Silva Fennica 35(2) research articles Can Physiological and Anatomical Characters Be Used for Selecting High Yielding Hybrid Aspen Clones?

Stomatal, CO2 exchange parameters and several leaf and growth traits were recorded in a fi ve-year-old hybrid aspen clone trial. The fi eld trial consisted of four aspen hybrid clones (Populus tremula L. × P. tremuloides Michx.) and one local Populus tremula seedling source. The mean estimated height of hybrid aspen clones was 1.6 times higher than for P. tremula. Similarly, basal diameter was 1.5 times and breast diameter 1.8 times higher in the hybrids. Differences were observed for physiological and growth traits among hybrid clones and P. tremula, but, only stomatal characters of hybrid clones differed signifi cantly from those in P. tremula. Hybrid clones had larger guard cells (22.9 μm) than P. tremula (19.2 μm), whereas P. tremula had a higher stomatal density (211.3/mm2) than the hybrid clones (164.4/mm2). Among four hybrid clones, net photosynthesis was strongly correlated with foliar nitrogen. Height correlated signifi cantly with foliar nitrogen, but negatively with leaf fresh weight, leaf dry weight and stomatal density. The results suggested that yield components could be controlled by many genes, specifi c to each clone. No single gas exchange or morphological variable can provide a reliable indicator of yield potential.


Introduction
Aspen hybrids (Populus tremula L. × P. tremuloides Michx.), mainly produced from crosses made in the 1950's have displayed hybrid vigor in Finland (Beuker 1989).The phenomenon of hybrid vigor in aspen hybrids is well documented in the US (Li and Wu 1996, 1997, Li et al. 1998).
To improve the yield of forest crops, plant and environmental variables that infl uence and control growth should be fully understood (Michael et al. 1990).Yield is a complex trait, which crop physiologists partition into components.It is important to identify genotypes with traits associated Silva Fennica 35(2) research articles with high yield and to understand the relationships among the traits.Yield involves various morphological, physiological, and biochemical components that are each regulated by different genes.The analysis of yield components and their genetic control in Populus has featured centrally in many studies (Hinckley et al. 1989, Stettler and Ceulemans 1993, Stettler et al. 1996).Biomass yields and their physiological basis, as well as the economic importance and practical aspects of intensively cultured poplar plantations, have been thoroughly examined (Zsuffa et al. 1993, Heilman andStettler 1985).
In tree breeding the use of yield components is new, but essential for understanding why certain breeds and hybrids exhibit superior growth.Signifi cant variation in yield components among Populus clones have been found in many studies (Ceulemans et al. 1987, Orlovic et al. 1998, Thomas et al. 1997a, 1997b).These studies have sought for physiological and anatomical measures that could be used to select superior genotypes (Ceulemans and Impens 1983, Ceulemans et al. 1987, Orlovic et al. 1998).However, the correlations between net photosynthesis and growth have been ambiguous (Barigah et al. 1994, Gatherum et al. 1967, Okafo and Hanover 1978, Reighard and Hanover 1990).Most physiological studies have been made in greenhouses, or based on 1-2 year old plants in the fi eld (Ceulemans et al. 1987, Orlovic et al. 1998).The correlation between performance in the greenhouse and in the fi eld has been variously proved signifi cant (Ceulemans et al. 1987) or non-signifi cant (Thomas et al. 1997).
Photosynthetic capacity is dependent on the concentration of N-containing enzymes, pigments, and electron transport components (Evans 1989).An association of foliar nutrient concentration with light-saturated photosynthesis has been found in deciduous and herbaceous species (Field andMooney 1986, Reich et al. 1995).Foliar nitrogen (N) concentration has been used to estimate net photosynthesis because of the strong coupling between the two (e.g., Field and Mooney 1986).
According to our knowledge, these types of studies have not been undertaken in aspen hybrid clones.We began a study to explore the physiological basis of heterosis by comparing yield components between hybrid aspen clones and a local Finnish P. tremula seedling source.The objectives of the study are: 1) to assess variation in physiological and anatomical characters among hybrid aspen clones and local P. tremula and 2) to establish relationships among growth, photosynthesis, leaf and stomatal traits and 3) to analyze whether some of these characters could be used to predict aspen hybrid growth.

Plant Material
The material used in the study of 1998 was a clone trial established by the Foundation for Forest Tree Breeding and the University of Helsinki.The trial is located in Viikki, Helsinki University (lat. 60°14´N,long. 25°05´E,alt. 10 m).The trial was planted in May 1994 with oneyear-old plants, and consists of four aspen hybrid clones (Populus tremula × P. tremuloides) and one P. tremula seedling source (Table 1).Clones 1-4 are known to be species hybrids between P. tremula and P. tremuloides, but in the case of clones 2, 3 and 4 there is uncertainty about the direction of the cross, and regarding the exact origin of the parental.Each clone was planted as a plot consisting of 4 × 4 plants spaced at 2.5 m × 2.25 m.The plots were laid out in a design of fi ve randomized complete blocks, giving a total of 16 trees × 5 entries (4 clones + one P. tremula) × 5 blocks = 400 trees.In each plot, all the characters were measured on the fi ve tallest trees.

Measurement of Growth
For observation of dynamic seasonal growth patterns, height, breast diameter at height 1.3 m and basal diameter 15 cm from the ground were measured every 2 or 3 weeks (8 measurements) over the period May 31 till October 19 in 1998.Measuring positions were marked on the stems to assure that repeated measurements were taken at exactly the same position.The last measurement on October 19, 1998 was used together with other traits for analysis of variance and correlation analysis.

Measurement of Gas Exchange Parameters
Observations of gas exchange parameters were recorded on July 23, 1998.Net photosynthetic rate (µmol CO 2 m -2 s -1 ), stomatal conductance (mol m -2 s -1 ), intercellular CO 2 concentration (µmol CO 2 /mol), and transpiration rate (mol H 2 O m -2 s -1 ) were measured with a Li-6400 portable photosynthesis system (Li-Cor Inc.).The measurements of gas exchange parameters were made on a branch on the south side of the lowest third of the crown.The fi rst three fully expanded leaves from the top of the shoot that had emerged in spring were sampled.In measuring, the parameters, we wished to examine the response of the trees to conditions actually prevailing in the fi eld.Accordingly, measurements were made under natural ambient light and temperature levels.The leaves were measured in direct sunlight.The measurement was made between 1100 and 1600 h at 1650-1850 µmol m -2 s -1 photosynthetically active radiation (PAR).The temperature stayed within the range of 26-27 °C and the relative humidity varied from 62-68%.Flow rate through the sample cell was 500 µmol s -1 with a CO 2 concentration of 325-335 µmol mol -1 .The measured leaf area taken from near the centre of the lamina was 6 cm 2 .

Measurement of Leaf Characters
Leaf characters were measured immediately after the gas exchange characters.Leaf area (cm 2 ), and the specifi c leaf fresh mass (mg/cm 2 ) and specifi c leaf dry mass weight/area (mg/cm 2 ) were recorded.Leaf shape was defi ned as the ratio of maximum width and maximum length of the leaves.For the measurements of leaf characters, 10 leaves were taken from the same branch that was used to measure gas exchange parameters.Leaf area and shape were measured with a LI-3000A portable area meter (Li-Cor Inc.).Leaf dry mass was obtained after the leaves were dried for 48 h at 105 °C.

Measurement of Foliar Nitrogen
The same 10 leaves used for photosynthetic and leaf character measurements were analysed for total nitrogen using a CHN-900 carbonhydrogen-nitrogen analyser (LECO Corporation, St. Joseph, MI, U.S.A).Foliar nitrogen was expressed as a percentage of total dry mass in the leaves.

Measurement of Stomatal Properties
The samples on which stomatal characters were measured were the same as for the measurements of CO 2 exchange parameters, but only one leaf from each individual tree was observed.Leaves were kept fresh until observation.Stomatal density (stomata/mm 2 ) and mean guard cell length (µm) of fresh leaves were observed with a Scanning Electron Microscope JSM-820 (JIOL, Japan).A piece of leaf about 1 cm 2 in area was cut from the middle of the lamina close to the main vein between two sub-veins.The leaf specimen was coated with platinum, and then observed under 400× magnifi cation.Sixteen fi elds were observed for stomatal density and one fi eld was transferred to a computer as an image for the measurement of guard cell length per unit area (µm/mm 2 ).Total guard cell length (µm) was calculated as the mean guard cell length multiplied by stomatal density.Plot means of the observations were used in the data analysis.Stomata occurred only on the abaxial surface of the leaf.

Statistical Analyses
All calculations were based on plot means.The differences among the hybrid clones and the P. tremula were examined by an analysis of variance (ANOVA) using the PROC GLM procedure of the SAS statistical software package (SAS Institute Inc. 1989) with the type III estimation of sum of squares.Entry means of gas exchange parameters, stomatal and leaf characters, and growth traits were separated by Tukey's multiple range test at level of signifi cance of p ≤ 0.05.Pearson's correlations were calculated using the SAS  PROC CORR procedure to assess the linear relationships between the studied traits among hybrid clones.

Growth and Gas Exchange Parameters
Seasonal growth patterns are illustrated in Fig. 1 as means of height, breast and basal diameter of the hybrids and P. tremula.Height, breast and basal diameter of the four hybrid clones were signifi cantly higher than for the P. tremula (Table 2).P. tremula gave higher coeffi cients of variation for growth and gas exchange parameters than the hybrid clones.There were no statistically signifi cant differences in stomatal conductance, intercellular CO 2 concentration or transpiration rate.There was signifi cant difference in net photosynthesis among the four hybrid clones and P. tremula, but no difference in net photosynthesis between mean of four hybrid clones and P. tremula.

Stomata and Leaves
Among the stomatal and leaf traits, only stomatal density and total guard cell length of P. tremula gave higher coeffi cients of variation than the hybrid clones (Table 3).Some differences were found among clones for stomatal and leaf characters among four hybrid clones and P. tremula (Table 3).However, there were no signifi cant differences in leaf fresh weight or dry weight

Relationship of Gas Exchange Parameters with Foliar Nitrogen and Growth
Non-signifi cant correlation was found between gas exchange parameters and growth traits of height, breast and basal diameter.Net photosynthesis correlated signifi cantly with foliar nitrogen, negatively with leaf area and stomatal density (Fig. 2).Foliar nitrogen was correlated with transpiration rate (r = 0.49, P = 0.02), and negatively correlated with stomatal density (r = -0.55,P = 0.01).

Discussion
In the present study, signifi cant variations were found in the wide array of growth, photosynthetic, stomatal and leaf traits among the hybrid  clones and P. tremula.The existence of such variation indicates that clones could rather easily be selected for further breeding and practical cultivation.High levels of clonal variation have been found for morphological, growth and molecular traits in trembling aspen (Chong et al. 1994, Lund et al. 1992, Reighard and Hanover 1990;Thomas et al. 1997aThomas et al. , 1997b)).Nelson and Ehlers (1984) noted that net photosynthesis was under strong genetic control in two hybrid Populus clones.Parameters for gas exchange and growth traits in P. tremula showed higher variation than parameters in hybrid clones.Because the P. tremula was a half-sib family, the coeffi cient of variation of P. tremula was higher than for the hybrid clones.A similar pattern was found for stomatal density and total guard cell length, but not for length of stomata or any of the leaf traits.It appears, characters like growth and gas exchange parameters, including length of stomata and total guard cell length, that show a higher coeffi cient of variation for the P. tremula than for hybrid clones, are better predictors of yield than characters (e.g.leaf traits) where the coeffi cient of variation for P. tremula is smaller than in the hybrids.
The relationships between carbon fi xation and yield for aspen indicate that no correlation or occasionally a negative one, exists between leaf or whole plant net photosynthesis and productivity (Okafo and Hanover 1978, Reighard and Hanover 1990, Thomas et al. 1997a).However, a signifi cant positive correlation between net photosynthesis and biomass production was reported in hybrid poplars (P.euramericana) and P. deltoides by Orlovic et al. (1998).For aspen, in particular, previous studies reported a poor or negative correlation between either leaf or net photosynthesis of whole plants and productivity (Okafo and Hanover 1978;Reighard and Hanover 1990).
Our results showed that net photosynthesis was not correlated with height, breast diameter or basal diameter.For aspen clones in growth chamber experiments, Thomas et al. (1997a) showed a positive relationship between net photosynthesis and dry mass or height growth, but correlations were not signifi cant between physiological characters measured in the growth chamber and fi eld measurements of either physiological or growth characters.
In our study, a signifi cant correlation was found between foliar nitrogen and net photosynthesis among the four hybrid clones.This indicates that foliar nitrogen might be used to predict net photosynthesis in aspen.The result is in agreement with a fi nding by Field and Mooney (1986).Foliar nitrogen has proved a good predictor of net photosynthesis (Reich et al. 1995).The biochemical basis for the relationship between foliar N and net photosynthesis is known (Evans 1989).For C 3 species, Reich et al. (1995) described the relationship between photosynthetic capacity and leaf nitrogen concentration with one general equation.
Variations in stomatal density and mean guard cell length might refl ect plant growth.A positive correlation was found between stomatal density and fast growth in Betula pendula Roth (Wang et al. 1995) and Azadirachta indica A. Juss (Kundu and Tigerstedt 1998).Likewise, a strong correlation was demonstrated between the number of stomata on the leaf adaxial surface and biomass in Populus hybrids (Orlovic et al. 1998).However, Ceulemans et al. (1987) found no correlation between stomatal density (either adaxial or abaxial) and growth.In our study stomatal density was correlated negatively with height, but did not correlate with breast or basal diameter.Stomatal density decreased linearly with mean guard cell length (Fig. 4).The phenomenon is known for many plants, e.g.oak trees (Abrams and Kubiske 1990) and peach (Loreti et al. 1993).Since stomatal density increased as fast as the mean guard cell length decreased, there was no signifi cant difference between total guard cell length of the mean of four hybrid clones and P. tremula.Nonetheless, signifi cant differences existed between hybrids and P. tremula for mean guard cell length and stomatal density.The hybrid clones had a lower stomatal density but a larger mean guard cell length compared to P. tremula.For example, the hybrid clone 1 and P. tremula both have almost the same values of total guard cell length, but stomatal density of P. tremula was the highest, and mean guard cell length was the smallest among the entries (Table 3).Leaf anatomical characters were affected by variations in the light regime (Garcia Nunez et al. 1995).At high light intensities stomatal density normally increased with the time the leaves are exposed to continuous light (Koike et al. 1998, Zacchini et al. 1997).
The high stomatal density and small mean guard cell length in P. tremula may be associated with the adaptation of this species to long day-length during the summer.In addition, stomatal characters could be also affected by different soil type.Guzina et al. (1995) showed that the differences in the numbers of stomata between Populus deltoides and hybrid poplar (Populus × euramericana) were statistically signifi cant, and a genotype × environment interaction (GE) was shown in fi eld experiments on three types of soil.
In our study, most leaf traits failed to correlate with growth traits.However, leaf fresh mass and leaf dry mass showed a moderate negative correlation with height, breast diameter and basal diameter.Wang et al. (1995) found a negative correlation between leaf specifi c dry mass and net photosynthesis in birch.They suggested that leaves with high dry mass might have high photosynthetic accumulates that inhibit photosynthetic effi ciency.Cain and Ormrod (1984), studying two hybrids and two Populus clones, observed no signifi cant differences among clones in ratio of leaf mass to leaf area.In our study, there were no signifi cant differences among the clones for leaf area, leaf fresh or leaf dry mass.
It is apparent that the correlation between net photosynthesis and growth rate in forest trees is complex and is dependent on many factors.These include the tree population being studied, the age of the trees, the time(s) of year that net photosynthesis is measured, and site conditions (Johnsen and Major 1995).

Conclusions
The mean estimated height of hybrid aspen clones was 1.6 times higher than for P. tremula.Similarly, basal diameter was 1.5 times and breast diameter 1.8 times higher in the hybrids.Because the P. tremula was a half-sib family, the coeffi cient of variation of P. tremula was higher than for the hybrid clones for most traits in this study.Among the physiological and morphological characters examined here, only stomatal characters of hybrid clones differ from P. tremula.Leaf fresh and dry weight was correlated negatively with these growth traits (height, breast diameter and basal diameter).None of the other traits showed signifi cant correlations with all the growth traits.Considering the complex interactions among factors governing yield, it is doubtful whether any single gas exchange or morphological variable can be a useful and reliable indicator of yield potential.Our analyses were based on a small number of clones measured over only fi ve years.The results of the study need to be followed up by larger and longer experiments.Further studies are needed to confi rm results found in this study.
In a separate paper concerning the same material (Yu et al. 2001) we investigated the relationships between growth and phenological traits.We found that the correlation between growth period and yield was highly signifi cant.The growth period varied from 143-158 days for the four hybrid clones, but was only 112 days for P. tremula.Thus so called hybrid vigor seems here to be mainly attributable to a longer growth period rather than to physiological, leaf morphological or stomatal characters.The long growth period of the hybrids can be at least partly explained by the relatively southern ecotypes of P. tremuloides used in the hybrids (Table 1).Physiological processes are important in accounting for hybrid vigor, but their expression is in turn regulated by daylength and temperature responses of the genotype.

Fig. 1 .
Fig. 1.Growth patterns of mean height, breast diameter and basal diameter for the hybrid clones and P. tremula.Each point is based on a plot mean.

Fig. 2 .
Fig. 2. Relationship between height and leaf specifi c dry mass (a), foliar nitrogen (b), and stomatal density (c) for the hybrid clones.Each point is based on a plot mean.The symbols are as in Fig. 1.

Fig. 3 .
Fig. 3. Relationship between net photosynthetic rate and foliar nitrogen (a), leaf area (b) and stomatal density (c) for the hybrid clones.Each point is based on a plot mean.The symbols are same as Fig. 1.

Fig. 4 .
Fig. 4. Relationship between mean guard cell length and stomatal density for the hybrid clones.Each point is based on a plot mean.The symbols are as in Fig. 1.

Table 1 .
Material used in the study.

Table 2 .
Means and coeffi cients of variation (CV) of growth and gas exchange parameters among the hybrid clones and P. tremula seedling source.Means followed by the same letters are not signifi cantly different at P < 0.05 (Tukey´s HSD test).The mean over hybrid clones and for P. tremula are followed by letters in brackets, which indicate whether or not these means are signifi cantly different at P < 0.05.

Table 3 .
Means and coeffi cients of variation (CV) of the stomatal and leaf traits among the hybrid clones and P. tremula seedling source.Means followed by the same letters are not signifi cantly different at P < 0.05 (Tukey´s HSD test).The mean over hybrid clones and for P. tremula are followed by letters in brackets, which indicate whether or not these means are signifi cantly different at P < 0.05.