Pisum Genetics
2010-Volume 42
Research Papers
Field quantification of foliar chlorophyll content in Pisum
Ambrose, M.J. John Innes Centre, Norwich, UK
Variation in the chlorophyll content of the foliage of peas has been long documented. Traditional quantification of chlorophyll levels in leaves is by acetone extraction and spectrophotometer analysis (1) which takes time and requires laboratory equipment and facilities. In the classification of cultivated germplasm, the variation in the colour of foliage is graded based on morphological descriptor states. The UPOV guidelines for distinctness, uniformity and stability for Pisum (2) recognises three descriptor states, yellow green (J), green (2) and blue green (3). State 2 (green) is further broken down into light (3), medium (5) and dark (7). Three descriptor states are used in recording on the John Innes Pisum Collection namely, 1. yellow green, 2. green, 3. dark green. While these scales are clearly discernable by eye, a quick and reliable objective method of quantifying this variation could be useful in quantifying this character. The portable Minolta SPAD 502 chlorophyll meter determines the relative chlorophyll in leaf tissue by measuring absorbance at two wavelengths, namely in the regions of 400-500nm and 600-700nm which are characteristics of chlorophyll absorption peaks. Initially developed for monitoring the nitrogen status of wheat crops, the meter has subsequently been deployed on a range of monocot crop and woody species where good linear relationships between SPAD readings and leaf chlorophyll content were obtained (3). The method has also been used in crop nitrogen studies in pea (4, 5, 6) and in studies in chickpea (7, 8). This is the first deployment of the meter on pea germplasm in order to establish whether its utility could be extended to studies of pea germplasm and mutation stocks.
1. Survey of descriptor states for leaf colour
Readings were collected from a reference set of morphological variation in pea growing in the field against wire. A representative accession for each of the three descriptor states used when recording on the John Innes Collection were all assessed on the same day in early June using the fully expanded leaflets and stipules 3-4 nodes below the shoot apex. The reading showed a range of 32 SPAD units between the lowest (27 SPAD) and the highest (59 SPAD) with clear separation between each of the three colour classes (Fig. 1).
Figure 1. Variation in foliage colour for threeJI accessions growing in the field. a. yellow green (JI799), b. green (JI1889) and c. dark green
Pisum Genetics
2010-Volume 42
Research Papers
Readings were taken from the same accessions two weeks after the initial set at the end of flowering (end of June). The two sets of readings were very similar with the same levels and differentials between lines evident throughout (data not shown).
The area of leaf tissue that is clamped in the meter has a diameter of 13mm and an aperture for the light beam to pass through of 2mm x 3mm. This raised the question as to whether the variable degrees of grey flecking on foliage which is varies greatly between different accessions might possibly interfere with readings thus leading to high deviations across a leaf surface in the readings proved unfounded. The meter produced consistent readings between leaflets and stipules where there is frequently a differential degree of flecking. Flecking is caused by air spaces underlying the outer epidermis (9). The expression of flecking varies from totally absent which is encountered infrequently, through to a near continuous airspace as in the mutant argentum (Arg) characterised by the even silvery appearance of all foliage. Leaflets of the type line for Arg (JI 1397) gave SPAD readings for intact leaflets of 53, 46.4 and 48 which were in the middle to high range for peas. Reading across an individual leaflet where the outer epidermis was intact provided readings of 48, 44 and 47 SPAD compared to SPAD readings of 44, 45 and 46 across the area where it had been removed. The very slight reduction in the readings was not significant and converse to what might have been expected as the removal of the epidermis to expose the underlying mesophyll cells results in a significantly greener looking tissue.
2. Survey across the JI Pisum Test Array
The opportunity was taken to take readings from a test array of broad Pisum taxonomic diversity that was growing in the field at the same time (10). This consisted of 6 plants of 56 lines grown as clumps. Two measurements were taken on the 16/06 (all lines were in flower) from different plants of each accession which were then averaged. The range of SPAD values over the lines ranged from a minimum of 22.65 units (JI 102) to a maximum of 53.8 units (JI 399) with a mean of 41.46 units and a standard deviation of 6.39 units. The lines were then grouped on the basis of taxon or geographic region of origin (Fig. 2).
This highlighted the relative high readings obtained for the four accessions of Pisum abyssinicum which as a group had a mean of 45.29 units ± st. dev.7.10. All 5 accessions of this taxon share an absence of grey flecking on the foliage. To the naked eye, this material appeared lighter than the SPAD readings indicated
Pisum Genetics
2010-Volume 42
Research Papers
suggesting this taxon is different in some way for the epidermal cell layer or in the epicuticular wax. The 4 accessions of Pisum sativum spp. transcaucasicum were noticeably paler green as a group which was reflected in the readings (34.81 units ± st. dev. 2.66). The sativum lines from the Asiatic highlands were separated out as they clearly form a distinct ecogeographic group which included 4 accessions of the 'Afghanistan type'. This distinct form of cultivated pea is noted for variation in their ability to nodulate with European strains of Rhizobium Lines resistant to nodulation are easily noted as becoming precociously yellow during the pod filling stage due to the total reliance on available soil nitrogen (ll, 12). This is regularly observed in such material in the low fertility sandy loam of the experimental plots at the Jic. Four 'Afghan type' accessions scored by Young and Matthews (12) were present in the test array and one line in particular (JI 102) was noticeably yellow. The data for these four accessions is presented in Table l and shows the two accessions resistant to nodulation provided lower SPAD values.
The number of data points was too low to perform significance tests but the readings obtained for the two groups were non-overlapping.
3. Survey of genetic stocks of leaf chlorophyll mutants
In the autumn a glasshouse survey was undertaken on a range of type lines and mutants for genes associated with foliage chlorophyll content. This included four lines that had been measured in the field earlier in the summer (section l). For this survey, three readings were taken from separate leaflets (2nd true leaf) from three separate plants. The plants were expanding their leaflets at the fifth vegetative node with the exception of JI 35 (yellow green) where the measurements were taken from the youngest expanded leaflets at the top of the plant (node 4) where the phenotype is expressed (13, 14).
No significant differences were found between the readings obtained in the field or glasshouse showing the SPAD reading to be consistent for these lines across these independent sowings and environments (Table 2).
In this survey of 12 lines (Fig. 3), a threefold range of values obtained from the palest line (JI 2414) to the darkest (JI 1405). JI 2414 is a genetically uncharacterised stable chlorophyll b mutant where the whole plant is a uniform bright light green (15). This was lower than the type line for o (JI 799) which is a pale yellow green and gave an mean value of 31.4 SPAD units. The highest SPAD readings obtained were for the cov type line (JI 2729) and JI 1405 which is equally dark green in appearance came with mean SPAD values of 67.46 and 61.8 respectively.
Pisum Genetics
2010-Volume 42
Research Papers
While this is a small survey of mutant stocks from the total number of chlorophyll mutants isolated in pea, they serve to demonstrate the range of greens of foliage observed in chlorophyll contents. The studies outlined in this paper demonstrate the suitability of the SPAD-520 chlorophyll meter for working with Pisum over a wide range of intensity of green coloured foliage. The meter is highly portable and proved easy to calibrate and quick and reliable to use. While differences at the extremes of the range and the mid point are easy to score by eye, variation within material in the mid-range is much harder. It is in this range or having an objective measure of chlorophyll across sites and/or years where this device might prove particularly useful.
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2. UPOV Code PISUM SAT, Pisum sativum L. TG/7/10.
3. Bullock, D.G. and Anderson, D.S. 1998. Journal of Plant Nutrition 21: 741-755.
4. Zhao, F.J., Wood, A.P. and McGrath, S.P. 1999. Plant and Soil 212: 209-219.
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11. Govorov, I.I. 1928. Bull. Appl. Bot. Genet. Pl. Br. 19: 497-522.
12. Young, P.J. and Matthews, P. 1982. Heredity 48: 203-210.
13. Kellenbarger, S. 1953. J. Genetics 51: 41-46.
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15. Stummann, B.M. and Henningen, K. W. 1984. Photosynthesis Research 5: 275-292.