HortScience 21(6): 1449-1450. 1986.
Before taking
A Rapid and Nondestructive Method
to Determine Chlorophyll in Intact
Leaves
Umedi L. Yadava
Agricultural Research Station, School of Agriculture, Home Economics,
and Allied Programs, Fort Valley State College, Fort Valley, GA 31030
Additional index words, extraction, portable chlorophyll meter
The important role that chlorophyll (CHL)
plays in plants necessitates its estimation in
various types of studies. Singh and Anantrao
(6) realized the need for a reasonably simple,
rapid, and accurate method of CHL deter­
mination capable of accommodating small
quantities of leaf tissue and a large number
of quantitative estimates in a short time, and
thus, in 1937, they developed a photocon-
ductive photometer for quantifying CHL in
80% methanol. Since then, CHL determi­
nation techniques have improved consider­
ably, but complete extraction of CHL is
laborious, slow, and inconvenient in some
plant species (3). Conventional methods used
for isolating and measuring chlorophyll in
aqueous acetone or similar organic solvents
are sometimes cumbersome and slow, and
always destructive to leaf tissue (1, 4, 7).
Furthermore, steps involved in sample prep­
aration, pigment extraction, and dilution re­
sult in pigm ent loss and contribute to
variability. Moran (5) developed an efficient
method for extracting small quantities of CHL
from intact cotyledons with A,./V-dimethyl-
formamide (DMF). Inskeep and Bloom (3)
further improved this technique by using ex­
tinction coefficients of chlorophyll a and b
extracted in DMF. Evans (2) extracted CHL
in 80% acetone and adapted Amon’s modi­
fication of the method of McKinney (5), who
provided equations (jimol CHL/1 = 22.22
D645 + 9.057 D663) to evaluate the molar
concentrations of total chlorophyll (CHL a
and b) in the tissue extracts. Nevertheless,
these procedures are still time consuming since
they require tissue extraction and spectro-
photometric measurement. The objective of
this investigation was to determine area con­
centrations of total chlorophyll (CHL a +
b) using a portable chlorophyll meter (SPAD-
501) and to compare and correlate these data
with area concentrations of total CHL ob­
tained by conventional methods.
Fresh leaf samples were collected from the
Received for publication 5 Dec. 1985. I thank
Richard E. MacCarty and the Minolta Corporation
for lending the SPAD-501 chlorophyll meter. The
cost of publishing this paper was defrayed in part
by the payment of page charges. Under postal reg­
ulations, this paper therefore must be hereby marked
advertisement solely to indicate this fact.
HortScience, Vol . 21(6), D ecember 1986
following 22 species representing 14 plant
families: cabbage {Brassica olerácea L. var.
capitata L.), camellia {Camellia japónica L.),
cassava {Manihot esculenta Crantz.), sick-
lepod {Daubentonia esculentus L.), collard
{Brassica olerácea L. var. acephala), mus­
cadine grape {Vitis rotundifolia Michx.), bell
pepper {Capsicum annuum L. var. grossum
L.), guar {Cymopsis tetragonoloba L.), ki-
wifruit {Actinidia chinensis Planch, and Ac-
tinidia argüía Sieb. & Z ucc., Planch.),
morning glory {Ipomea purpurea L.), okra
{Abelmoschus esculentus Moench.), papaya
{Carica papaya L.), peach [Prunus pérsica
(L.) Batsch], pecan [Carya illinoensis (Wen-
genh.) Koch.], plum [Prunus americana (L.)
Marshall], poinsettia {Euphorbia pulcher-
rima Willd. ex Klotzsch.), soybean [Glycine
max (L.) Merr.], sweet potato [Ipomea ba­
tatas (L.) Lam.], strawberry [Fragaria X
ananassa (L.) Duchesne], sugarcane {Sac-
charum ojficinarum L.), and yam {Diosco-
rea alata L.). Leaf samples were collected
between 0800 and 1000 hr from plants
growing in field plots, a greenhouse, or out­
doors as weeds. Two 1.35 cm diameter disks
were cut from each of four adjacent leaves,
which were fully expanded, healthy, turgid,
and flat. Soon after cutting, each disk was
individually measured four times for CHL
concentration using a chlorophyll meter
SPAD-501 READINGS
Fig. 1. Linear regression with 95% confidence limits for total extracted chlorophyll (|xmol*m'2) de­
termined by spectrophotometry vs. SPAD-501 readings.
(SPAD-501, Minolta Corp.).
individual measurements, the meter was set
to zero by pressing the same button that is
used for data taking. Following measure­
ments on SPAD-501, both disks from each
leaf were homogenized in 8 ml of 80%
aqueous acetone using a polytron tissuemizer
(Brinkmann Instruments). Optical density
(OD) of filtered aqueous acetone supernatant
was measured at 645 nm and 663 nm on a
spectrophotometer (UV-D spectronic 21,
Bausch & Lomb), using 10-mm path length
cuvettes. These values were used to deter­
mine concentration of total chlorophyll in
micromoles per square meter as described by
Evans (2). Area concentrations of CHL, thus
determined, were plotted against the respec­
tive mean values of SPAD-501 readings for
the same leaf disk pairs. From these corre­
lations, the regression equation (Y = a +
bx) was developed.
Figure 1 shows a linear regression with
95% confidence limits of extracted CHL vs.
SPAD-501 readings. A significant correla­
tion (r = 0.692) was found between SPAD
readings and CHL concentration determined
by conventional techniques. This correlation
might have improved if fewer closely related
plant species or a single species had been
employed, as evident from the relatively high
correlation (r = 0.843) between CHL con­
centration and SPAD readings from 40 leaf
samples of several peach cultivars with widely
variable colors, including a yellow leaf mu­
tation and plum (Table 1). It is revealed from
this table that concentration of SPAD CHL
varied with the color intensity of leaves as
did extracted CHL from the same leaves.
Thus, CHL determination by transforming
SPAD readings into CHL concentration us­
ing the regression equation appears to be sat­
isfactory. Since, SPAD-501 reads leaf CHL
directly, it is convenient for a large number
of samples. The nondestructive nature of this
procedure is particularly useful for such
studies as gas exchange activities, including
photosynthesis and CHL development and
1449
 Table 1. Leaf CHL area concentrations from peach and plum leaves determined by spectrophotometric measurement of leaf extracts
and direct reading of intact leaves on SPAD-501 chlorophyll meter.

Plant Optical density x 1000 Ext CHL concn Spad-501 Spad CHL concn
species 645 nm 663 nm (|jLmol-m“2) readings (|jimol-m-2)
Dwarf peach 391 (26) 749 (51) 432.6 (29.4) 48.0 (2.1) 426.2 (19.4)
Winblo peach 328 (40) 674 (84) 374.3 (46.2) 42.5 (1.5) 376.1 (13.7)
Lovell peach 745 (65) 1351 (93) 387.8 (22.8) 51.8 (1.6) 460.4 (14.9)
Wild peach 624 (24) 1196 (40) 346.8 (08.9) 43.8 (2.7) 387.5 (24.5)
Peach2 417 (39) 998 (91) 183.1 (16.9) 25.5 (0.5) 221.0 (04.6)
Peachy 174 (11) 368 (43) 072.1 (06.3) 08.3 (2.2) 063.7 (19.8)
Peachx 270 (19) 531 (36) 302.1 (20.8) 46.0 (3.7) 407.9 (34.1)
Peachw 307 (47) 642 (97) 353.1 (53.6) 46.8 (0.4) 414.8 (03.9)
Peachv 129 (47) 293 (96) 154.6 (53.7) 22.3 (2.9) 191.4 (26.9)
Plum 349 (16) 658 (28) 383.8 (17.1) 41.3 (2.1) 365.2 (19.1)
zNew yellow leaves on a Red Skin mutation.
yNew yellow leaves from a one-month-old greenhouse seedling of Red Skin mutation.
xGreen leaves from the nonmutated limb of Red Skin tree with mutation.
wYellow leaves turned green on the mutated limb of a Red Skin peach tree.
vYellow leaves turning green on greenhouse seedling of Red Skin mutation.

degradation, that need to be carried out on
the same leaves over a period of time. The
low weight (350 g) of the SPAD-501 CHL
meter, and its battery operation and small
size (7 x 21.15 x 3.7 cm) make it con­
venient for use in the field, greenhouse, or
laboratory. There are a few limitations cur­
rently associated with the use of this instru­
ment; these include a small area (12.57 mm2)
of measurement, maximum measurable leaf
thickness of 1.2 mm, measurement of total
CHL only, and the need for a frequent re­
charging of the Ni-Cd battery (although the
instrument also works on AC current).
It is concluded that CHL determination with
the SPAD-501 chlorophyll meter was rapid,
convenient, reliable, and nondestructive to
leaf tissue. Furthermore, this procedure may
be useful for acquiring data on leaf chloro­
phyll to be incorporated with gas exchange
data from portable systems (as in our work
with LI-COR 6000) for easy handling by
computer.
Literature Cited
1. Bruinsma, J. 1961. A comment on the spec­
trophotometric determination of chlorophyll.
Biochim. Biophys. Acta 52:576-578.
2. Evans, J.T. 1983. Nitrogen and photosyn­
thesis in the flag leag of wheat (Triticum aes-
tivum L.). Plant Physiol. 72:297-302.
3. Inskeep, W.P. and P.R. Bloom. 1985. Ex­
tinction coefficients of chlorophyll a and b in
A,A-dimethylformamide and 80% acetone.
Plant Physiol. 77:483-485.
4. McKinney, G. 1941. Absorption of light by
chlorophyll solutions. J. Biol. Chem. 140:315-
322.
5. Moran, R. 1982. Formulae for determination
of chlorphyllous pigments extracted with N,N-
dimethylformamide. Plant Physiol. 69:1376-
1381.
6. Singh, B.N. and N.K. Anantrao. 1937. A
photoconductive photometer—A new method
and apparatus for the quantitative estimation
of chlorophyll. Current Sci. 5:416-418.
7. White, R.C., I.D. Jones, andE. Gibbs. 1960.
Determination of chlorophylls, chlorophyl-
lides, pheophytines, and pheophorbides in plant
materials. J. Food Sci. 28:431-436.

HortScience 21(6): 1450-1451. 1986.

Transplanting Matured Chinese
Cabbage Plants for Seed Production
M.W. McCaffery, K.A. Keyes, and S. Honma
Department of Horticulture, Michigan State University, East Lansing,
Ml 48824
Additional index words. Brassica campestris (Pekinensis group), cole crops, vegetable
breeding
Since Chinese cabbage, Brassica campes­
tris L. (Pekinensis group), is normally grown
as a fall crop in the northern United States,
it is impossible to obtain seeds from these
plants. The transplanting of selected matured
plants from the field to the greenhouse for
seed production has not been successful, be­
cause a high percentage of the plants suc-
Received for publication 14 Mar. 1986. Michigan
State Univ. Agricultural Experiment Station Jour­
nal no. 11939. The cost of publishing this paper
was defrayed in part by the payment of page
charges. Under postal regulations, this paper
therefore must be hereby marked advertisement
solely to indicate this fact.
cumb to bacterial soft rot, Erwinia carotovora
(Jones).
The cut stem of Chinese cabbage is more
susceptible to bacterial soft rot than that of
common cabbage (1). Shinohara (1) reported
that successful transplanting of matured
Chinese cabbage depends on minimal dam­
age to the root and stem and the removal of
the outer leaves that readily detach at the
abscission zone on the stem. This investi­
gation was conducted to compare three
methods for transplanting selected mature
Chinese cabbage plants for seed production.
In this method, herein called method I (Fig.
1A), the outer dead and senescing leaves are
removed carefully, followed by decapitation
of the upper leaf of the plant, avoiding dam­
age to the growing point and the yellow heart
leaves. We modified Shinohara’s head prun­
ing technique by avoiding the downward
splitting of the opposite sides of the head to
the yellow heart leaves. The plant is care­
fully dug with a large ball of soil, potted,
and moved to a cool house for recovery and
flowering. Method II (Fig. IB), with a mod­
ification in the pruning method, has been
described to the authors by O. Takahashi
(personal communication). This method in­
volves removal of the white outer leaves two-
thirds from the top of the plant, while the
heart leaves are left intact; only the heart
leaves are capable of regrowth (1). Method
HI (Fig. 1C) is described by a visiting scholar
as used in northern China. Matured plants
are lifted without a ball of soil and stored in
a cool storage room with medium humidity.
At a convenient time, the plants are removed
from the storage and the plants are prepared
for planting by trimming the upper portion
of the leaves forming a pyramid around the
heart leaves of the plant.
Seeds were sown in flats in the greenhouse
and were transplanted into No. 24 polyvi­
nylchloride (PVC) growing trays when the
cotyledons were fully expanded. Seedlings

1450 HortScience , V ol . 21(6), D ecember 1986