In: Pollen: Structure, Types and Effects
Editor: Benjamin J. Kaiser, pp. 65-99
ISBN: 978-1-61668-669-7
©2010 Nova Science Publishers, Inc.
Chapter 2
POLLEN BIOLOGY AND HYBRIDIZATION PROCESS:
OPEN PROBLEM IN WALNUT
Paola Pollegioni,1, Keith Woeste,2,† Irene Olimpieri,1
Fulvio Ducci 3,‡ and Maria Emilia Malvolti,1,
1
C.N.R. Institute of Agro-environmental and Forest Biology,
Porano, Terni, Italy
2
U.S.D.A. Forest Service, Hardwood Tree Improvement and Regeneration Center,
Department of Forestry and Natural Resources, Purdue University, Lafayette IN, USA
3
C.R.A. Research Centre for Silviculture, Arezzo, Italy
ABSTRACT
This review focuses on the pollen biology of Juglans, and in particular Juglans nigra
(Eastern Black walnut) and Juglans regia (Persian or English walnut), which are
economically important species in Europe, Asia and North America. Both species are
monoecious, heterodichogamous and wind –pollinated. Their mating system is
predominantly outcrossing, although under particular environmental conditions selfpollination is possible. Hybrids between the two species, Juglans × intermedia (Carr) can
occur naturally, although they often have reduced fecundity. Compared to the parental
species, most J. × intermedia (J. nigra × J. regia) hybrids show increased vegetative
vigor, distinct disease resistance, high wood quality, and greater winter-hardiness. For
these reasons here is great demand for J. × intermedia for forestry, especially in Northern
Europe. We review several aspects of Juglans pollen biology that frustrate the production
of J. × intermedia and limit the progress of researchers and plant breeders who work with
this genus. We also discuss the ways in which scientists and breeders are working to
overcome problems related to pollen storage and viability testing, pistillate flower
abscission (PFA), fertilization and embryogenesis in Juglans, and the use of
microsatellites to monitor gene flow, ploidy, parentage, and hybridogenesis all with an
E-mail: [email protected]
E.mail: [email protected]
‡
E-mail: [email protected]
†
[email protected]
66
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
eye toward practical solutions to the current shortage of J. × intermedia for research and
applied forestry.
Keywords: J. × intermedia, Persian walnut, black walnut, hybrid
INTRODUCTION
Juglans is one of eight genera composing the family Juglandaceae and consists of 21
species of deciduous, monoecious trees distributed in North and South America, Southeastern Europe, Eastern Asia and Japan (Manning, 1978). Juglans species are traditionally
divided into four distinct sections mainly based on leaf architecture, wood anatomy, pollen
and fruit morphology: Dioscaryon Dode (traditionally Juglans), Rhysocaryon Dode (black
walnuts), Cardiocaryon Dode (Asian butternuts) and Trachycaryon (American butternut)
(Dode, 1909).
Dioscaryon contains just one species, Juglans regia L. (Persian or English walnut) which
is native to Eurasia from the Balkans to southwest China. Persian walnut bears four-celled
nuts singly or in pairs, with smooth, thin shell and a dehiscent husk that separates easy from
the nut at maturity. Section Juglans also includes the iron walnut, Juglans sigillata Dode, a
type from Southern China and Tibet, with thick, rough-shelled nuts and very dark-colored
kernels. The iron walnut has been considered as an ecotype of J. regia for long time, but it is
also accepted as a separate species by some botanists (Manning, 1978). The Rhysocaryon
section is endemic to the Americas and includes approximately 16 species: seven North
American species, Juglans californica S. Wats. (Southern California black walnut), Juglans
hindsii (Jeps) Rehder, Juglans major (Torr. Ex Sitsgr.) Heller, Juglans microcarpa (Texas
black walnut) Berl., Juglans jamaicensis C.DC (West Indies black walnut), Juglans mollis
Engelm., and Juglans nigra L. (Eastern Black walnut); four Central America species, Juglans
olanchana Standl. & L.O. Willimas, Juglans steyermarkii Mann., and Juglans guatemalensis
Mann, Juglans pyriformis Liebm.; and five South American species, Juglans australis
Griesb., Juglans boliviana (C.DC.) Juglans soratensis Mann., Dode, Juglans neotropica
Diels, and Juglans venezuelensis Mann. All members of Rhysocaryon section exhibit fourchambered nuts with thick, ridged or striate, not completely smooth shells and indehiscent
and persistent husks. These species are so closely related that their discrimination is often
difficult. Section Cardiocaryon (Oriental butternuts) includes three species all native to East
Asia: Juglans ailantifolia Carr., Juglans cathayensis Dode, and Juglans mandshurica Mahim.
Asian butternuts produce two chambered nuts with 4-8 prominent ridges and indehiscent
husks, and are borne in long racemes of up to 20 nuts. Their susceptibility to walnut bunch
disease has limited their horticultural diffusion in the eastern U.S. Section Trachycaryon
consists only of J. cinerea L. butternut, a North America species, characterized by twochambered nut with high prominent ridges on the shell and an indehiscent husk. A Complete
description of ecological distribution and the morphological variation in Juglans genus are
found in two extended reviews, Manning, (1978) and McGranahan & Leslie, (2009).
Earlier molecular studies based on nuclear RFLPs (Fjellstrom & Parfitt, 1995) and matK
and ITS sequences (Stanford et al., 2000) confirmed the traditional taxonomic classification
of Juglans and are consistent with biogeography and fossil history. Fossil evidence supported
the ancient divergence of sections Cardiocaryon and Rhysocaryon almost simultaneously
Pollen Biology and Hybridization Process: Open Problem in Walnut
67
with the origin of the genus in the middle Eocene (~ 45 Ma) in North America (Manchester,
1987). Black walnut spanned from the West to East coast of North America extending into
the Southern Hemisphere as far as Ecuador, whereas members of Cardiocaryon section
crossed the Bering land bridge, existed from the early Eocene (55 million years before
present) until the late Miocene, and spread into Eurasia; this theory implied that Dioscaryon
section evolved from a common ancestor with Cardiocaryon. Nevertheless, recent study
based on non-coding intergenic spacer (NCS) regions of chloroplast DNA supported section
Juglans as the oldest lineage within the genus Juglans and the section Rhysocaryon as the
youngest, in contrast to fossil evidence (Aradhya et al., 2007): Juglans section may be an
independent, monophyletic clade sister to sections Cardiocaryon and Rhysocaryon. However
the authors also postulated that the evolutionary history of Juglans section may have been
confounded by geographic isolation, bottleneck events, human selection and introgression
among isolated population during the post Pleistocene glaciations.
Walnuts are among the most important trees in the world for nut and wood production. In
particular two species, J. nigra L. (Eastern black walnut) and J. regia L. (Persian or English
walnut) are widely cultivated. Most of the member of Juglans are of low economic value and
are used only occasionally as timber or in the brown dye industry.
JUGLANS REGIA L. (PERSIAN OR ENGLISH WALNUT)
Juglans regia, the Persian or English walnut, is one of the most economically important
member of the genus Juglans. Persian walnut is widely cultivated throughout the temperate
regions of the world for its high quality wood and edible nuts. Persian walnut wood has a
light yellow color and is characterized by a hard and homogenous grain. It is used for the
production of furniture, panels and other manufactured products. Its non-edible parts, such as
leaves and husks, find broad application in cosmetic and dye industries, and in traditional
medicine (Amaral et al., 2008). For example, leaf extracts have a remarkable capacity to
scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can at least
partially justify the therapeutic use of J. regia leaves in folk medicine (Almeida et al., 2008).
In addition, during the last decade several studies described the biochemical composition of
walnut nutmeats, mainly with respect to their nutritional and health benefits. Walnuts are rich
in -6 (linoleic acid) and -3 (linolenic acid) essential polyunsaturated fatty acids which
cannot be produced in the human body and must be taken up through food (Caglarirmak
,2003; Amaral et al., 2003; Pollegioni et al., 2006). An inverse relationship between the
relative risk of coronary heart disease and the frequent daily consumption of small amounts of
walnut nuts was found. Feldman (2002) reported that:
―Compared to most other nuts, which contain monounsaturated fatty acids, walnut are
unique because they are rich in ω6 and ω3 acids‖.
Walnuts also contain significant amounts of tocopherols, in particular - tocopherol,
which protects storage lipids and proteins from oxidation (Verardo et al. 2009).
Persian walnut is considered native from South-Eastern Europe to North-Western China
(Xinjiang province) through Turkey, Caucasus, Iran, Pakistan, Northern India, Pakistan,
68
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
Nepal and Tibet (Huntley & Birks, 1983). In recent decades, the origin of the European
walnut has been a debated subject among foresters, botanists and bio-geographers. According
to a traditional theory, the diffusion of the walnut species in Europe followed the ancient trade
routes, passing from China into India, Persia and Greece (Forte, 1993). Nevertheless, it is still
debated if the species was extinguished during the Pleistocene glaciations or if it survived the
rigours of the cold, dry glacial intervals in refugia in Southern Europe and the Balkans, as
suggested by some paleopalynologic studies (Huntley & Birks, 1983; Carrión & SànchezGomez, 1992; Fornari et al., 1999). Without regard to this debate, the first post-glacial
appearances of Persian walnut pollen in Europe occurred around 1500-2500 yr BP and
corresponded to the establishment of the Greek and Roman settlements (Huntley & Birks,
1983; Beer et al., 2008; Chester, 2009). From Greece, the cultivation of walnuts spread to
Rome where walnuts were called Jovis Glans (Jupiter‘s acorn), from which comes the name
of Juglans genus. From Italy, J. regia was exported to France, Spain, Portugal and Southern
Germany (McGranahan & Leslie, 2009).
Although there is evidence that environmental change could influence its expansion
(Winter et al., 2009), J. regia generally grows wherever the climate is temperate from the 10th
to about 50th parallel Northern latitude. J. regia grows best where the mean annual
temperature is in the range of 10.5-15°C and annual precipitation is up to 700 mm. Persian
walnut grows at altitudes from sea level to 1000-1200 m. a. s. l. (Forte et al., 1993). It is
considered a frost-sensitive species because it is threatened by the occurrence of both early
and late-season frost. As observed by Fady et al., (2003), late spring frosts have a negative
impact on architectural traits and thus on wood quality. Early budbreak leads to loss of apical
dominance and defective stem form when late spring frosts occur. Recently, Loacker et al.,
(2006) found a striking positive correspondence in alpine meadows between the number of
germinated Persian walnut seedlings and higher average minimum temperatures during
winter; conversely, germination rate was negatively associated with the number of days with
severe frost They reported that in the last thirty years, climate warming promoted the
expansion of J. regia in the South- and South-West-facing forests of inner Alpine valleys
(Tyrol, Austria) which are often dominated by Scots pine (Pinus sylvestris L.). Persian walnut
is also sensitive to soil conditions, developing best on deep, well-drained, moist and fertile
soils rich in Calcium with a pH range from 6 to 7.5 (McGranahan and Leslie, 2009). Walnut
is known to have very low tolerance for drought and flooding, which cause root system
anaerobiosis (Mapelli et al., 1997) and enhance susceptibility to several Juglans diseases,
including walnut blight (Belisario et al., 1997), anthracnose (Belisario et al., 2008) and
root/collar infection by Phytophthora cinnamomi (Belisario et al., 2009).
Persian walnut is cultivated in Southern and Western Europe, but also in Central Asia,
Northern India, China, South Africa, Argentina, Chile, USA, Australia, New Zealand and
Japan (McGranahan & Leslie, 1991). China leads world production, followed by the USA,
Iran, Turkey, Ukraine, Romania, France and India. (FAOSTAT data, 2004). The major
exporters are the USA, which exports 115.000 Metric Tons, followed by France (23.000 MT),
China (22.000 MT) and India (17.000 MT). In the United States, 99% of the walnut crop is
produced in California, where the crop has been grown since the 18th century when plants
were imported from South America by Spanish missionaries (Potter et al., 2002). China has
encouraged J. regia production and expects to have over 1 million hectares of walnut by 2012
(McGranahan and Leslie, 2009). In Europe J. regia is considered one of the most valuable
broadleaved tree species. For example, in Italy, Campania is traditionally the most important
Pollen Biology and Hybridization Process: Open Problem in Walnut
69
region for walnut cultivation.Walnut cultivation in Italy decreased after the Second Word
War (from 80 to 10 MT) because of land abandonment and the mechanization of agricultural
lands (Di Vaio & Minotta, 2005). Local varieties/accessions were increasingly neglected
because of their irregular fruit size and limited market demand. Never the less, in the last
twenty years almost 100,000 ha of forest tree plantations were established on former
agricultural lands with grants from the European Union. In a large percentage of these
plantations (40–50%), Persian walnut (Juglans regia L.) was planted as the main species, due
to the high value of walnut wood in the European market (Paris et al., 2005).
JUGLANS NIGRA L. (EASTERN BLACK WALNUT)
Juglans nigra L. (Eastern black walnut) is one of the most valuable hardwood species. It
grows as scattered individual trees or in small, spatially distinct groves throughout the
deciduous forests of eastern North America. It is a fast growing species, producing high
quality timber on a relatively short rotation of about 60 years (Beineke, 1983). Eastern black
walnut is native to most of the eastern U.S. from New Hampshire south to Georgia and west
to Texas. Its western border includes parts of the states of Oklahoma, Kansas and Nebraska,
with the northern limits crossing Minnesota, Wisconsin, Michigan and Ontario, Canada. On
the western border in Kansas, in locations where environmental conditions are favorable for
black walnut cultivation, it is abundant and occupies 50 percent or more of the basal area in
stands of several hectares (Grey & Naughton, 1971). According to Williams et al., (2004),
black walnut probably re-colonized the Midwest as a single, large population from a glacial
refugium in the Lower Mississippi valley between 14,000 yr BP and 12,000 yr BP. Beginning
in the 17th century, J. nigra was imported from the Eastern and Central hardwood forests of
the United States to the European continent for ornamental purposes, and subsequently for its
rapid growth, which led to its use for wood and as rootstocks. It is cultivated in Central
Europe, the Balkans, Caucasus, Russia and Eastern-Central Asia. For example in Italy, black
walnut is usually found in private and public parks of Pianura Padana, where is also used for
reforestation and recovering degradated areas (Fenaroli & Gambi, 1975).
In its native range, the vast majority of black walnut occurs in natural stands. Walnut
plantations only cover about 13,800 acres in the United States, which represents about 1 % of
all black walnut volume in U.S. (Shifley, 2004).
According to Rink et al., (1994), intense harvesting pressure in the first part of the 20th
century resulted in severely fragmented black walnut populations and consequently in
significant losses of genetic diversity. Recently, a broad-scale study of the genetic structure of
43 indigenous populations of J. nigra, using (neutral) microsatellite markers indicated that the
large deforestation and fragmentation that occurred across the range of black walnut after
European settlement had little effect on the neutral genetic diversity of the species (Victory et
al., 2006). In spite of differences in adaptative traits observed in provenance tests, high
genetic homogeneity was found among American walnut populations. The authors postulated
that because strong adaptative differences can persist in the face of high levels of gene flow,
the use of functional markers, tightly linked to trait of interest, could be more useful for
detecting regional adaptation in black walnut. Furthermore, because walnut trees can live to
70
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
greater than 200 years of age, it is possible that an insufficient number of generations have
passed to detect the effects of recent forest fragmentation.
J. nigra is most valued for its lumber and veneer. The wood is used for multiple
purposes, including the production of fine furniture, interior panelling, plaques and gunstocks.
The wood machines easily, though it is hard, and when finished it has a dark luminous
beauty. Uniformity of color is an extremely important factor in wood quality that is not
present in black walnut because of the contrast in color between the sapwood and the darklycolored heartwood (Cassens, 2004). The heartwood of J. nigra is markedly darker than J.
regia heartwood. Beritognolo et al., (2002) studied the role of transition zone (innermost
sapwood) in the transformation of sapwood to heartwood and in the accumulation of phenolic
substances in J. nigra heartwood. Although the mechanisms underlying heartwood formation
are not completely elucidated, their results supported the hypothesis that flavonols are
synthesized de novo in J.nigra in aged xylem tissues during the transformation of sapwood to
heartwood. In addition flavonol accumulation appeared to be regulated mainly at the
transcription level by the expression of chalcone synthase (CHS), flavanone 3-hydroxylase
(F3H) and dihdroflavonol 4-reductase (DFR) enzymes.
The nut produced by the black walnut has a furrowed, hard and thick shell that protects
the edible seed. Well-managed seedling black walnuts produce nuts averaging 20% kernel but
after shelling only 6 to 10% usable kernel is recovered. Nevertheless each year, American
consumers use 2 million pounds of black walnut kernels in cookies, cakes and ice cream
products (Reid et al., 2004). In addition, ground black walnut shell is extremely valuable for
industrial applications such as metal cleaning and polishing and oil well drilling (Cavender,
1973).
More than 400 black walnut cultivars have been named and released during the past
century (Woeste 2004). Twenty of the most popular have been analysed and showed
considerable genetic variation in nut quality, blooming date, leafing date, age of first bearing
and growth rate (Reid et al., 2004). Despite its wide and geographically diverse native range,
J. nigra is generally considered by silviculturists to be site sensitive; it only competes well
against other temperate forest species on a limited number of site types. The growing season
of J. nigra ranges from 140 to 280 days. Black walnut is tolerant of annual precipitation and
temperature variations. For example, annual precipitation is less than 640 mm in northern
Nebraska and about 1780 mm in the Appalachians of Tennessee and North Carolina. Mean
annual temperatures range from about 7°C at the north of J. nigra‘s range, to 19°C at the
south (Schlesinger & Funk, 1977). Black walnut generally requires moist, well drained,
loamy, deep, nearly neutral soils; it grows best on sandy loam, loam or silt loam soils that
hold a large amount of water that can be used by the tree during dry periods of the vegetative
season (Beineke, 1983). J. nigra plants can reach a height of 45 m and a trunk diameter of 2
m; the root system is deep and wide spreading, with a definite taproot, at least in early life. As
reported by Burke & Williams (1973), the taproot of 9-year-old black walnut trees, excavated
from an Indiana plantation, was 2,3 m long, with lateral roots extended more than 2.4 m. In
comparison with J. regia, J. nigra appeared to be more tolerant to water logging (Mapelli et
al., 1997) and resistant to some walnut diseases, including, bacteriosis and infection by
Phytophthora cinnamomi (Belisario et al., 1997; 2009; 2008). The most serious foliar disease
of black walnut is anthracnose, caused by Gnomonia leptostyla (Fr.) Ces. Symptoms of
walnut anthracnose develop on leaves, stem and fruit as irregular necrotic areas that are often
surrounded by small chlorotic halos. In severe cases, these lesions may cause premature
Pollen Biology and Hybridization Process: Open Problem in Walnut
71
defoliation, fruit drop, or poorly filled nuts (Funk et al., 1981). The selection of resistant
genotypes toward the anthracnose disease could represent a valid alternative to cultural and
chemical (fungicides) management (Woeste and Beineke, 2001). A wide range in
susceptibility to walnut anthracnose appears to exist in J. nigra, but. no specific genotype has
been reported to be immune. Two black walnut cultivars, ―Thomas‖ and ―Ohio‖, have been
noted for their anthracnose resistance, although both cultivars could contract the disease
under condition of high pressure (Berry, 1960). As reported by Mielke et al., (2004), trees in
adjacent J. nigra plantations located in North America frequently exhibit different levels of
disease incidence. Genotypes derived from the western edge of the natural range of black
walnut (Kansa and Oklahoma) appeared most susceptible, perhaps because of low selective
pressure for anthracnose resistance in this relatively arid region. These observations clearly
suggested the existence of natural resistance to anthracnose. Studies also indicated that the
natural resistance to G. leptostyla is highly heritable (Beineke & Masters, 1973), encouraging
genetic breeding programs in walnut. The genetics of the pathogen have never been
researched, and it is possible that the fungus has multiple races and local or regional variation
in virulence.
INTERSPECIFIC HYBRID (JUGLANS × INTERMEDIA CARR)
Although phylogenetic analysis based on nuclear RFLP, matK and ITS sequence has
demonstrated that black walnut and Persian walnut belong to different sections of genus
Juglans, Rhysocaryon and Dioscaryon respectively (Stanford et al., 2000), a hybrid between
them, Juglans × intermedia (Carr), can occur naturally. Generally, the female parent of J. ×
intermedia is J. nigra and the male parent is J. regia (J. nigra × J. regia). In fact, because J.
nigra pistillate flowers usually mature at least two to three weeks later then J. regia catkins,
there is a considerable phenological barrier to hybridization which is overcome only rarely in
nature. The percentage of hybrid progeny in a mixed population is usually less than 10 %
(Funk, 1970). The difficulty obtaining hybrids of the two species could be the result of an
incompatibility in flowering phenology or some mechanism(s) of genetic incompatibility
(Sartorius 1990), failure of fertilization (pre-zygotic factors), or embryo abortion (postzygotic factors). In addition to synchrony of flowering, hybridization rate may be affected by
air temperature, which influences pollen germination and penetration through the stigma and
the style to the J. nigra ovary. As described in the next section, Luza et al. (1987) found clear
differences in temperature optima for pollen germination and tube grow in J. nigra and J.
regia.
Compared to the parental species, most J. × intermedia hybrids show increased
vegetative vigour, distinct disease resistance, good wood quality, and greater winter-hardiness
than Juglans regia (Fady et al., 2003). In particular they showed strong apical dominance, late
budbreak and resistance to spring frost damages. They were superior to the parents in growth
at sites with medium to low fertility and were moderately tolerant of flooding. As reported by
Mapelli et al., (1997), some walnut hybrid genotypes could be exposed to anoxia stress for 10
to 12 days before they showed visible signs of injury. For these reasons there is a great
demand for J. x intermedia for forestry, especially in Northern Europe. Recent investigation
on the resistance to anthracnose infections of J. regia, J. nigra and inter-specific hybrids (J.
72
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
nigra × J. regia) plants proved that J. regia is susceptible, J. nigra is relatively resistant,
while hybrids showed an intermediate behavior toward Gnomonia leptostyla infection
(Anselmi et al 2005). In general, J. × intermedia hybrids flower profusely but never bear
much seed; inadequate chromosome pairing in megaspore and microspore mother cells can
frequently occur (McKay 1941) Often the seed produced is not able to germinate well,
averaging only 27 percent (Funk, 1970). Walnut trees that show a particular aptitude for
producing hybrids are defined as ―hybridogenic‖ plants. The identification and selection of
hybridogenic parents is the first step toward obtaining hybrid progeny in walnut. In addition,
although some trees appear to be hybridogenic under natural conditions, it has been difficult
to produce hybrids using controlled crosses (McKay 1965; Scheeder 1990). As reported
below, breeders have encountered difficulties obtaining sufficient Persian walnut pollen at the
time J. nigra pistillate flowers are receptive. Suitable and relative simple method for pollen
storage and viability testing is now available for Juglans (Luza & Polito, 1985, 1988b). In
addition, pistillate flower abscission (PFA), caused by excessive pollen load, has been
reported in Persian (Catlin et al. 1987) and black walnut (Beineke and Masters 1976). PFA
may decrease the final nut set (Figure 1).
Figure 1. Summary of the hallmark events (the low pollen viability, the pistillate flower abortion,
fertilization and embryogenesis) of inter-specific hybridization between Eastern black walnut and
Persian walnut.
Pollen Biology and Hybridization Process: Open Problem in Walnut
73
Thus, the production of hybrid plants depends mostly on successful natural hybridization.
In practice, forest nurseries commonly collect seeds from J. nigra trees that are expected to be
pollinated by J. regia. After one or two years of cultivation, the hybrid genotypes are
distinguished mainly by phenotypic traits such as leaf and bud shape. As suggested by
Hussendorfer (1999), ―the natural variation of phenotypic traits sometimes leads to the
problem of miss-identification of hybrids‖. In the past, several methodologies have been
developed to distinguish between J. nigra and J regia and to identify French and German
inter-specific walnut hybrids. They were based on morphological traits (Jay-Allemand et al.,
1990), biochemical markers such as isozymes (Germain et al., 1993; Hussendorfer, 1999),
PCR-markers as Restriction Amplified Polymorphisms (RFLPs) (Tanzarella & Simeone,
1996) and Random Amplified Polymorphic DNA (RAPDs) (Malvolti et al., 1997). Although
isozymes and RFLPs are codominant markers, they are not frequently used for hybrid
identification because they are time-consuming, expensive and characterized by low levels of
polymorphism. Malvolti et al., (1997) reported that a subset of twenty selected RAPDs
markers were a powerful tool to discriminate between J. x intermedia genotypes and backcross plants ((J. nigra x J. regia) x J. regia ) and ((J. nigra x J. regia) x J. nigra).
Nevertheless, RAPDs (dominant markers) can show a low reproducibility and are not useful
for pedigree and parentage analysis.
Vegetative propagation of the identified walnut hybrids, by cutting or micropropagation,
has proved difficult. Numerous juvenile and mature clones cannot be propagated at a
commercial scale because of their limited ability to form adventitious roots. Claudot et al.,
(1993) detected a strong accumulation of hydrojuglone glucoside (precursor of juglone) in
phloem and parenchymal cells in seedling and rejuvenated material, whereas a high content of
flavonol glycosides (myricitrin and quercitrin) in the peripheral zone of mature shoots. It was
postulated that these polyphenols may inhibit adventitious root generation in microcuttings.
The expression of antisense chalcone synthase RNA (key enzyme in flavonoid biosynthesis)
in transgenic hybrid walnut microcuttings confirmed the previous results: decreased flavonoid
content in stems of antisense chs transformed lines was associated with enhanced adventitious
root formation (Euch et al., 1998). In addition the widespread use of micropropagation in
order to produce hybrid walnuts has been limited by the low survival of shoots cultured in
vitro during acclimatization. An antagonism between the number of roots and the number of
leaves in the walnut plantlet was observed (Cheneval et al., 1995; 1997). They noted that a
low sucrose concentration in the propagation medium promotes photosynthetic activities of
shoots and consequently the establishment of photoautrophy but also reduces the
development rate of root system.
Somatic embryogenesis was developed from cotyledons of immature nuts of J. ×
intermedia hybrids (Cornu, 1988). Unfortunately only few propagated somatic embryos
completed their growth and produced whole plants. Nevertheless recent increases in industrial
demand for wood have led to expanded planting areas and the establishment of new seed
orchards for production of J. × intermedia trees.
74
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
FLORAL AND POLLINATION BIOLOGY IN WALNUT
Black and Persian walnut are wind-pollinated, monoecious, dichogamous and
hypothetically entirely self-compatible species, with the same number of chromosomes (2n =
32). Male (staminate) and female (pistillate) flowers are on the same tree but separated from
each other. Both species are characterized by a dichogamous bloom habit: the period of the
female flower receptivity does not overlap the period when male flowers shed pollen. As
discussed by Bertin & Newman (1993), dichogamy represents an evolutionary mechanism to
encourage an outcrossing mating system, reducing or preventing self-pollination.
Nevertheless this bloom habit does not eliminate the possibility of self-fertilization in walnut
because the temporal separation of female and male flower bloom is sometimes incomplete
(Forde & Griggs, 1975).
The mating system of walnuts exhibits a phenotypic dimorphism defined as
―heterodichogamy‖: if the male flower shed their pollen before the pistillate flowers are
receptive, the genotypes are classified as ―protandrous‖, whereas if the mature pollen is
released after the period of the female flower receptivity, the genotypes are classified as
―protogynous‖. Most J. regia trees are protrandrous, only a few cultivars, such as ―Chico‖
and ―Amigo‖, are protogynous. On the contrary, a high incidence of protogyny is detected in
J. nigra species (Funk et al., 1970). According to Gleeson (1982), heterodichogamy in
Persian walnut is regulated by two dominant-recessive alleles at a single locus, with
protogyny as a dominant phenotype. In addition, the mode of dichogamy in Juglans seems to
be correlated with the extent of both staminate and pistillate flower differentiation that occurs
prior to the onset of the dormant season. As is typical for many winter-deciduous tree species,
floral organogenesis and differentiation in Juglans begins in the growing season prior to
dormancy and ends in the spring during the weeks before bloom. Luza & Polito (1988a)
showed that in each protandrous tree, the staminate flower primordia entered the dormant
season with anthers having all wall layers and four microsporgia fully differentiated; in the
protogynous tree, anthers presented only as undifferentiated structures. Subsequently, Polito
& Pinney (1997) observed that pistillate floral primordia in protogynous individuals
progressed to the initiation of a perianth (four sepal primordia), whereas in protandrous
individuals development stopped at an early stage corresponding to initiation of the involucral
ring.
STAMINATE FLOWER AND POLLEN STRUCTURE
In walnut the staminate (male) flowers are small and densely grouped in catkins, 10-15
cm long, borne laterally on 1-year-old wood. Each catkin includes up to 40 sessile petaless
florets surrounded by green sepals. The individual flowers lack petals and are characterized
by numerous stamens. Each stamen terminates in a pollen-bearing anther (Figure 2). At
maturity, each catkin is able to release two million pollen grains that are subsequently
dispersed by wind over long distances (Impiumi, & Ramina, 1967). Emergence of the
staminate inflorescence and shedding of pollen increase with rising temperatures and are
associated with lower relative humidity: cold weather has the opposite effect and reduces
pollen dispersal. In addition, the pollen mother cells of anthers are usually very sensitive to
Pollen Biology and Hybridization Process: Open Problem in Walnut
75
spring frost. Frost frequently causes partial or full abortion of normal meiosis, causing catkins
to shed sterile pollen (Kvaliashvili et al., 2006).
Pollen grains consist of three distinct portions (Polito et al., 1998a). The central, living,
cytoplasm in which is found the nuclei responsible for fertilization, is surrounded by two
distinct layers that compose the pollen wall: the inner layer, the ―intine‖, and the outer layer,
the ―exine‖.
Figure 2. Male walnut catkins borne laterally on 1-year-old wood. Each catkin includes up to 40 sessile,
petaless florets surrounded by green sepals. The individual flowers lack petals and are characterized by
numerous stamens. Each stamen terminates in a pollen-bearing anther.
The intine is a thin inner wall made of mostly pectin and cellulose. The exine is
composed of ―nexine‖ and ―sexine‖, and is perforated by numerous pores (germination
apertures). The pollen wall is resistant to degradation and treatment with intense heat; strong
acids and bases usually have little effect upon the pollen wall. In particular, the walls of the
pollen grains of J. nigra (the exine) include a sexine three time thicker than the underlying
nexine (Calzoni et al., 1990). Of the structural elements of the sexine, the ―tectum‖ appears
strongest, crossed by thin channels and decorated by spinulose extroflections. Bacula are
differently shaped and irregularly distributed; thin lamellar structures are rarely present. In J.
regia the exine is not as thick as in J. nigra, although the sexine/nexine ratio remains
unvaried. In both species, the intine is widely spread through the oncus and nexine is
homogeneous and broken at the pores without opercula.
Meiosis in the pollen mother cells, and maturation of pollen grains, occur before
(protandrous) or after (protogynous) pistillate flowers bloom. The pollen grains, which
contain the male gametes, are transferred to the sticky stigmatic surface of receptive female
flowers by wind. After 7 to 8 hours, in warm and sunny conditions, or 24 to 36 hours in cold
and humid weather, the pollen grain germinates (Kvaliashvili et al., 2006). Pollen germination
requires hydration of the dry cytoplasm followed by expansion of the inner wall through one
of the pores in the outer wall. As described by Polito et al., (1998a), the cytoplasm of pollen
grain moves into the long pollen tube defined by the growing wall. After pollen germination
on the surface of stigma, multiple pollen tubes grow through the style; some of them penetrate
the ovary but only one reaches the embryo-sac and fertilizes the egg cell. Within the pollen
tube, two non-motile sperm cells are ultimately formed and are conveyed through the tube,
76
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
keeping pace with tip growth. Fertilization occurs one week from the time of pollen
germination on the stigmatic surface.
The ability of walnut pollen to germinate can vary among years and during the same
vegetative season. Understanding this critical process is not only important for deciphering
the basic mechanism of sexual reproduction in walnut but also has value for the potential
manipulation of nut production. In particular the barriers underlying the partial
incompatibility between J. nigra and J. regia are still unclear. In order to prevent the ―wrong‖
cross, many plants have developed barriers that operate in the pistil either before fertilization,
inhibiting pollen tube germination and elongation, or after fertilization, causing abortion of
the illegitimate embryo. The barriers in interspecific-crosses are mostly referred to as
―incongruity‖, indicating the lack of communication due to the absence of co-evolution of
two species (Hogenboom, 1984). Detecting the signals that regulate the compatible
interaction between a pollen tube and all the female cells in its path is crucial for breeders to
break species barriers and produce J x intermedia hybrids.
Pollen germination and pollen tube growth involve a high number of signalling events,
including cell-environment interaction, intercellular and intracellular communications. It‘s
very well known that pollen germination and tube growth are significantly regulated by the
temperature, the transport of inorganic ions such as Ca+2 and K+ across the plasma
membranes of pollen, and by the synthesis of signal molecules such as gametophyte-specific
flavonol diglycosides (Taylor & Hepler, 1997).
Clear differences in temperature optima for pollen germination and tube grow were found
in J. nigra and J. regia: maximum germination occurred at 32°C and 28°C respectively (Luza
et al., 1987). Pollen germination percentage increased with temperature in both species but
declined abruptly and approached zero at approximately 40°C; no germination of pollen
occurred below 14°C. In addition, a positive linear correlation between staminate bloom date
and optimum temperatures for pollen germination was detected; higher optimum temperatures
were associated with late blooming dates. No differences in optimum temperature (33°C) for
pollen tube elongation in vitro were detected between black and Persian walnut. Nevertheless
the minimum temperature that would support pollen tub elongation in J. regia was lower than
in J. nigra. According to Luza et al., (1987), although some degree of phenotype plasticity
may influence the responses to the temperature, differences in the ability of pollen to
germinate at various temperatures could be genetically fixed. Significant variations in the
mineral ion composition of pollen were also identified between black and Persian walnut.
Notable differences were observed in P, N, Mg+2, Ni, and K+ content. As proposed by Calzoni
et al., (1990), the capacity of the sporophyte parent to accumulate mineral elements into
pollen grains during dehydration can be considered as species-specific. In particular, studies
in Arabidopsis demonstrated that an inward K+ current across the plasma membrane may play
a role in the activation of the osmotic water influx required for pollen germination and the
regulation of pollen turgor pressure during tube elongation (Fan et al., 2001). Extracellular
acidification induced by a H+-ATPase pump and high concentration of external Ca+2, typical
of the micropylar apparatus and the receptive synergid cell, may negatively regulate the
pollen inward K+ channels, inhibiting tube growth. Significant variation in macro- and microelements found in walnut pollen could represent a discriminating factor between J. nigra and
J. regia and negatively affect the ability of pollen tube to grow through the style and ovary
tissue when interspecific pollinations occur.
Pollen Biology and Hybridization Process: Open Problem in Walnut
77
The low probability of fertilization between Persian and black walnut also may be caused
by inefficient pollen –pistil recognition during germination and pollen tube elongation.
Successful fertilization depends on specific pollen –pistil interactions and only ―compatible‖
pollen grains are able to complete the passage through stigma, style and ovary (Geitmann &
Palanivelu, 2007). Pollen tube growth takes place in the extracellular matrix (ECM) of the
stigmatic and stylar transmitting tissues (TT) and along the ovule surface. Pollen tube growth
has been described as a specialised form of plant cell movement in which the pollen
cytoplasm moves forward, leaving behind cell wall materials connecting the tube to the empty
pollen grain that remains anchored on the stigmatic surface. This process involves
cytoskeletal elements such as actina, myosin, microtubules and the synthesis of wall
degradating enzymes (Taylor & Hepler, 1997). The pistil ECM provides chemical and
physical support as well as directional cues for pollen tube elongation toward the ovules. The
ECM is enriched with secretory materials such as free sugars, polysaccharides, glycoproteins
and glycolipids. Arabinogalactan proteins (AGPs), which are ubiquitous to plants, represent
the major class of proteins in the ECM of the transmitting tissue and in the stigmatic
exudates. AGPs are a class of hydroxyl-proline-rich glycoptoteins characterized by a high
carbohydrate content that include arabinose and galactose residues (Bacic et al., 1988). In the
last fifteen years, numerous studies demonstrated that AGPs of the transmitting tissues play a
major role in pollen recognition and adhesion on the stigma: they serve as nutrients and
adhesive substrates for the tube pollen elongation (Cheung et al., 1995; Taylor & Hepler,
1997; Sanchez et al., 2004; Geitmann & Palanivelu, 2007). Within the receptive female
flower, TTS proteins display a gradient of increasing concentration and glycosylation from
the stigmatic surface to the ovarian transmitting tissue. The increase in acidity associated with
increased TTS protein glycosylation may have a chemotropic effect, guiding pollen tube from
the stigma to the ovary (Wu et al., 1995). TTS proteins are also deglycosylated and then
incorporated into the pollen tube wall, providing nutrient and energy for tube elongation
process. Recently Sanchez et al., (2004) reported that new, interesting signalling systems are
involved in pollen tube growth, including ethylene and GABA. Furthermore, Geitmann &
Palanivelu, (2007) suggested a putative ovule—based pollen repulsion mechanism during
inter-specific crosses. This short-range repulsion of the pollen tube is used to inhibit the
access of multiple pollen tubes to an ovule, but it also prevents intra genomic conflicts that
would rise from the egg cell being fertilized by genetically distinct sperm. As demonstrated
by Palanivelu & Preuss (2006), the repulsion initiated prior to tube reception in the female
gametophyte maybe mediated by synthesis of nitric oxide (NO). This study also showed that
in Arabidopsis the repellent signal from ovule was less effective than in closely related
species.
According to Calzoni et al., (1990), the pattern of soluble cytoplasm, membrane and cellwall proteins of J. nigra and J. regia pollen vary quantitatively and qualitatively. In particular
the affinity chromatography of salt-soluble proteins of the pollen wall revealed a glycoprotein
fraction eluted with 300 mM of methyl- -D-mannopyranoside present in J. regia and
completely absent in J. nigra. We can postulate that these differences in the chromatography
profiles may reflect differences in enzymatic activities critical for hydration during pollen
germination, for adhesion and penetration through the stigmatic and stylar transmitting tissue,
and for proper pollen tube guidance. Which molecules, structures and interactions are relevant
for the expression of the incongruity in Juglans during inter-specific pollination are not yet
78
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
understood. Therefore functional and genetic redundancy among molecules involved in the
pollen-pistil recognition / fertilization permits rare crosses among hybridogenic plants.
POLLEN VIABILITY AND CONSERVATION
Taking into consideration the wide variability associated with walnut flower phenology, a
suitable method of pollen storage is essential. One of the main problems in black and Persian
walnut production is that it is difficult for breeders to obtain sufficient amounts of desired
pollen at the time pistillate flowers are receptive because of the dichogamous nature of the
species. For example, J. regia pollen matures and is usually shed about one month in advance
of J. nigra pistillate receptivity. In this case, short-term storage of Persian walnut pollen is
needed in order to carry out the inter-specific cross. In black walnut the situation is frequently
reversed, and the pistillate flowers are receptive a week in advance of pollen maturation,
requiring a pollen storage for one year (Griggs et al., 1971). As reported by Luza & Polito
(1985), the life span of walnut pollen appears to be very short under natural conditions and its
vitality can be affected by temperature, relative humidity and maturity. Studies of pollen
germination in vitro and of tube growth revealed differences among 21 Persian walnut
genotypes for ability to germinate. The samples were collected in the experimental fields of
the University of California, Davis (Luza & Polito, 1985). In a different study, similar results
were observed between 32 walnut cultivars from different sites of Turkey (Sütyemez, 2007).
The most remarkable indication of low vitality of Persian walnut pollen was given by
observations after 24 hours of incubation at 24°C. Under these conditions, pollen lost the
ability to germinate in vitro within two days for all cultivars tested. Black walnut pollen
seems to be viable at least 24 hours at 24°C, with an average of 21% of pollen germination
(Beineke & Masters, 1983). Polito et al., (1998a) concluded that rapid desiccation was the
probable cause of pollen death, and this factor may be a serious problem for breeders who
wish to store pollen for an extended period and for production of J. × intermedia hybrid
progeny.
Few methods have been developed for storing walnut pollen, and none of them are easy
to apply. In black walnut, refrigeration (14°C), without desiccation provided satisfactory
short-term storage for one to three weeks. According to Beineke & Masters (1983), freezer
storage and treatment in desiccators were inconsistent and for the most part damaging.
Nevertheless in this study the maximum pollen germination was 36.2 % after only one week
of storage. Persian walnut pollen can also be stored at typical freezer temperatures (-20°C)
but only with careful control of the relative humidity (RH) of the storage environment. As
described by Luza & Polito (1995), most of the Persian walnut pollen did not germinate after
three months of storage at -20°C when RH was not controlled. Pollen storage for three
months up to one year at -20°C is possible if the RH remains near 33 percent. This can be
achieved by storing pollen over a saturated solution of magnesium chloride (MgCl·6H2O),
although under these conditions the germination ability may vary and be near zero for some
cultivars. Hall and Farmer (1971) proved that liquid nitrogen storage (-196°C) of black
walnut pollen was effective and suggested the possibility of long-term viability retention.
Results of Luza & Polito (1988b) study indicated that Persian walnut pollen may be stored at
-196°C and pollen germinability can be maintained if the pollen grain moisture content is
Pollen Biology and Hybridization Process: Open Problem in Walnut
79
controlled and reduced to a value between 7.50% and 3.20% by gentle drying for 24 hours
after collection. They concluded that excessive moisture (more than 30%) may be lethal,
inducing ice-crystal formation in the pollen cell during freezing. Intracellular ice formation
can induce fractionation of organelles and disruption of membranes. As described in the
previous section, the viability and germinability of pollen depends strongly on the state of the
vegetative cell membranes. Obviously the apparatus for liquid nitrogen long-term storage are
not widely available and this method is not easy to apply in many instances.
PISTILLATE FLOWER STRUCTURE AND RECEPTIVITY
Pistillate flowers of both, J. regia and J. nigra, are borne at the tips of terminal shoots on
current season‘s wood, in spikes of typically two to three flowers. McGranahan and Leslie
(2009) reported that female flowers are also produced on the tips of lateral shoots in some
cultivars. This type of flowering is called ―lateral bud fruitfulness‖ and is often correlated
with high nut yield in young trees.
Pistillate flowers lack visible sepals and petals, are pubescent, small and green. In
particular, the entire basal portion of the flower is enclosed with a hairy sticky involucre fused
to four sepals. The husk of the mature walnut fruit is derived mainly from the tissues of the
involucre and sepals (Figure 3).
Figure 3. Longitudinal section of walnut pistillate flower. Pistillate flowers lack visible sepals and
petals, are pubescent, small and green. In particular entire basal portion of the flower is enclosed with a
hairy sticky involucre fused to four sepals. The ovary is the enlarged basal portion of the pistil which
also includes a short style and bilobular stigma. At the base of the locule is a one ovule which is
surrounded by a single integument. Surrounded by the integument is a region called nucellus in which
is present the embryo-sac that contains an egg cell and two synergid cells at micropylar pole, two polar
nuclei in the centre, and three antipodal cells at chalazal pole.
80
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
The ovary is the enlarged basal portion of the pistil which also includes a short style and
a large feathery bilobular stigma. The structure of the stigma facilitates the interception of
wind-borne pollen, the recognition of walnut pollen and the exclusion of pollen of other
species. The surface of the stigma secretes a thin layer of exudates providing a suitable
medium for pollen germination and the initial growth of the pollen tube (Polito et al., 1998a).
The shell of fully matured fruit is derived from the ovary wall. At the base of the locule is one
ovule which is surrounded by a single integument. At the end of fruit development, the
integument becomes the seed coat, the brownish pellicle that enclosed the kernel at maturity.
A region called nucellus is surrounded by the integument. The nucellus constitutes the bulk of
the ovule during fertilization. The cells of the nucellus degrade as the fruit develops. Within
the nucellus is present the embryo-sac (seven-celled structure) containing the female germ
cell (egg cell). At the time of pollination most of the embryo-sac structure contain an egg cell
and two synergid cells at the micropylar pole, two polar nuclei in the center, and three
antipodal cells at chalazal pole (Figure 3). When pollen enters through the embryo-sac, one
sperm cell is discharged and fuses with the egg cell to form the zygote and subsequent
embryo. The second sperm cell fuses with two polar nuclei to provide a nutritive tissue called
endosperm (triploid tissue). In the early stages of fruit growth the endosperm is consumed and
disappears in mature fruit. One week after fertilization, the zygote has already started cell
division and the proembryo is composed of a maximum of eight cells.
Polito et al., (1998a) reported that fertilization in walnut usually occurs five to seven days
after pollination. Luza and Polito (1991) showed that porogamy and chalazogamy are
alternate pathways in walnut flowers. In angiosperms, pollen tubes typically enter the ovule
through the micropyle, a phenomenon referred as porogamy. Chalazogamy refers to pollen
tube entry through funiculus and chalaze tissues rather than though the micropyle. According
to Luza and Polito (1991), in J. nigra and J. regia the occurrence of chalazogamy / porogamy
is correlated with the developmental stage of pistillate flower.
They observed that during the earlier stages of anthesis, pollen tubes arrive at the ovary
when the integument is less developed and a considerable space is present between the apex
of the nucellus and the base of the stylar canal. In this case the pollen tubes may be unable to
cross this open space and bypass the micropyle. They grow along the surface of winged
outgrowths to the chalazal end of the ovule. When the development of ovary progress to the
point that integument is close to the bottom of the style, then porogamy occurs. Nevertheless
in J. regia cv. Franquette, Tadeo et al., (1994) observed that five days after the time of
pollination one of the synergid cells had a normal structure whereas the other usually was
degenerated. In porogamy, the pollen tube contents are discharged into one of the synergids
prior to fertilization, causing the breakdown of this cell. Since only one of the synergid cell
survived in all embryo-sacs analysed in this study, Tadeo and colleagues (1994) suggested
that pollen tubes might have entered the ovule mainly via the micropyle.
Pistillate flowers that are not fertilized continue to grow for the next three weeks, at
which point they drop. In the embryo-sac of unpollinated ovaries, fusion of the two polar
nuclei occurs in the early development stages, leading to a 2n endosperm tissue. The absence
of pollination accelerates cellularization of the 2n endosperm, causing degeneration of
embryo-sac.
Tadeo et al., (1994) evaluated the putative role of endogenous gibberellins (GAs) in
walnut fruit development. A wide body of evidence suggested that pollination process may be
particularly dependent on GAs. They observed different patterns of GA change in pollinated
Pollen Biology and Hybridization Process: Open Problem in Walnut
81
and unpollinated ovaries of ‘Franquette‘. In particular, gibberellin A1 (GA1), which is thought
to be an active GA controlling vegetative growth in higher plants, showed a transitory
increase prior to and immediately after fertilization, and a gradual decrease subsequently in
pollinated ovaries. In unpollinated ovaries, the transitory GA1 peak occurred at the same
moment but was higher (2-fold) than in pollinated ovaries. Thirteen days after pollination,
GA1 levels were much lower in unpollinated flowers than in pollinated flowers. It has been
postulated that GAs may preserve embryo-sac viability and extend the period of maximum
pollen receptivity. GA1 may also postpone the beginning of senescence in unpollinated
ovaries and protect the reproductive structures of the ovary before fertilization. According to
Tadeo et al. (1994), fertilization induced immediately the gradual reduction of GA1 at the
beginning of embryo cell division. They proposed that GA1 may be a critical component for
embryogenesis. Growth arrest and flower abscission coincided with very low amounts of
gibberellins.
For a long time the design of walnut orchards has been focused on maximizing pollen
density during pistillate flower bloom to improve nut yield. Moreover there were major
breeding efforts to modify the quality and quantity of walnut production by selecting suitable
genotypes and/or carrying out controlled crosses between useful parental trees with a handpollination into receptive female flowers. The pistillate flower is usually receptive to pollen
for a short time, seven days at most, if conditions are ideal. Generally hot and dry
environmental conditions reduce the period of optimal receptivity. Before the expansion of
the stigma, female flowers are not able to retain wind-borne pollen and to produce the layer of
exudates in which pollen can germinate and tube growth occurs. Considering the low pollen
viability observed at room temperature, pollen that lands on stigmas does not have a good
chance to survive until the female flowers become receptive. Polito et al., (1998a) reported
that pistillate flowers were highly receptive when the two stigmatic lobes were separated from
one another to form a V-shape. Once the stigmatic lobes were orientated at more than 45
degree angle to the longitudinal axis of the ovary, the surface began to dry out and the female
flowers were not longer receptive.
Nevertheless, as described in the next section, some evidence suggests that a large and
uncontrolled amount of pollen can adversely affect the final nut set by inducing pistillate
flower abscission.
PISTILLATE FLOWER ABSCISSION (PFA)
Pistillate flower abscission (PFA), was reported for the first time in Persian and black
walnut by Catlin et al., (1987) and Beineke and Masters (1976) respectively. Pistillate flower
abscission is the loss of the pistillate flowers early in the season, typically two to three weeks
after bloom and prior to fruit drop due to lack of pollination. According to Catlin et al.,
(1987), the ovary enlargement in PFA-type flowers stops at a diameter of 3 to 4 mm, leading
to abscission 10 to 14 days later. The abscission of un-pollinated flowers occurs three to four
weeks after bloom, and their ovaries have been enlarged to more than 7 mm in diameter. Two
different areas of separation were also detected: the distal and proximal area of the peduncle.
PFA-type abscission of flowers occurs at the zone between the peduncle and vegetative apex,
causing drop of flowers still attached to the peduncle, in contrast to separation between the
82
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
ovary and peduncle, which was typical of non-fertilized flowers. In addition PFA-type
flowers showed cell and tissue necrosis at the tip of the stigma, in the inner wall of the ovary,
the integuments and throughout the placental evaginations (Catlin & Polito, 1989).
Pistillate flower abscission has emerged rapidly as a major non-pathogenic problem of
walnut production, reducing final yield. In particular, Catlin et al. (1987) recorded levels of
PFA in excess of 90 percent in ‗Serr‘ orchards planted in the Sacramento Valley of
California. ‗Serr‘ appears to be the most susceptible cultivar. Other J. regia cultivars, such as
‘Chandler‘, ‘Howard‘ and ‘Vina‘, are affected by PFA but usually with less loss (Catlin &
Olsson, 1990; Rovira & Aletà, 1997; Polito et al., 1998b). Moreover this phenomenon may
vary among varieties and sites, and it is not consistent between years. Rovira & Aletà (2001)
evaluated the incidence of PFA in 19 different cultivars and selections of J. regia: five
California cultivars (‗Chandler‘, ‗Chico‘, ‗ Hartley‘, ‗Serr‘, and ‗Vina‘), four Chilean
selections (‗AS-0‘, ‗AS-1‘, ‗AS-5‘ and ‗AS-7‘) five French cultivars (‗Franquette‘, ‗Lara‘,
‗Mayette‘, ‗Marbot‘ and ‗Parisienne‘) and five selections from Spain (‗MBT-49‘, ‗MBT-31‘,
‗MBT-247‘, ‗MBT-119‘, and ‗MBT-122‘) located at IRTA-Mas Bovè (Spain). Significant
differences were observed between years, among groups of cultivars of different geographic
origin and within cultivars. The Spanish selections were the most affected group with 73.4%
PFA, compared to Chilean selections that showed only 6.8%. Unexpectedly, French and
Californian cultivars presented an intermediate behaviour, showing lower mean values of
PFA in ‗Chandler‘, ‗Chico‘, ‗Franquette‘, ‗Hartley‘ and ‗Serr‘ cultivars than those observed
in California. Nevertheless in all cases the number of dropped flowers due to PFA was
negatively correlated with final nut set. In addition, although PFA incidence is difficult to
predict and control, the heritability of this trait seems high (narrow sense heritability = 0.61);
selection of parents with low abortion could produce offspring with lower levels of PFA
(Hassani et al., 2006). Finally a bias in the measures of PFA incidence could have occurred in
the previous studies. PFA level was usually quantified as a percentage of the necrotic
pistillate flowers 3-4 mm in diameter dropped while attached to the peduncle. In a recent
study, Gonzàlez et al. (2008) reported two separation areas in PFA-type flowers of ‗Serr‘
walnut. The distal separation area of the peduncle was present in 36 % of the cases, causing
flower drop without the peduncle; the remaining 64% showed an attached peduncle. The
absence of the peduncle may be attributed to abscission from lack of pollination and may
have misled the researchers. They also noted a new and interesting symptom useful for
discriminating between these two types of abscission. The scar caused by PFA presented an
irregular surface, was brown, and 1-2 mm in diameter, versus the scar caused by lack of
pollination, which had a smooth surface, was chalky and 4 to 5 mm in diameter.
During the late 1980s several studies were conducted to determine the cause of this
disorder. Mineral nutrient deficiency, the phytotoxic effect of copper sprays used for control
of blight/anthracnose disease, unmet chilling requirement, tree age, water stress,
environmental conditions, defective ovarian development (Catlin et al., 1987), low nitrogen
content and competition for carbohydrate (Deng et al., 1991) were excluded as plausible
causes of PFA. In the walnut orchards of Balatonboglàs Winery (Hungary), Pór & Pór (1990)
observed that the nut yield decreased significantly as distance from the pollenizer decreased.
After noting PFA-type drop when large amounts of pollen were applied to flowers in the
course of making crosses for breeding, McGranahan et al. (1994) proved that pistillate flower
abscission was caused by the presence of excess pollen on the stigma. They discarded the
hypothesis that the high number of pollen tubes growing through the stigma and the style to
Pollen Biology and Hybridization Process: Open Problem in Walnut
83
the ovary may influence the fertilization rate. Dead pollen induced the same amount of PFA
as live pollen. Polito et al., (1998b) confirmed the previous findings, detecting a positive
correlation between PFA and pollen load. High PFA was always associated with high
numbers of pollen grains on stigmas. They combined data from walnut orchards at different
sites in California and deduced that 50 % PFA occurred in ‗Serr‘ when an average of 85
pollen grains per flower were present.
Polito et al., (2006) evaluated the putative involvement of dichogamy in pollen load.
Analysis of pollination dynamics in a California ‗Chandler‘ walnut orchard, using
microsatellite markers, permitted them to discriminate the effective sources of pollen during
the dichogamous bloom cycle of the trees. The most likely source of excess pollen necessary
for PFA induction was the self-pollen shed from catkins at the beginning of female flower
receptivity. Therefore the extent of bloom overlap (self-pollination) may also have a role in
the evolution of walnut dichogamy and PFA may have been a mechanism to improve progeny
fitness. As reported by McGranahan et al., (1994):
―Trees with overlapping male and female blooms would be at a reproductive
disadvantage and thus dichogamy would be favored. In forest tree competition, this
phenomenon would tend to discourage walnut trees crowded by other walnut from producing
a heavy nut set, instead they could put their energy into vegetative growth ‖.
They suggested that comparing ―bloom overlap‖ and PFA levels in some cultivars might
prove meaningful. In the last decade, data supporting the theory that excess pollination causes
PFA were collected in central Chile where ‗Serr‘ pollination was insufficient due to poor
overlap of male and female flowers. The consequent reduction in self-pollination led to a
correspondingly lower percentage of PFA (Gratacòs et al., 2006). On the other hand no
significant correlation was detected between the incidence of pistillate flower abscission and
bloom overlap for 19 walnut cultivars and selections planted in Spain (Rovita et al., 2001).
As indicated by Kruger (2000), a reduction of pollen density in the orchard and
minimization of the losses caused by pollen-induced pistillate flower abscission may be best
achieved by removing pollinizer trees from the site and/or mechanical shaking of the trees
with the objective of removing some of the catkins. This suggestion requires careful
consideration of how many pollinizers are adequate and which orchard configuration is
suitable to provide sufficient but not excessive pollen loads. Preliminary experiments carried
out by Polito et al., (1998b) demonstrated that removing some pollinizers from a ‗Serr‘
orchard improved walnut yield from 20 to 86 percent. An alternative approach implies the use
of tree shakers at the beginning of the male bloom when the first catkins fall from the trees.
At that time, most of the catkins are half size or longer, and easy to remove without damaging
the trees by injuring shoot tips. Considering that the density of walnut pollen is constant for
160m around a pollenizer, even in the absence of wind (Impiumi & Ramina, 1967), Polito et
al., (2006) suggested that entire rows of trees be shaken if they are within 47m of the cultivar
affected by PFA. Lemus (2005) and Gratacòs et al., (2006) have successfully applied
mechanical shaking treatments in Chilean ‗Serr‘ orchards. By shaking walnut trees when 15%
and 50% of female flowers were receptive, they produced an increase in nut yield of 25-30%.
Both of these techniques are time-consuming and require information about phenology and
the history of the orchard. These methods can fall short for practical reasons as well. Many
‗Serr‘ growers do not own shakers and find it difficult to coordinate the required activities in
84
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
the necessary time frame. Other growers have obtained mixed results from these methods
because their proximity to other orchards increases their pollen load (Beede et al., 2008).
Recent research has focused on the physiology and efficacy of ethylene antagonists as
management tools for controlling PFA in walnut orchards, especially in ‗Serr‘ orchards. It is
well known that the final stages of fruit development are controlled by hormones such as
ABA and ethylene. In particular ethylene is a natural hormone associated with organ
senescence and dehiscence of flowers and fruit. Although ethylene has been extracted and
identified in walnut fruit, its precise role is unclear. Polito et al. (2005), has postulated that the
overloading of pollen on stigmas may increase ethylene biosynthesis, inducing pistillate
flower abscission. As proved by Johnson (2008), a peak in ethylene production was detected
approximately 12-30 hours after pollination in excised pistillate flowers, with pollinated
flowers producing more ethylene than non-pollinated ones. It‘s also interesting to note that
ethylene production was inducted by both live and dead pollen. An increasing number of
researchers have focused their attention on the potential for reducing the effect of pollinationinduced ethylene by applying two inhibitors, aminoethoxyvinylglycine hydrochloride (AVG)
and 1-methylcycloproane (1-MCP). The modes of action for these two molecules are distinct.
AVG, as Retain® (Valent Bioscience), inhibits ethylene synthesis, while 1-MCP, as an
isopropanol-based adjuvant or as a gas, is a competitive inhibitor of ethylene action. Johnson
(2008) observed that AVG and 1-MCP produced a significant decrease and increase,
respectively, in ethylene biosynthesis by pollinated flowers. In the latter case, the observed
increase in ethylene production may have been due to a feedback mechanisms triggered when
1-MCP blocked the ethylene receptors. In recent studies, the effect of AVG and 1-MCP
application was tested with mixed results. Early application of AVG (125ppm) consistently
reduced PFA in ‗Serr‘ orchards located in different Chilean walnut production areas (Lemus
et al., 2007) and in San Joaquin County, California (Beede et al., 2008; Johnson, 2008). In
particular, AVG-treated trees showed a 57 to 70% yield increase over the untreated controls.
Flowers treated in the pre-receptive and early stages of stigma development performed better
than flowers at peak receptivity. The AVG residual must be sufficient to inhibit ethylene
production caused by excessive pollen load during the 5 to7 day receptivity period.
Surprisingly, a field experiment in a ‗Chandler‘ orchard showed no reduction of PFA using
AVG, but the effectiveness of 1-MCP (1-10ppm) against pistillate flower abscission was
verified (Johnson, 2008).
Although these studies are not conclusive, the role of ethylene in the regulation of
fertilization and fruit development deserves thorough investigation. The use of AVG and 1MCP could represent powerful tools to overcome PFA in the orchard management but also in
breeding programs in order to carry out controlled crosses. Rovita and Aletà (1997) reported
that an artificial load of pollen applied to female flowers raised significantly the percentage of
PFA, compared to open-pollinated reference flowers. Gonzàlez et al., (2008) observed that
only very low concentration of ‗Serr‘ pollen (maximum 5 grains per mm-2 of applied surface)
could prevent PFA in controlled crosses.
The theory that hand–pollinations using pollen from a single source may influence the
rate of pistillate flower abscission and fertilization in walnut has not been evaluated until
recently. In all experiments previous described, a single pollen source was used, either self
pollen or pollen from a single donor. McGranahan et al., (1994) also noted that PFA was first
discovered in commercial walnut orchards where low pollen diversity is expected. In
addition, investigation of self pollinated flowers proved that some Georgian walnut varieties
Pollen Biology and Hybridization Process: Open Problem in Walnut
85
were self-sterile. Kvaliashvili et al.,(2006) proposed that self pollen promoted pistillate flower
abscission.
There is a growing but conflicting body of evidence that high pollen diversity can
enhance plant fecundity, although the mechanisms underlying such results are still poorly
understood (Kron and Husband, 2006). For many plants, the number of pollen genotypes
deposited on a stigma is positively correlated with reproductive success. In controlled crosses,
increasing the diversity of the pollen source increases the probability that a female flower will
receive pollen from a genetically compatible donor, it enhances the number of ovules
fertilized per tree (pre-zygotic factors) and/or reduces embryo abortion (post-zygotic factors).
INVESTIGATION OF GENETIC HYBRIDIZATION IN WALNUT
The importance of intra- and inter-specific hybridization for the genetic improvement of
forest trees has been evident for at least 50 years (Schreiner 1960). Nevertheless, tree
improvement often has been narrowly focused on selection and breeding within a single
native species. As suggested by Schreiner (1963), inter-specific hybridization also provides
the maximum genetic diversity needed for greatest genetic improvement. Sometimes interspecific hybrids may be difficult to obtain, however, even with the use of controlled
pollination. This is the case for hybridization between Juglans nigra L. and Juglans regia L.
that produces Juglans × intermedia Carr. As reported in the previous sections, the
hybridization between black and Persian walnut species is rare under natural conditions and
difficult using controlled pollination because of phenological and genetic incompatibilities. It
requires the overlapping of the bloom time for the two parental trees, an appropriate
temperature for pollen germination and penetration though the stigma and the style to the J.
nigra ovary (Luza & Polito, 1987), and genetic compatibility pre- and post-pollination
(Sartorius, 1990).
In the last thirty years, seed orchards for hybrid production have been designed; generally
one plus tree as a female parent and several plus trees as fathers were deployed to ensure
enough pollen pressure. The oldest and best known European J. × intermedia (NG23 × RA)
was obtained in France by the open-pollination of the mother J. nigra NG23 with four J.
regia plus trees RA984, RA996, RA331, RA295 as male parents (Becquey 1990). However,
selection by phenological observations and clonal (graft) propagation of hybridogenic parent
trees required more than ten years (Jay-Allemand et al. 1990).
In these studies (Pollegioni et al, 2009a, b), we reported a new method based on (neutral)
microsatellite markers which permitted the identification of new interspecific hybrids and, at
the same time provided a rough idea of which walnut genotypes might be useful for
establishing new seed orchards for inter-specific F1 hybrid production. The use of genotypes
with demonstrated compatibility may increase the efficiency of F1 production. This method
should also provide a powerful tool to evaluate the barriers to hybridization between Juglans
species and to detect the factors that reduce hybrid fertility
86
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
RETROSPECTIVE IDENTIFICATION OF HYBRIDOGENIC WALNUT
TREES
The identification and selection of genotypes with a spontaneous ability to cross
(hybridogenic parent trees) is a simple and efficient method for obtaining hybrid progeny.
Pollegioni et al. (2009b) reported a new method for retrospective identification of
hybridogenic walnut trees based on microsatellite (SSR) fingerprinting and parentage analysis
in order to establish new seed orchards for hybrid production.
Woeste et al. (2002) developed a panel of thirty nuclear microsatellites in J. nigra L. as
markers for a wide range of genetic investigations. A subset of these markers has been
successfully used for clonal identification (Robichaud et al., 2006) and a broad-scale study of
the genetic structure of J. nigra populations in the Central Hardwood Region of the United
States (Victory et al., 2006). At the same time, a subset of microsatellites were also selected
and screened in J. regia L. as a starting point for the genetic characterization of walnut
cultivars (Dangl et al., 2005) and the variety ‗Sorrento‗ (Foroni et al.,2005). Microsatellites,
known as simple sequence repeats (SSRs), are short (1-6 bp long), tandemly repeated DNA
sequences widely dispersed throughout eukaryotic genomes. These markers require the
design of primers for the conserved flanking regions of the microsatellite and the PCR
amplification of the repeat region. The single-locus markers are characterized by
hypervariability, abundance, high reproducibility, Mendelian inheritance, and co-dominant
expression. These positive features make them suitable tools for parentage analysis (Streiff et
al., 1999) and molecular fingerprinting of hybrids (Nandakumar et al., 2004). Nevertheless, a
detailed study of the inter-species transportability of the microsatellite markers in walnut was
not yet available. Peakall et al. (1998) demonstrated that the successful cross-species
amplification of SSRs does not prove the maintenance of the repeat motif in the non-source
species. Studies employing cross-species amplification should therefore be accompanied by
knowledge of the underlying DNA sequence.
Over the last six years under the framework of the national Project RI.SEL.ITALIA
(financially supported by the Italian Ministry of Agricultural Policy, Sottoprogetto 1.1
―Biodiversità e Produzione di Materiale Forestale di Propagazione‖, coordinator Dr. Fulvio
Ducci CRA-Arezzo), the C.N.R. Institute of Agro-environmental and Forest Biology
(Porano) has been intensively evaluating walnut germplasm in Italy. As a result of these
efforts, a promising mixed population, including J. nigra, J. regia and some J. × intermedia
hybrids, was discovered in Northern Italy, Veneto region, Villa Mezzalira Park, Bressanvido
(Pollegioni et al. 2009a). Ten microsatellites tested in the mixed walnut population collected
in Villa Mezzalira‘s Park amplified in both species, producing fragments of variable size;
eight (7.14 %) were common, 68 (60.7 %) amplified in J. nigra and 36 (32.1 %) in J. regia
only (private alleles). Indices of genetic diversity revealed a high level of variability. DNA
fingerprinting analysis divided the total sample set (138 plants) into three main groups: J.
nigra (82), J. regia (49) and diploid (2n = 32) J. x intermedia hybrids (7). Forty-nine J. regia,
8 J. nigra, 3 diploid hybrids, are adult trees growing in the Park (Table1); 15 J. nigra adults
plants (J. nigra NC) were located outside the park; 59 J. nigra and 4 diploid hybrids were sixyear old plants grown at the Veneto regional nursery (Montecchio Precalcino, Vicenza) from
seeds collected in the Park.
Pollen Biology and Hybridization Process: Open Problem in Walnut
87
Table 1. Characteristics of 139 plants sampled in Villa Mezzalira Park, Bressanvido
(Northern Italy 45° 39′ 0′ ′ N, 11° 38′ 0′ ′ E) genotyped using SSR markers (Pollegioni
et al., 2009a).
Species
Group
Adult
Trees (N)
Genotype label
Six year old
Treesb (N)
Genotype Label
J. nigra N
8
N3, N4, N5, N17, N18,
N22, N23, N24
59
N25-N83
67
J. nigra
NC a
15
NC1-NC15
-
-
15
J. regia
49
R6-R16, A.E., B2-B20,
V1-V17
-
-
49
Diploid
hybrid
3
H1, H2, H19
4
IMP3, IMP4,
IMP9, IMP18
7
Triploid
hybrid
1
N21
-
-
1
Total
J. nigra L.
J. regia L.
J. ×
intermedia
Carr.
Total
a
b
76
63
139
Fifteen black walnut adults plants located outside the park were labelled J. nigra-NC.
Six-year old plants growing at the Veneto regional nursery (Montecchio Precalcino, Vicenza), Italy,
from seeds collected inside the park.
By genotyping the adult trees in the population with microsatellites, a triploid hybrid
plant with two genome parts from J. nigra and one part from J. regia was identified (N21
tree). Cytological analysis proved that the N21 tree is triploid and that it contains 48 somatic
chromosomes (Figure 4).
The analysis to identify the maternal parents of the seedling trees from the population in
the park (exclusion method) indicated that J. nigra N17 was the ―putative‖ hybridogenic
mother plant of the seven diploid hybrids. Analysis of the sequence of the amplified
fragments confirmed the cross-species amplification of the SSRs, but inter-specific
differences in allele sizes were due not only to simple changes in the number of repeats but
also to mutations in the flanking regions: insertion and deletion events in the flanking regions
contributed to the variation in allelic size among and within Juglans species.
The same battery of 10 SSR primer pairs was used to perform the DNA fingerprinting
and parentage tests of eight half-sib families collected in the Villa Mezzalira‘s Park with the
specific objectives of 1) detecting the presence of J. × intermedia in these progenies, 2)
identifying J. nigra mother trees that spontaneously crossed with J. regia (hybridogenic
mothers), and 3) verifying the differential reproductive success (DRS) of J. regia male
parents (hybridogenic fathers) for production of hybrid offspring genotypes. (Pollegioni et al.,
2009b). Seeds were collected from seven adult J. nigra trees and the triploid hybrid plant in
Villa Mezzalira Park. The seeds were planted in a field at the CRA Institute for Silviculture
(Arezzo), and eight open-pollinated progenies (461 total seedlings) were obtained: forty-one
88
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
seedlings from plant N3; 29 from N24; 88 from N17; 24 from N18; 76 from N22; 71 from
N23; 114 from N24, and 18 from the triploid N21 (Table 2).
Figure 4. Somatic chromosome number (3n = 48) of developing (premeiotic mitosis) pollen mother
cells of N21 hybrid genotype was evaluated microscopically after traditional aceto-carmine staining.
The high levels of polymorphism (129 alleles) detected positively influenced the
exclusion and identity probabilities described in the study. The allelic richness and the
observed heterozygosity measured for each locus in the tested samples provided high
combined power of exclusion and low probability of identity. The study clearly demonstrated
the power of SSR markers for DNA fingerprinting and parentage analysis. Principal
Coordinate Analysis, which was performed on the Simple Match‘s similarity coefficient was
computed using 129 alleles. It revealed distinct J. nigra and J. regia clusters and the presence
of several intermediate individuals. Three main groups were detected (Figure 5); two that
included 49 J. regia and 82 J. nigra trees were clearly separated by the first principle
coordinate. The second principal coordinate divided the J. nigra trees in two subgroups. J.
nigra-NC plants, located outside the site area, were found to be genetically distinct from the
other eastern black walnut trees planted inside the Park. Seven diploid hybrids (H1, H2, H19,
IMP3, IMP4, IMP9 IMP18) were incorporated in the third main group, located in an
intermediate position between black and common walnut. As expected, the triploid hybrid
plant (N21), with two genome parts of J. nigra and one part of J. regia, was placed between
black walnut and the hybrid groups. Cluster analysis showed that the third group was
composed of genotypes genetically distinct from individuals of the two parental species, but
this placement does not prove the trees in this group are all interspecific hybrids.
The identification of diploid hybrids was definitively performed by assigning the459
offspring genotypes to four putative classes: two black walnut (J. nigra N, J. nigra NC), one
J .regia, and one J. × intermedia. The assignment analysis by the Paetkau et al. (1995)
frequency method and Rannala & Mountain (1997) partial Bayesian method, combined with
the exclusion-simulation significance test of Cournet et al. (1999), revealed the presence of
198 diploid J. x intermedia hybrids among the total of the progeny seedlings (42.9%).
Maternity checks were performed on all individuals. A few errors of sampling (0.06 %) were
found. These probably resulted from accidental mixing of seeds during collection of
Pollen Biology and Hybridization Process: Open Problem in Walnut
89
progenies (Table 2). Four distinct hybridogenic J. nigra mother trees were identified,
including N17 as expected, but also N23, N24 and the triploid hybrid plant N21. The three
hybridogenic black walnut plants had different reproductive success rates.
Figure 5. Pollegioni et al., 2009b. Principal Coordinate Analysis of 600 Juglans individuals based on
genotypic similarity as determined by simple match coefficients based on 10 SSR loci. J. regia (N =
49), J. nigra N (N = 67), J. nigra NC (N = 15), N21 triploid hybrid, ◊ diploid hybrids (N = 7) and
J. nigra offspring (N = 461).
The identification of three distinct hybridogenic J. nigra mothers was an important
practical result. Indeed, genetic improvement, especially of long living plants, requires the
availability of selected ―plus‖ genotypes able to produce a consistent quantity of hybrid
progeny. The authors‘ approach also permitted the quantification of the differential
reproductive success of each mother. Thus, even though these results should be confirmed by
observations over additional years, it should be possible to focus breeding research on two
plants with a relatively high rate of hybrid production: N24 (87%) and N17 (70%). The
authors also showed that the triploid hybrid plant N21 produced fertile female flowers,
although the number of progeny was limited (18 total seedlings: 15 hybrid and 3 J. nigra
genotypes). As described by Funk (1970) some J. × intermedia trees flower profusely but
never bear much seed. In addition two hybrid plants out of 15 displayed an unusual and fatal
karyotype. The most likely explanation for the unusual microsatellite profiles in some of the
progeny is irregular meiosis in the original triploid hybrid parent and subsequent elimination/
addition of chromosomes.
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
90
Table 2. Maternity analysis and identification of hybridogenic mother trees (Pollegioni
et al., 2009b).
Maternal
tree
Number of putative
offspring
Non-maternity
(seed mixture)
Maternity
assignment b
Total
number of
offspring
Hybrid progeny J.
×intermedia Carr c (N)
N3
41
0
-
41
0
N4
29
0
-
29
0
N17
88
0
-
97
68 (70%)
N18
24
9
N17 (9)
15
0
N21
18
0
-
18
15 (83.3%) a
N22
76
3
N23 (3)
73
0
N23
71
0
-
74
17 (22.9%)
N24
114
0
-
114
100 (87.7%)
a
Two hybrids offspring, N21- 14 and N21-15, triploid for one locus, were included.
b
The maternity was re-assigned combining the exclusion method based on Mendelian segregation rules
with maximum-likelihood approach (Marshall et al. 1998).
c
Based on genotyping eight half-sib progenies using ten microsatellite loci.
Paternity of 198 diploid hybrids detected in four open-pollinated families was inferred by
using a likelihood-based approach (Marshall et al. 1998) based on nine microsatellite loci.
Differential male reproductive success was observed among pollen donors within the research
site (Figure 6).
Figure 6. Pollegioni et al., 2009b. Number of hybrid offspring produced by each J. regia male that
pollinated J. nigra females,
N17,
N21,
N23,
N24, and ---- total. Assignment
was based on greatest likelihood. The successful pollinations corresponded to the number of times a
pollen donor (J. regia) pollinated a mother tree (J. nigra).
Pollen Biology and Hybridization Process: Open Problem in Walnut
91
In the production of hybrid progeny male reproductive success was unevenly distributed
both in amount and in space. In particular 49 (47.5 %) of the total diploid hybrids detected in
four half-sib families were sired by only three J. regia genotypes (B6, V15 and B7).
Although phenological data was not recorded for the individuals at the research site and
the authors‘ experimental design could not differentiate among all possible reasons for
unequal paternal success, the results do guide speculation. Juglans nigra generally blooms
later than J. regia, so the amount and timing of pollen shed, distance of pollen donor from
seed trees, plant size, and weather conditions, may have had a profound effect on the
distribution of male reproductive success.The authors did not find a significant correlation
between reproductive success of Persian walnut trees and the distance from black walnut
mother plants. Spatial factors may have influenced pollination in their study, but they were
probably not a major determinant of male success. The timing of pollen release and the
presence of some mechanisms of genetic incompatibility could be plausible explanations for
the observed fertilization pattern. The paternal plants may have been the only trees releasing
pollen when the maternal trees had receptive stigma (synchronous flowering). On the other
hand, as reported previously, pre-zygotic factors, such as pollen germination and tube growth
rate, or post-zygotic factors, such as genetic complementation, could have affected male
reproductive rate and may have been particularly relevant in this case where inter-specific
crosses were made (Wheeler et al. 2006).
In conclusion, although fluctuations in pollen production can occur among years, and the
experiment was carried out on a relatively small sample of parent trees, parentage analysis of
half-sib families based on microsatellite markers permitted the identification of new
interspecific hybrids and, at the same time provided a rough idea of which walnut genotypes
might be useful for establishing new seed orchards for inter-specific F1 hybrid production.
The use of genotypes with demonstrated compatibility may increase the efficiency of F1
production. This method should also provide a powerful tool to evaluate the barriers to
hybridization between Juglans species and to detect the factors that reduce hybrid fertility.
Finally the retrospective selection of hybridogenic trees is a valid approach for the
identification of new parental combinations when no phenological and morphological data of
the trees are available.
ACKNOWLEDGMENTS
The authors thank Dr. Agnes Major, Susanna Bartoli, Giovanni De Simoni, Claudia
Mattioni, Marcello Cherubini and Daniela Taurchini for their support in statistical and
laboratory analysis. A warm thank to Prof. Chuck Leslie (Walnut Breeding
Department,University of California, Davis) for the critical review of the manuscript.
The use of trade names is for the information and convenience of the reader and does not
imply official endorsement or approval by the United States Department of Agriculture or the
Forest Service of any product to the exclusion of others that may be suitable.
92
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
REFERENCES
Almeida, I.F., Fernades, E., Lima, J.L.F.C., Costa, P.C., Bahia, M.F. (2008). Walnut (Juglans
regia) leaf extracts are strong scavengers of pro-oxidant reactive species. Food
Chemistry, 106, 1014-1020.
Amaral, J.S., Casal, S., Pereira, J.A., Seabra, R.M., Oliveira, B.P. (2003). Determination of
sterol and fatty acid compositions, oxidative stability, and nutritional value of six walnut
(Juglans regia L.) cultivars grown in Portugal. Journal of Agriculture and Food
Chemistry, 51, 7698-7702.
Amaral, J.S., Valentão, P., Andrade, P.B., Martins, R.C., Seabra, R.M. (2008). Do cultivar,
geografic location and crop season influence phenolic profile of walnut leaves?.
Molecules, 13, 1321-1332.
Anselmi, N., Mazzaglia, A., Scaramuccia, L. De Pace, C. (2006). Resistance attitude of
Juglans regia L. provenances towards anthracnose (Gnomonia leptstyla (fr.) ces et de
not.). Acta Horticulturae. 705, 409-416.
Aradhya, M.K., Potter, D., Gao, F., Simon, C.J. (2007). Molecular phylogeny of Juglans
(Juglandaceae): a biogeography perspective. Tree Genetics & Genomes, 3, 363-378.
Bacic, A., Gell, A.C., Clarke, A.E. (1988). Arabinogalactan proteins from stigmas of
Nicotiana alata. Phytochemistry, 27, 679-689.
Becquey, J (1990) Quelques précisions sur les noyers hybrides. Forêt enterprise, 69, 15-19.
Beede, R:H., Grant, J., Anderson, K.K., Hasey, J. (2008). Managing pistillate flower abortion
(PFA) in walnut. Pacific Nut Producer, March, 28-32.
Beer, R., Kaiser, F., Schmidt, K., Ammann, B., Carraro, G., Grisa, E., Tinner, W. (2008).
Vegetation history of the walnut forests in Kyrgyzstan (Central Asia): natural or
anthropogenic origin?. Quaternary Science Review, 27, 621-623.
Beineke, W.F. & Masters, C.J. (1973). Black walnut progeny and clonal tests at Purdue
University Proc. 12th South. Forest Tree Improvement Conf., Baton Rouge, LA. p. 233242.
Beineke, W.F. (1983). Genetic improvement of black walnut for timber production. Plant
Breeding Review, 1, 236-266.
Belisario, A., Are, M., Palangio, C.S. and Zoina, A., (1997). Walnut blight resistance in the
genus Juglans. Acta Horticulturae, 442, 357-360.
Belisario, A., Scotton, M., Santoni, A., Onori, S. (2008). Variabilità in the Italian population
of Gnomonia leptostyla, homothallism and resistance of Juglans species to anthracnose.
Forest Pathology, 38,129-145.
Belisario, A., Galli, M., Wajnberg, E. (2009). Evaluation of Juglans species for resistance to
Phytophthora cinnamomi: differences in isolate virulence and response to fosetyl-Al. .
Forest Pathology, 39,168-176.
Beritognolo, I., Magel, E., Abdel-Latif, A., Charpentier, J.P., Jay-Allemand, C., Breton, C.,
(2002). Expression of genes encoding chalcone synthase, flavanone 3-hydroxylase and
dihydroflavonol 4-reductase correlates with flavanol accumulation during heartwood
formation in Juglans nigra. Tree Physiology, 22, 291-300.
Berry, F.H. (1960). Etiology and control of walnut anthracnose. Bull. A-113. University of
Maryland Agriculture Experiment Station. 22 p.
Pollen Biology and Hybridization Process: Open Problem in Walnut
93
Bertin, R.I. & Newman, C.M. (1993). Dichogamy in angiosperms. Botanical Review, 59,
112-152.
Burke, R. D., & Williams, R. D. (1973). Establishment and early culture of plantations. In
Black walnut as a crop. Proceedings, Black Walnut Symposium, August 14-15,
Carbondale, IL. (pp 36-41). USDA Forest Service, General Technical Report NC-4.
North Central Forest Experiment Station, St. Paul, MN.
Cağlarirmak, N. (2003). Biochemical and physical properties of some walnut genotypes
(Juglans regia L.). Nahrung/Food, 47, 28-32.
Calzoni, G.L., Speranza, A., Caramiello, R., Piccone, G., Tannini, P. (1990). Wall
ultrastructure and biochemical features of the Juglans regia L. and Juglans nigra L. male
gametophyte. Sexual Plant Reproduction, 3, 139-146.
Carrión, J.S. & Sànchez-Gomez, P. (1992). Palynological data in support of the survival of
walnut (Juglans regia L.) in the western Mediterranean area during last glacial times.
Journal of Biogeography, 19, 623-630.
Cassens, D.L. (2004). Factors affecting the quality of walnut lumber and veneer. In C.H.
Michler, P.M. Pijut,; J.W., Van Sambeek,; M.V. Coggeshall, J. Seifert, K. Woeste, R.
Overton, F. Jr. Ponder (Eds). Black walnut in a new century, proceedings of the 6th
Walnut Council research symposium July 2528 (pp 161-167) Lafayette, IN. Gen. Tech.
Rep. NC-243. St. Paul, MN: U.S. Department of Agriculture, Forest Service, North
Central Research Station.
Catlin, P.B., Ramos, D.E., Sibbett, G.S., Olson, W.H., Olsson, E.A. (1987). Pistillate flower
abscission of the Persian walnut. HortScience, 22, 201-205.
Catlin, P.B.& Polito, V.S. (1989). Cell and tissue damage associated with Pistillate flower
abscission of Persian walnut. HortScience, 24, 1003-1005.
Catlin, P.B. & Olsson, E.A. (1990). Distillate flower abscission of walnut-―Serr‖,
―Sunland‖, ―Howard‖ and ―Chandler‖. HortScience, 25, 1391-1392.
Cavender, C.C. (1973). Utilization and marketing of shells. In ―Black walnut as a crop‖
USDA Forest Serv. Gen. Tech. Rpt. NC-4. pp 77-78.
Chenervard D., Jay-Allemand C., Gendraud M., Frossard JS., (1995). The effect of sucrose on
the development of hybrid walnut microcutting (Juglans nigra x Juglans regia).
Consequences on their survival during acclimatization. Annales des sciences forestières,
52, 147-156.
Chenervard D., Frossard JS., Jay-Allemand C. (1997). Carbohydrate reserves and CO2
balance of hybrid walnut (Juglans nigra no. 23 x Juglans regia) plantlets during
acclimatisation. Scientia Horticulturae, 68, 207-217.
Chester, P.I. (2009). Pollen analysis of a soil sample associated with a Greek cult statue as a
guide to province. Journal of Archaeological Science, 36, 1424-1429.
Cheung, A.Y., Wang, H., Wu, H.M., (1995). A floral transmitting tissue-specific glycoprotein
attracts pollen tubes and stimulates their growth. Cell, 82, 383-393.
Claudot, A.C., Jay-Allemand, C., Magel, E.A., Drout, A. (1993). Phenylalanine ammonialyase, chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid
walnut tree. Trees, 2, 92-97.
Cornu, D., (1988). Somatic embryogenesis in tissue cultures of walnut (Juglans nigra, J. major
and hybrids J. nigra x J. regia). In M.R., Ahuja (Ed), Somatic Cell Genetics of Woody
Plants (pp 45-49). Kluwer Academic Publishers.
94
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
Cournet, J.M., Piry, S., Luikart, G., Estoup, A., Soligna, M. (1999). New methods employing
multilocus genotypes to select or exclude populations as origins of individuals. Genetics
153, 1989-2000.
Dangl, G.S., Woeste. K., Ardhya. M.K., Koehmstedt. A., Simon C. (2005). Characterization
of 14 microsatellite markers for genetic analysis and cultivar identification of walnut.
Journal of the American Society for Horticultural Science, 130, 348-354.
Di Vaio, C., Minotta, G. (2005). Indagine sulla coltivazione del noce da legno in campania.
Forest@ 2: 185-197.
Deng, X., Weinbaum, S.A., DeJong, T.M., Muraoka, T.T. (1991). Pistillate flower abortion
in 'Serr' walnut associated with reduced carbohydrate and nitrogen concentrations in
wood and xylem sap. Journal of the American Society for Horticultural Science, 116,
291-296.
Dode, L.A. (1909). Contribution to the study of the genus Juglans (English translation by
R.E. Cuendett). Bull Soc Dendrol France, 11, 165-215.
El Euch, C., Jay-Allemand, C., Pastuglia, M., Doumas, P., Charpentier, J.P., Capelli, P.
(1998). Expression of antisense chalcone synthase RNA in transgenic hybrid walnut
microcutings. Effect on flavonoidi content and rooting ability. Plant Molecular Biology,
38, 467-479.
Fady, B., Ducci, F., Aleta, N., Becquey, J., Diaz Vazquez, R., Fernandez Lopez, F., JayAllemand, C. (2003). Walnut demonstrates strong genetic variability for adaptive and
wood quality traits in a network of juvenile field tests across Europe. New Forests 25:
211-225.
Fan, L.M, Wang, Y.F., Wang H., Wu, W.H. (2001). In vitro Arabidopsis pollen germination
and characterization of the inward potassium currents an Arabidopsis pollen grain
protoplasts. Journal of Experimental Botany, 52, 1603-1614.
Feldman, E.B. (2002) The scientific evidence for a beneficial health relationship between
walnuts and coronary heart disease. Journal of Nutrition, 132, 1062S-1101S.
Fenaroli, L., Gambi, G. (1976). Alberi. Dendroflora italica. Museo trentino di Scienze
Naturali. Trento.
Fjellstrom, R.G. & Parfitt, D.E. (1995). Phylogenetic analysis and evolution of the genus
Juglans (Juglandaceae) as determined from nuclear genome RFLPs. Plant Systematic
Evolution, 197, 19-32.
Fornari, B., Cannata, F., Spada, M., Malvolti, M.E., (1999). Allozyme analysis of genetic
diversity and differentiation in european and asiatic walnut (Juglans regia L.)
populations. Forest Genetics, 6, 115-127.
Foroni, I., Rao, R., Woeste, K. Gallitelli, M. (2005). Characterization of Juglans regia L.
with SSR markers and evaluation of genetic relationships among cultivars and the
‗Sorrento‘ landrace. Journal of Horticultural Science and Biotechnology 80, 49-53.
Forde, H.I. & Griggs, W.H. (1975). Pollination and blooming habits of walnuts. Div. Agr.
Sci. Univ. Calif. Lflt 2753.
Forte, V. (1993). Il noce (Quarta edizione), Bologna, Italy: Ed agricole.
Funk D., (1970). Genetics of black walnut. USDA For. Serv. Res. Paper (WO), Washington,
DC, ii p. 1-13.
Funk, D. & David, T. (1979). Black walnuts for nuts and timber. In Nut tree culture in North
America (pp 51-73). Northern Nut Growers Association, Hamden, CT.
Pollen Biology and Hybridization Process: Open Problem in Walnut
95
Funk, D.T., Neerly, D., Bey, C.F. (1981). Genetic resistance to antharacnose of black walnut.
Silvae Genetica, 30, 4-5.
Geitmann A. & Palanivelu R. (2007). Fertilization requires communication: signal
generation and perception during pollen tube guidance. Floriculture and Ornamental
Biotechnology, 1, 77-89.
Germain, E., Hanquier, I., Monet, R. (1993). Identification of eight Juglans spp. and their
interspecific hybrids by isoenzymatic electrophoresis. Acta Horticulturae, 311, 73-81.
Gleenson, S.K. (1982). Heterodichogamy in walnut: inheritance and stable ratios. Evolution,
36, 892-902.
González, C.R., Lemus, G.S., Reginato G. (2008). Pistillate flower abscission symptoms of
―Serr‖ walnut (Juglans regia L.). Chilean Journal of Agricultural Research, 68, 183-191.
Gratacós, E., Brauchi, P., Herrera, R. (2006). Characterization and management of flowering
in walnut (Juglans regia) cv. Serr, for increased productivity in central Chile. Acta
Horticulturae, 705, 515-520.
Grey, G.W., & Naughton, G.G. (1971). Ecological observations on the abundance of black
walnut in Kansas. Journal of Forestry, 69, 741-743.
Griggs, W. H., Forde, H. I., Iwakiri, B. T., Assay R. N. (1971). Effect of subfreezing
temperature on the viability of Persian walnut pollen. Horticultural Science 6, 235-237.
Hall, G.C. & Farmer, R.E. (1971). In vitro germination of black walnut pollen. Canadian
Journal of Botany, 49, 799-802.
Hassani, D. Eskandari, S., Jarrahi, K. (2006). Pistillate flower abscission of walnut genotypes.
Acta Horticulturae, 705, 257-260.
Hogenboom, N.G. (1984). Incongruity: non functioning of inter-cellular and intracellular
partner relationships through non-matching of information. In H.F. Linskens, J. HeslopHarrison (Eds. Cellular interaction (pp 641-654), New York: Encyclopedia of plant
physiology, NS, vol 18, Springer, Berlin Heidelberg.
Huntley, B. & Birks, H. J. B. (1983). an atlas of past and present pollen maps for Europe: 013000 Years Ago. Cambridge University Press. N.Y., pp 238-242.
Hussendorfer, E. (1999). Identification of natural hybrids Juglans x intermedia Carr. – using
isoenzyme gene markers. Silvae Genetica, 48, 50-52.
Impiumi, G. & Ramina, A., (1967). Ricerche sulla biologia fiorale e di fruttificazione del
noce (J. regia). Rivista dell‘Ortifrutticoltura italiana pp: 538-543.
Jay-Allemand, C., Dufour, J., Germain, E. (1990) Detection précoce et rapide des noyers
hybrids interspécifiques (Juglans nigra x Juglans regia) au moyen de critères
morphologiques. PHM- Revue Horticole, 311, 39-41.
Johnson, H.A. (2008). Pistillate flower abortion and ethylene production in English walnut.
PhD thesis in Plant Biology, University of California.
Kron, P. & Husband, B.C. (2006). The effects of pollen diversity on plant reproduction:
insights from apple. Sexual Plant Reproduction, 19, 125-131.
Krueger, W.H. (2000). Pollination of English walnuts: practices and problems.
HortTechnology, 10, 127-130.
Kvaliashvili, V., Samushia, M., Vashakidze, E., Abashidze, E., Marghania, M. (2006).
Studies of pollination, fertilization and embryogenesis of Georgian walnut varieties. Acta
Horticulturae, 705, 275-279.
Lemus, A.S. (2005). Control de la caída de flores en nogal ―Serr‖. Tierra Adentro, 63, 18-21.
96
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
Lemus, G., González, C., Retamales, J. (2007). Control of pistillate flower abortion in ―Serr‖
walnuts in Chile by inhibiting ethylene biosynthesis with AVG. In A. Ramina, C. Chang,
J. Giovannoni, H. Klee, P. Perata & E. Woldering (eds.). Advances in plant ethylene
research: Proceedings of the 7th International Symposium on the Plant Hormone Ethylene
(pp. 305-307). Dordrecht, The Netherlands: Springer.
Loacker, K., Kofler, W., Pagitz, K., Oberhuber W. (2006). Spread of walnut (Juglans regia
L.) in an Alpine valley is correlated with climate warming. Flora, 202, 70-78.
Luza, J.G. & Polito, V.S. (1985) In vitro germination and storage of English walnut pollen.
Science Horticulturae, 27, 303-316.
Luza, J.G., Polito, V.S., Weinbaum, S.A. (1987). Staminate bloom date and temperature
responses of pollen germination and tube growth in two walnut (Juglans) species.
American Journal of Botany, 74, 1898-1903.
Luza, J.G. & Polito, V.S. (1988a). Microsporogenesis and anther differentiation in Juglans
regia L.: a development basis for heterodichogamy in walnut. Botanical Gazette, 149, 3036.
Luza, J.G. & Polito, V.S. (1988b). Cryopreservation of English walnut (Juglans regia L)
pollen. Euphytica 37, 141-148.
Luza, J.G. & Polito, V.S. (1991). Porogamy and chalazogamy in walnut (Juglans regia L.).
Botanical Gazette, 152, 100-106.
Malvolti, M.E., Spada, M., Beritognolo, I., Cannata, F. (1997). Differentation of walnut
hybrids (Juglans nigra L. x Juglans regia L.) through RAPD markers. Acta Horticulturae,
442, 43-52.
Manchester, S.R. (1987). The fossil history of Juglandaceae. Monographs in Systematic
Botany from the Missouri Botanical Garden, 21, 1-137.
Manning, W.E. (1978). The classification within the Juglandaceae Annual report Missouri
Botanical Garden, 65, 1058-1087.
Mapelli, S., Lombardi, L., Brambilla, I., Iulini, A., Bertani, A. (1997). Walnut plant selection
to hypoxic soil resistance. Acta Horticulturae 442: 129-136.
Marshall, T.C., Slate, J., Kruuk, L.E.B., Pemberton, J.M. (1998). Statical confidence for
likelihhod-based paternity inference in natural populations. Molecular Ecology 7, 639655.
McGranahan, G. & Leslie, C. (1991).Walnuts (Juglans L.). In J.N. Moore & J.R. Ballington
(Eds), Genetic resources of temperate fruit and nut crops (pp. 907-951). Wageningen,
The Netherland: International Society for Horticultural Science.
McGranahan, G.H., Voyiatzis, D.G., Catlin P.B., Polito V.S. (1994). High pollen loads can
cause pistillate flower abscission in walnut. Journal of the American Society for
Horticultural Science, 119, 505-509.
McGranahan, G. & Leslie, C. (2009). Breeding walnuts (Juglans regia). In S.M. Jain & P.M.
Pryadarshan (Eds), Breeding Plantation Tree Crop: Temperate Species (pp. 249-273).
New York: Springer Science + Business Media.
McKay, J.W., McKay, H.H. (1941). Microsporogenesis in Juglans intermedia Carr.
American Journal of Botany, 28, 4s.
McKay, J.W. (1965). Progress in black × Persian walnut breeding. Annual Report of the
Northern Nut Growers Association, 56, 76–80.
Mielke, M.E. & Ostry1 M.E. (2004). Disease of Intensively managed eastern black walnut. In
C.H. Michler, P.M. Pijut,; J.W., Van Sambeek,; M.V. Coggeshall, J. Seifert, K. Woeste,
Pollen Biology and Hybridization Process: Open Problem in Walnut
97
R. Overton, F. Jr. Ponder (Eds). Black walnut in a new century, proceedings of the 6th
Walnut Council research symposium July 2528 (pp 188-192) Lafayette, IN. Gen. Tech.
Rep. NC-243. St. Paul, MN: U.S. Department of Agriculture, Forest Service, North
Central Research Station.
Nandakumar, N., Singh, A.K., Sharma, R.K., Mohapatra, T., Prabhu, K.V., Zaman F.U.
(2004). Molecular fingerprinting of hybrids and assessment of genetic purity of hybrid
seeds in rice using microsatellite markers. Euphytica, 13,: 257-264.
Paetkau, D., Calvert, W., Stirling, W., Strobeck, C. (1995). Microsatellite analysis of
population structure in Canadian polar bears. Molecular Ecology, 4, 347-354.
Palanivelu R. & Preuss, D., (2006). Distinct short-range ovule signals attract or repel
Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biology, 6, 7.
Paris, P., Pisanelli, A., Todaro, L., Olimpieri, G., Cannata, F. (2005). Growth and water
relations of walnut trees (Juglans regia L.) on a mesic site in central Italy: effects of
understorey herbs and polyethylene mulching. Agroforestry System, 65, 113–121.
Peakall, R., Gilmore, S., Keys, W., Morgante, M., & Rafalski A. (1998). Cross-species
amplification of Soybean (Glycine max) simple-sequence-repeats (SSRs) within the genus
and other legume genera: implications for the transferability of SSRs in plants.
Molecular Biology and Evolution, 15: 1275-1287.
Polito, V.S. & Pinney, K. (1997). The relationship between phenology of pistillate flower
organogenesis and mode of heterodichogamy in Juglans regia L. (Juglandaceae). Sexual
Plant Reproduction, 10, 36-39.
Polito, V.S. (1998a). Floral biology:structure, development and pollination. In D.E. Ramos
(Ed), Walnut Production Manual (pp. 127-132). Oakland, California: Univ. Calif. Div.
Agr. Natural Resources Publ. 3373.
Polito, V.S., Sibbett, G.S., Grant, J.A., Kelley, K.M., Catlin, P.B. (1998b). Pistillate flower
abortion and pollination management. In D.E. Ramos (Ed), Walnut Production Manual
(pp. 133-138). Oakland, California: Univ. Calif. Div. Agr. Natural Resources Publ. 3373.
Polito, V., Grant J., Johnson H. (2005). Walnut Pollination and pistillate flower abortion..
Available from: http://walnutresearch.ucdavis.edu/2005/2005_133.pdf
Polito, V.S., Pinney, K., Weinbaum, S., Aradhya, M.K., Dangl, J.,Vaknin, Y., Grant, J.A.
(2006). Walnut pollination dynamics: pollen flow in walnut orchards. Acta Horticulturae,
705, 465-472.
Pollegioni, P., Batoli, S., Malvolti, M.E., Mapelli, S., Bertani, A., Cannata, F. (2006).
Identificazione di ecotipi italiani di J. regia mediante marcatori molecolari, morfologici
e biochimici. Forest@ 3: 598-609.
Pollegioni, P., Woeste, K., Major, A., Scarascia Mugnozza, G., Malvolti, M.E. (2009a).
Characterization of Juglans nigra (L.), Juglans regia (L.) and Juglans x intermedia
(Carr.) by SSR markers: a case study in Italy. Silvae Genetica, 58, 68-78.
Pollegioni, P., Woeste, K., Scarascia Mugnozza, G., Malvolti, M.E. (2009b). Retrospective
identification of hybrodogenic walnut plants by SSR fingerprinting and parentage
analysis. Molecular Breeding, 24, 321-335.
Pór, E., & Pór, J. (1990). The effect of the excess of pollen on the fruitset of walnuts in
Balatonboglár. Acta Horticulturae, 284, 253-260.
Potter, D., Gao, F., Aiello, G., Lesile, C., Mc Granahan, G. (2002). Inter simple sequence
repeat markers for fingerprinting and determining genetic relationships of walnut
98
Paola Pollegioni, Keith Woeste, Irene Olimpieri et al…
(Juglans regia) cultivars. Journal of American Society of Horticultural Science, 127, 7581.
Rannala, B. & Mountain, J.L. (1997). Detecting immigration by using multilocus genotypes.
Proceeding of National Acaemy of Scence, USA, 94, 9197-9201.
Reid, W., Coggeshall, M.V., Hunt, K.L . (2004). Cultivar evaluation and development for
black walnut orchards. In C.H. Michler, P.M. Pijut,; J.W., Van Sambeek,; M.V.
Coggeshall, J. Seifert, K. Woeste, R. Overton, F. Jr. Ponder (Eds). Black walnut in a new
century, proceedings of the 6th Walnut Council research symposium July 2528 (pp 161167) Lafayette, IN. Gen. Tech. Rep. NC-243. St. Paul, MN: U.S. Department of
Agriculture, Forest Service, North Central Research Station.
Rink, G., Zhang, G., Jinghua, Z., Kung, F.H., Carrol, E.R. (1994). Mating parameters in
Juglans nigra L. seed orchard similar to natural population estimates. Silvae Genetica,
43, 261-263.
Robichaud, R.L., Glaubitz, J.C., Rhodes, O.E. Jr, Woeste, K. (2006). A robust set of black
walnut microsatellites for parentage and clonal identification. New Forest 32, 179-196.
Rovira M. & Aletà N. (1997). Pistillate flower abscission on four walnut cultivars. Acta
Horticulturae, 442, 231-234
Rovira, M., Ninot, A., Aletà, N. (2001). Pistillate flower abortion in walnut (J. regia L.). Acta
Horticulturae, 544, 287-293.
Sanchez, A.M., Bosch, M., Bots, M., Nieuwland, R., Feron, R., Mariani, C. (2004). Pistil
factors controlling pollination. The Plant Cell, 16, S98-S106.
Sartorius, R. (1990) Anatomische histologische und cytologische Untersuchungen zur
Samenentwicklung bei der walnut (Juglans regia L) unter besonderer Berücksichtigung
der apomoxis Dissertation, Fakulät III, Agrarwissenschaften I der Universität
Hohenheim, p 123.
Scheeder, T. (1990). Juglans intermedia in einem Bestand am Kaiserstuhl. AFZ Der Wald,
45, 1236-1237.
Schlesinger, R. C., Funk, D.T. (1977). Manager‘s handbook for black walnut. USDA Forest
Service, General Technical Report NC-38. North Central Forest Experiment Station, St.
Paul, MN.
Schreiner, E.J. (1960). Objectives of pest-resistance improvement in forest trees and their
possible attainment. 5th World Forestry Cong Pro, 2, 721-727.
Schreiner, E.J. (1963). Some suggestions for plus-tree selection and seedling seed orchards
Northeast Forest Tree Improve, Conf Proc 10, 53-60.
Shifley, S.R.. (2004). The black walnut resource in the United States. In C.H. Michler, P.M.
Pijut,; J.W., Van Sambeek,; M.V. Coggeshall, J. Seifert, K. Woeste, R. Overton, F. Jr.
Ponder (Eds). Black walnut in a new century, proceedings of the 6th Walnut Council
research symposium July 2528 (pp 168-176) Lafayette, IN. Gen. Tech. Rep. NC-243. St.
Paul, MN: U.S. Department of Agriculture, Forest Service, North Central Research
Station.
Stanford, A.M., Harden, R., Parks, C.R. (2000). Phylogeny and biogeography of Juglans
(Juglandaceae) based on matK and ITS sequence data. American Journal of Botany, 87,
872-882.
Streiff, R., Ducousso, A., Lexer, C., Steinkellner H., Gloessl J., Kremer A. (1999). Pollen
dispersal inferred from paternity analysis in a mixed oak stand of Quercus robur L. and
Q. petraea (Matt.) Liebl. Molecular Ecology 8, 831-841.
Pollen Biology and Hybridization Process: Open Problem in Walnut
99
Sütyemez, M. (2007). Determination of pollen production and quality of some local and
foreign walnut genotypes in Turkey. Turkish Journal of Agriculture and Forestry, 31,
109-114.
Tadeo, F.R., Talon, M., Germain, E., Dosba, F. (1994). Embryo sac development and
endogenous gibberellins in pollinated and unpollinated ovaries of walnut (Juglans
regia). Physiologia Plantarum, 91, 37-44Tanzarella, O.A. & Simeone, M. (1996). Final Report Project UE ―W –Brains‖ AIR3 –
CT92-01429.
Taylor, L.P. & Hepler, P.K. (1997). Pollen germination and tube growth. Annual Review of
Plant Physiology and Plant Molecular Biology, 48, 461-491.
Verardo, V., Bendini, A., Cerretani, L., Malaguti, D., Cozzolino, E., Caboni, M.F. (2009).
Capillary gas chromatography analysis of lipid composition and evaluation of phenolic
compounds by micellar electrokinetic chromatography in Italian walnut (Juglnas regia
L.): irrigation and fertilization influence. Journal of Food Quality 32, 262-281.
Victory, E.,. Glaubitz, J.C, Rhodes, Jr O.E., Woeste, K., (2006): Genetic homogeneity in
Juglans nigra (Juglandaceae) at nuclear microsatellites. American Journal of Botany,
93, 118- 126.
Wheeler, N., Payne, P., Hipkins, V., Saich, R., Kenny, S., Tuskan, G. (2006). Polymix
breeding with paternity analysis in Populus: a test for differentiation reproductive
success (DRS) among pollen donors Tree Genetics & Genomes, 2, 56-60.
Williams, J.W., Shuman, B.N., Webb III, T., Bartlein, P.J., Leduc, P.L. (2004). LateQuaternary vegetation dynamics in North America: scaling from taxa to biomes.
Ecological Monographs, 74, 309-334Winter, M.B., Wolff, B., Gottschling, H., Cherubini P. (2009). The impact of climate on
radial growth and nut production of Persian walnut (Juglans regia L.) in Southern
Kyrgyzstan. European Journal of Forest Research, 128, 531-542.
Woeste, K. 2004. An online database of Juglans names and origins. HortScience 39:1771.
Woeste, K.E., & Beineke W.F., (2001). An efficient method for evaluating black walnut for
resistance to walnut anthracnose in field plots and the identification of resistant
genotypes. Plant Breeding 120, 454-456.
Woeste, K., Burns, R., Rhodes, O., Michler C. (2002). Thirty polymorphic nuclear
microsatellite loci from black walnut. Journal of Heredity, 93, 58-60.
Wu, H.M., Wang, H., Cheung, A.Y. (1995). A pollen tube growth stimulatory glycoprotein is
deglycosylated by pollen tubes and displys a glycosylation gradient in the flower. Cell,
82, 395-403.