Advance in Forestry Research February 2014, Volume 2, Issue 1, PP.

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Seedling Cultivation Techniques and Genetic Variation in Growth Traits and Their Relationships to Toona Ciliata Families
Shijun Wu 1, Jianmin Xu 1#, Guangyou Li 1, Chao Han 1, Yang Hu 2, Xinxian Hu 1
1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangdong Guangzhou 510520; 2. Xinhui Research Institute of Forestry Science, Guangdong Jiangmen 529100 #Email: wushijun0128@163.com

Abstract
The height of seedling, ground diameter and number of leaves were assessed for seedling cultivation techniques and151 Toona ciliata families. Analysis of variance showed that there were significant differences on studied traits among treatments. The medium treatment 1-2 (Yellow Subsoil: Vermiculite=1:1) and 2-6 (Light Medium) could improve the growth traits of seedling, suggesting that this two treatments could be popularized in seedling cultivation for Toona ciliate. The results showed that there were significant differences on studied traits among families, indicating clear differences among families. Coefficients of genotypic variation were 32.96% for height, 24.27% for ground diameter, 48.23% for number of leaves. Heritabilities were 0.96 for height, 0.81 for ground diameter, 0.91 for number of leaves. The strongly positive phenotypic correlations suggested that selection on one traits can be effective to predict other studied traits of Toona ciliata families. Keywords: Cultural Techniques; Genetic Variation; Toona Ciliata Families; Growth Traits

1 INTRODUCTION
As the global population grows and areas of native forest decreases, the protection of forest species has become more and more important for ecosystem to meet the increasing demand for wood products, particularly in developing countries such as China (Xu and Dell 2002; Wu et al. 2012; Wu et al. 2013) having become a leading country in the production, consumption, import and export of forest products in general, and the total output value of forestry sector in China, reaching US$180 billion in 2007, has developed rapidly along with the overall growth of the national economy (Ma et al. 2009; FAO 2009). Among the genera selected for protection and timber, Toona ciliata seems to be a suitable tree species to supply the wood on a long-term sustainable basis because of fast-growing, well adaptability, wide-distribution, excellent wood properties, vigorous sprout and wide-use (Xie 2010; Zhao et al. 2005; Liang et al. 2011; Wu et al. 2006; Wu et al. 2011; Cheng and Cui 2010; Wang et al. 1999). Toona ciliate, belonging to Toona of Meliaceae, being a rare and native timber species in China is distributed from 2421 N to 3031N and 10016 E to 11940 E, mainly including Gunagdong, Guangxi, Fujian, Sichuan, Guizhou and Yunnan provinces (Wu et al. 2011). However, Toona ciliate has been listed in Red Book of China Plant and became the national second class endanger species because of the human activity, biological characteristics and environment and so on (Xie 2010; Liang et al. 2011). Unfortunately, very little information about breeding and seedling cultivation techniques of Toona ciliate in China has been published. Liang (2011) summarized the reasons of endanger and brought forward some protection strategies for Toona ciliate. Cheng and Cui (2010) analyzed the main utilization value and cultivation techniques of Toona ciliate. Wu (2011) reported that there was significant difference in the rooting rate among different Toona ciliate clones and the cuttings with two pieces of leaves could improve the rooting rate significantly. The objectives of this study were to: (1) compare growth traits of different container, medium and families (2) estimate among-family variation and heritability of traits (3) examine the genotypic relationships between growth traits, (4)
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explore the implications and techniques for Toona ciliate improvement. This information will be used to develop appropriate selection strategies for Toona ciliate protection and utilization in China.

2 MATERIAL AND METHODS

2.1 Trial Description
The trial was established at Xinhui Research Institute of Forestry Science in Jiangmen City of Guangdong (2233N, 11302E, 45m asl), and affected by the south subtropical monsoon with annual mean temperature of 22.3 C and annual mean rainfall of 1750 mm. Mean January temperature at this location is 6.7 C, mean for July 28.3 C and the minimum temperature is 2.9 C. There is not frost period and average annual mean accumulated temperature is 7653 C.

2.2 Methods Description

At least 10 g Toona ciliate seeds of each family were collected from 151 plus trees distributed in Yunnan, Fujian and Jiangxi provinces from July to December 2011. The sowing, transplant seedlings and test were carried out in March, May and October. The height of seedling (HGT) was measured by ruler, and the ground diameter (GD) was measured by calipers. The number of leaves (NL) was also counted. Mixed families were used for cultivation techniques, and then measured at 5-month. Two types of containers and six treatments were used with four replicates, and each treated with 30 seedlings (Table 1). The size of black container is 17 cm 7.5 cm, and the white one is 17.0 5.0 cm. Compound fertilization with N15-P15-K15 was used in medium, and the volume percentage was 0.25%. Light medium contained coconut chaff, perlite and peat soil.
TABLE 1 DETAILS OF DIFFERENT CONTAINER AND MEDIUM TREATMENT

Container

Black Container

White Container

Treatment No 1-1 1-2 1-3 1-4 1-5 1-6 2-1 2-2 2-3 2-4 2-5 2-6

Treatment Yellow Subsoil: peat Soil =1:1 Yellow Subsoil: Vermiculite=1:1 Vermiculite: Peat Soil =1:1 Yellow Subsoil: Peat Soil: Vermiculite =1:1:1 Yellow Subsoil: Peat Soil: Vermiculite =3:2:1 Light Medium Yellow Subsoil: Peat Soil =1:1 Yellow Subsoil: Vermiculite =1:1 Vermiculite: Peat Soil =1:1 Yellow Subsoil: Peat Soil: Vermiculite =1:1:1 Yellow Subsoil: Peat Soil: Vermiculite =3:2:1 Light Medium

Seedling Number 30 30 30 30 30 30 30 30 30 30 30 30

Replicates 4 4 4 4 4 4 4 4 4 4 4 4

Total Seedling 120 120 120 120 120 120 120 120 120 120 120 120

2.3 Statistical Analysis

The significance of fixed effects was assessed using F-tests. Meanwhile, the analysis of variance was performed using the PROC ANOVA. yij =+i+i +ij (1) where yij is the performance of the ramet of ith families (container or treatment) within the jth replicate, and is the general mean, i is the random effect of the ith families (container or treatment), j is the random effect of the jth replicate ,ij is the random error. The heritability of the clone mean was calculated following Hai et al. (2008a): R = 2a/(2a + 2e), where 2a is the additive genetic variance of clone, 2e is the residual variance. The genotypic coefficient of variation (CV) was calculated as (Pliura et al. 2007; Hai et al. 2008a): CV=(100c/x) 100%, (3) where x is the phenotypic mean. The equation expresses a standardized measure of the genetic variance relative to
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(2)

the mean of trait.

3 RESULTS AND DISCUSSION

3.1 Variance among Containers, Medium Treatments and Replicates
The analysis of variance of studied traits among containers, medium treatment and replicates is presented in Table 2. The results showed that there were highly significant differences on HGT and GD except NL among container, indicating that the size of container could affect the HGT and GD of seedling. The differences among medium treatment were also highly significant on HGT, GD and NL, indicating medium treatment could affect the HGT, GD and NL of seedling. However, there were significant differences on HGT at 0.01 level and significant differences on GD and NL at 0.05 level among replicates, indicating that the seedling of different replicates had different growth.
TABLE 2 ANALYSIS OF VARIANCE OF THE STUDIED TRAITS WITH DIFFERENT CONTAINERS, DIFFERENT MEDIUM TREATMENT AND DIFFERENT REPLICATES

Traits HGT

GD

NL

Type Container Medium Treatment Replicates Container Medium Treatment Replicates Container Medium Treatment Replicates

DF 1 5 29 1 5 29 1 5 29

F 31.06 21.76 7.07 270.65 19.45 1.49 1.2 5.36 1.64

Pr>F <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0459 0.2736 <0.0001 0.0172

HGT height; GD ground diameter; NL number of leaves

TABLE 3 AVERAGE VALUE OF STUDIED TRAITS AMONG CONTAINERS AND MEDIUM TREATMENTS

Treatment 1-1 1-2 1-3 1-4 1-5 1-6 Average Value 2-1 2-2 2-3 2-4 2-5 2-6 Average Value

HGT (cm) 14.46 21.94 20.15 18.59 16.61 20.51 18.71 16.78 21.19 20.26 19.92 18.31 22.57 19.84

GD (mm) 3.527 4.306 4.077 3.975 3.612 3.568 3.844 3.079 3.661 3.53 3.705 3.39 3.583 3.491

NL 6.035 7.033 6.913 6.396 5.889 6.164 6.405 4.767 5.805 5.753 5.655 5.867 6.37 5.703

HGT height; GD ground diameter; NL number of leaves

3.2 Average Value of Studied Traits among Containers and Medium Treatments
Mean average values of HGT, GD and NL with two different containers and six medium treatment are presented in Table 3. The significant variance due to medium treatment found in joint analysis for all growth traits (Table 2) indicated clear differences between medium treatment in growth traits. The medium treatment 1-2 (Yellow Subsoil: Vermiculite=1:1) could improve the HGT, GD and NL of seedling, whereas the studied traits with medium treatment 1-1 (Yellow Subsoil: peat Soil =1:1) were the lowest, suggesting that medium treatment 1-2 (Yellow Subsoil: Vermiculite=1:1) could be popularized in seedling cultivation with first black container for Toona ciliate. The medium treatment 2-6 (Light Medium) could improve the HGT, GD and NL of seedling, whereas the studied traits with medium treatment 2-1 (Yellow Subsoil: Vermiculite=1:1) were the lowest, suggesting that medium treatment 2-6 (Light Medium) could be popularized in seedling cultivation with second white container for Toona ciliate. It was interesting to note that the average value of HGT and GD of the second white container (17.0 5.0 cm) were higher than that of the first black one (17cm 7.5 cm), however, the seedling with first black container had more NL
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than second white one. Many papers have been published about the seedling cultural techniques of Eucalyptus (Shang et al. 2010), Pinus Kesiya var langbianecsis (Xu et al. 2007) and Taxus wallichiana var. Willichiana (Yuan et al. 2012).

3.3 Variance among Families and Replicates

The analysis of variance of studied traits among families and replicates is presented in Table 4. The results showed that there were significant differences on HGT, GD and NL among families, indicating that clear differences among families. Furthermore, there were significant differences on HGT and GD among replicates, whereas the differences on NL among replicates were not significant, suggesting the less environmental effects on these traits.
TABLE 4 ANALYSIS OF VARIANCE OF THE STUDIED TRAITS AMONG FAMILIES AND REPLICATES

Traits HGT GD NL

Type Families Replicates Families Replicates Families Replicates

DF 150 29 150 29 150 29

F 31.01 11.36 5.13 1.8 11.59 0.66

Pr>F <0.0001 <0.0001 <0.0001 0.0052 <0.0001 0.9138

HGT height; GD ground diameter; NL number of leaves

3.4 Family Variation and Heritability

Mean value and ranges, coefficient of variation and heritabilities of HGT, GD and NL are presented in Table 5. The mean, minimum and maximum of HGT were 22.30, 3.00 and 74.00 cm respectively. The mean value of GD and NL were 2.98 mm and 5.92 mm. The coefficient of variation of HGT, GD and NL were 32.96%, 24.27% and 48.23%, respectively. The coefficient of variation of NL was bigger than others, indicating that the scope for selection among clones would be considerable. These results were bigger than the previous studies by Liu et al. (2008), in which coefficients of variation of HGT and GD were 27% and 21% for Toona ciliata var. Pubescens in Jiangxi province. Liu et al. (2011) reported that the coefficients of variation of Phoebe bournei were 20.3% for HGT, 13.8% for GD and 32.0% for NL. Estimates of heritabilities for HGT, GD and NL were 0.96, 0.81 and 0.9 respectively. This conclusion was consistent with previous studies in Toona sinensis (Liu et al. 2010) and bigger than that in Toona ciliata var. Pubescens (Liu et al. 2008). Liu et al. (2010) reported that the heritabilities of HGT and GD were 0.76 and 0.78 for Toona sinensis collected from 17 provinces and cultivated in Fujian province.
TABLE 5 MEAN VALUE, RANGES, COEFFICIENT OF VARIATION AND CLONAL REPEATABILITY OF THE STUDIED TRAITS AT 21, 52, 71 AND 96 MONTHS OLD

Traits HGT (cm) GD (mm) NL

Mean 22.30 2.98 5.92

Minimum 3.00 0.43 2.00

Maximum 74.00 10.35 16.00

SD 10.96 1.11 1.54

SE 0.17 0.02 0.02

CV% 32.96 24.27 48.23

H 0.96 0.81 0.91

HGT height; GD ground diameter; NL number of leaves; SD standard deviation; SE standard error; CV genotypic coefficient of variation; H heritability

3.5 Phenotypic Correlations between Studied Traits

Phenotypic correlations among traits at three ages are listed in Table 4. In this study, HGT had strongly positive phenotypic correlations with GD and NL, ranging from 0.25 to 0.62. The phenotypic correlation between GD and NL was 0.25. The correlations between HGT and GD, however, were generally higher than for those between other studied traits, indicating the importance of HGT in selecting for GD. It was interested to find that the phenotypic correlation between GD and NL had the same value between HGT and NL. Liu et al (2008) reported that the phenotypic correlation between GD and HGT of Toona ciliata var. Puescens was nearly 1.00, indicating that HGT can be used as prediction for GD. Liu et al (2011) summarized that HGT can be use as primary trait for Toona ciliata var. Puescens family selection. Chen et al (2011) found positive phenotypic correlation (0.754) between GD and HGT of Betula alnoides in Yunnan province.

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TABLE 6 PHENOTYPIC CORRELATIONS AMONG STUDIED TRAITS

GD NL
*

HGT 0.62 0.25

GD 0.25

, **means significant differences at 5% and 1% level respectively. HGT height; GD ground diameter; NL number of leaves

4 MAJOR CONCLUSIONS AND IMPLICATIONS FOR BREEDING PROGRAMS

In the present study, the variance among treatments, replicates and families, the genotypic parameters of the studied traits and relationships among height, ground diameter and number of leaves were examined. Joint analysis in the present study showed four implications for tree improvement in Toona ciliate in China. Primarily, the medium treatment 1-2 (Yellow Subsoil: Vermiculite=1:1) and 2-6 (Light Medium) could improve the growth traits of seedling, suggesting that these two treatments could be popularized in seedling cultivation for Toona ciliate. Secondly, the results showed that there were significant differences on studied traits among families, indicating clear differences among families. Thirdly, the coefficient of variation and estimates of heritabilities of studied traits were medium to higher, implying that the family selection would be high-efficiency. Furthermore, the correlations between height and ground diameter was 0.62, indicating the importance of height in selecting ground diameter.

ACKNOWLEDGMENTS
The authors gratefully thanked Prof. K. Harding and RE. Pegg for their earlier passional help to improve our writing ability, Dr. Siming Gan and Dr. Jie Zeng for their suggestions and helps and Hongjian Huang and Peitao Tan from Xinhui Research Institute of Forestry Science for their painstaking assistance in sample investigation. Comments from anonymous reviewers are also appreciated.

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AUTHORS
Dr. Wu Shijun, was born in 1984, postgraduated from Chinese Academy of Forestry in Beijing in 2012. He was majored in Forest Genetics and Breeding. This study was undertaken as a project for the Resource Investigation, Collection and Introduction of Toona ciliata (2011KJC007-01). Currently, he is working in Research Institute of Tropical Forestry Chinese Academy of Forestry in Guangzhou. His many finished papers were published in New Forests, Silva Genetica, Journal of Tropical Forest Research and Journal of Forestry Research. His email: wushijun0128@163.com.

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