to multicellular embryo were found in fruits at 4-6 MAP.
At the fertilization stage (2-2.5 MAP), the ovule had two double integuments surrounding the embryo sac. The micropylar end turned towards the placenta (anatropic ovule). At the early embryogenesis stage (3 MAP), the inner part of the inner integument is covered with a cutinized layer. This layer was red-brown when stained with FDA (Fig. 1C). The cutinized shell was thin at the micropylar end and thicker at the chalazal end (Fig. 1D, E).
Seeds with suspensor inside and outside the embryo sac were identified on 4 MAPs (Fig. 1D). The first category included smaller multicellular embryos with vesicle suspensor. The second category included larger multicellular embryos with a branched unicellular suspensor. Living cells of the inner integument lining died at 5 MAP. Suspensor desorption and the formation of a multicellular autonomous embryo were observed at 6 MAP (Fig. 1F). The protoplasts of the inner layer of the outer integument died at 7 MAP, and the formation of a full-fledged seed coat from the outer layer of the outer integument occurred at 8 MAP. Table 1 shows the parameters of seeds before immersion in LN and after cryogenic storage. The immature seeds at the initial stages of embryogenesis poorly retained moisture.
Large water losses are associated with intensive morphostructural processes, in particular, the suspensor formation and its exit outside the embryo sac (Fig. 1D). The immature seeds stabilized the ability to retain moisture after suspensor resorption (6 MAP).
The ability of embryos to stain with vital dyes depended on seed age. At 3 MAP, TTC and FDA stained only 28-30% Fig. 1 Flowers, fruits and seeds of D. nobile. A Flowers; B mature fruits at 12 MAP; C the suspensor of the few-celled embryo does not extend the embryo sac, the cutinized inner layer of the inner integument fluoresces red-brown (3 MAP) (FDA); D the suspensor extends beyond the embryo sac, the nuclei of the proper embryo fluoresce yellow, the nucleus of the suspensor fluoresces blue, the dark cutinized shell of the inner layer of the inner integument does not fluoresce (4 MAP) (FDA); E the branched suspensor fluoresces blue after elimination of the inner integument (5 MAP) (unstained specimen); F elimination of the inner layer of the outer integument and the suspensor, nuclei of the proper embryo fluoresce yellow (6 MAP) (FDA). A, B Canon G16 (Japan); C-F FV1000D confocal microscope (Olympus, Japan). Scale bars: A = 20,000 μm; B = 10,000 μm; C = 20 µm; D-F = 100 μm. cl, cutinized layer; ep, proper embryo; es, embryo sac; ii, inner integument; mc, micropyle; n, nucleus; oi, outer integument; olii, outer layer of inner integument; oloi, outer layer of outer integument; sp, suspensor of the embryos. Maximum staining was observed at 4-5 MAP (59-77%). At this stage, the suspensor extends beyond the embryo sac (Fig. 1E). At 6 MAP, the number of TTC stained embryos decreased to 44%. The staining dynamics did not correlate with the in vitro germination. In the 3-6 MAP range, the percentage of FDA and TTC stained embryos was noticeably higher than the percentage of seeds that germinate in vitro.
During the germination of immature seeds at the stage of the first embryonic divisions (3-3.5 MAP), the embryos swelled and formed protocorms without rhizoids ( Fig. 2A). No further development occurred. Seeds at 4-5 MAP retained germination after drying (16%) but lost germination after LN storage. At the age of 5 MAP, after drying in a chamber with a laminar flow and cryogenic storage, a small number of protocorms with rhizoids were obtained (Fig. 2B, C). At 6 MAP, 30% of the seeds germinated in vitro after dehydration in airflow and only 11% of the seeds germinated after storage in LN. At the age of 6 MAP, protocorms with rhizoids and an apical bud were developed (Fig. 2D). At 7 MAP, 81-94% of seeds acquired cryostability and retained their germination after cryogenic storage. Over 80% of seeds had stable germination, both after drying and after cryogenic storage until full ripening and fruit opening at 7-12 MAP.
The earliest time that immature D. nobile seeds can be successfully stored in LN in a viable state and germinated if necessary is 6 MAP. A visual sign of seed maturation is the appearance of a yellow colour. The proportion of protocorm formation and further organogenesis is higher in seeds older than 6 MAP. The proportion of percentage embryos decreased as the seeds matured. Staining decreases after suspensor desorption at 6 MAP. The cells of the inner integument died, and their periclinal walls became denser, forming an impermeable membrane around the embryo (Fig. 1F). Thus, the multicellular embryo acquired autonomy, and the percentage of seed germination and cryostability increased.
For the immature seeds culture, the most important thing is not the time of full maturation, but the time of passing the stage of embryonic development, at which germination is possible using special methods. In D. nobile and its hybrids, the ability of seeds of different ages to germinate in vitro varies according to different authors from 3 to 4 MAP (Vasudevan and van Staden 2010;Udomdee et al. 2014) to 6 MAP (Poddubnaya-Arnoldi 1964). Arditti (1992) considers such stages of fruit formation as (i) seed formation at 180 days after pollination and (ii) fruit ripening at 310-365 days after pollination.
In our work, immature seeds (without LN storage) germinated in vitro at the age of 3 MAP. Early stages of development of the ovule were observed during this period, but mainly the formation of the embryo sac and fertilization. Our study confirmed the ability of D. nobile seeds to germinate at 3 and 4 MAP, but the development stopped at the stage of protocorms without rhizoids ( Fig. 2A). At the age of 5 MAP, after drying in a chamber with a laminar flow and cryogenic storage, a small number of protocorms with rhizoids were obtained (Fig. 2B), and from the age of 6 MAP protocorms with rhizoids and an apical bud developed (Fig. 2C, D).
Vital dye staining is often used to assess the viability of orchid seeds. The methods using TTC and FDA were described by Wood and Pritchard (2004) and Sawma and Mohler (2002), respectively. These tests are often used due to a simple assessment by counting coloured embryos. However, the time of seed staining, as well as the pre-soaking in distilled water or other substances (for example, when  Vujanovic et al. 2000). Our study showed that FDA and TTC staining did not correlate with the germination ability of D. nobile seeds. The increase in the number of FDA-stained embryos at the stage of active embryogenesis (4 MAP) in our work corresponds to the data of authors who used TTC (Vasudevan and van Staden 2010). Compared to most crop seeds, mature seeds of epiphytic orchids are short-lived. Today, the most long-term preservation of orchid seeds is possible in LN, at -196 °C (Engelmann 2004). Optimal seed moisture is crucial to maintain viability during cryopreservation (Hirano et al. 2005;Flachsland et al. 2006). According to our data, in immature seeds of D. nobile, resistance to both stress factors-dehydration and freezing-first appears at the age of 6 MAP (initial germination rate, 34%; after cryopreservation, 11%), and only in seeds aged 7 MAP and older resistance becomes sufficient (over 80%). In this work, we have shown that the method of dehydration in laminar airflow can be successfully used for cryogenic storage of immature orchid seeds, provided that they have achieved morphophysiological resistance to drying. The optimal period for cryopreservation of immature seeds is 7 MAP, which reduces the time to achieve technical maturity by 5 months.
Author contribution statement The GLK and ONV conceived the project; GLK, TVN, and ONV performed the experiments; GLK, AVB, and ASR analyzed the data and wrote the paper.

Funding
The study was carried out under Institutional research project №118021490111-5 at the Unique Scientific Installation "The Fund Greenhouse" of the Main Botanical Garden of the Russian Academy of Sciences, and the research was carried out within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (Theme No. 121041200194-7) with the partnership of Unique scientific installation: the Experimental Plant Cryobank at K.A. Timiryazev Institute of Plant Physiology (RAS, Russian Academy of Sciences). We thank the Ministry of Science and Higher Education of Russia for support of CCU "Herbarium MBG RAS", Grant 075-15-2021-678. A Enlarged protocorms without rhizoids (FDA, confocal microscopy); B the protocorm with rhizoids; C the protocorm with pointed shoot apex and rhizoids (cultivation in the dark for 4 weeks); D protocorms with pointed shoot apex and rhizoids, germinated 3 weeks in the dark and 1 week in the light. A-D VHX-1000E light microscope (Keyence, Japan). Scale bars: A-C = 100 μm; D = 500 μm