Báo cáo khoa học: "Evaluation of some markers of vigor in seedlings of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco)

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Evaluation of some markers of vigor in seedlings of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) J.B. Zaerr V.D. Hipkins K. Forry Department of Forest Science, Oregon State University, Corvallis, OR 97331,U.S.A. dates throughout the planting season and di- Introduction vided into lots of 70 seedlings for each treat- ment. The following treatments were applied: timely identification of damaged seed- The drying seedlings with exposed roots in a warm, lings or seedlings of low vigor is an impor- dry room (30°C, 30% relative humidity) for 0.25, tant aspect of forest regeneration in the 0.5 or 1 h; freezing to -9, -12 or -15°C; and storage at 2°(; for 0, 2 or 4 mo. There was a Pacific Northwestern United States. When total of 30 treatments. Survival in the field plantation fails because seedlings die, a indicated that a wide range of survival percent- replanting is necessary. If damaged or ages was achieved and that there were fairly seedlings of low vigor could be identified well distributed intermediate values. before they are planted, considerable For each treatment, 20 seedlings were potted labor savings could be realized as well as in soil and placed in a greenhouse for measure- improved productivity of the forest (Cleary ment of RGC (number and length of new roots et al., 1982). A practical test of seedling after 30 d), 20 were potted in soil and placed in vigor must be easy to conduct and should a growth chamber (21 °C, 16 h photoperiod) to measure survival and bud-burst, 20 were provide results quickly. At the present outplanted at a field site near Corvallis (30 x 30 time, the root growth capacity (RGC) test cm spacing), and 10 were used for samples of is used extensively, but results require 2-4 plant material. Xylem sap was extracted with a wk and do not always predict field survival pressure chamber apparatus and the osmotic accurately. The objective of this study was concentration of the sap was measured with an osmometer immediately. The roots were ex- to evaluate 3 recently suggested alterna- cised, steamed for 5-10 min to inactivate the tive indicators of seedling vigor: the starch native enzymes, frozen with liquid nitrogen, lyo- content of roots (Puttonen, 1986), the philized and stored at -80°C until analyzed for osmotic concentration of xylem sap and (n . Starch was analyzed by the starch )= 1 0 biochemical markers of seedling vigor amylase/amyloglucosidase method of Haissig and Dickson !1982). The apical 2 cm of the (Duryea, 1985). shoot were excised, ground with methanol in a polytron, extracted 5 times with hexane to remove chlorophyll, dried and stored at -80°C Materials and Methods until analyzed by high performance liquid chro- matography (FIPLC). For analysis by HPLC, the Two yr old seedlings of Douglas fir (Pseudo- dried sample was taken up in 1 ml of methanol tsuga menziesii (Mirb.) Franco) were lifted on 4 and a 25 pl sample was injected into a Varian
related to survival. Comparable results model 5000 HPLC fitted with B C, a reverse phase column (Spherical ODS, 0.46 x 2 cm, obtained using either the presence were 5 ;um), and a Beckman 164 variable wavelength or the area of peaks. Fig. 1 shows a ca- detector set at 254 nm in tandem with a nonical plot of field survival using peak fre- Perkin-Elmer 650-10S fluorescence spectro- quency data. In this case, the discriminate photometer (ex/em 290/360 nm). The flow rate was 1 ml/min and the solvent gradient was analysis using 9 peaks completely sepa- linear from 10% methanol in 20 mM triethyla- rated the 3 classes of field survival. An mine acetate (pH 3.37) to 65% methanol at 20 even better separation can be achieved by min to 85% methanol at 25 min to 100% metha- using additional peaks (up to 20). If fewer nol at 27 min holding at 100% until 40 min. Data peaks are used, the classes of field survi- were collected with a Keithley DAS 500 con- nected to an IBM XT and controlled by MAXIMA val tend to overlap. Similar results were software. Statistical analysis was conducted obtained when peak presence or peak with SAS programs and included cluster analy- area was tested against survival in the sis and stepwise discriminate analysis (Afifi and growth room or against RGC. Clark, 1984). Results Discussion and Conclusion Measurements of osmotic concentration of The osmotic concentration of xylem sap is xylem sap varied widely within treatments clearly a poor marker of vigor in Douglas and between treatments. There was no fir seedlings except in the case of freshly apparent overall trend. For individual treat- frozen plants. In the latter case, osmotic ments there were no apparent trends concentration measurements could proba- except for seedlings which had been bly detect badly frozen seedlings (high damaged by freezing but had not been low survival) and osmotic concentration = stored. For freshly frozen seedlings, might be useful in situations where recent increased osmotic concentration was an freezing damage was suspected. indicator of low survival. The concentration of starch in roots The starch content of roots increased might be used as an indicator of date of from a low on the first lifting date (October, lifting, but it is not a useful indicator of 2%) to a high on the last lifting date plant vigor. Common sense suggests that (March, 11%). This increase in starch plants low in starch might have difficulty content did not match the overall field sur- surviving when outplanted, particularly on vival of those plants. Root starch content harsh site. However, data show that our a was not related to survival in the growth plants low in starch can survive, at least room or to survival in the field. Even when on some sites. Although we measured considering the overall increase in starch field survival, we do not yet have a mea- content with lifting date, root starch surement of growth for these seedlings. It content did not predict growth room or could be that root starch content is more field survival. closely related to growth than to survival. Cluster analysis of the chromatography The chromatography data were found to data indicated a strong effect of lifting be related to RGC, survival in the growth date, but stepwise discriminate analysis room and survival in the field. The most was more useful in relating chromatogra- important relationship was that found be- phy peaks to survival data. Of the 230 dis- tween a small number of peaks (up to 20 tinct peaks observed, 20 were found to be
can be used but only 9 were used suggested by this study as being predic- peaks in the example shown in Fig. 1 ) and field tive ofvigor and to test the ability of peaks survival. This result implies the possibility on an HPLC chromatogram to predict the vigor of outplanted seedlings. After suffi- of using a few peaks on an HPLC chroma- togram to predict vigor of outplanted seed- cient field testing, this technique might be developed into a reliable operational lings. Efforts currently underway to are chemically the compounds method for evaluating seedling vigor. characterize
Abilities of Major Tests. Forest Research Labo- References ratory, Oregon State University, Corvallis, OR, pp. 143 Afifi A.A. & Clark V. (1984) In: Computer-aided & Dickson R.E. (1982) Glucose mea- Haissig B. Muttivariate Analysis. Lifelong Learning Publica- surement errors in enzymatic starch hydroly- tions, Belmont, CA, pp. 246-286; 379-411 1 sates at high enzyme-glucose weight ratios. Cleary B.D., Greaves R.D. & Hermann R.K. Physiol. Plant. 54, 244-248 (1982j In: Regenerating Oregon’s Forests. Ore- gon State Univ. Extension Serv., Corvallis, OR, Puttonen P. (1986) Carbohydrate reserves in . 287 PP Pinus sylvestris seedling needles as an attri- bute of seedling vigor. Scand. J. For. Res. 1, Duryea M.L. (1985) In: Evaluating Seedling 181-193 Quality: Principles, Procedures, and Predictive