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Rapid Screening of ‘Satsuma’ Mandarin Progeny to Distinguish Nucellar and Zygotic Seedlings1

Authors:
S. A. Weinbaum
S. A. Weinbaum
S. A. Weinbaum
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E. Cohen
E. Cohen
E. Cohen
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P. Spiegel-Roy
P. Spiegel-Roy
P. Spiegel-Roy
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Abstract

Nucellar and zygotic offspring of ‘Satsuma’ mandarin ( Citrus reticulata Blanco) were differentiated by gas chromatographic analysis of gaseous emanations from fragmented leaves of 6-month-old seedlings. Analysis was rapid (< 3 minutes/sample), could be performed on individual leaves, and required virtually no tissue processing. Thus, the method is amenable to the screening of large progeny at the young seedling stage.
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1 1
.
Table 2. Cl index in extracts from transverse sec
tions of mesocar of avocado stored for dif
ferent periods at 5°C. Extracts from non-stored
control fruit had zero absorbance.
Cl index (absorbance)7
Storage time (wks)
Fruit section no.y 2 6 8
1 0.10ax 2.27c 3.46de
20.29a 2.90cd 3.70ef
31.35b 4.20fg 5.15h
4 1.41b 4.53gh 5.08h
'Absorbance based on a 1:5 (w/v) extraction. Each
value is a mean of 5 fruit sections.
yTransverse sections about 25 mm thick. Section
1 at the pedicel end and section 4 at the stylar end.
xMeans separation by Duncan’s multiple range test,
5% level.
within individual fruit. Disadvantages of vi
sual rating methods, additional to those de
scribed above, were also evident. For example,
cut fruit darken on exposure to air (6) and
Cl ratings made 1 hour after cutting were
about 40% higher than those made im me
diately after the fruit were cut. This addi
tional darkening was confined to the surface
layer and had a negligible e ffect on absorb
ance values obtained by the objective method.
Al so, visual rating values given by various
assessors varied by up to 2 rating units in
individual fruit.
This objective method overcomes the lim
itations o f visual rating method s and there
fore provid es a more accurate, s ensitive, and
reproducible measure o f Cl in stored avo
cados. The method may als o have application
in studies with other fruit which display in
ternal browning respon ses.
Literature Cited
1. Chaplin, G. R. and K. J. Scott. 1980. As
sociation of calcium in chilling injury sus
ceptibility of stored avocados. HortScience
15:514-515.
2. Eaks, I. L. 1976. Ripening, chilling injury,
and respiratory response of ‘Hass’ and ‘Fuerte’
avocado fruits at 20°C following chilling. J.
Amer. Soc. Hort. Sci. 101:538-540.
3. Fogerty, A. C., A. R. Johnson, and J. A.
Pearson. 1971. Examples of lipid method
ology. p. 337-360. In:A. R. Johnson and
J. B. Davenport (eds.). Biochemistry and
methodology of lipids. Wiley, New York.
4. Golan, A. and A. Y. Sadovski. 1977. Eval
uation of browning potential in avocado me-
socarp. J. Food Sci. 42:853-855.
5. Hatton, T. T. and W. F. Reeder. 1965. Rip
ening and storage of Florida avocados. U.S.
Dept. Agr. Market Res. Rpt. 697.
6. Kahn, V. 1975. Polyphenol oxidase activity
and browning of three avocado varieties. J.
Sci. Food Agr. 26:1319-1324.
7. Lieberman, M., C. C. Craft, W. V. Audia,
and M. S. Wilcox. 1958. Biochemical stud
ies of chilling injury in sweet potatoes. Plant
Physiol. 33:307-311.
8. Lyons, J. M. 1973. Chilling injury in plants.
Annu. Rev. Plant Physiol. 24:445-466.
9. Patterson, B. D., T. Murata, and D. Gra
ham. 1976. Electrolyte leakage induced by
chilling passiflora species tolerant to differ
ent climates. Austral. J. Plant Physiol. 3:435-
442.
10. Scott, K. J. and G. R. Chaplin. 1978. Re
duction of chilling injury in avocados stored
in sealed polyethylene bags. Trop. Agr.
(Trinidad) 55:87-90.
Spalding, D. H. and W. F. Reeder, 1972.
Quality of ‘Booth 8’ and ‘Lula’ avocados
stored in a controlled atmosphere. Fla. State
Hort. Soc. 85:337-341.
H o r t S c i e n c e 1 7 (2 ): 2 3 9 -2 4 0 . 1 98 2 .
Rapid Screening of ‘Satsuma’
Mandarin Progeny to Distinguish
Nucellar and Zygotic Seedlings1
S. A. Weinbaum ,2 E. Cohen, and P. Spiegel-Roy3
Agricultural Research Organization, The Volcani Center, Bet Degan,
Israel
Additional index wor ds. Citrus reticulata, apom ictic, gas chromatography, hybrid, leaf
volatile s, polyembryony
A b st ra c t. Nucellar and zygotic offspring of ‘Satsuma’ mandarin (C itr us re ticu lat a
Blanco) were differentiated by gas chromatographic analysis of gaseous emanations
from fragmented leaves of 6-month-old seedlings. Analysis was rapid (< 3 minutes/
sample), could be performed on individual leaves, and required virtually no tissue
processing. Thus, the method is amenable to the screening of large progeny at the
young seedling stage.
Nucellar em bryony represents a twofold
obstacle in Citrus breeding. It is often dif
ficult to distinguish between nucellar and
zyg otic progen y on the basis o f vegetative
chara cters. S econd, many polyem bryo nic
cultivars produce very lo w percentages of
hybrids. The gen e combinations in somatic
offsp ring are already present in the seed par
ent, and resources invested in their culture
could be more efficien tly redeployed if rapid
means for early identification were available.
‘Satsuma’ mandarin is highly desired as a
parent because it is comm ercially seedless.
However, ‘Satsu ma’ is m ale-sterile, and nu
cellar seedlings typically constitute 95% o f
the offspr ing. Thus, the problem o f poly em
bryony is particularly acute in breeding pro
grams that attempt to incorporate ‘Satsum a’
gen es into sexual progenies.
Various approaches have b een used to dis
tinguish between sexual and nucellar o ff
spring (7) . However, among these, only
electr ophoretic analysis o f isozymic variants
is being empl oyed routinely in Citrus breed
ing programs (1, 5, 6).
A satisfactory screening system must be
‘Received for publication Oct. 22, 1981.
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.
2On leave from the Department of Pomology, Uni
versity of California, Davis.
^The authors gratefully acknowledge stimulating
discussions with Aliza Vardi and Jonathan Gressel
and the technical expertise of Ida Rosenberger.
nond estructiv e, require minimal tissue pro
ces sing, and be amenable to rapid sample
analysis. T he impetus to our work was the
fact that we could seem ingly distinguish be
twee n z ygot ic and somatic seedlings of ‘Sat
sum a’ by means of the aroma o f fragmented
leaves. Two grams of fragmented fresh leaves
(2 to 6 leave s depending on leaf size) were
enc losed in 30-ml vials, which we re then
rendered gas-tight with serum stoppers. Gas
samples were withdrawn from the head-space
and injected into a Packard 7 400 series gas
chromatograph design ed to separate volatile
mixtures. Eight-fo ot glass columns, 4 mm in
diameter, were packed with Carbowax 20 M
(terminated with terephthalic acid). Injector
and detector temperatures wer e 150°C; the
column temperature was 110°. The gas chro
matograph w as attached to a Unicorder U-
125 recorder.
Our inten tion was that the v olatiles d e
tected should reflect the aromatic differen ces
we had noticed previously. Following initial
suc cess , we selected 11 nuce llar ‘Satsuma’
seedlings and 15 ‘Satsuma’ hybrids as pre
viou sly distin guished by means of root per
oxidas e patterns (1). These 6-month-old plants,
25 cm in height, were repotted and placed
under optim um greenhouse condit ions in late
May (30°C day/23° night, 70% relative hu
midity). By mid-July, all plants were grow
ing vigo rously and had produced 6 or 7 new
leav es. Fu lly expanded young leaves were
exc ised, f olded, and broken to release v ol
atiles and then were placed in gas-tight via ls.
Analysis o f the head-space gas was per
formed within 1 hr of samp ling, and virtually
identical patterns were achieved when sam
ples were retested periodically during the
Hor tSci enc e, V o l . 1 7 (2 ), April 1 9 8 2 . 239
J L L -
ABC
Fig. 1. Volatile emanations from fragmented
leaves of nucellar ‘Satsuma’ (A) and represen
tative ‘Satsuma’ hybrids (B-F): (A) nucellar
‘Satsuma’; (B, C) ‘Satsuma’ (9) X ‘Fortune’
(c?) (C. reticulata Blanco X C. sinensis [L.]
Osbeck); (E) ‘Satsuma’ (9) x Poncirus trifol-
iata (8); and (F) ‘Satsuma’ (9) x “Avana’ (8)
(C. deliciosa Tenore). The attenuation setting
on the gas chromatograph in these examples
was 64 x 10"n, and the origin (left-hand
margin) of each chromatogram tracing repre
sents the same point in time immediately fol
lowing sample injections.
subseq uent 5 hr. Under our conditions, the
vola tiles had a retention time of less than 2
min.
‘Satsu ma’ exhibited 2 distinct peaks, and
the peak with the slightly longer retention
time w as slig htly larger (Fig. 1A). All 11
nucellar see dlings exhibited this pattern with
exc ellen t reproducibility. Most of the hybrids
exhibited volatile patterns, which were con
spic uously different from that characteristic
of ‘Satsuma’ (Fig. IB -IF ). Of 18 plants tested,
only 1 ‘Satsu ma’ x ‘Tem ple’ [C. reticulata
Blanco x C. sinensis (L.) Osbeck] hybrid and
1 ‘Satsuma’ X ‘Ponkan’ (C. reticulata Blanco)
hybrid could not be differentiated from ‘Sat
sum a’ (Table 1). So me of the zygotic prog
eny appeared to emanate considerably greater
quantities of volatiles than ‘Sats uma’ (data
not presented).
Table 1. Gas chromatographic analysis of vola
tile emanations from fragmented leaves of 18
‘Satsuma’ hybrids representing 10 different
matings.
No. hybrids
No. hybrids distinguished
Male parent tested from Satsuma
Fortune 66
Tankan 1 1
Michal 1 1
Temple 21
Avana 22
Wilking 22
Clementine 11
Ponkan 10
Ortanique 11
Trifoliate orange 1 1
Thus, 90% of the 18 presumed hybrids
received further confirmation on the basis of
the evolved volatiles. Under the highly fa
vorable conditions for seedling growth, many
of the 18 zygotic seedlings also could be
differentiated morpholog ically from the nu
cellar type.
Volatile emanation patterns of recently
matured leaves from fruiting ‘Satsuma’ trees
in the field were virtually identical to those
of leaves from juvenile ‘Satsum a’ seedlings
in the greenhou se. Thus, the characteristic
volatile pattern was not affected by differ
enc es in environment or by the ontogenetic
age (juvenile vs. mature) of the tree sampled.
However, physiologica lly old leaves sam
pled from the field in May did exhibit a re
du ced lev el o f v ol at ile s, and often the
characteristic pattern itsel f was altered.
Release of volatiles from intact leaves was
insu fficient, even after prolonged incubation
times, and volatile emanations were greatly
reduced i f leaves were sliced into 1 -mm strips
instead o f being folded and broken. Appar
ently, the pressure on the leaves that accom
panied manual fragmentation effected a greater
rupturing of the oil glands (4).
Several workers have reported differences
betw een parents and hybrids and among cit
rus varieties in volatile leaf oil compositi on
(2, 3, 8). These workers, howev er, employed
such a large leaf mass and such extensive
processing before analysis that they pre
cluded applicability of the method to large-
scale screening in breeding programs. Be
cause we have not yet tested the method in
other than ‘Satsuma’ progeny, the potential
usef ulness o f the method should be reas
sess ed using progeny o f other polyembryonic
seed parents.
Literature Cited
1. Button, J., A. Vardi, and P. Spiegel-Roy.
1976. Root peroxidase isoenzymes as an aid
in Citrus breeding and taxonomy. Theor. Appl.
Gen. 47:119-123.
2. Cameron, J. W. and R. W. Scora. 1973. Leaf
oils and fruit characters in relation to genetic
identity among twin and triplet citrus hybrids.
Lloydia 36:410-415.
3. Kamiyama, S. 1970. Leaf oils of citrus spe
cies. V. Leaf oil compositions of Citrus Pon
cirus hybrids in comparison with their parents.
Bui. Brew. Sci. 16:1-14.
4. Schneider, H. 1968. The anatomy of citrus,
p. 47. In: W. Reuther, L. D. Batchelor, and
H. J. Webber (eds.). The citrus industry, Vol.
II. Div. of Agr. Sci., Univ. of Calif., Berke
ley.
5. Soost, R. K., T. E. Williams, and A. M.
Torres. 1980. Identification of nucellar and
zygote seedlings of Citrus with leaf isozymes.
HortScience 15:728-729.
6. Spiegel-Roy, P., A. Vardi, and A. Shani. 1978.
Peroxidase isoenzymes as a tool for early sep
aration of nucellar and zygotic citrus seed
lings. Proc. Intern. Soc. Citriculture, 1977,
2:619-626.
7. Vardi, A. and P. Spiegel-Roy. 1978. Citrus
breeding, taxonomy and the species problem.
Proc. Intern. Soc. Citriculture, 1978. p. 51-
57.
8. Wolford, R. W. 1964. The identification of
citrus species and varieties by instrumental
analysis of citrus leaf oils. Proc. Amer. Soc.
Hort. Sci. 84:204-212.
240 HortScience, Vol. 17(2), April 1982.
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... Various methods have been attempted to discriminate the zygotic seedlings from nucellar's such as rootstock colour test (Furr and Reece 1946), flow cytometry (Tusa et al. 2002), thin layer chromatography (Tatum and Berry 1974), gas chromatography (Weinbaum et al. 1982), morphological and put in a liquid nitrogen box for inactivation of enzymes. The leaves were washed and midribs and thick veins of the leaves were removed. ...
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Full-text available
  • Feb 2018
El desarrollo de variedades tolerantes puede ayudar a enfrentar y reducir los impactos de enfermedades que amenazan al cultivo de limón mexicano [Citrus aurantifolia (Christm) Swingle]. Se han generado plantas hibridas interespecíficas e intergenéricas, en busca de genotipos con resistencia o mayor tolerancia a VTC, HLB y antracnosis. Debido a que la mayoría de los cítricos son apomícticos y poliembriónicos se requiere la identificación de plantas híbridas por medio de técnicas tales como los marcadores genéticos moleculares. El objetivo del estudio fue identificar híbridos de limón mexicano mediante marcadores moleculares denominados microsatélites (SSR). Se utilizó una población de 203 individuos de cruzas realizadas de 2009 a 2012, en el Campo Experimental INIFAP-Tecomán. El ADN genómico se realizó de acuerdo con el método CTAB 2%. La reacción de PCR se hizo con un volumen de 20µl conteniendo 10 µl de iTaq™ Universal Probes supermix y 2.5 µl de cada iniciador Forward y Reverse. Los iniciadores utilizados fueron TAA41, TAA45, cAGG9 y TAA52. Los productos amplificados, se analizaron en gel de poliacrilamida al 8% y se tiñeron con nitrato de plata al 0.2%. La identificación de plantas hibridas se realizó mediante la comparación de bandas producidas por los híbridos con las de los progenitores. Los cuatro iniciadores permitieron la identificación de plantas híbridas. El mayor número de plantas identificadas se obtuvieron con los iniciadores TAA45 y cAGG9 con 145 y 130 respectivamente. La identificación de los híbridos de la población en estudio a través del análisis de marcadores moleculares, permitirá que los programas de mejoramiento genético sean eficientes, lo cual implica la obtención de un gran número de plántulas derivadas de las cruzas realizadas.
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Current status and prospects of molecular marker development for systematic breeding program in citrus
Article
Full-text available
  • Sep 2016
Citrus is an economically important fruit crop widely growing worldwide. However, citrus production largely depends on natural hybrid selection and bud sport mutation. Unique botanical features including long juvenility, polyembryony, and QTL that controls major agronomic traits can hinder the development of superior variety by conventional breeding. Diverse factors including drastic changes of citrus production environment due to global warming and changes in market trends require systematic molecular breeding program for early selection of elite candidates with target traits, sustainable production of high quality fruits, cultivar diversification, and cost-effective breeding. Since the construction of the first genetic linkage map using isozymes, citrus scientists have constructed linkage maps using various DNA-based markers and developed molecular markers related to biotic and abiotic stresses, polyembryony, fruit coloration, seedlessness, male sterility, acidless, morphology, fruit quality, seed number, yield, early fruit setting traits, and QTL mapping on genetic maps. Genes closely related to CTV resistance and flesh color have been cloned. SSR markers for identifying zygotic and nucellar individuals will contribute to cost-effective breeding. The two high quality cit rus reference genomes recently released are being efficiently used for genomics-based molecular breeding such as construction of reference linkage/physical maps and comparative genome mapping. In the near future, the development of DNA molecular markers tightly linked to various agronomic traits and the cloning of useful and/or variant genes will be accelerated through comparative genome analysis using citrus core collection and genome-wide approaches such as genotyping-by-sequencing and genome wide association study.
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Identification of Nucellar and Zygotic Seedlings of Citrus with Leaf Isozymes1
Article
Full-text available
  • Dec 1980
The distinction between apomictic nucellar and zygotic 5-month-old seedlings of Citrus was made using two genetically defined isozyme markers: phosphoglucose isomerase and phosphoglucose mutase. Of 123 seedlings of attempted crosses of ‘King’ (seed parent) × ‘Parsons Special’, 18 had the ‘King’ genotype and were therefore nucellars. Segregation ratios for both markers were as expected among the zygotics.
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Leaf oils and fruit characters in relation to geneticidentity among twin and triplet citrus hybrids
Article
  • Jan 1973
Relative quantities of leaf oil components within and among 7 sets of identical twins and triplets in Citrus and Citrus X Poncirus were compared by gas chromatography. Among all 7 sets, F values indicating greater similarity within sets than among sets were significant for 16 of 18 components tested. Among 3 of the sets with the same parents, there were significant F values for 10 of the 18 components. Duncan separations showed a wide range of patterns among twin sets, from one terpene component to another; however, each set was separated from all others by at least 2 components at the sampling date chosen. Fruits were obtained from some of these sets and related ones, and fruit characters substantiate earlier vegetative evidence that the genotypes within sets are identical. The leaf oil data are consistent with this conclusion, but their use to prove complete identity within such sets would be limited.
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Root peroxidase isoenzymes as an aid in Citrus breeding and taxonomy
Article
  • May 1976
A rapid method for differentiating between Citrus clones as well as between zygotic and nucellar plants has been developed. It is based on determination of peroxidase isoenzyme patterns in small root samples. Ten to 14 bands could be distinguished, in contrast to the small number of bands present in leaves. Tetraploid seedlings gave uniform patterns closely resembling the diploid, while triploids showed segregation in isozyme pattern.
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Leaf oils of citrus spe cies. V. Leaf oil compositions of Citrus Pon cirus hybrids in comparison with their parents
  • Jan 1970
  • 1-14
  • S Kamiyama
Kamiyama, S. 1970. Leaf oils of citrus spe cies. V. Leaf oil compositions of Citrus Pon cirus hybrids in comparison with their parents. Bui. Brew. Sci. 16:1-14.
The anatomy of citrus
  • Jan 1968
  • 47
  • H Schneider
Schneider, H. 1968. The anatomy of citrus, p. 47. In: W. Reuther, L. D. Batchelor, and H. J. Webber (eds.). The citrus industry, Vol. II. Div. of Agr. Sci., Univ. of Calif., Berke ley.
Peroxidase isoenzymes as a tool for early sep aration of nucellar and zygotic citrus seed lings
  • Jan 1977
  • 619-626
  • P Spiegel-Roy
  • A Vardi
  • A Shani
Spiegel-Roy, P., A. Vardi, and A. Shani. 1978. Peroxidase isoenzymes as a tool for early sep aration of nucellar and zygotic citrus seed lings. Proc. Intern. Soc. Citriculture, 1977, 2:619-626.
Citrus breeding, taxonomy and the species problem
  • Jan 1978
  • 51-57
  • A Vardi
  • P Spiegel-Roy
Vardi, A. and P. Spiegel-Roy. 1978. Citrus breeding, taxonomy and the species problem. Proc. Intern. Soc. Citriculture, 1978. p. 51-57.