Effects of Nutrients and Growth Regulators on Seed Germination and Development of Juvenile Rhizome Proliferation of Gastrodia elata In Vitro

: Two prevalent issues with Gastrodia elata growing in a natural wild environment are the necessary symbiotic seed germination with Mycena spp. and the long growing cycle of protocorms into mature rhizomes with Armillaria mellea . This study explored the most suitable nutrients to germinate seeds and the developmental stages and propagated protocorm/juvenile rhizomes (jrhs) through a tissue culture approach. Adding humic acid, mashed potato, peptone, and (indole-3-acetic acid) IAA to the 2 g/L Hyponex 7-6-19 basal medium accelerated seed germination into protocorms with an average length of 2 mm. The nutrients best for the in vitro propagation of jrhs consisted of 1 g/L Hyponex 7-6-19 and MS salts with 1/2 NH 4 NO 3 and KNO 3 , 2 mg/L BA, 2 mg/L NAA, 2 g/L tryptone, 2 g/L glutamic acid, 1 g/L chitosan, 10 g/L apple puree, 20 g/L mashed potato, 100 mL/L coconut water, and 1.5 g/L A. mellea powder, at a pH level of 6. The maximum length and number of jrhs were 17.2 ± 0.8 mm and 76.6, respectively, in two subcultures. The in vitro propagation system started from seeds of G. elata can be employed as a potential method to propagate protocorm/jrhs for the cultivation of mature rhizomes from indoor or outdoor cultures.


Introduction
Gastrodia elata Blume, also known as Tian Ma in Chinese, is the largest plant from the genus Gastrodia, family Orchidaceae.As a medicinal herb, it can be used as an analgesic and a sedative [1], an anti-depressant [2], and an anticonvulsant [3], and it helps regenerate and protect nerves from damage [4,5].It is also considered as a potential herb to develop medicine for Alzheimer's disease and other brain diseases [6].
G. elata is a perennial and terrestrial orchid which is leafless and rootless, having fleshy, thickened underground rhizomes (rhs) bearing an apical buds, several nodes and lateral buds (Figure 1a,d).Most of the time, the rhizomes of G. elata are buried deep in the ground, where only the inflorescence stalk is exposed to the ground, and sunlight is helpful for bees to carry out pollination [7].In just 2 months of reproductive growth of Gastrodia, it is completely dependent on nutrients from mature rhizomes.The stored nutrients complete the whole process from bolting to seed maturation.Tian Ma flowers are naturally cross-pollinated by insects, epigynous with an inferior ovary and composed of three fused carpels containing many tiny ovules which develop into seeds after fertilization.It normally takes 20~21 days for seeds to mature.The ovary typically develops into a capsule that is dehiscent.Since it is a non-photosynthetic orchid, most of the life cycle of G. elata takes place underground as it does not need any sunlight and it can be cultivated indoors or in the wild.Although a single plant of Gastrodia is estimated to produce more than 2 million seeds, naturally, the germination rate of the seeds is extremely low, and the yield is also unstable [8].The reason is that the seeds of G. elata are extremely small, simple in structure, and do not have the ability to store nutrients.Seed germination needs to rely on germination fungi to complete.In nature, G. elata needs symbiotic seed germination with Mycena spp., and the step-by-step development of protocorms into mature rhizomes with the symbiotic fungus of Armillaria mellea.
Scientists have made breakthroughs in the last two decades in their studies of Tian Ma.They found that it is a mycoheterotrophic plant which promotes the development of techniques for the cultivation of G. elata mature rhizomes [7,9].Seed germination of G. elata can be successfully achieved by seed dressing with Mycena osmundicola, M. dendrobii, M. orchicola or M. anoectochila [10,11].Afterwards, germinated seeds are co-cultured with A. mellea either indoors or in the wild, and grow into juvenile, immature and finally mature rhizomes (mrhs).Rhizome is a storage organ found in the genus Gastrodia orchids.It is derived from a thickening of the part of a stem between nodes and may be composed of several internodes (Figure 1a,d).By asexual reproduction, a protocorm derived from a germinated G. elata seed can soon reproduce several juvenile rhizomes (jrhs), which will further proliferate and grow into several immature rhs (irhs) (Figure 1d).When these irhs grow mature enough with a dormant inflorescence in apical bud (Figure 1a), the mrhs can be adopted for medicinal use.The whole life cycle from seed germination to mrhs takes about 3 years.However, the extended asexual propagation and cultivation of jrhs, irhs and mrhs by artificial cultivation will cause the degeneration of mrhs with poor quality.Thus, sexual reproduction that started from seeds of G. elata with or without the help of Mycena is necessary for the commercial mass cultivation of mrhs.However, using Mycena seed dressing, a seed germination rate of less than 50% will result in the low productivity of jrhs.The propagation of jrhs through the traditional culture method is low, and, ultimately, mrhs is also the same [12].In recent years, some studies examined the effects of lignin-modifying enzymes and abscisic acid on the germination of G. elata, or used fungus material and soil microorganisms to continuously culture G. elata; however, some technical breakthroughs are still needed [13][14][15].
Due to the minute size of the seeds and undifferentiated immature nature of orchid embryos, orchid seed germination in situ was for a long time practically impossible to observe, creating an obstacle towards understanding seedling site requirements and developmental growth of rhizomes in Gastrodia.Gastrodia is a not only a terrestrial orchid from temperate regions, but also an orchid which counts on rhizome reproduction which is much more difficult to germinate in vitro [16].The proliferation rate of rhizomes is usually not much in nature; it also depends on the strains of A. mellea that are infected.Because of the long period from seed germination to mrhs formation to inflorescence development, propagation of Gastrodia with the help of symbiotic fungi was considered difficult and timeconsuming.However, aseptic germination of other orchid seeds through the tissue culture method has been very successful for lots of species such as Cymbidium, Cattleya, Dendrobium, Phalaenopsis and many others which have protocorm formation but not rhizome formation in nature.Asymbiotic propagation of G. elata through the tissue culture method is worth studying toward understanding the physiology and morphogenesis of seed germination, protocorm and rhizome development through in vitro seeding.
Little attention was given to the basic studies of in vitro propagation and culture of G. elata in recent studies.This study aimed to explore the most suitable nutrients to germinate G. elata seeds and reproduce protocorms or jrhs using a tissue culture approach.This study may address the problem of time-consuming G. elata cultivation and serve as a foundation for further studies on the development of outdoors mass production system from in vitro seed germination and jrhs propagation.

Floral Induction and Artificial Pollination of G. elata
Mrhs (Figure 1a) with thickened stems with short internodes, dormant lateral buds and inflorescence in apical bud were kept at 4 • C for two months to break the dormancy of inflorescence bud.In February 2011, twelve mrhs were planted and floral induction was conducted in the cool room at 20 ± 2 • C.During its flowering, artificial pollination was carried out by hand (Figure 1b).Capsules with the seeds inside were harvested at different ages for the germination test.

Fruit Capsule Collection after Pollination
Overall, 15 to 25 days after pollination (DAP), each of the fruit capsules (Figure 1c) was carefully collected and labeled, surface-sterilized by 1% sodium hypochlorite solution and washed thoroughly with aseptic water.After that, the capsules were open and seeding was carried out on various media containing different basal salts, plant growth regulators and additives in a laminar flow hood.Seed cultures were kept in the dark at 21 • C to assess the nutrients and age required for germination.The seed germination percentage was recorded after one month in culture.

Medium for Seed Germination In Vitro
For seed germination, Vacin and Went medium (V) [17] or Hyponex 7-6-19 (Y) were used as the main salts and supplemented with D nutrient (100 mg/L inositol, 0.3 g/L B complex*, 2 g/L charcoal, 8 g/L agar, and pH was adjusted at 5.2) as the basal medium.Various additives, growth regulators, 100 mg/L humic acid (H), 10~30 g/L glucose (Gc), 10~30 g/L sucrose (S) were added to the basal medium to investigate their effects on the seed germination after one month in culture.The culture media were sterilized at 121 • C for 20 min and used when cooled and solidified.(B complex*: A mixture of 34 mg B1(thiamine), 22 mg B2(riboflavin), 16 mg B3 (niacinamide), 58 mg B5(pantothenic acid), 6 mg B6(pyridoxine), 35 mg B7(biotin), 0.04 mg B9(folic acid), 37 mg inositol, 159 mg taurine, and 39 mg choline bitartrate).S1) Va as a seeding medium consisted of V salts and D nutrient.The seeds tested were 15 and 20 days old.

Plant Growth Regulators Tested for Seed Germination
In basal medium YH, different plant growth regulators including IAA (indole-3acetic acid), GA 3 (gibberellin) and 2iP (N6-∆2-isopentyl-adenine) were used alone or in combination to test the seed germination of G. elata in vitro.
YH basal medium contained 2 g/L Y, D nutrient, 100 mg/L humic acid, 10 g/L glucose and 10 g/L sucrose.
(A) Tests of Single and Combination of Plant Growth Regulators for Seed Germination (Table S2).
H basal medium supplemented with one of 0.5, 1, 2 mg/L GA 3 (G); 0.2, 0.5, 1, 2 mg/L IAA (I); 0.1, 0.2 mg/L 2iP (P) or 150 mL/L CW to test the effect of single growth regulator on seed germination.Two media (YIPa, YIPb) containing both IAA and 2ip were also evaluated.CW at 150 mL/L was tested at different agar concentrations.Seeds tested were 20 days old.
(B) Test of IAA, 2iP and Their Combinations on Seed Germination (Table S3).VH basal medium supplemented with combinations of 0.5, 1, 2 mg/L IAA and 0.2, 0.4 mg/L 2iP were tested.The seeds tested were 18, 21 and 22 days old.
(D) Test of GA 3 and 2iP for Seed Germination (Table S5).
VHb culture medium (VH + 2 mg/L B6) along with plant growth regulators at different concentrations of 0.5, 1, 2 mg/L GA 3 and 0.2, 0.4 mg/L 2iP were assessed for seed germination in vitro.Seeds used were 23 and 24 days old.

Protocorms/Juvenile Rhizomes (Jrhs) Proliferation Test
Five replicates were set for each treatment, with ten protocorms/Jrhs per replicate were cultured on media composed of different plant growth regulators, various additives and basal salts, and kept in dark at 21 • C to assess the effect on protocorms/jrhs proliferation.Cultures were subcultured once every 4 weeks.Data for the proliferation rate and size of jrhs were recorded after 8 weeks in culture.S6)

Test of Chitosan on Jrhs Proliferation (Table
For testing the chitosan on proliferation of jrhs, basal medium YH3 and TM was supplemented with 1 or 2 g/L chitosan (CHT1 or CHT2), at a medium pH of 5.6.

Combination of BA with IAA or NAA on Jrhs Proliferation
In order to understand the difference of IAA and NAA on jrhs proliferation, 2 mg/L BA was combined with either 1 or 2 mg/L IAA or NAA to determine their effect by using YH4 (CHT1 medium without BA and IAA) medium as the basal medium.
The results were recorded after 2 months in 2 subcultures.

Test of Different pH in Culture Medium on Proliferation of Jrhs
The Y1 + 1/2 AB medium added to 10 g/L apple puree and 20 g/L mashed potato adjusted the pH to 5.6, 6.0 and 6.5, respectively, to evaluate the proliferation rate of jrhs.

Statistical Analysis
All the data were analyzed by SPSS PASW Statistics V18 software (version 18.0; SPSS Inc., Chicago, IL, USA) with an Analysis of Variance (ANOVA) approach considering the difference of average value with the least significant difference (LSD) test.Means within each column followed by the different letter(s) are significantly different at p < 0.05 using Fisher's protected LSD test.

Aseptic Seeding of G. elata on the Culture Medium
Figure 1 shows the commercial mature rhizome bearing a dormant inflorescence in apical bud (Figure 1a), artificial pollination by hand (Figure 1b), capsule enlargement (Figure 1c), immature rhizome with growing lateral buds (Figure 1d), seeds (Figure 1e) and seed germination (Figure 1f-h) of G. elata.The seeds appeared fusiform under the scanning electron microscope (SEM), with a length of 0.4-0.7 mm (Figure 1e), and began to absorb nutrients after sowing (Figure 2a).The primitive embryo in the seeds underwent cell divisions, and burst the seed coat to give rise to the protocorms (Figures 1f and 2b).The protocorm is described specifically for the young seedling produced from the seeds of various orchids that have just germinated.
The embryo germinated to a yellowish white protocorm (Figures 1g-h and 2b,c), which was regarded as germination.Germinated seeds were studied for their protocorm and jrhs development (Figure 2).The germinating seeds of G. elata was observed under a stereomicroscope or a SEM (Scanning Electronic Microscope).At the very beginning of germination, the embryo underwent cell division (Figure 2a), broke through the seed coat (Figure 2b,c), and soon formed a globular-embryo-like structure (Figure 2c-e).The lower part of the globular-embryo-like structure resembled a suspensor during embryonic development (Figure 2d-f).Orchid seeds usually have a round or oval undifferentiated embryo.During the germination process, the embryo seems to continue following the embryonic developmental stage.The suspensor is traditionally believed to be a supporting structure during plant embryo development.The formation of jrhs on CHT1 medium gradually showed gravitropism with the apical bud growing downward (red→); the lateral buds were formed at the lower part of jrhs (→), and the secondary branching of the lateral buds was developed as well (yellow→).(p) Protocorm proliferation on TM medium.(q) Jrhs induced more lateral buds on 2Glu medium, each rhizome bearing an apical bud and several lateral buds.
Dian and Liang [19] reported that seeds of G. elata were collected 12 days after pollination (DAP) when the embryos of a few seeds began to mature, and after 15 days, the germination rate of the seeds was maintained at 85% until the capsule split.In present studies, the aseptic seed sowing of G. elata started from the 15 DAP. Figure 3 showed that seeds collected 18 DAP had the highest germination rate, reaching 88.2%.The germination rate was 84.5% on the 20th DAP, 62.4% on the 21st day, and the germination rate decreased to 10.4% on the 22nd day (Figure 3).The protocorm size was about 0.5-2 mm.Details of seed germination were shown in Table 1.Afterwards, the germination rate was decreasing until it reached 0% on the 23rd day.Thus, seeds harvested at 18~20 days old were the most suitable for germination.It is quite common that Orchidaceae seeds harvested late will become dormant.It happened that seeds of G. elata older than 21days decreased germinability rapidly.Many cold-hardy terrestrial orchids exhibit low seed germination, which is often attributed to low viability or dormancy.Immature seeds of many orchid species have been shown to germinate more readily than mature seeds [20][21][22].Mature seed mentioned in orchids actually refer to the seeds from dehiscent capsules.Several factors may contribute to the increased germinability of immature seeds.Immature seeds may be more water permeable than mature seeds [23].Mature seeds still contain undifferentiated embryo and may have chemical inhibitors, such as ABA, or lack certain germination-promoting hormones [24], or have an impermeable testa [25].During the best germination time period (18~20 DAP), the germination of seeds was recorded in series of Hyponex medium.A medium containing only basic D nutrient (100 mg/L inositol + 0.3 g/L B complex* + 2 g/L charcoal + 8 g/L agar, pH 5.2), 10 g/L glucose + 10 g/L sucrose, and 2 or 3 g/L Hyponex (Y2 or Y3) provided seed germination (Table 1).The germination of orchid seeds can be promoted by adding coconut water, potato, and fruit puree, etc.The additive supplements provided nutrients and natural growth hormones, might also have a buffering effect on stabilizing the pH of medium, and can even be regarded as a chelating agent, maintaining the solubility of minerals and other ingredients [26,27].The results of this study also demonstrated that the medium which contributed to the earlier germination and larger protocorms of G. elata seeds was those with 2 g/L Hyponex supplemented with humic acid (Y2H), peptone (Y2Pp) or mashed potato (Y2Pt), followed by apple puree (Y2A, Y3A) and Na-glutamate (Y2MSG), or bee larva (Y2L, Y3L).The average protocorm length was twice the size in medium Y2H, Y2Pp and Y2Pt (Table 1, Figure 2) than all of the other media tested.Therefore, it is recommended to add 100 mg/L humic acid, 2 g/L peptone, or 20 g/L mashed potato to the medium with 2 g/L Hyponex, respectively, to promote earlier seed germination.The protocorms derived are the largest, having an average length of 2 mm (Table 1).Agar at 6 or 8 g/L in the medium did not show significant difference after seed germination.Seeds collected on 15th~18th DAP did not germinate on V series medium added with glucose or sucrose at 10~30 g/L.Seeds at 20 days old were germinated on V medium only with the help of 200 mL/L CW or 30 g/L sucrose.In Y basal medium the recorded amount of carbohydrates for seed germination is 10 g/L glucose + 10 g/L sucrose.Without the help of IAA, seeds at 20 days old and younger did not germinate on medium containing V, but did germinate on medium containing Y. Seeds growing a day older, between 21 and 22 days old, needed 0.5~2 mg/L IAA to germinate on both Y and V series media.
Certainly, IAA is necessary for germination on Y as well as V series medium when seeds are 21~22 days old.Nevertheless, without IAA in the medium, seeds that are 20 days old or younger could germinate on Y basal medium but not V basal medium.It can be determined that 2 g/L Y is more conducive to seed germination than 3 g/L Y because of the larger size of the protocorm derived.The amount of carbohydrate in Hyponex series medium is supposed as 10 g/L glucose + 10 g/L sucrose, which makes G. elata seeds germinate (Table 1).As far as the basal medium for seed germination is concerned, it is concluded that Hyponex was superior to V medium.Hyponex at 2 g/L was more suitable for seed germination than 3 g/L.
If CW is added to the seeding medium, seeds harvested on the 25th DAP germinated, but seeds harvested on the 15th to 18th DAP did not germinate (Table 1; Figure 3).CW is not beneficial to the germination of seeds younger than 20 day old.At the late harvest, 23~25 days after pollination, the seeds enter a dormant state.The medium with 100 mL/L CW supported seed germination after keeping seeds at 4 • C for one month in dark to break dormancy.Coconut water at 200 mL/L reinforced germination when the seeds were 20 days old using V as the basal medium.When seeds are 25 days old, 100 mL/L of coconut water helped them to germinate.
Nevertheless, Yeh et al. [27] showed the best treatment for the germination of G. elata seeds was adding 25 mL CW to the medium.Adding 5-20% CW to the culture medium could slightly promoted protocorm of Pleione formosana development in the early stage [28].The culture medium contained 100 mL/L CW was the best for stimulating germination of Paphiopedilum primulinum orchid seed [29].
Seeds collected on the 23rd day and later did not germinate on the media with GA 3 and 2ip, which might be due to the common seed dormancy of orchid family.GA 3 and cytokinins are generally considered to be good to orchid seed germination [30]; however, this was not concluded in seeds of G. elata (Table 1).
It is feasible that the non-germinated seeds kept at 4 • C in the dark for one month to break dormancy, and then rose to 20 ± 2 • C to continue growing.One month later, more seeds of G. elata germinated.Our results showed that the time of seed collection would affect its germination, and seeds collected between 18 and 20 DAP were the easiest to germinate.Seeds harvested and sown at 18th DAP had the highest germination rate reaching 88.2%.When seeds reached 21~22 days old, IAA was required for seed germination on both Hyponex and VW basal medium.The results were not exactly similar to those reported by Yeh et al. [27], who indicated that the germination rate was 3% for the seeds collected 14 DAP, and the rate gradually increased to 67% from day 17 to day 20; however, after 23 DAP, the germination rate began to decline and lowered to only 10% after 26 DAP.The pollination of G. elata is recommended to be allogamous pollination, which can significantly improve the quality of seeds.Such seeds have strong vitality and a high reproduction rate, and the resultant G. elata rhs has a high resistance to disease and cold [8].
Seeds of terrestrial orchids from temperate regions are much more difficult to germinate in vitro [16].Gastrodia is not only a terrestrial orchid from temperate zones but also an orchid without leaves, roots and photosynthesis in nature.
Artificial cultivation for the production of mature rhizomes can be carried out indoors or outdoors, because sunlight is not required except for the flowering period.All the nutrients needed through the lifetime of Gastrodia come from the symbiosis with different fungi; one is for seed germination, and the other is for the growth and development of rhizomes.After seed germination, protocorms were observed to proliferate, soon to form several protocorms under a stereomicroscope or a scanning electron microscope (SEM) (Figure 2g-j).TM medium containing 1 g/L chitosan induced the multiplication of many protocorms (Figure 2p).The lower part of protocorms elongated to form jrhs, with terminal bud, nodes and lateral buds continuing to grow and branch (Figure 2i-l); the mediums with amino acids and glutamic acid or MSG were the most efficient.After the elongation of the rhizomes, the terminal buds grew upward and enlarged (Figure 2k-n).The jrhs had several nodes, and then the apex gradually showed the physiology of gravitropism in vitro (Figure 2o).Observation was also found in terrestrial Cymbidium, which had rhizome in transition [16,31], but the rhizomes are not elongated and swollen like Gastrodia elata.
Humic substances could enhance the absorption of several nutrients in plants and combine with metal cations to affect the absorption capacity of roots and promote plant growth and yields.Low molecular weight humic substances (containing humic acid) can be absorbed by plants.These components increase the permeability of cell membranes and have hormone-like activity [32].Humic substances have an influence on seed germination and seedling development, can increase the enzyme activity in seed tissues, and increase seed water absorption, respiration and germination rate [32].In our research, humic acid was found to affect the elongation and proliferation of G. elata protocorms into jrhs (Figure 2c,k,l), and peptone and mashed potato (Figure 2b,h) as well as to accelerate the formation of jrhs.
Chitosan was beneficial to the protocorm proliferation (Figure 2p).We see that protocorms continuously regenerated multiple protocorms when basal medium was added with 1 g/L chitosan.
Three kinds of growth regulators, IAA, GA 3 and 2iP, were studied for their effect on germination of G. elata seed at 20 days old.When YH was used as basal medium, YHI0.5 (0.5 mg/L IAA), YHI 1 (1 mg/L IAA), YHI2 (2 mg/L IAA) and YIPb (2 mg/L IAA + 0.2 mg/L 2iP), determined seed germination.Medium VI0.5, VI1 and VI2 which containing V and 0.5~2 mg/L IAA could make seeds germinate, too.It is obvious that seed germination required a medium containing 0.5~2 mg/L IAA.Media containing GA 3 , or 2ip alone did not give rise to seed germination in Y or V basal medium.However, seed could germinate on medium YIPb (2.0 mg/L IAA + 0.2 mg/L 2iP).It meant that IAA could overcome the inhibitory effect exerted by low concentrations of 2ip, whereas Hyponex was the basal medium; this did not happen in the VIPe medium (2.0 mg/L IAA + 0.2 mg/L 2iP), whereas V was the basal medium.
Seed germination was not found in the V medium with 2ip alone or the medium with both IAA and 2iP (Table 1).When V was supplemented with 2 mg/L IAA and 0.2 mg/L 2iP, the same concentration did not support seed germination like in the Hyponex medium.Therefore, again, it was concluded that Hyponex was more suitable for seed germination than the V medium.For medium solidification, gerlite was better for protocorm and rhizome proliferation than agar.
In the Hyponex basal medium, seed germination was observed from those harvested 18~20 DAP when IAA was not added to the medium.However, seeds older than 20 days needed IAA to germinate in Hyponex medium.Seeds did not germinate on all the V medium containing 2ip even higher IAA concentration was presented.It was certain that IAA was helpful but 2ip was inhibitory for seed germination of G. elata.IAA could assist seed germination no matter if GA 3 was present (YIG1~YIG6) or not (Table 1).
Seeds could not germinate with the presence of 0.5~1 mg/L GA 3 only or with different concentrations of GA 3 + 2iP (Table 1).Only IAA at 0.5~2.0mg/L would help seed (on 20~22 DAP) germination, but not IAA at 0~0.1 mg/L (YH, YHI0.2).Seeds also germinated when the culture medium containing only IAA and IAA+GA 3 as well.It meant that GA 3 was neither helpful nor harmful to seed germination.However, the inhibitory effect of 2ip on seed germination was assured.
In tests of growth regulators on the seed germination of G. elata, it is concluded that IAA is beneficial to seed germination, and that seeds between 21 and 22 days old need IAA to germinate, 2ip inhibits seed germination and GA 3 was neither harmful nor helpful to seed germination.

Effect of Different Cytokinins on Proliferation of G. elata Jrhs
Observation and records were made after eight weeks when protocorms or jrhs were cultured on different basal media containing various cytokinins.In Figure 4, the jrhs with the significantly largest size and proliferation were those cultured in YH1 medium supplemented with 1mg/L BA (p < 0.05), and the average length and the number of proliferated jrhs were 4.5 ± 0.7 mm and 8.9, respectively, followed by 2iP and CPPU.Cytokinin is divided into two types: one is the purine type (adenine), such as zeatin ((E)-2-methyl-4-(7H-purin-6-ylamino) but-2-en-1-ol) and derivatives, kinetin, BA (N6benzyladenine), and 2iP (N6-∆2isopentenyl-adenine); the other is phenylurea type, such as TDZ (thidiazuron) and CPPU (N-phenyl-N -[2-chloro-4-pyridyl]urea) [33].These cytokinins have different activities, which affect the induction and number of adventitious buds of various plants [34].Centaurium erythraea cultivated in phenylurea cytokinins, TDZ and CPPU were more effective than purine cytokinins for inducing morphogenesis in adventitious bud.The largest number of adventitious buds induced among them was the treatment with 1.0 µM CPPU [35].Huang and Chung [36] studied Lycaste hybrids and found that 1/2 MS supplemented with 0.5 mg/L BA and 1.0 mg/L TDZ could promote the callus forming protocorm-like bodies after culture for 2 months.Vitex trifolia was cultivated on MS medium with different cytokinins, and the data showed that the largest number of buds were obtained on medium with 5.0 µM BAP [37].In our study, the adenine-type BA treatment could make G. elata jrhs reach a significantly larger size and the most proliferative effect (p < 0.05), followed by 2iP and CPPU (Figure 4).

Effect of BA and IAA Concentrations on the Proliferation of G. elata Jrhs
The effect of different ratios of cytokinin and auxin on the proliferation of G. elata jrhs were investigated.YH1 medium of containing 2 g/L Hyponex, combining with different concentration ratios of BA and IAA was investigated the effect on jrhs proliferation after eight weeks in culture.Table 2 showed that the culture medium YBI 10 (2 mg/L BA + 1 mg/L IAA) promoted the best growth of G. elata jrhs, with a size of 5.1 ± 0.4 mm and the highest proliferation number of 5.4 (p < 0.05).Based on the above results, BA is recommended to be joined to the culture medium to multiply jrhs and only when the amount added was 1-2 mg/L, especially when the ratio of BA: IAA was 2:1 (Table 2).Basal medium YH1: 2 g/L Y+ E nutrient.BA: 6-benzyladenine; IAA: indole-3-acetic acid.Each value is expressed as mean ± SD (n = 10).Means within each column followed by the different letter(s) are significantly different at p < 0.05 by Fisher's protected LSD test.
Auxin/cytokinin antagonism could affect plant morphology; when auxin levels are higher, they promote root growth and differentiation, and inhibit the growth of lateral buds and bud differentiation.When cytokinin is higher, it promotes shoot production, bud growth and differentiation, and inhibits root growth and differentiation.When auxin/cytokinin is approximately the same, it is best to promote cell division and form calluses.Therefore, in tissue culture, for achieving the experimental goals and expected results, choosing the right type, concentration and ratio of growth regulators was very important [38][39][40].Cai et al. [41] found that adding 1 mg/L BA, 0.5 mg/L NAA and 50 mg/L banana to the modified 1/2 MS medium could help induce the formation of G. elata jrhs and the best germination rate.The modified 1/2 MS medium containing 1/2 amounts of ammonium nitrate and potassium nitrate of the original MS media was optimal for the growth of rhizomes.In our observations, 2 mg/L BA and 1mg/L IAA together were adequate for the growth and proliferation of jrhs (Table 2).
3.2.3.Effect of Jrhs Proliferation on Media Containing Proteins, Amino Acids, B Vitamins, and Plant Growth Regulators Nitrogen or amino acids brought many benefits to growth or morphogenesis, including increased callus formation, prevention of browning, and increased protocorm-like bodies, seedlings and the yield of orchids [17,[42][43][44].
The effect of proteins on the growth and proliferation of G. elata jrhs after 8 weeks in culture was shown in Figure 5. Jrhs that cultured with 1 g/L tryptone obtained significantly (p < 0.05) larger size and better proliferation rate, which were 8.9 ± 2.0 mm and 12.4, respectively.Tryptone is mainly composed of amino acids and vitamins [42].Related research data showed that 2 g/L tryptone in the culture medium could help the development of Paphiopedilum primulinum and Phaius tankervilliae seeds [29,45] as well.Figure 6 showed the results of testing 11 kinds of amino acids.The size of jrhs was the largest (p < 0.05), up to 8.1 ± 1.4 mm, when treated with 1 g/L glutamic acid.However, the maximum number of jrhs reached 17.0 with 2 g/L glutamic acid (p < 0.05) in the medium.Glutamic acid at 1~2 g/L is advantageous for jrhs growth and proliferation.The higher number of jrhs obtained with a smaller size is acceptable.When culture media with various B vitamins were investigated, the results were demonstrated in Figure 7.The addition of B6 and B9 resulted in the largest size (p < 0.05) of jrhs, which were 7.2 ± 1.0 and 7.1 ± 1.1 mm, respectively.When the number of multiplied jrhs was concerned, B6 treatment had the highest number (p < 0.05) of jrhs reaching 24.4,followed by B9 treatment of 20.0.MS medium containing 50 mg/L nicotinic acid (B3), 50mg/L pyridoxine-HCl (B6), 10 mg/L thiamine-HCl(B1) and 4 mg/L BAP helped potatoes to proliferate large numbers of lateral shoots [46].The use of 50 µM folic acid (B9) as exogenous growth enhancers could increase seed vigor, germination rate, shoot weight, shoot height and root length of Pisum sativum [47].
As noted above, we propose that adding 1g/L tryptone, 1~2 g/L glutamic acid and 0.5 mg/L B6 and B9 to the culture medium was very valuable for the proliferation of G. elata jrhs.

Effect of Chitosan Concentrations on the Proliferation of G. elata Jrhs
In the cultivation of Orchidaceae, chitosan could be used to promote the growth of orchid protocorm, seedlings, leaf quantity, plant height, fresh weight, and dry weight [48].Table 3 showed the observation recorded eight weeks after culturing G. elata jrhs.The length of jrhs grown in the YH medium without chitosan was about 5.4 ± 0.4 mm, whereas medium with chitosan like TM, CHT1, and CHT2, the protocorms were bigger with a size of 9.0 ± 1.0, 12.8 ± 1.5, and 10.0 ± 1.4 mm, respectively.When 1 g/L chitosan was added, not only the size was the largest (p < 0.05), but the number of multiplied rhizomes was also the largest (p < 0.05), reaching 34.8, and the number decreased to 24.6 when 2 g/L chitosan was added.To conclude, adding chitosan at 1 g/L enhances the growth and proliferation of G. elata jrhs (Table 3).In agriculture, chitosan treatment can increase the germination rate of corn seeds, the length and dry weight of shoots and roots, and is beneficial to the growth of seedlings under low temperature stress [49].Chitosan could effectively increase the total weight, vitamin C content and hardness of soybean sprouts [50].Soaking peanut seeds in different concentrations of chitosan solution could increase peanut seeds' germination energy, germination rate, lipase activity, and endogenous hormones GA 3 and IAA content [51].In our study, 1 g/L chitosan increased the size of G. elata jrhs, which is relatively thick in appearance, and could also increase the proliferation of jrhs.
Comparing the fortified medium CHT1 (YH3 + 1 g/L chitosan) shown in Table 3, or YH4 medium (Table 4: YBI 1, YBI 2, YBN 1, YBN 2) with unfortified medium YH1 medium (Table 2, YBI 10), the size of jrhs was tripled and the number of proliferations was increased more than six-fold.It is very clear that chitosan together with humic acid, tryptone, glutamic acid and vitamin B6 and B9 are very powerful in increasing the size and number of jrhs proliferated.After the chitosan test, we tried to understand the effects of different auxins on the proliferation of G. elata jrhs.Hyponex at 2 g/L with 1g/L chitosan and 2 mg/L BA was added with 1 or 2 mg/L IAA or NAA to investigate the difference between IAA and NAA after 8 weeks in culture.Table 4 showed that jrhs had significantly (p < 0.05) the larger size when 1~2 mg/L IAA was replaced with NAA in culture medium.However, the number of jrhs proliferation was not affected.
NAA at 1 ppm or low concentration of 2,4-D in tissue culture of Pleione formosana helped protocorm proliferation [52]; the MS medium consisted of 2 µM NAA and 4 µM BA contributed to the induction and multiplication of shoots [53].
It is certain that the effect of adding NAA to the medium could increase the size but not the number of G. elata jrhs more than that of IAA.

The Effect of Medium Containing Various Basal Salts and pH on the Proliferation of G. elata Jrhs
In the tissue culture of orchids, different macro elements in the medium were confirmed to affect the growth.The seedlings of Pleione formosana treated with 1/2 MS have the best growth and development [54]; Hyponex 7-6-19 medium contributed to seedling development of Phaius tankervilliae about 4~5 months after germination [45].In the present study, the protocorms of G. elata were inoculated and subcultured twice on proliferation medium having different salt formula, the results recorded after 4 months were shown in Table 5. Y1, Y1 + 1/2 AB and 1/2 AB were compared with each other.In the Y1 + 1/2 AB medium, the length and the number of multiplications of G. elata jrhs were the best, at 13.4 ± 1.1 and 45.0, respectively.Basal medium containing 1 g/L Hyponex and half amount of ammonium nitrate and potassium nitrate of the original MS media (1/2 AB) was applied for pH test.The YS 6.0 medium at a pH of 6.0, was particularly beneficial to the proliferation of jrhs, and the maximum length and the number of multiplications of jrhs were 17.2 ± 0.8 mm and 76.6, respectively (p < 0.05) (Table 5), followed by the YS 5.6 medium, and the worst was YS 6.5.
The design of the proliferation medium combined the factors that had accelerated growth in each experiment.The experimental data declared that the basal salt with 1 g/L Hyponex and 1/2 AB was the most constructive for the growth and proliferation of G. elata jrhs; the additive parts included tryptone, glutamic acid, vitamin B6, and B9 to provide sufficient nutrition to help the growth and development of jrhs.When IAA was replaced with NAA, the combination of 2 mg/L BA and 1~2 mg/L NAA in the medium could produce the larger size of jrhs.Natural additives such as humic acid, potato, apple and coconut water contain some natural ingredients and hormones which may help seed germination and jrhs to grow.The addition of potato could sustain the survival time of the culture and treat the occurrence of vitrification in tissue culture plants, in which the variety and concentration of the formula content resulted in the osmotic pressure unaccommodated [55].In addition, this research also simulated the growth of G. elata in nature.When plants are invaded by pathogens or in stress environments, plant cells will initiate a defense response and release enzymes to break down the cell wall.It made chitosan or oligosaccharides release that triggered plants to synthesize many special proteins and plant defensins to resist diseases and environmental changes [50,56].Adding chitosan and A. mellea powder to the culture medium can imitate the state of A. mellea invading the G. ealta jrhs in nature, and provide substantial amounts of nutrients from above various experiments will promote the rapid growth of jrhs.Finally, the effect of medium pH on the proliferation of G. elata jrhs was found to be optimal at pH 6.0.
In summary, seeds collected and sown 18 DAP have the highest possibility to geminate, reached 88.2%.The best seed germination medium is 2 g/L Hyponex 7-6-19 medium added with one of 100 mg/L humic acid, 2 g/L peptone or 20 g/L mashed potato, and 0.5~2 mg/L of IAA which accelerates seed germination into protocorms with an average length of 2 mm.For seed germination, IAA is helpful but 2ip is inhibitory.IAA at higher concentrations might overcome the inhibitory effect of 2iP at low concentrations, whereas Hyponex worked with the basal salts but not the V salts.GA 3 was neither helpful nor detrimental to seed germination.Without the help of IAA, seeds at 20 days old and younger did not germinate on medium containing V salts, but did germinate on medium with Hyponex as the basal salts.For certain, seeds between 21 and 22 days old needed IAA to germinate on Hyponex as well as the V medium.
Many experiments were carried to obtain the most suitable culture conditions for growing G. elata protocorm into many jrhs.Protocorms multiplication is induced on TM medium with 1 g/L chitosan.The best way to enhance proliferation of jrhs was to use a growth medium containing 1 g/L Hyponex 7-6-19 and 1/2AB together with 2 mg/L BA, 2 mg/L NAA, 2 g/L tryptone, 2 g/L glutamic acid, 10 g/L apple puree, 20 g/L mashed potato, 100 mL/L coconut water, 1 g/L chitosan, and 1.5 g/L A. mellea powder, pH at 6.0.The length of the jrhs was about 17.2 ± 0.8 mm, and the maximum number of rhs derived could be as many as 76.6 in 4 months with two subcultures.
Comparing the fortified nutrient medium, that was developed step by step like CHT1 (YH3 + chitosan) shown in Table 3, or YH4 medium added with BA and NAA (Table 4) to the unfortified medium YH1 (Table 2), the size of jrhs was tripled and the number of proliferations was increased almost seven folds.It is very clear that chitosan, tryptone, glutamic acid or MSG, vitamin B6 and B9 together with BA and NAA were very favorable to increase the size and number of jrhs proliferated.The most effective pH medium is at 6.0.

Conclusions
This study explored the best time and nutrient compositions to germinate seeds and grow G. elata protocorm into juvenile rhizome through experiments of combining different media.The result may challenge the coexistence of discordant symbiotic fungi, but more importantly, help seeds to germinate and proliferate a large amount of jrhs in vitro without the help from A. mellea.It is expected that the seeding procedure in vitro may not be required every year for the mass production of jrhs since artificial pollination is also quite laborious.The aseptic culture of jrhs can be continued for years to sustain the production of jrhs for traditional artificial cultivation.The results of this study may be applied for aseptic seeding and multiplication of juvenile rhizomes in the future to be ready for the mass production of commercial mature rhizomes of G. elata outdoors.This method is also useful for the germplasm preservation of G. elata.

Figure 1 .
Figure 1.The rhizomes, flowers, pollination, capsules and seed germination of G. elata.(a) Mature rhizome bearing a dormant inflorescence in bud which was a typical commercial product; (b) hand pollination; (c) capsules on the flower stalk; (d) immature rhizome having buds breaking dormancy; (e) seeds under SEM; (f) seeds germinating in vitro bursting the seed coat under SEM; (g) seed germination on culture medium; (h) a germinated seed grow into a protocorm with seed coat still attached beneath, under a stereo microscope.

Figure 2 .
Figure 2. The in vitro asymbiotic germination of seeds of G. elata into protocorms and the development of juvenile rhizomes (jrhs).(a) The undifferentiated embryo in seeds started cell division and swelled.(b) Germinating seeds with seed coat attached, under SEM.(c) Protocorm on the left (→) started elongating.(d) The lower part of two protocorms began to elongate, forming a structure similar to globular embryo.(e) Cells were clearly seen in the head when the globular embryo-like structure grew more in advance, the lower part grew into a suspensor like structure.(f) The lower part of the two protocorms were elongated, and the left one grew more forward than the right one, which already showed a differentiated terminal bud.(g) A germinated seed multiplied to form more protocorms, under SEM.(h) One seed germinated into tween protocorms, the third protocorm was initiating (red →).(i) A germinated seeds with seed coat still attached, proliferated and developed several protocorms.(j) Many protocorms formed from one germinated seed which had seed coat (→) still attached.(k) Protocorms generated several protocorms (→) on the Y2H medium; the photo on the right showed that the protocorms elongated into jrhs (red→).(l) Multiple jrhs under SEM.(m) Jrhs proliferated on medium added with 2 g/L glutamic acid.(n) The tip of the apical bud of jrhs enlarged (→) on medium with 4 g/L MSG.(o)The formation of jrhs on CHT1 medium gradually showed gravitropism with the apical bud growing downward (red→); the lateral buds were formed at the lower part of jrhs (→), and the secondary branching of the lateral buds was developed as well (yellow→).(p) Protocorm proliferation on TM medium.(q) Jrhs induced more lateral buds on 2Glu medium, each rhizome bearing an apical bud and several lateral buds.

Figure 3 .
Figure 3. Changes in the germination rate of G. elata seeds after one month in culture.DAP: days after pollination (carried in March 2012).

Figure 4 .
Figure 4. Effect of different cytokinin on proliferation of G. elata juvenile rhizomes after 8 weeks in YH1 culture medium.Each value is expressed as mean ± SD (n = 5).Means followed by the different letter(s) are significantly different at p < 0.05 by Fisher's protected LSD test.

Figure 5 .
Figure 5.Effect of nitrogen source (1 g/L) on the growth and proliferation of G. elata juvenile rhizomes after 8 weeks in YH2 culture medium.Basal medium YH2: YH1 + 3.5 g/L gelrite (to replace 8 g/L agar) CH: casein hydrolysate; ME: malt extract; Pep: peptone; Try: tryptone; YE: yeast extract.Each value is expressed as mean ± SD (n = 5).Means followed by the different letter(s) are significantly different at p < 0.05 by Fisher's protected LSD test.a,b Mean values with unlike letters were significantly different.

Figure 7 .
Figure 7. Effect of addition B vitamins on growth and proliferation of G. elata juvenile rhizomes after 8 weeks in YH2 basal medium without B complex added.Each value is expressed as mean ± SD (n = 5).Means followed by the different letter(s) are significantly different at p < 0.05 by Fisher's protected LSD test.a,b,c,d Mean values with unlike letters were significantly different.YH2: YH1 + 3.5 g/L gelrite (to replace 8 g/L agar) B vitamins: 0.1 mg/L B1; 0.5 mg/L of B2, B3, B6, B9 each; and 1 mg/L B7.

Table 1 .
Effect of carbohydrate, protein, and organic additives tested for germination of G. elata seeds harvested at different days after pollination (DAP) and cultured for one month.

Table 2 .
Effect of BA and IAA concentration on growth and proliferation of G. elata juvenile rhizomes in the culture medium for 8 weeks.

Table 3 .
Effect of chitosan concentration on growth and proliferation of G. elata juvenile rhizomes in culture medium for 8 weeks.

Table 4 .
Effect of BA combined with IAA or NAA on growth and proliferation of G. elata juvenile rhizomes in culture medium for 8 weeks.Basal medium YH4: CHT1 without growth regulators.MSs*: MS (Murashige & Skoog, 1962) basic salts without NH 4 NO 3 and KNO 3. BA: 6-benzyladenine; IAA: indole-3-acetic acid; NAA: 1-Naphthaleneacetic acid.Each value is expressed as mean ± SD (n = 5).Means within each column followed by the different letter(s) are significantly different at p < 0.05 by Fisher's protected LSD test.3.2.5.Effect of IAA or NAA Concentrations on the Proliferation of G. elata Jrhs