GUAN Hong-jiao, SUN Bing, YANG Yu-jie, HU Die, FEI Yong-jun

Evaluation and Innovation Center of Phoebe Germplasm Resources, Yangtze University, Jingzhou 434025, PRC

Abstract To study the leaf variation and morphological diversity of Phoebe bournei, the leaves of two leaf types and one wild type in the same growth environment, which were marked as Type A, B, and C with wild type being marked as B, were used as testing materials. 40 healthy mature leaves collected from healthy plants were taken as research samples and phenotypic characteristics, such as leaf shape, leaf length, leaf width, leaf area, and leaf vein were observed and analyzed. The leaves of Type A were oblanceolate, the base of the leaves was gradually narrow, and the tip of the leaves was gradually sharp. There were white pubescences on the back of the leaves, which were arranged in order. The pubescences were evenly distributed on the mesophyll and vein, in the same direction. The length, width, perimeter, and area of the leaves were smaller than those of the other two types. The leaves of Type B were narrow, oblanceolate, wedge-shaped at the base, long acuminate at the tip, with yellow brown villi on the back of the leaves, mainly distributed along the vein and the grid vein between the veins; the villi on the main veins were longer, and the villi between the mesophylls were less numerous and shorter. The length, perimeter, and aspect ratio of the blade were the largest of the three types and the whole blade was slender. Type C leaves were elliptical with a nearly truncated base and a sharp tip. There were white pubescences on the back of the leaves and less mesophyll pubescence along the veins. The length-width ratio of the blade was small, the width was large, and the whole blade was round. There were significant differences among the three leaf types of P. bournei and preliminary judgement was that Type A and Type C were spontaneous mutants.

Key words Phoebe bournei; Leaf shape; Spontaneous mutants

1. Introduction

Phoebe bournei

is an evergreen tree of the genus

Phoebe Nees

in the family Lauraceae, which is a national class II key protected wild plant in China.

P. bournei

is a unique and precious timber tree species that is an excellent ornamental tree.

P. bournei

prefers humidity and is shade tolerant, has few plant diseases and insect pests, grows well at the slope bottom with good water and humidity conditions, and is naturally distributed in Fujian, Jiangxi, Hunan, Guangxi, Guangdong, Guizhou, Zhejiang, Hubei, and Sichuan and among others.

P. bournei

is famous in China for its fragrant and durable wood, compact and tough material, smooth surface, and beautiful texture. It has a high ecological and economic value.The leaf is the main organ of photosynthesis, and it is the energy converter of a primary producer in an ecosystem. In addition to their main role in light harvesting, leaves are also important for nutrient storage, defense, and stress responses. Spontaneous mutants are mutations caused spontaneously under natural conditions. They are an important source of biological variation and the basis of natural evolution. The mechanism of spontaneous mutation is the change of genetic material, including chromosome aberrations and gene mutations. The results of these mutations can be morphological or physiological changes. Leaf mutations are mainly divided into leaf color and leaf type. WANG P

et al

. found novel natural yellow leaf mutants (YR) of “Rougui” with the same genetic background. Some researchers have found a new spontaneous phenotype of the rice cultivar 'L-202'. Its distinguishing features are: dark blue-green colour, short and narrow leaves, high tittering and relatively short height. In this study, three leaf types of

P. bournei

were examined morphologically through phenotypic observation and data analysis to verify whether they were spontaneous mutants.

2. Materials and Methods

2.1. Sample collection

The research materials came from 7-year-old

P. bournei

plants located in the Agricultural Science and Technology Industrial Park of Yangtze University, Jingzhou National High-tech Zone, Hubei Province. Jingzhou City is located in the south-central part of Hubei Province, in the middle reaches of the Yangtze River, and the hinterland of the Jianghan Plain. It has a subtropical monsoon climate. The total annual solar radiation in Jingzhou City is 104~110 kcal/m, the annual sunshine hours are 1 800~2 000 h, the annual average temperature is 15.9°C~16.6°C, the annual frost free period is 242~263 d, and rainfall in most years is 1 100~1 300 mm.

2.2. Experimental method

40 healthy mature leaves were randomly selected as experimental samples. A Scanmaker i800 plus scanner (Zhongjing Technology Co., Ltd., China, resolution 300 dpi) was used to obtain pictures of each sample for analysis. Leaf length, leaf width, leaf area and leaf circumference were obtained using the WANSHEN LA-S plant image analysis system (Hangzhou WANSHEN Testing Technology Co., Ltd, China). The vein logarithm of each leaf was counted on the image, and the length width ratio of each leaf was calculated by the obtained data. The shape, tip, base, and vein of three different leaf types were compared and analyzed and the number of veins on each leaf was counted. A DFC550 body microscope (Beijing Groupca Technology Co., Ltd., China) was used to collect the microscopic images of three typical leaf variations.

2.3. Data analysis

Data and graphics were processed by Origin Pro 2019 software. Analysis of variance was performed by SPSS 23.0 software and the Duncan method was used for multiple comparisons.

3. Results

3.1. Comparison of overall plant morphological characteristics

According to the measurement results of the tree height and diameter at breast height of the three leaf types of

P

.

bournei

in the test samples, the plant height of the

P

.

bournei

Type A leaves was 2.5 m and the base diameter was 3.68 cm. The plant height of the

P

.

bournei

Type B leaves was 3.3 m and the base diameter was 4.02 cm. The plant height of the

P

.

bournei

Type C leaves was 2.2 m, and the base diameter was 2.81 cm. Compared with Type B (the wild-type plant), Type A had a smaller plant shape, while Type C had a larger base diameter, a smaller crown, and round and thick leaves.

3.2. Comparison of leaf morphological charac- teristics

As shown in Fig. 1a, the Type A leaves were oblanceolate with a leaf length of about 6 cm and a width of about 2.5 cm, and the leaves were smaller overall. The Type B leaves (The normal leaf shape of

P. bournei

) were narrow and oblanceolate with a length of 15 cm and a width of 3 cm, and the leaves were relatively slender. The Type C leaves were elliptical with a length of about 10 cm, a width of about 5 cm, and they were relatively wide. To summarize, Type A leaves were oblanceolate and smaller; Type B leaves were narrow and oblanceolate; and Type C leaves were elliptical and wider.Fig. 1b showed a principal component analysis graph (PCA) with the 6 index data of the three leaf types of

P. bournei

, including leaf length, leaf width, and leaf area, as parameters. From the figure, it can be clearly seen that the scattered points of the three leaf types were more concentrated and each of the three leaf types form clusters, which showed that the three types of

P. bournei

had obvious differences.

Fig. 1 Comparison of three typical leaves of P. bournei and a principal component analysis diagram

3.3. Comparison of leaf local morphology

As shown in Fig. 2, there were differences in leaf microstructure phenotypes. The leaf base of the Type A leaves (Fig. 2a) was gradually narrow, the Type B leaves (Fig. 2d) were wedge-shaped and narrow, and the edge of the leaf base was slightly curled towards the back of the leaf, and the Type C leaves (Fig. 2g) were nearly truncated and wider. From the aspect of leaf apex morphology, the leaf apex of the Type A (Fig. 2b) and Type B (Fig. 2e) leaves were all acuminate, but the leaf apex of the Type B leaves was more slender, and the leaf apex of the Type C leaves (Fig. 2h) was acute. In terms of the difference in leaf attachment, there was white pubescence on the back of the Type A leaves (Fig. 2c), which was evenly distributed on the mesophyll and vein, arranged in order and in the same direction. There was yellow brown pubescence on the back of the Type B leaves (Fig. 2f), which was mainly distributed along the vein and grid vein. The villi on the main vein were longer, and the villi between the mesophyll were less numerous and shorter. The Type B leaves (Fig. 2i) had white pubescence on the back, which was distributed along the vein with less on the mesophyll. Comparing the distribution of leaf veins, the veins of the Type C leaves (Fig. 2i) were prominent and the grid veins between the lateral veins were clear and prominent. The lateral veins of the Type A leaves (Fig. 2c) were shallow and the grid veins between the lateral veins were almost invisible. The Type B leaves (Fig. 2f) had lateral veins and grid veins and the depth of the veins was between that of the Type A and Type C leaves.

3.4. Comparative analysis of morphological indices of the three leaf types

As shown in Fig. 3, the Type A leaves had the smallest leaf width, leaf length, leaf perimeter, and leaf area among the three types of leaves, with a moderate aspect ratio. The leaf size of the Type A leaves was the smallest among the three types but the width was moderate. The Type B leaves had the largest leaf length, perimeter, and aspect ratio, and moderate leaf width and area. The Type B leaves were long and narrow, and moderate in size. The Type C leaves had the largest leaf width and area among the three types of leaves, with moderate leaf length and perimeter. The Type C leaves were the widest among the three types of leaves, the length was moderate, the shape was elliptical, and the leaf area was the largest. As shown in Fig. 3F, the number of vein pairs of the Type A leaves ranged from 9 to 14, among which 12 was the most common number. The number of vein pairs of the Type B leaves ranged from 10 to 14, among which 12 and 14 were the most common numbers. The number of vein pairs of the Type C leaves ranged from 7 to 11, among which 8 to 10 were the most common numbers. There were no significant differences in the number of veins between the Type A and Type B leaves but the number of veins in the Type C leaves was relatively small.

Fig. 2 Leaf microphenotypes diagram of the three types of P. bournei leaves

Fig. 3 Comparative analysis of morphological indices of three types of blades

In terms of the degree of dispersion of the data, except for the relatively discrete logarithm data of the leaf vein, the shape of the Type A leaves was relatively stable. The Type B leaves were discrete in leaf length, width, circumference, area, and logarithm of veins, which indicated that the size of the Type B leaves changed drastically. Apart from leaf length, leaf area, and vein logarithm, The Type C leaves were also discrete, which showed that the size of the Type C leaves was also unstable. The degree of dispersion of the aspect ratio of the three types of leaves was small, indicating that the three types of leaves could maintain a stable basic leaf shape.

To summarize, the Type A leaves were the smallest and the perimeter and area were significantly smaller than those of the Type B and Type C leaves. However, they had a stable leaf shape and size. The Type B leaves were the longest and narrowest, with a length longer than that of the Type A and Type C leaves, in other words, a typical narrow oblanceolate leaf. However, the size of the Type B leaves was not stable and there was a difference in leaf size. The Type C leaves had the smallest aspect ratio, and were the widest and most oval. Comparative analysis of the sample data confirmed the morphological differences in the three leaf types of

P. bournei

.

4. Conclusion

In this study, two phenotypic variant leaves of

P. bournei

were compared with wild-type leaves. Morphological identification was carried out through observation of leaf morphology, leaf local microscopic observation, and leaf morphological index data analysis. The results showed that leaf types A, C and wild type B had significant differences in leaf size, leaf shape, and leaf attachments. The mutation of Type A was manifested in the overall reduction of the leaf size, and the leaf pubescence was white and uniformly distributed. The mutation of Type C showed that the shape of the leaf became oval, the length of the leaf became shorter, the width of the leaf became wider, and the yellow-brown pubescent hairs were mostly distributed along the vein. It was preliminarily judged that

P. bournei

leaf types A and C were spontaneous mutants.

5. Discussion

At present, more than 94 varieties of

Phoebe

are known and 37 are present in China. According to the “Flora of China”,

P. bournei

leaves are lanceolate or oblanceolate with apex tapering or long tapering, base tapering or shaped, and with 10~14 lateral veins on each side. Through observation and comparative statistical analysis, the leaf phenotype and records of the three plants in this study were different. Preliminary judgement was that the Type A and Type C leaves of

P. bournei

were mutants.The phenotypic variation of leaves was a common reflection of plant genetic variation and environmental differences. SUN B

et al

.compared the morph- ological characteristics of the typical

Phoebe

with the fission of the leaves, and finally identified a new variety-Phoebe

Phoebe

. This was similar to the research point of this research. The

P

.

bournei

in this research underwent leaf variation under natural conditions, and its surface characteristics were significantly different, hence it was initially identified as a natural mutant.Because morphological identification cannot detect all the mutants, in order to further verify the mutation of

P. bournei

leaves, it was necessary to combine physiological identification and molecular identification, and in-depth study of its mutations from the perspective of cytology, histology, and genetics.As for the cause of leaf mutation, further research is needed. The molecular mechanism of leaf formation is mainly regulated through gene level control, hormone regulation control and environmental factor control. Since the

P. bournei

in this study grew in the same environment without the addition of artificial hormones, we believed that the plant leaf mutations were largely due to gene level control. Genes participate in various developmental processes of leaves, such as polar cell expansion, transduction of hormonal signals, gene regulation, plastid biogenesis, and chromatin remodeling, among others. In the genetic analysis of rice with a narrow-leaf phenotype mutant, it was found that the

nal1

single mutation and the

nal2 nal3

double mutation had a severe effect on leaf width, resulting in very narrow leaves. As the basic unit of plant growth and development, cells in different states constitute different tissues and morphologies. Tsukaya’snew cell theory holded that the morphology of plant leaves was determined by the different states of cells. Therefore, we can think that the size of plant leaves may be caused by mutations in genes that determine cell state. From the cellular behavior involved in organ size control and leaf-shape patterning, mutants showed changes in leaf size caused by specific change in the number and/or size of cells. Due to the complexity of leaf ontogeny, the specific reasons for the mutation of

P. bournei

leaves need to be further studied from such aspects as physiological indicators, cell morphology, genes, and so forth.