Study on the Effects of Different Media on the Seeding of F. cirrhosa at Plateau

Bulbus Fritillaria cirrhosa is a precious wild herb of Liliaceae Fritillaria which grows in Tibetan area of China. Ripe seeds and oneyear bulbs of Fritillaria cirrhosa D. Don were planting materials. Sandy soils, humus soils and humus soils with the dung of Hepialidae Larvae (Lepidoptera,Hepialidae) were selected as nursery materials.The experiments were conducted at XinduqiaoTown of Kangding County (Latitude: 101°, 34′N, longitude: 30°, 03′ E, altitude: 3500 m) from March to Augustin 2011. The research was aimed to select the optimum medium of productive seeding and to study on effects of different media on growth of F. cirrhosa seeding. The results were: (1) Sowing with seeds and one-year-old bulbs of F. cirrhosa, S1 (100% humus soils with dung of Hepialidae larvae) was the best nursery media for sprouting. (2) The integrated performance was considered, including transverse diameter, longitudinal diameter, the ratio of longitudinal diameter to transverse diameter, 30-grain fresh weight and 30-grain dry weight. Sowing with F. cirrhosa seeds, S1 (100% humus soils with dung of Hepialidae Laravae) was the best medium for promoting the seedlings and getting larger size of bulbs. Sowing with one-year-old F. cirrhosa bulbs, S1 (100% humus soils with dung of Hepialidae Laravae) and S2 (100% humus soils) were advantageous for the growth of plantlet bulbing and getting larger size of bulbs. Adding sandy soil to substrates or 100% sandy soil asnursery media could inhibit thegrowth of F. cirrhosa bulbs, getting better shape of F. cirrhosa bulbs.

The cultivation of medicinal plant can effectively solve the shortage of herbal source drugs or remedies and largely reduce pressure on excess exploitation of wild herbs (WHO et al., 1993). Germplasm resources are not only an important part of biological resources, but also a key to success for cultivation (Srivastava et al., 1996). At present, more than 250 species of medicinal plants are commercially cultivated on about 380,000 ha in China (Canuto et al., 2012). Media are the base of the material needed for plant growth, providing moisture and nutrients for crop root, affecting plant physiology and yield heavy (Sonneled, 1991). So a key to achieve industrial development of Chinese herbal medicines is to get a suitable media. Currently, the herbs had been studied on nursery media, such as Carthamus tinctorius L. and Atractylodes macrocephala Koidz (Rao-sheng, 2011, 2010. Bulbuls Fritillariae cirrhosae is one of the most widely used traditional Chinese medicines with the function of relieving cough and eliminating phlegm (Chinese Pharmacpopoeia Commission, 2010). Bulbs are not only medicinal parts of Bulbuls Fritillaria cirrhosa, but also a breeding material. The quality of bulbs is related to yield and quality of the Bulbuls Fritillaria cirrhosa. Bulbuls Fritillaria cirrhosa (Liliaceae, Fritillaria) is a kind of endemic and rare perennial herbs which grows in a limited area in the Tibetan plateau. Severe plateau climate causes to slow growth and reproduction. One time "sprouting" and so-called "sprout tumble"were observed within a year (Editorial Committee of Chinese Academy of Sciences 'Flora of China', 1980). The artificial cultivation of Bulbuls Fritillaria cirrhosa has been carried out in 1960s. 3 years cultivation for Bulbuls Fritillaria cirrhosa could ensure its economic characteristics (Jun et al., 2009;Da-yong et al., 2010). In recent years, studies on cultivation of Bulbuls Fritillaria cirrhosa are concerned with those aspects: (1) contrasting with wild materials in efficacy of active ingredient (Congetal, 2010;Xiao-yan et al., 2009), (2) cultivation techniques. Cultivation techniques of Bulbuls Fritillaria cirrhosa have a breakthrough. Current research has defined the basic ecological factors closely related to quality of Bulbuls Fritillaria cirrhosa, including soil, climate and the surrounding biological community ecology (Hui et al., 2008). Bulbuls Fritillaria cirrhosa have different qualities because of that the significant factors are different in the contents of soil elements K, Mn, Zn and P (Shi-lin et al., 1990). Humus soil has been selected as a better media for the growth of seeding, because Bulbuls Fritillaria cirrhosa have achieved comparatively high germination rate and comparatively good quality in humus soil (Ying-ying, 2008).
In this paper, based on previous studies, under the nursery mode of shed, author selected 4 kinds of media and adopt the ripe seeds and one-year-old bulbs of Fritillaria cirrhosa D.Don as plant materials to study on the effects of different media on growth of F. cirrhosa breeding bulbs.The result of this paper is to provide a research base for the industrial development of Bulbuls Fritillaria cirrhosa.

Nursery Media
Author chose media which was common used for cultivation. Sandy soils, humus soil sand humus soils with the dung of Hepialidae Laravae were selected as nursery materials. Sandy soils were collected from Xinduqiao Town. Humus soils were purchased from Yi Bin, Sichuan Province. Humus soils with the dung of Hepialidae Laravae were collected from Chengdu Enwei Group.

Local Conditions
The experiment was carried out in Mar. 27 2012 with the shed (span: 8 m, length: 25 m, height: 3.8 m, light transmittance: 30%) in Xinduqiao Town, Kangding Country, Sichuan Province. The climate is the plateau continental monsoon. The annual mean air temperatures were 209.56°F. The annual rainfall was 922.7 mm. The annual frost free time was 2525.9 h. The annual frost free time in a year was 95 days (Jun et al., 2009).

Breeding Experiment
The experimental design was a single factor with 4 level sand randomized in complete blocks with three replicates. Three kinds of media were sifted with sieve (10 meshes). Four nursery media were S1 (100% humus soils with dung of Hepialidae Laravae), S2 (100% humus soils), S3(50% humus soils +50% sandy soil)and S4 (100% sandy soil). The test numbers for sowing with seeds were marked as SS1, SS2, SS3, and SS4. The test numbers of sowing with one-year-old bulbs were SB1, SB2, SB3, and SB4. Seedbed (length: 7 m, width: 1 m, height: 0.06 m) was set up in the shed. Seedbed was split up into plots (length of each plot: 1.00 m, width of each plot: 0.25 m, size of each plot: 0.25 m 2 ) with bricks. Organic mulch was put at the bottom of each seedbed. The time of sowing was Mar 2011. The density of seeds was 10000 per square meter. The density of one-year bulbs was 5000 per square meter. Irregularly watering and weeding, monthlyobserving and recording the growth status of seeding were until 'sprout tumble', in other words, the aboveground part of plant had the state of being wilted and lodging.

Observations
Bulbs weight and size were recorded after 'sprout tumble'. To record the size and to record the fresh biomass yield (Table 1), 30 bulbs were randomly selected from each plot, wiped with a clean, damp cloth or gauze pad, and menstruated by caliper. The bulbs were dried at 1,859°F to a constant mass for 48h. The media simples were taken back to Sichuan Agriculture University in March 31, 2012. Substrates samples were taken at the beginning of Mar with cutting ring V100 cm (Ф50.46×50).
Soil physic factors were measured as described by Guang-song (1996): Water content in soils was measured with oven drying method; soil bulk density (BD), total porosity (TP), aeration porosity (AP) and water retaining porosity (WRP) were measured with cutting ring method. In addition, the soil chemical factors were measured as described by Ru-kun (1999): soil pondus hydrogenii (pH) in water was measured with potentiometer; organic matters were measured with potassium dichromate oxidation and heating method; the cation exchange capacity (CEC) was determined by the ammonium saturation method; total nitrogen (N) was with semimicro-Kjeldahl determination; total phosphorus (P) in the extract was determined by the ascorbic acid-molybdate blue; total potassium (K) was with the flame atomic emission spectrophotometer; hydrolysable nitrogen was with determination of hydrolysable nitrogen; available P was with Mo-Sb colorimetric method; available K was with neutral ammonium acetate digestion and flame photometers method.

Statistical Analyses
Statistical analyses were carried out by SPSS v19 (SPSS Inc., Chicago, USA). One-way ANOVA with Fisher's LSD tests were performed to examine sprouting percentage, shapeindexes and biomass of bulbs for the different treatments. The Correlativity Analyzing tests were used to test the relations between pH with sprouting percentages. Correlativity Analyzing tests were used to test the relations among BD, Hydrolysable N with shape of bulbs. Significance was determined at p= 0.05.

The Physicochemical Properties of Media
The physical properties of the media have a great influence on the cultivation of crops growth. Bulk density (BD), total porosity (TP), aeration porosity (AP) and water retaining porosity (WRP) were important traits (Li-hong, 2004). In this test, the physicochemical properties of media were showed in Tables 2 and 3.
The pH values of all treatments were in the suitable range (5.3-7.0) of main crops growing in media (Shi-zhe, 2001). The highest value of organic matter, total nitrogen and hydrolysable nitrogen was S2 (100% humus soils). The highest value of total phosphorus (P), totalpotassium (K), available phosphorus (P) and available potassium (K) was S1 (100% humus soils with dung of Hepialidae Laravae). The data from the determination of cation exchange capacity (CEC) showed: S2 (71.9%)>S1 (48.5%)>S3 (39.2%)>S4 (4.12%). The CEC was used to evaluate the capacity of soil fertility conservation. So the data of CEC indicated that humus soil contained soluble salt, and the sandy soils have very low capacity of nutrient supplying.

Sprouting Percentage
The germination rates varied in different nursery media (Table 4). The greatest degree of sprouting (63.25%) was observed of F. cirrhosa in sowing with seeds for SS1 followed by SS2 and SS3. SS4 showed very poor sprouting with mean values of 48.40%. The greatest degree of sprouting (91.30%) was observed for SB1 of F.cirrhosa in sowing with one-year-old bulbs-it was significantly higher than all other treatments. The application of SB2, SB3 and SB4 had no significant effect on the sprouting rateof F.cirrhosain sowing with one-year-old bulbs. The study revealed that the germination rate of ripe seeds was significantly less than one-year bulbs. Sowing with seeds and one-year-old of F. cirrhosa, S1 (100% humus soils with dung of Hepialidae Laravae) was the best nursery media for sprouting rate.
pH had high significant negative correlation at the 0.05 level with sprouting rate of F. cirrhosa seeds with correlation coefficient of 0.966 and also had significant negative correlation at the 0.01 level with sprouting percentage of F. cirrhosa one-year-old bulbs with correlation coefficient of 0.960 (Table 5). It explained that pH had significant influence onsprouting percentage of F. cirrhosa seeds and one-year-old bulbs. Based on former researches in cropssuch as cole and paddy, the best pH for seed germination of different crops was different and pH had significant effects on amylase, protease and lipase during seed germination, thereby affecting sprouting percentage of seeds (Cheng-cang & Fa-shui, 1998). The pH values of four treatments were in the suitable range (5.3-7.0) of main crops growing in media, in contrast, S1 (100% humus soils with dung of Hepialidae Laravae) with pH value of 5.3 was the best nursery media for sprouting percentage.

The Influence of Nursery Substrates on Shape of F. cirrhosa Bulbs
Bulbuls Fritillaria cirrhosa were divided into three groups (Chinese Pharmacopeia, 1980) Vol. 5, No. 12;2013 Therefore, the R of Bulbuls Fritillaria cirrhosa was 0.95~1.13. R could directly express the appearance of Chinese herbal medicines, in addition, R was usedas the index for commercial evaluationfor controlling the appearance quality standards of Bulbuls Fritillaria cirrhosa (Jun et al., 2009). So if R of Bulbuls Fritillaria cirrhosa were in this rang (0.95~1.13), the shape of bulbs was regard as good. Transverse diameter represents the size of bulbs in the traditional commercial market of Bulbuls Fritillaria cirrhosa. In this test, the influence of nursery substrates on shape of F.cirrhosa one-year-old and two-year-old bulbs were showed in Table 6 and  Table 7, respectively. The correlation analyses of the shape of F. cirrhosa with BD and Hydrolysable N were showed in Table 8.

One-Year-Old Bulbs
The study revealed that SS1 achieved average of transverse dia. (4.17 mm) -this was significantly more than the other treatments in the test.It indicated that S1 (100% humus soils with dung of Hepialidae Laravae) was the best medium for promoting the seedlings and getting the large size of bulbs.Viewed the SS4, it achieved average of longitudinal dia. -this was significantlyless than the other treatments ,but it achieved average of transverse dia.-this was just significantly less than SS1and was no significantly difference with the others in the test. It indicated that S4 (100% sandy soil) could inhibit the growth of bulbs, getting the better shape of bulbs. In addition to SS4, R (the ratio of longitudinal dia. to transverse dia.) of the other treatments were not in the range of 0.95~1.13. It explained that the growth of bulbs' longitudinal dia. were greater than the growth of bulbs' transverse dia. in SS1, SS2 and SS3. R of SS4 was significantly less than others and the transverse dia. of SS4 was the least.

Two-Year-Old Bulbs
SB1 (6.05 mm) and SB2 (5.77 mm) had no significantly difference with each other in average of transverse dia., but both were significantly larger than SB3 and SB4. It indicated that S1 (100% humus soils with dung of Hepialidae Laravae) and S2 (100% humus soils) were advantageous for the growth of plantlet bulbing and getting the large size of bulbs. SB3 (6.05 mm) and SB4 (5.77 mm) had no significantly different with each other in average of transverse dia. and longitudinal dia., but both were significantly less than SB1 and SB2. It indicated that adding sandy soil to substrates or 100% sandy soil asnursery media could inhibit the growth of bulbs, getting the better shape of bulbs. R of all treatments were not in the range of 0.95~1.13. It explained that the growth of bulbs' longitudinal dia. were greater than the growth of bulbs' transverse dia. in all media. The Rand transverse dia. of SB3 and SB4 were both significantly less than SB1 and SB2.

Correlation Analysis of the Shape of F. cirrhosa with BD and Hydrolysable N
BD had significant negative correlation at the 0.05 level with R of F.cirrhosa one-year-old bulbs and two-year-old bulbs with correlation coefficient of 0.968 and 0.960. It explained that BD had significant influence on R of F. cirrhosa one-year-old bulbs and two-year-old bulbs. Hydrolysable N had significant positive correlation at the 0.05 level with R of F. cirrhosa one-year-old bulbs and two-year-old bulbs with correlation coefficient of 0.954 and 0.956. It explained that Hydrolysable N had significant influence on R of F. cirrhosa one-year-old bulbs and two-year-old bulbs.

The Influence of Nursery Substrates on Biomass of F. cirrhosa Bulbs
In production, the same weight of bulbs was consistent with their nutrient content (Jun et al., 2009). Therefore, when the amount of bulbs was same, the heavier weight of bulb was, the more nutrient content of bulbs contained.

One-Year-Old Bulbs
The study revealed that SS1 achieved 30-grain fresh weight (1.25 g) and 30-grain dryweight (0.2923 g) -this was significantly more than the other treatments in the test. It explained that S1 (100% humus soils with dung of Hepialidae Laravae) was advantageous for the seeding growth and getting the large size of bulbs. The bulbs were dried at 1,859°F to a constant mass for 48 h. The moisture rates of all treatments had no significantly differentwith each other.The mean value of moisture rates of SS1, SS2, SS3 and SS4 were 76.58%, 74.16%, 74.98% and 74.30%, respectively.

Two-Year-Old Bulbs
Viewed 30-grain fresh weight and 30-grain dry weight, both SB1 and SB2 were significantly more than SB3 and SB4. The 30-grain fresh weight of SB1 and SB2 were 5.15 g and 4.29 g, respectively. The 30-graindry weight of SB1 and SB2 were 1.3058 g and 1.8750 g, respectively. It indicated that S1 (100% humus soils with dung of Hepialidae Laravae) and S2 (100% humus soils) were advantageous for the growth of plantlet bulbing and www.ccsenet.org/jas Journal of Agricultural Science Vol. 5, No. 12;2013 getting larger size of bulbs. SB3 (72.73%) and SB2 (71.61%) had no significantly difference with each other inmoisture rate, but both were significantly less than SB3 and SB4. It indicated that bulbs contained relatively less free water, which was plant with adding sandy soil to substrates or 100% sandy soil.

Discussion
Considered the result of media nutrients content in this test (Table 3) and the classification of the soil nutrient status of second soil investigation in China (1992 , Table 11), the study showed that the classifications of the available nutrient of S1，S2，S3 and S4 were grade1, grade 2, grade 3 and grade 6, respectively. Consequently, the media of better available nutrient were S1 and S2, and the medium of the poor available nutrient was S4. Studied dry weight of bulbs, SS1 was significantly more than the other treatments, and both SB1 and SB2 were significantly more than SB3 and SB4. According to the survey, F. cirrhosa seedings could be stored on S4 which contained the poorly available nutrient. It explained that seedling growth possibly needed fewer nutrients. S1 was made of humus soils with dung of Hepialidae Laravae. Dung was one of agricultural wastes. Carlisle and Wilson (1991) considered that the main direction of choosing media was taken full advantage of the agricultural wastes and realized resources reusable utilization. In this test, S1 (100% humus soils with dung of Hepialidae Laravae) achieveddry weight of one-year-old and two-year-old bulbs -these were significantly more than the other media. It inferred that the excretion of Hepialidae Laravae contained physiological active substance except N, P, and K, which were played an important role on enhancing dry weight of bulbs. Currently, the physiological active substance of Vermi-compost had been studied (Albanell et al., 1998).
Based on field investigation of cultivating Bulbuls Fritillariae cirrhosae, predecessors and author found that bulbs had been obviously produced, but the reasons of produced bulbs were unclear. There were only data for one year, besides the test of single factor was absent. So the causes of produced Bulbuls Fritillariae cirrhosae bulbs were not explicated by the results of this test. If combining the results of this test with the causes of tuberization by predecessors studying, the reasons of produced Bulbuls Fritillariae cirrhosae bulbs were several possible explanations as followings: Firstly, plant cell division and produced growth of plant cell were sensitive to water. Plant growth needed a certain degree of tougor pressure. Shortage of water would make tougor pressure reduce or even disappear, and it serious affected cell division andcell produced growth to inhibit the growth of plants (He-sheng, 2002). The main condition which made produced growth of cell was water.
Water of environment would automatically enter from extracellular to intracellular ( ∆φ 0 )-protoplast expanded-promoted turgor pressure-expandedprimary cell wall-produced growth of cell (Ji-shu, 1999). In this test, Bulbuls Fritillariae cirrhosae bulbs were in media environment. Consequently, the unsuitable management measures probably made media contain too much water. Meanwhile based on field investigation of cultivating Bulbuls Fritillariae cirrhosae by author, the produced Bulbuls Fritillariae cirrhosae bulbs were found in soil with shortage of water. It inferred cell produced growth of Bulbuls Fritillariae cirrhosae bulbs were sensitive to shortage of water. This phenomenon was supported by the findings of He-sheng (National Soil Survey Office, 2002) that shortage of water hardly affect produced growth of roots-growing areas. To sum up the above arguments, the morphogenesis of Bulbuls Fritillariae cirrhosae bulbs was stick in water of media, and this result was used for reference for suitable management measures of Bulbuls Fritillariae cirrhosae.
Secondly, N was contained in stems and leaves in the prophase and metaphase of growth of crops (He-sheng, 2002). Therefore, N could promote division and growth of stems. The bulb of Bulbuls Fritillariae cirrhosae was a kind of modified subterranean stems. In this test, Hydrolysable N had significant positive correlation at the 0.05 level with R of F. cirrhosa one-year-old bulbs and two-year-old bulbs with correlation coefficient of 0.954 and 0.956. The total N of S1, S2 and S3 (1.061%, 1.530%, 0.865%, respectively) and the hydrolysable N of S1, S2 and S3 (599 mg/kg, 673 mg/kg, 374 mg/kg, respectively) were higher than the nutrients level of general soils.
To sum up the above arguments, one of causes of produced Bulbuls Fritillariae cirrhosae bulbs was probable nursery media contained higher nutrient (N). This inference was supported the findings of Hui-jun et al. (2006) and Yuan et al. (2012) that nitrogen supplying had impact on produced growth of several crops stems.
Thirdly, harvested organ was the underground parts of crops such as sweet potato and the findings of Kazuyukiand Toshio (1964Toshio ( , 1965 were that increasing the soil bulk density would stunt sweet potato roots swelled. In this test, media bulk density had significant negative correlation at the 0.05 level with R of F.cirrhosa one-year-old bulbs and two-year-old bulbs with correlation coefficient of 0.968 and 0.960. Therefore, www.ccsenet.org/jas Journal of Agricultural Science Vol. 5, No. 12;2013 media bulk density was probableone of the causeof the growth of bulbs' transverse dia. less than the growth of bulbs' longitudinal diameter. Be perpendicular to longitudinal diameter R The ratio of longitudinal diameter to transverse dia. One-year-old bulbs Sow with seeds and reap bulbs after 'sprout tumble' in the same year Two-year-old bulbs Sow with one-yaer-old bulbs and reap bulbs after 'sprout tumble' in the same year   Mean value followed by the same letter are not significant difference at p≤0.05.
R=the ratio of longitudinal dia. to transverse dia. Mean value followed by the same letter are not significant difference at p≤0.05.
R=the ratio of longitudinal dia. to transverse dia. R=the ratio of longitudinal dia. to transverse dia. Mean values followed by the same letter are not significant difference at p≤0.05.  Note: the worse the quality of soil, the greater grade number.

Conclusion
Under the nursery mode of shed at Plateau, Sowing with seeds and one-year-old bulbs of F. cirrhosa, S1 (100% humus soils with dung of Hepialidae larvae) was the best nursery media for sprouting.