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Section of Plant Breeding
PUBLICATION no. 11
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ACADEMIC DISSERTATION
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HELSINKI 2002
Supervisors:
Professor (emer.) P.M.A. Tigerstedt
Department of Applied Biology
University of Helsinki, Finland
Professor Pertti Pulkkinen
Department of Applied Biology
University of Helsinki, Finland
Reviewers:
Professor Heikki Kallio
Department of Biochemistry and Food Chemistry
University of Turku, Finland
Dr. Tapani Repo
Finnish Forest Research Institute
Joensuu Research Centre, Finland
Opponent:
Professor Jaakko Kangasjärvi
Plant Physiology and Molecular Biology
Department of Biology
University of Turku, Finland
ISBN: 952-10-0542-4 (Printed Book)
ISBN: 952-10-0543-2 (PDF)
ISSN: 1457-8085
University Press, Helsinki 2002
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1.4.1 Cold acclimation
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1.4.2 Inheritance of cold hardiness
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1.4.3 Cold hardiness of sea buckthorn
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1.5.1 Development of sea buckthorn fruits
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1.5.2 Chemical composition of sea buckthorn berries
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1.5.3 Sensory properties of berries
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3.3.1 Berry physical characters
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3.3.2 Chemical analyses
23
3.3.3 Evaluation of sensory related attributes
23
3.3.4 Observations on phenology
24
3.3.5 Field evaluation of winter hardiness and berry yield
24
3.3.6 Determination of levels of dehydrin mRNA
24
3.3.7 Determination of cold hardiness by controlled freezing
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4.1.1 Dynamic variation of berry quality properties
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4.1.2 Variation in quality among origins
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4.1.3 Variation in quality between years
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4.1.4 Berry sensory properties
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4.2.1 Crosses between origins
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4.2.2 Heritabilities and repeatabilities
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4.2.3 Genetic and phenotypic correlations
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4.2.4 Correlations of yield with flowering and maturity events 31
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4.3.1 Genetical basis of winter hardiness and yield
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4.3.2 Freezing tolerance with controlled freezing
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4.3.3 Changes in biochemical during cold acclimation
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The success of a breeding programme is dependent on two main factors: the available
variation and the breeder’s ability to manipulate this variation to produce a stable
cultivar. Therefore, a realistic assessment of the variation of characters and their
heritable characteristics is a prerequisite for efficient breeding work.
Large genetic variation was observed for concentrations of sugar components,
titratable acidity, vitamin C, water percentage and size of sea buckthorn berries, as
well as for their dynamics, among subspecies. Glucose, the main sugar component
analysed, generally accumulates as the berries enlarge. The ratio of glucose to fructose
varied greatly among subspecies. Other traits: vitamin C, titratable acidity and berry
hardiness decrease during maturation.
Juice samples of sea buckthorn were characterised by astringency, sourness and
bitterness. These attributes were masked considerably by sweetness. Addition of sugar
significantly improved overall acceptance of sea buckthorn juice. The sugar:acid ratio
related most closely with pleasantness.
Most quality related traits, including vitamin C, berry size and titratable acidity, were
quantitatively inherited. A high concentration of glucose appeared to be dominant
while fructose was recessive. This dominant tendency also extended to cold hardiness,
flowering and maturity related characters of inter-subspecific crosses. The dominant
tendency may also be due to cytoplasmic inheritance. Flowering and maturing related
traits tend to be subject to environmental effects, and their heritabilities ranged from
weak to rather strong. The flowering and maturing events displayed different degrees
of intra-correlations.
Yield showed a genetic correlation with all characters pertaining to flowering and
maturity, indicating that selection for early flowering or early maturity should result in
a gain in yield. Yield showed a weak heritability. The productive potential of sea
buckthorn can only be achieved under conditions without winter injury.
Differences in cold hardiness among genotypes of various origins have underlying
biochemical bases. Cold acclimation increases the concentration of sugars and levels
of dehydrin which, in turn, result in elevated freezing tolerance. Both levels of
dehydrin mRNA and sugar components appeared to correlate with cold hardiness
among genotypes.
5
/,672)25,*,1$/38%/,&$7,216
I.
Tang, X. and Tigerstedt, P.M.A. 2001. Variation of physical and chemical
characters within an elite sea buckthorn (+LSSRSKDsUKDPQRLGHV L.) breeding
population. Scientae Horticultae, 88: 203-214.
II.
Tang, X. 2002. Intrinsic change of physical and chemical properties of sea
buckthorn (+LSSRSKDs UKDPQRLGHV) and implications for berry maturity and
quality. Journal of Horticultural science & Technology, 77: 177-185.
III.
Tang, X., Kälviäinen, N. and Tuorila, H. 2001. Sensory and hedonic
characteristics of juice of sea buckthorn (+LSSRSKDs UKDPQRLGHV L.) origins
and hybrids. Food science and technology (lwt), 34:102-110
IV.
Tang, X. and Tigerstedt, P.M.A. 2001 Inheritance of flowering, maturity, yield
and hardiness of sea buckthorn (+LSSRSKDs UKDPQRLGHV L.). Journal of the
American Society for Horticulture, 126: 744-749
V.
Tang, X and Pappinen, A. Changes in dehydrin mRNA levels, carbohydrates
and cold hardiness during cold acclimation in sea buckthorn (+LSSRSKDs
UKDPQRLGHV L.) (submitted manuscript).
6
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Sea buckthorn (+LSSRSKDs UKDPQRLGHV, L.) is a multipurpose tree-shrub native to
Europe and Asia. Its berries were recorded in traditional Tibetan medicine more than a
millennium ago (Li and Gao 1989). They have also been used as table fruits in China
and for making jam and juice in Finland and Russia for centuries (Rousi 1971, Yao
1994). In ancient Greece, leaves of sea buckthorn were used as horse fodder for
improving weight and shiny hair, thus gaining the sea buckthorn genus a Graeco-Latin
name ‘+LSSRSKDs’ meaning shining horse (Lu 1992).
Nowadays, this plant is appreciated in many more aspects. With its fast-developing,
strong root system and nitrogen fixing capability, sea buckthorn has been used as a
frontier species in water and soil conservation and reforestation in the eroded areas
(Lu 1992, Zhang 2000). Its young green leaves have high nutrient, carotene and
flavonoid contents and are suitable for processing health tea (Zhao et al. 1999).
Colourful berries and good tree shape make this plant a good ornamental plant.
However, the most important part of sea buckthorn are still the berries. Their high
nutritional and medicinal value has stimulated great interest in researching and
breeding sea buckthorn for berry production in the Eurasia and Northern America (Lu
1992, Yao 1994, Jeppsson 1999, Li 1999, Yang 2001).
An ideal cultivar for berry production of sea buckthorn should have high yield, high
berry quality and good adaptability to environmental stress. Sea buckthorn berries
contain different kinds of nutrients and bioactive substances including vitamins, fatty
acids, free amino acids and elemental components (Yao 1994, Beveridge et al. 1999,
Yang 2001, Yang and Kallio 2001) These components vary substantially among
individuals, populations, origins or subspecies (Beveridge et al. 1999, Yang 2001,
Yang and Kallio 2001).
Sea buckthorn have a wide distribution and have adapted to different environments. In
a northern climate, frequent winter injury causes yield and economic losses and is the
primary factor limiting extension of the crop northwards (Prokkola 2001). In the
present study, I examine components pertaining to berry yield including cold
hardiness and phenological events, and berry quality involving nutritional and sensory
properties in order to create a better understanding of the genetic patterns of
inheritance of these characters, as well as some of the physiological phenomena
involved.
7
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Sea buckthorn is a deciduous and dioecious species forming a shrub or small tree
between 2 and 4m high, with yellow, orange or red berries containing a single seed.
Sea buckthorn is a typical wind pollinated species and is easily propagated by seeds,
cutting and suckers. It is able to grow in arid to very wet conditions, and is widely
distributed (Lu 1992, Yao 1994). In the present study, sea buckthorn refers primarily
to +LSSRSKDs UKDPQRLGHV L., which is the most important species in the genus
+LSSRSKDs.
+LSSRSKDs belongs to the family (ODHDJQDFHDH. Classification within this genus is
still controversial. Rousi (1971) divided +LSSRSKDs into three species based on
morphological traits: + UKDPQRLGHV L., + VDOLFLIROLD D. Don and + WLEHWDQD
Schlecht. + UKDPQRLGHV was further divided into nine subspecies: FDUSDWLFD
FDXFDVLFD J\DQWVHQVLV PRQJROLFD VLQHQVLV WXUNHVWDQLFD \XQQDQHQVLV UKDPQRLGHV
andIOXYLDWLOLV Rousi. Liu and He (1978) described a fourth species, +QHXURFDUSDLiu
& He, found on the QingHai-Xizang Plateau of China. Lian (1988) upgraded +
UKDPQRLGHV, subsp. J\DQWVHQVLV to an independent species, thus there were 5 species
and 8 subspecies in the genus. Lu (1999) introduced a sixth species +JRQLRFDUSD.
Different opinions on the classifications of genus have appeared in a variety of studies
(Avdeev 1983, Eliseev 1983, Lian 1988, Hyvönen 1996). However, analyses of
isozyme markers confirmed that variation among the three putative species (+
UKDPQRLGHV, +WLEHWDQD and +QHXURFDUSD) exceeds that among subspecies within +
UKDPQRLGHV, which, in turn, is larger than that within subspecies (Yao and Tigerstedt
1993). The difference between subsp UKDPQRLGHV and subsp PRQJROLFD was also
confirmed with DNA markers (Bartish et al. 1999).
Among the six species in the genus +LSSRSKDs, + UKDPQRLGHV is the most widely
distributed The distribution of + UKDPQRLGHV ranges from Himalayan regions
including India, Nepal, Bhutan, Pakistan and Afghanistan, to China, Mongolia,
Russia, Kazakstan, Hungary, Romania, Switzerland, Germany, France and Britain,
and northwards to Finland, Sweden and Norway (Lu 1992, Yao 1994). This plant has
also been introduced to North America (Li and Schroeder 1996, Li 1999). The wide
distribution of sea buckthorn is reflected in its habit-related variation not only in
morphology, yield, growth rhythms and cold hardiness, but also in berry related
characters such as fresh weight, chemical and sensory attributes (Lu 1992, Yao 1994,
Yang 2001).
3KHQRORJLFDOHYHQWV
In sea buckthorn, information on phenological events is quite limited. Yao and
Tigerstedt (1995) observed the onset and cessation of leaf growth, and found large
variations for these characters among different subspecies or populations. However,
8
for a crop or fruit tree, the most important phenological events are time of flowering
and maturity, which are important agronomic traits and directly related to yield and
yield stability (Aksel and Johnson 1961, Aitken 1974, Wallace 1985).
The times of flowering and maturity, measured in days, are affected by interactions
between genetic control and environmental variables, primarily photoperiod and
temperature. It is believed that a few subsets of a few genes control maturity in many
plant species. Each gene subset is associated with a physiological component of
maturity. These components interact with each other and are subject to environmental
modifications (Aggarwal and Poehlman 1977, Stout et al. 1981, Wallace 1985).
Some plants are photoperiod-sensitive and only start flowering after a specific nightlength is attained (Spence and Williams 1972, Wallace 1985). These plants are more
location-specific than photoperiod-insensitive plants or cultivars. Crosses between
photoperiod-sensitive plants of various origins give rise to progenies with
unpredictable flowering and maturity characteristics. This tendency also extends to
introducing photoperiod-sensitive plants (Halloran 1976, Hunt 1979, Wallace 1985).
The period of flowering is prolonged by low temperatures but shortened under high
temperatures (Murfet 1977, Stout et al. 1981). In peach (de Souza et al. 1998) and
almond (Dicenta 1993), some maturity related events are highly heritable. The high
heritability for date of maturity in peach is attributed to major gene action (Hesse
1975).
Generally, crop yields will increase as the time over which plants are actively growing
and developing is extended (Kaufmann 1961). Consequently, later-maturing cultivars
are usually thought to yield more than early ones, since the former can allocate more
resources into development. However, many factors may limit the period available for
crops to ripen. In a northern temperate region, earlier flowering my increase the risk of
frost damage to the flowers. Later maturing cultivars may be exposed to unduly low
temperatures before they have fully ripened (Wallace 1985).
&ROGKDUGLQHVV
Cold hardiness, resistance to low temperature, is a complex phenomenon in plant. It
involves both environmental and endogenous factors, and changes seasonally (Levitt
1980). In a northern climate, cold hardiness is the capability for a plant to resist
freezing temperatures, whereas winter hardiness includes also biotic and abiotic
stresses characterising the winter environment (Levitt 1980, Sakai and Larcher 1987,
Palonen 1999, Väinölä 2000).
Plants’ response to freezing temperatures generally include two mechanisms:
avoidance and tolerance. Plants can avoid freezing initially by depressing the freezing
point of living tissues by accumulating solutes such as antifreeze proteins and other
cryoprotectants, or by dehydration (Levitt 1980, Sakai and Larcher 1987). The effect
of freezing point depression is limited, and generally ranges between –1 to -2°C and
9
seldom beyond -4°C. Most woody plants survive harsh winter conditions by deep
supercooling, another avoidance system that allows the water in cells to remain
unfrozen at very low temperatures in the absence of nucleators. Pure water nucleates
at -38°C, whereas the solutions in plant cells may reach as low as -47°C (Levitt 1980).
The capacity for deep supercooling limits the northern distribution of plants, such as
apple (0DOXV GRPHVWLFD Borkh) and pear (3\UXV FRPPXQLV L.), for crop production
(Quamme 1976).
The role of freezing avoidance is primarily to protect a cell from internal ice formation
which is fatal to a cell. As temperatures fall below freezing, ice generally starts to
form in the extracellular spaces of plants. Since the chemical potential of ice is less
than that of liquid water, unfrozen intracellular water moves towards extracellular
spaces where it freezes, until an equilibrium in chemical potential is achieved. Plants
resist ice formation in extracellular spaces by tolerance. Cold hardiness in woody
plants usually involves either tolerance to extracellular freezing, or an avoidance
mechanism such as deep supercooling, or both (Levitt 1980, Sakai and Larcher 1987,
Thomashow 2001).
1.4.1 Cold acclimation
A preceding period of low, non-freezing temperatures improves freezing tolerance in
many plants (Levitt 1980, Sakai and Larcher 1987, Palonen 1999). This process,
known as cold acclimation, involves a series of alterations in the metabolism and
composition of lipids, proteins and carbohydrates (Levitt 1980, Sakai and Larcher
1987, Thomashow 1993).
The ability of a plant to cold acclimate is controlled by multiple cold response genes
which are characterised by promoters containing the C-repeat (CRT)/ dehydration
responsive element (DRE). The low temperature and dehydrative signal induce
transcriptional activators (CBFs) that bind to the CPT/DRE to transcribe the
corresponding gene (Gilmour 2000, Thomashow 2001). Certain cold response genes
encode proteins that share certain distinctive properties (Thomashow 1993, Close
1996).
Dehydrins form such a family of proteins induced during cold acclimation,
dehydrative stress, ABA and water stress in many plants. The proteins contain one or
several copies of highly conserved lysine-rich 15 amino acid consensuses, and are
hydrophilic and boiling stable (Close et al. 1989, 1993, Close 1996). These proteins
display not only LQ YLWUR cryoprotectant properties (Lin and Thomashow 1992,
Wisniewski et al. 1999) but also a directLQYLYR relationship with freezing tolerance
(Jaglo-Ottosen et al. 1998). Correlations between dehydrins and freezing tolerance
have been established for diverse plant species, such as in alfalfa (Mohapatra et al.
1989), wheat (Houde et al. 1992), blueberry (Arora et al. 1997) and 5KRGRGHQGURQ
(Lim et al. 1999). Since cold and freezing temperatures denature a variety of proteins
10
(Tamura et al. 1991), dehydrins may also act as molecular chaperones (Thomashow
1993).
During cold acclimation, elevated levels of sugars have been illustrated in a wide
variety of plants (Levitt 1980, Sakai and Larcher 1987, Palonen 1999). The increased
levels of different sugars associate with enhanced cold hardiness in different plant
tissues and organs (Kaurin et al. 1981, Stushnoff et al. 1993, Palonen 1999). The
contribution of sugars to enhanced freezing tolerance are thought to act, in part, by
stabilising membranes (Santarius 1982, Koster and Lynch 1992). Sugar accumulation
can also lower the freezing point, elevate intracellular osmotic potential to protect
cells from drying (Levitt 1980) and interfere with ice-crystal growth to reduce
mechanical injury associated with freezing (Antikainen and Griffith 1997).
Plant cold acclimation also increases the proportion of phospholipids and unsaturated
fatty acids in cell membranes, which account for the decreased fluidity of membranes
at low temperatures (Graham and Patterson 1982, Sakai and Larcher 1987,
Thomashow 1990, 1999, Steponkus et al. 1993).
1.4.2 Inheritance of cold hardiness
Cold hardiness is a very complex trait that is controlled by many genes (Guy 1990,
Bourne and Moore 1992). The majority of studies have shown that cold hardiness is
inherited in an additive manner, progenies being intermediate to the parents, in both
herbaceous and woody plants (Hummel et al. 1982, Timmis et al. 1991, Lim et al.
1999).
Bourne and Moore (1992) found in blackberries (5XEXV) that the cold hardiness of
progenies with a hardy paternal parent is comparable with their half sibs in which both
of whose parents are hardy. The same tendency extends also to 5KRGRGHQGURQPLQXV
var. PLQXV (Cox 1991).
Cytoplasmic or maternal inheritance is another inheritance pattern documented for
cold hardiness. In apples, Wilner (1965) observed greater frost-hardiness in progenies
with a hardy mother than in their reciprocal full sibs with a hardy father. A similar
finding was also obtained for 5 EUDFK\FDUSXP subsp. WLJHUVWHGWLL (Uosukainen and
Tigerstedt 1988).
The inheritance patterns of plant cold hardiness are complicated by the trait’s own
complex properties. Cold hardiness in the same plant varies in different tissues or
organs which may have different cold acclimation mechanisms (Väinölä 2000). Cold
hardiness in the non-acclimated state and cold acclimation capacity are inherited
separately and may not correlate genetically (Stone et al. 1993, Teutonico et al. 1995).
The freezing conditions may also affect the direction of dominance (Rohde and
11
Pulham 1960, Eunus et al. 1962, Guy 1990, Thomshow 1990). The heterozygotic state
confers more diversity in allozymes which could assume different temperature optima,
and hence hold tolerance or thermostability over a wider range of temperatures
(Tigerstedt 1985).
1.4.3 Cold hardiness of sea buckthorn
Sea buckthorn with its wide distribution has evolved to adapt to varying
environments. Native Finnish sea buckthorn plants are adapted to a semi-maritime
climate and sustain no winter injuries, while Russian cultivars, adapted to a
continental climate, suffer winter injury in Finland (Pietilä and Karvonen 1999,
Lindén et al. 1999).
Finnish selections of sea buckthorn have cold hardiness to temperatures ranging from
–30 to –35°C and –40 to –45°C for male and female plants respectively during
dormancy (Pietilä and Karvonen 1999). These temperatural ranges of cold hardiness
could assume a progressive decline for increasingly southern origins, since a clinal
variation of frost and winter hardiness was observed among populations of three
subspecies of +UKDPQRLGHV (Yao and Tigerstedt 1995). However, short exposure to 40.4°C in Northern China, or -43°C in the Gorky State of the former USSR, was
found not to cause any long-term damage in sea buckthorn plants (Lu 1992).
A positive association between cold hardiness and early growth cessation was
documented (Yao and Tigerstedt 1995). Physiological and biochemical aspects
pertaining to cold hardiness of sea buckthorn plants remain largely unknown.
4XDOLW\FRPSRQHQWV
Berry quality involves a series of components pertaining to physical, chemical and
sensory properties. Different breeding tasks have different priorities as to quality. In
the present study, I investigated berry size, vitamin C, sugars, acidity and sensory
characteristics for table fruit production or for juice preparation. However, various
other nutrients were also reviewed in the present thesis.
1.5.1 Development of sea buckthorn fruits
The growth of sea buckthorn berries generally undergoes three phases. The first phase
is characterized by a period of accelerating seed growth, followed by a short transition
period with declined rate of seed growth. The third phase is highlighted by the fast
growth of the berry flesh, and initial fast growth of seed with seed weight
subsequently declining Demenko et al. 1986). The latter phase is generally referred to
as berry maturation. Since a seed accounts for only about 3 – 4% fresh weight of a
12
berry, the growth curve of a sea buckthorn berry assumes a sigmoid shape (Rousi and
Aulin 1977, Demenko et al. 1986, Berezhnaya 1993, Yao 1993). In Finland, sea
buckthorn fruits generally ripen around the beginning of September (Rousi and Aulin
1977, Yao 1993). Fully ripe berries generally range in weight from 4 – 60 g/100
berries, with the color varying from yellow, through orange to red (Rousi and Aulin
1977, Yao 1994, Li et al. 1998).
1.5.2 Chemical composition of sea buckthorn berries
Sea buckthorn fruits of subsp. VLQHQVLV have been revealed as containing much higher
concentrations of vitamins A, B2, C than other fruits and vegetables such as carrot,
tomato, orange, hawthorn (&UDWDHJXV VS.) and kiwi fruit ($FWLQLGLD VLQHQVLV). Sea
buckthorn berries also show appreciable levels of vitamin B1, P and K (Lu 1992). The
high vitamin concentrations, especially of vitamin C (VC), make sea buckthorn fruit
highly suitable for the production of nutritious soft drinks.
In + UKDPQRLGHV, extesive variations in vitamin C (VC) have been revealed among
individuals, populations and subspecies (Table 1). The vitamin C concentration ranges
from 28 to 310 mg/100g of berries in the European subspecies UKDPQRLGHV (Rousi and
Aulin 1977, Wahlberg and Jeppsson 1992, Yao at al. 1992), from 40 to 300 mg/100g
of berries in Russian cultivars belonging to subspecies PRQJROLFD (Plekhanova 1988),
from 460 to 1330 mg/100g of berries for subsp. IOXYLDWLOLV (Darmer 1952), and from
200 to 2500 mg/100g of berries in Chinese subspecies VLQHQVLV (Ma et al. 1989, Yao et
al. 1992, Zheng and Song 1992).
Harvesting time affects the concentration of vitamin C considerably. Dynamic
variations in vitamin C have been well exploited and can be grouped into two kinds of
curves. The first is characterized by a declining trend (Rousi and Aulin 1977,
Wahlberg and Jeppsson 1990), while the other shows a single rise (Darmer 1952, Liu
et al. 1990) or two peaks occurring in the VC concentration during berry maturation
(Yao 1993).
Sugar components and organic acids are important ingredients of sea buckthorn juice
(Table 1). Total soluble sugars reported for Chinese origins ranged from 5.6 – 22.7 %
in raw juice (Ma et al. 1989, Tong et al. 1989, Zhang et al. 1989, Kallio et al. 1999).
Chinese origins show higher concentrations of total sugars than Russian ones (Shyrko
and Radzyuk 1989, Kallio et al. 1999) which, in turn, are higher in sugars than Finnish
origins (Kallio et al. 1999). Glucose is a major sugar component in all origins tested
(Table 1). Both glucose and fructose account for around 90% of the total sugar content
for Chinese and Russian origins (Ma et al. 1989, Kallio 1999), but only for about
60% for Finnish ones (Kallio 1999).
13
502-1061
200-780
600-2500
1348 (single value)
513-1676
165.7-293.3
150-310
27.8-201
460-1330
40-300
10.83-15.55
10.19-22.74
6.4-12.7 (reducing sugar)
5.6-20.1
0.9-3.2
2.85-4.79
2.8-10.4
0.4-1.6
3.6-6.0
49.5-62.1
48-62
34-63
59-82
2.1-8.1
0.1-0.4
0.6-3.0
Vitamin C (mg/100g)
Soluble sugars (°Brix)
Fructose (g/100ml)
(% of total)
Glucose (g/100ml)
(% press juice)
Range
Attributes
5.5
0.9
5.02
54.2
53
52
74
3.8
0.2
1.49
13.51
15.98
9.0
1038
233
709
Average
14
Tong et al. (1989)
Zhang et al. (1989)
Ma et al. (1989)
Kallio et al .(1999)
Kallio et al .(1999)
Shyrko and Radzyuk (1989)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Ma et al. (1989)
Kallio et al. (1999)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al. (1999)
Kallio et al .(1999)
Kallio et al .(1999)
Ma et al. (1989)
Zheng and song (1992)
Yao et al. (1992)
Liu and Liu (1989)
Zhang et al. (1989)
Rousi and Aulin (1977)
Darmer (1952)
Yao et al. (1992)
Darmer (1952)
Plekhanova (1988)
subsp. VLQHQVLV
subsp. VLQHQVLV
subsp. VLQHQVLV
Chinese sea buckthorn
Chinese sea buckthorn
subsp. UKDPQRLGHV
subsp. UKDPQRLGHV
subsp. UKDPQRLGHV
subsp. IOXYLDWLOLV
Subsp. PRQJROLFD
Chinese sea buckthorn
Chinese sea buckthorn
subsp. VLQHQVLV
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
References
Identification/var.
Table 1. Vitamin C, sugar components and organic acids in sea buckthorn berries/juice of different origins
Citric acid (%)
Tartaric acid (%)
Succinic acid (%)
D-malic acid
Quinic acid (g/100ml)
Malic acid (g/100ml)
Organic acid (% malic)
Xylose (% of total)
Mannitol (µg/g)
Sorbitol (µg/g)
Xylitol (µg/g)
Attributes
Fructose (% total )
Table 1. continued
3.5-4.4
4.61-7.35
4.1-9.1
4.2-6.5
2.1-3.2
1.11-2.34 (L-malic)
2.82-6.08
1.7-4.8
2.0-4.1
0.7-1.2
1.0-4.9
0.8-1.7
0.9-2.3
0.042-0.234
0.013-0.014
0.236-0.643
0.015-0.054
Range
37.3-50.4
30-40
6-11
9-37
0.1-0.7
17 (single value, unripe berry)
13-640 (varies with maturity)
15-19 (varies with maturity)
1.85
4.57
3.3
3.4
1.0
2.8
1.3
1.5
0.111
0.0135
0.474
0.033
4.0
6.05
314
39.2
Average
45.4
36
8
21
0.42
Ma et al. (1989)
Zhang et al. (1989)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Ma et al. (1989)
Zhang et al. (1989)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Ma et al. (1989)
Ma et al. (1989)
Ma et al. (1989)
Ma et al. (1989)
subsp. VLQHQVLV
Chinese sea buckthorn
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
Chinese sea buckthorn
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
subsp. VLQHQVLV
subsp. VLQHQVLV
subsp. VLQHQVLV
15
References
Ma et al. (1989)
Kallio et al .(1999)
Kallio et al .(1999)
Kallio et al .(1999)
Ma et al. (1989)
Makinen and Soderling (1980)
Makinen and Soderling (1980)
Makinen and Soderling (1980)
Identification/var.
subsp. VLQHQVLV
subsp. VLQHQVLV
subsp. UKDPQRLGHV
Russian sea buckthorn
subsp. VLQHQVLV
subsp. UKDPQRLGHV
subsp. UKDPQRLGHV
subsp. UKDPQRLGHV
Large variations in concentrations of acids have been also reported among different
origins. Russian berries showed relatively lower concentrations of total acidity (2.1 –
3.2g/100 ml), Finnish genotypes were intermediate with a range of 4.2 - 6.5g/100 ml,
while Chinese genotypes showed the highest concentrations of organic acid with a
range of 3.5 – 9.1g/100 ml (Ma et al. 1989, Zhang et al. 1989, Kallio 1999).
Of all organic acid components in sea buckthorn juice, malic and quinic acids were
major acids together constituting around 90% of all the fruit acids in different origins
(Kallio et al. 1999). However, to what extent the variations in the above mentioned
traits have a genetic base is unknown (Kallio et al. 1999).
Sea buckthorn juice is rich in various free amino acids. Chen (1988) detected 18 kinds
free amino acids in juice of Chinese sea buckthorn (Table 2). Of these, eight free
amino acids (threonine, valine, methionine, leucine, lysine, trytophan, isoleucine, and
phenylalanine) are essential for the human body.
Table 2. Contents of various free amino acids in juice of + UKDPQRLGHV subsp.
VLQHQVLV (Chen 1988)
Free Amino Acids
mg/100g
Free Amino Acids
mg/100g
Aspartic acid
Threonine
Serine
Glutamic acid
Glycine
Alanine
Cysteine
Valine
Methionine
Trytophan
3.72
6.24
5.31
2.65
0.64
2.50
0.82
2.85
1.12
0.51
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Proline
0.97
1.94
1.79
3.21
1.06
3.49
0.47
12.28
Total
51.57
Elemental compositions of sea buckthorn are listed in Table 3. Potassium is the most
abundant of all the elements investigated in berries or juice (Chen 1988, Tong et al.
1989, Zhang 1989, Kallio et al. 1999). More than tenfold variations of elemental
concentrations were observed for Mo and Fe in juice as well as for Fe in dry mass
within Chinese sea buckthorn. Kallio et al. (1999) compared eight elements between
Chinese and Finnish sea buckthorn and found that Finnish berries had less iron,
calcium and lead but more cadmium than the Chinese berries. Bounous and Zanini
(1988) reported that fruit maturity affects N, Ca K, Na Mg, Cu, Fe, Zn and Mn
concentrations. The authors suggested that the elemental differences in sea buckthorn
berries were the results of the natural contents of elements in the soil as well as
contamination in both soil and air in which the plants were growing (Kallio et al.
1999). In liqueurs prepared from sea buckthorn, traces of Al, As, Ca, Cd, Cr, Cu, Fe,
K, Mg, Mn, Na, Li, Pb, Rb, and Zn were detected by Harju and Ronkainen (1984).
16
Table 3. Elemental composition of sea buckthorn juice /dried berries
Element
mg/L of juice
Chinese origin
Potassium
100-806
147-209
Calcium
Phosphorus
Magnesium
Sodium
Cobalt
Chromium
Copper
Manganese
Nickel
Strontium
Vanadium
Iron
Molybdenum
Zinc
Tin
Selenium
Boron
Barium
Aluminum
Titanium
Lithium
Cadmium
64-256
93.9-173
82.1-206
39.8-103
17.7-125
”
0.108-0.287
0.158-0.653
1.17-2.6
0.115-0.357
0.19-0.616
0.002-0.009
4.13-10.9
0.03-0.058
0.431-1.25
0.045-0.259
7.96-11.3
0.43-1.38
0.168-0.362
2.2-16.7
0.103-0.814
0.132-0.303
<0.05
53.3-165
18-89.9
0.01-0.09
0.47-1.00
0.81-3.86
0.39-0.09
0.08-0.45
5.93-161
1.18
2.09-6.31
0.06-0.15
0.0020.015
mg/kg of dried berries
Chinese origin
Finnish ori.
3119.3
959.62
2222.2
2.54
no trace
93.68
4.99
5.15
2.73
3264.3
7.29
30.44
8.66
5.02
no trace
11.66
2593.9
44.91
no trace
Lead
Beryllium
Silicon
Arsenic
Lanthanum
Zirconium
Yttrium
0.431
1.215
0.095
83.78
24.803
6.655
0.875
0.97
Reference
Tong et al. Zhang et al. 1989
1989
Chen
1988
644012200
800-1480
10300-14000
270-740
470-730
560-790
3.8-12
8.7-15
6.0-9.5
8.1-17
64-282
22-33
8.8-27
14-27
0.0160.055
0.044-0.105
Kallio et al. 1999
Sea buckthorn oil is an important component of certain medicines, and is rich in
lipophilic nutrients conferring protective, tissue-regenerative and anti-inflammatory
effects on skin and mucous membranes. Some of these substances may have positive
effects in regulating the immune functions, inhibiting oxidation, suppressing the
growth of cancer cells and reducing the onset of cardiovascular disease (Lu 1992,
Johansson et al. 2000, Yang et al. 2000, Yang 2001). A detailed review on oil
compositions, lipophilic nutrients and their physiological effects was recently made by
Yang (2001). Based on Yang’s review, the ranges of oil concentration in whole
berries, seeds and soft parts are listed in Table 4. The oil concentrations in seeds
17
appear to be relatively constant, with a value around 10% in seeds of natural stands of
sea buckthorn, while wider variations were observed in fresh berries or berry flesh
within or among subspecies (Table 4).
Table 4. Oil content range in whole berries (fresh
buckthorn of different origins (Yang 2001)
Subspecies
Fresh berry
Seed
FDXFDVLFD
1.9-6.2%
WXUNHVWDQLFD 3.9-13.7%
5.3-15.7%
PRQJROLFD
FDUSDWLFD
UKDPQRLGHV
1.4-10.5%
4.2-6.6%
2.5-7.9%
9.5-16.5%
VLQHQVLV
1.5-3.5%
4.2-12.3%
7.0-14.2%
weight), seed and flesh of sea
Fruit soft parts
23.2-34.0% (dry soft part)
17.8-26.7 % (dry soft part)
29.5-34.4% (dry pulp)
21.2-29.1% (peel)
16-28. % (peel)
3.2-5.4% (fresh soft part)
4.1-27.6 % (dry pulp)
5.4-20% (dry soft part)
Yellow and orange-yellow fruits were observed to have higher levels of oil than fruits
orange and orange-red in color (Daigativ et al. 1985). Yang and Kallio (2002) reported
that harvesting time significantly affected oil concentrations in soft parts and whole
berries. Berezhnaya et al. (1993) observed that dynamic accumulations of oil in seed
and mesocarp tissues parallel their respective growth curves. After berry ripening,
subspecies VLQHQVLV showed the lowest while subspecies WXUNHVWDQLFD the highest oil
concentrations in fresh berries or dry soft parts (Table 4).
Oil from the soft parts of sea buckthorn berries is dominated by palmitolic (16:1n7),
palmitic (16:0), oleic (18:1n9), linoleic (18:3n3) and vaccenic (18:1n7) acids, while
WKHRLOIURPWKHVHHGLVULFKLQOLQROHLFQ OLQROHQLFQROHLFSDOPLWLF
stearic (18:0), and vaccenic acids (Berezhnaya et al. 1993, Kallio et al. 1999, Yang
2001, Yang and Kallio 2001). Considerable variation of fatty acids occurs among
subspecies. Oil from soft parts of subsp. PRQJROLFD shows higher levels of palmitoleic
acid and lower levels of oleic acid more frequently than other subspecies investigated
(Yang 2001). Yang and Kallio (2002) reported that the effects of annual variations in
oil concentrations and fatty acid composition were less profound than the influence of
origin, indicating that there is a genetic basis for such variations.
Various fat-soluble nutrients or bioactive components have also been elucidated in sea
buckthorn berries (Ma and Cui 1987, Lu 1993, Lian 2000, Yang 2001, Kallio et al.
2002). Carotenoids and vitamin E are the two most abundant fat-soluble nutrients
IRXQG LQ VHD EXFNWKRUQ EHUULHV <DQJ 3XOS RLO VKRZV KLJKHU OHYHOV RI carotene than seed oil, and + VDOLFLIROLD DSSHDUV WR KDYH WKH KLJKHVW OHYHO RI carotene in both pulp and seed oil among species (Table 5). Within + UKDPQRLGHV,
subsp. PRQJROLFDVKRZVWKHORZHVW FDURWHQHOHYHOLQVHHGDQGSXOSRLO/LDQ
&RQFHQWUDWLRQVRI FDURWHQHFRQVWLWXWHRIWRWDOFDURWHQRLGVGHSHQGLQJRQWKH
RULJLQV /LDQ <DQJ +RZHYHU FRQFHQWUDWLRQV RI FDURWHQH DQG RI WRWDO
18
carotenoids are affected substantially by effects of berry maturity, years and practices
of fertilisation (Zhang et al. 1989, Yang 2001)
7DEOH 9LWDPLQ ( PJJ RLO /X DQG FDURWHQH FRQFHQWUDWLRQ PJJ
oil) (Lian 2000) in seed oil and pulp oil of different species of +LSSRSKDs
Species
subspecies
Seed oil
Pulp oil
Vitamin E
FDURWHQH Vitamin E
FDURWHQH
+VDOLFLIROLD
49.9
97.5
199.1
485.2
+UKDPQRLGHV VLQHQVLV
100.2
33.6
248.5
363.9
\XQQDQHQVLV
103.1
21.2
119.2
364.8
WXUNHVWDQLFD
159.4
18.4
87.2
169.2
PRQJROLFD
120.7
14.6
98.2
122.7
+J\DQWVHQVLV
85.3
22.1
71.7
271.6
+QHXURFDUS
13.0
148.6
VWHOODWRSLORVD 92.3
54.5
QHXURFDUSD
134.2
+WLEHWDQD
98.1
17.3
394.3
Concentration of vitamin E reported by Lu (1993) among species or subspecies are
also listed in Table 5. +UKDPQRLGHV tends to show higher levels of vitamin E in seed
oil than the other species listed. Alpha-tocopherol is the most active form of vitamin E
in humans, and is a powerful biological antioxidant (Farrell and Roberts 1994, Traber
1999). An extremely high level of alpha-tocopherol (1046 mg/100g pulp oil) was
reported for a cultivar of subsp. PRQJROLFD from Altai (Jablczynska et al. 1994). Large
variations among most nutrients within origin or between origins offer attractive
prospects for breeding work.
1.5.3 Sensory properties of berries
Sensory properties constitute a very important aspect of berry quality for a successful
fresh market. The term sensory is defined as relating to the senses. Sensory assessment
or evaluation is accordingly based on appearance, taste, aroma, sound and texture of
food as perceived though the senses (ASTM Committee E-18 1978).
Taste is perceived on the tongue where taste buds respond to certain types of
chemicals which are commonly recognised as four taste qualities (tastants): salt,
sweet, acid and bitter. Odour is detected by olfactory sense organs in the nose which
respond to chemicals in the gaseous phase (Cardello 1996). Taste and odour together
form the flavour of food, the most important sensory attribute of most foodstuffs.
Flavour is, therefore, mainly related to chemical composition but also, to some extent,
to texture (Laing and Jinks 1996).
Sensory evaluations generally involve subjective or objective assessment. The
subjective assessment is based on a large number of untrained assessors for their
19
personal preference for a food, whereas the objective assessment is performed by a
panel of trained assessors following strictly defined methods.
Nowadays, sea buckthorn berries are processed into a wide range of products
including juice, jam and food additives (Yao 1994, Beveridge et al. 1999). There is
little literature available pertaining to sensory properties. A dietetic butter, enriched
with ascorbic acid by addition of sea buckthorn juice, had a pleasant sweetish-sourish
taste, and a characteristic aroma of the juice (Pestryakova et al. 1978). The studies of
Mihelic and Vajic (1969) and Lange et al. (1991) showed negative opinions on taste
or flavor of sea buckthorn on its own. The causes underlying their findings are not
clearly indicated.
For the majority of fruits, sweetness is the most important tastant. A sugar:acid ratio
of 15-16 apple-juice produces the optimal balance of sweetness-sourness in this
product (Poll 1981). In citrus fruits, a sugar:acid ratio over 12 gives the most
acceptable product (Sistrunk and Moor 1983). Sweetness also correlates closely with
overall liking of soft fruit juice (Tuorila-Ollikainen et al. 1984). However, in sea
buckthorn berries, the highest value of sugar:acid ratio that has been analysed in a
Chinese genotype should be not over 5 (Kallio et al. 1999).
%UHHGLQJIRUKLJK\LHOGDQGTXDOLW\
The tasks of plant breeding can generally be grouped into yield and quality
improvement. Yield is the best indicator of integrated performance to a specific
environment and its complex nature has made yield-based selection difficult to adopt.
Most plant breeders have a conceptual model for an ideal plant type, i.e. an ideotype.
Traits of value are retained and only those of negative or little value can be discarded
(Austin 1993). Yield has, therefore, been traditionally selected for by modifying
factors which contribute to the harvest index, i.e. morphological characteristics as
branch growth, habit and shape, phenological events as flowering period and time of
fruit maturity, and physiological traits such as disease resistance and tolerance to
stress (Austin 1993, Robinson et al. 1994). Therefore, breeding for high yield and
quality can be simply viewed as improving different or multiple traits.
20
2EMHFWLYHV
The main objectives of the present study are to allow an understanding of the physiogenetic variation of berry quality and yield related traits in sea buckthorn. More
specifically, we set out to:
1) determine the dynamic variation of quality related traits during berry maturation of
different origins (II),
2) determine genetic variation of quality related traits in a breeding population (I),
3) assess examine berry sensory properties and examine their relationship with
biochemical traits (III),
4) examine the inheritable characteristics of flowering and maturity related events
and their relationship with yield (IV),
5) understand the changes in dehydrin mRNAs, sugar components and freezing
tolerance during cold acclimation and evaluate possible markers for cold hardiness
(V).
21
0DWHULDOVDQG0HWKRGV
3ODQWPDWHULDOV
The plant materials used in the present study were derived from the sea buckthorn
breeding orchard established in 1991 with 1-year-old seedlings at Arabia, Helsinki,
latitude 61°10´N and longitude 24°58´E. The orchard was composed initially of 4000
individuals belonging to several origins and sources: parental populations of subsp.
UKDPQRLGHV from Finland (Fin, latitude 60-65°N) and a Danish origin of the same
subspecies growing in Finland (Dan, latitude 60°N), subsp. VLQHQVLV from China (Chi,
latitude 36-41°N), subsp. WXUNHVWDQLFD from China (latitude 43-44°N), progenies of
crosses between Fin and Dan, hybrids between subsp.UKDPQRLGHV and subspVLQHQVLV,
WXUNHVWDQLFD or PRQJROLFD. An elite breeding population was marked out in the
breeding orchard up to 1997. More details of the material are provided in the separate
papers.
(I): During 1997 and 1998, berry samples were harvested after full maturation from 44
elite individuals comprising subsp. UKDPQRLGHV, subsp. VLQHQVLV and hybrids between
subsp.UKDPQRLGHV with subspWXUNHVWDQLFD or subsp.PRQJROLFD originating in Siberia
(Sib), Russia.
(II): Berries sampled during 1999 and 2000 were of various origin: subsp.
UKDPQRLGHV, subsp. VLQHQVLV and hybrids subsp. UKDPQRLGHV x subsp VLQHQVLV, and
showed varying degrees of maturity.
(III): Juice samples were extracted from berry samples harvested just after maturation
in 1999 from six genotypes of different origins: subsp. UKDPQRLGHV, subsp. VLQHQVLV,
hybrids subsp. UKDPQRLGHV x subsp VLQHQVLV and subsp. PRQJROLFD x subsp.
UKDPQRLGHV.
(IV): During 1999 and 2000, 468 female plants in the breeding orchard were observed
for studies on the inheritance of flowering, berry maturation, berry yield and plant
winter hardiness.
(V) Plants of three genotypes, Fin, Chi and Fin x Chi, derived from Finnish (subsp.
UKDPQRLGHV) and Chinese (subsp. VLQHQVLV) origins and a hybrid Fin x Chi, with
different cold hardiness, were initially raised by tissue culture, and then rooted and
grown in containers in the greenhouse. These plants were subjected to cold
acclimation and levels of cold hardiness, sugars and dehydrin transcripts assessed.
$FFOLPDWLRQWUHDWPHQWVDQGH[SHULPHQWDOFRQGLWLRQ9
The cold acclimation procedures consisted of two different temperature regimes as
showed in Fig. 1 (V). Cold acclimation process one (CA1) has a period with the
temperature held constant at 4ºC for about two weeks. CA2 was programmed with a
22
stepwise regime of lowering temperatures, with the lowest temperature held at –5ºC
for one day (April) or three days at –4ºC (September). Control plants were grown
under at the same conditions, except that the temperature was held at 20ºC throughout
the procedure. A controlled-climate regime was used for plant acclimation. The
photoperiod was 16-h, with a photo flux density of 135 µmol m-2s-1 and with moisture
kept at a constant level in the soil in the container.
0HWKRGV
3.3.1 Berry physical characters (I, II, III)
Berry fresh weight was determined as the average weight of 100 berries with four
replications before they were frozen. Berry dry mass was calculated as the average
percentage of fresh berry weight after oven drying at 105°C for 24 hours with two
replications (I, II).
Berry softness was measured with a Penetrometer PNR 6 (Berlin), with a 67g standard
cylindrical ram-type penetrator. Softness was defined as the depth, in tenths of a
millimeter, to which the ram-type penetrator vertically penetrates a berry in a standard
position under gravity. Berry color was estimated with an R.H.S. Color Chart 1966
(Later edition) (II)
3.3.2 Chemical analyses (I - III)
The determination of vitamin C (VC) was made according to the procedure developed
by Sparrman and Danielson (1969) (I, II). Titratable acidity, conventionally expressed
as citric acid monohydrate, was determined by titration with 0.1 N NaOH solution to
the end point of phenolphthalein (I - III).
Different methods were used to determine sugar components. Total soluble sugars,
reducing sugars and sucrose were estimated by the Lane-Eynon volumetric method
(AOAC, 1995) on berry samples in paper I. The estimation of glucose, fructose and
sucrose was based on an enzymatic method according to the guide for test-kits for
sucrose/D-glucose/D-fructose (Boehringer Mannheim) for both berry samples (II, III)
and leaf samples (V).
3.3.3 Evaluation of sensory related attributes (III)
The sensory attributes aroma, sweetness, sourness, bitterness, astringency and redness
of color, were assessed by trained panellists. First, the intensity of aroma was assessed
on a 100mm linear scale, with the ends verbally anchored with ‘weak - strong’. A
verbal description of aroma was also required for each sample. Then tastants
sweetness, sourness and bitterness as well as sensory attribute astringency were
sequentially rated on their respective 100mm linear scales with ends verbally anchored
accordingly. Two reference solutions with concentrations representing mild and strong
levels of each tastant were used to introduce taste qualities to the assessors. Tap water
and unsalted white bread were used for cleansing the palate between samples and to
23
remove aftertastes. The final task was to rate color of the same set of samples (yellow
- red, 100mm linear scale) under fluorescent light.
Pleasantness ratings were carried out by 40 subjects on two sets of the samples, one
sweetened and the other not, on a nine-point category scale anchored verbally at both
ends (extremely unpleasant - extremely pleasant) for taste and color.
3.3.4 Observations on phenology (IV)
Phenological observations mainly focused on flowering and berry maturation. The
start of flowering was defined as the number of days, from 1 May, at which the flower
stigmas of a particular bush elongated enough to be visible. The end of flowering was
recorded when all the stigmas had disappeared or wilted. The duration of flowering
was calculated as the difference between end of flowering and start of flowering. The
start of maturity was determined as the number of days, from May 1, on which 1% of
berries on a bush changed color, while half maturity and full maturity represented the
times when half and all berries changed color, respectively. Duration of berry
maturation was calculated as the difference between full maturation and initial
maturation, while entire duration of reproductive time was taken as the difference
between full maturation and the onset of flowering.
3.3.5 Field evaluation of winter hardiness and berry yield (IV)
Winter hardiness was scored on a scale of 1 to 5 with 1 indicating that most of the
shoots which had developed during the previous summer had died without producing
any of the current season’s young shoots and 5 representing no winter injury. Berry
yield was scored visually on a scale of 0 to 5 with 0 indicating no yield and 5 the
highest level of yield.
3.3.6 Determination of levels of dehydrin mRNA (V)
A appropriate DNA probe for dehydrin gene was generated based on an RNA
template coupled with a pair of primers derived from two consensuses of dehydrin
families. The reverse primer was 5´-AGATCAAAGAAAAACTCCCCGGA-3´ and
the forward primer 5´-GACGAGTACGGTAACCCAAT-3´. The reverse primer was
used for RT-PCR but both reverse and forward primers were included in subsequent
PCR’s. The annealing temperature was 50°C. The PCR products were separated on
1.2% agarose gel and the promising PCR bands were purified with a DNA
Purification Kit (Bio-RAD). Complementary DNAs (cDNAs) were then cloned by
using pGEM®-T Easy Vectors (Promega) according to the manufacturer’s guide.
A cDNA sequence of 482 bps was found to have 72% similarity with a dehydrin
cDNA of glycine max (AF004807). This cDNA sequence was labelled as a probe for
the dehydrin gene transcripts according to DIG-High Prime (Roche).
7RWDO51$ZDVLVRODWHGIURPWKHOHDYHVDFFRUGLQJWR&KDQJHWDO JWRWDO
RNA were used for northern blotting. The northern blotting was carried out with two
24
replications, or checked interactively. The RNA blots were then quantified by
densitometric scans.
3.3.7 Determination of cold hardiness by controlled freezing
Freezing tolerance was estimated in two ways. In the first, ten individuals of each
genotype had undergone cold acclimation process II (CA2) (V, Fig.1) down to -5°C
for one day in spring or down to -4°C for four days in autumn. The leaves of each
individual were evaluated visually during CA2 or deacclimation.
In the second procedure, leaves from non-acclimated (NCA) or cold acclimated (CA)
plants were subjected to controlled freezing in a programmable refrigerated ethylene
glycol bath (RC 6 CP Lauda, Germany). Tubes containing leaf samples were taken out
at -2ºC, -5ºC and then at 5ºC intervals down to - 30°C, placed on ice and thawed
overnight at 4ºC. Controls were always kept at 4ºC during the freezing procedures.
After thawing, 20 ml deionized water were added and the tubes were shaken at 200
rpm for one hour at room temperature. The initial conductivity of the diffusate was
measured with a conductivity meter. The samples were then killed by incubating the
tubes (without aqueous liquid) in liquid nitrogen for 10 min. The second conductivity
measurement was made after the tubes were shaken again at room temperature for one
hour. Relative electrolyte leakage (REL) was calculated as (initial conductivity/second
conductivity) x 100%. The temperature which caused 50% REL corresponding to 50
% lethal temperature (LT50) was at the inflection point of the sigmoid curve plotted
from REL of each genotype (Ingram and Buchanan 1984, Väinölä and Repo 1999).
6WDWLVWLFDODQDO\VHV
The correlations among traits and conformity of traits were assessed by Pearson’s
product-moment correlations or by Spearman’s rank-order correlations. The variation
of traits among the same genotypes in different years or at different level of maturity
was evaluated by the paired-difference Wtest with the PROC UNIVARIATE (I, II, V).
Variation of means among different groups were analyzed using ANOVA and Tukey’s
test (PROC GLM) (I-III).
PROC MIXED was used to estimate variance components and corresponding standard
errors. The MANOVA statement of PROC GLM provided the sum of cross products
for estimation of covariance. A weighted average for the variance components was
used to calculate the overall heritability or genetic and phenotypic correlations (IV).
All the above computer programmes were performed under the SAS soft package
(SAS Institute Inc. 1996)
Partial Least Squares Regression (PLSR) was carried out to establish spatial
correlations among samples, sensory attributes, mean pleasantness of samples and
subjects’ individual preferences. The software used was the Unscrambler version 7.5
(CAMO A/S, Trondheim, Norway) (III)
25
5HVXOWVDQG'LVFXVVLRQ
%HUU\TXDOLW\
4.1.1 Dynamic variation of berry quality properties (II)
In order to understand how different quality components vary throughout maturation,
one needs to evaluate genetic variation for the traits among different genotypes or
origins. In the present study, the period of berry growth investigated fell between 10
August and the end of September, which corresponded to the third phase of a threephase diagram of berry growth described by Demenko et al. (1986). This period is
characterized by rapid berry expansion, color alteration and berry softening. After
berry rapid expansion, berry growth in all genotypes tailed off around the beginning of
September during both years. Meanwhile, berry colors in all berries changed to the
fully ripe colors which remained steady for two or more weeks, and berry softening
accelerated in most genotypes.
The trend of color change in sea buckthorn in the present study was consistent with
the studies of Rousi and Aulin (1977) and Yao (1993). Changes in fruit color are often
accompanied by a loss of chlorophyll coupled with the biosynthesis of anthocyanins
and carotenoids (Rogiers and Knowles, 1997). Carotenoids are fat-soluble and
convertible to vitamin A, while anthocyanins are water-soluble and commonly present
in juice (Dauthy, 1995). Therefore a ripe color also represents value in nutritional and
sensory properties.
The date for all berries in a bush attaining fully ripe color (FRC) marked the ending of
berry rapid expansion which, in turn, signalled a sharp increase in berry softening in
most genotypes. The coincidences of dates of FRC, cessation of berry rapid expansion
and accelerating rate of softening highlighted that any of their occurrences could be
used as a predictor of berry maturity. However, since it is easy to follow, FRC has
been used more frequently as a suitable indicator of harvesting time in sea buckthorn
(Rousi and Aulin 1977, Yao 1993) and many other fruits. In addition, the strong rank
correlation for dates of FRC attainment among genotypes between the years (rs = 0.92)
shows that color is also a stable indicator of berry maturity.
Sugar components altered in a manner reflecting the degree of berry maturation.
Glucose, the major sugar component in genotypes of subsp. UKDPQRLGHV, accumulated
initially, but remained relatively steady or declined after mid-August. In contrast, Chi
showed a steady increase in glucose and fructose to the end of investigation. Its
glucose/fructose ratio remained at approximate unity after initial coloration of berries
during both years, without any effect of years. The inter-subspecific hybrid Fin x Chi
was similar to Chi for glucose but similar to Fin for fructose concentrations. This
suggests that the gene/s for high glucose is/are dominant, whereas those for high
26
fructose are recessive. However, further studies would be needed to make a definitive
conclusion on this point.
The changes in concentrations of titratable acid or vitamin C among genotypes were
more predictive than were sugar components during maturation. Both traits correlated
dynamically strongly and significantly, but negatively, with berry weight in each
genotype. This dynamic variation of VC confirmed the suggestion proposed in paper I
that VC was more affected by the degree of ripeness than by environmental effects.
Since the amount of vitamin C per 100 berries differs non-significantly regarding with
different degree of maturity among genotypes, this indicates that there are no effects
of harvest dates on the total amount of vitamin C during the period concerned.
Similar dynamic trends of VC were observed by Rousi and Aulin (1977) and Jeppsson
and Gao (2000). In contrast, a peak of vitamin C concentration was detected for
genotypes in late August in the study of Yao (1993).
4.1.2 Quality variation among origins (I-III)
Chi (subsp. VLQHQVLV) generally showed smaller fruits, higher sugar concentration,
higher titratable acidity and higher vitamin C than Fin and Dan (subsp. UKDPQRLGHV)
This trend is in accordance with the results of Kallio et al. (1999).
Inter-subspecific hybrids (Fin x Chi) were intermediate in most characters. Intrasubspecific hybrids (Fin x Dan) differed from parental origins only for berry weight
(I). This suggests that there is little opportunity for improving those biochemical traits
investigated in Fin and Dan by crossing within subspecies. However, according to
Karhu et al. (1999), high concentrations of vitamin C existed in populations of subsp.
UKDPQRLGHV and the trait was heritable.
The range of VC in the present study (I) is in good accordance with previous studies
for subspUKDPQRLGHV (Yao et al. 1992), but VC measurements for Chi fall into the
lower part of combined range of VC reported for subsp VLQHQVLV (Zheng and Song
1992, Beveridge et al. 1999).
Fin x Sib displayed the lowest concentration of VC and titratable acid but the highest
sugar/acid ratio. These values were more similar to those obtained by Shyrko and
Radzyuk (1989) on cultivars of subsp PRQJROLFD than those analysed for Fin in the
present study, but fall roughly between them.
Of three sugar components analysed on berries, glucose was the major sugar
component, followed by fructose for all genotypes (III). The ranking of the sugar
components is consistent with the results obtained for subspVLQHQVLV by Ma and Cui
(1987) and by Kallio et al. (1999). Chi displayed a glucose/fructose ratio of
approximately unity. This result agrees roughly with the ratio found for subsp
27
VLQHQVLV by Ma et al. (1989). In contrast, a very high glucose/fructose ratio was
analysed for subspUKDPQRLGHV and its related inter-specific hybrids (II, Fig.5).
4.1.3 Variation in quality between years (I-II)
Large fluctuations between years was found for the physical traits such as berry fresh
weight, dry mass and seed weight, and the chemical traits VC, titratable acid and sugar
components. The chemical traits generally showed a higher rank correlation between
years than the physical traits. This suggests that the physical traits are subject to a
larger interaction with years than chemical traits (I). The same tendency was also
observed in the dynamic variation of physical and chemical traits during berry
maturation (II) and other crops (Austin 1993). The differences in accumulative
patterns of berry fresh weights between years parallel the patterns of rainfall between
the two years during berry growing periods (II, Fig. 1, Fig. 3).
The concentration of sucrose was an exception and showed a very low rank
correlation between years. This may be because the concentration of sucrose was too
low for accurate determination (I – II).
4.1.4 Berry sensory properties (III)
Sensory attributes are important aspects of berry quality. In the present study, the
major focus is on the flavour of juice extracted from the berries. Sensory components
astringency, sourness, bitterness, sweetness, density of aroma and redness were
assessed as objectively as possible, whereas overall pleasantness was evaluated
subjectively. Of six genotypes in question, Sib x Fin showed significantly lower
intensities of astringency, sourness and bitterness, but a significantly higher intensity
of sweetness than the other genotypes (III, Fig. 1). Consequently, Sib x Fin had a
better pleasantness rating (III, Fig. 3).
In general, the flavor of sea buckthorn was characterized by astringency, sourness and
bitterness (III, Fig 1.). These three components correlate strongly with each other (III,
Fig. 3) and also with titratable acidity (III, Table 3). These findings are consistent with
the fact that astringency, sourness and bitterness can all arise from the presence of
acids and phenolic compounds (Arnold and Noble 1978, Lea 1990, Peleg and Noble
1995, Thomas and Lawless 1995).
Astringency, sourness and bitterness all show negative correlations with pleasantness
and are only favored by a few subjects, while sweetness confers a strong positive
correlation with pleasantness (III, Fig. 3). This phenomena are further supported by
the significant improvement of pleasantness ratings by artificial sweetening (III,
Fig.2). This agrees with the finding that sweetness correlated closely with overall
liking of soft drinks (Tuorila-Ollikainen et al. 1984).
28
Astringency is a tactile stimulus causing sensations of dryness or roughness (Ishikawa
and Noble 1995, Thomas and Lawless 1995) and although only favored at low
intensities is an important sensory attribute of many beverages and fruits (Haslam and
Lilley 1988). Sourness is elicited by organic acids which are common ingredients of
foods and contribute to a wide range of properties in them (Hartwig and McDaniel
1995). These taste qualities can be modified in processing or used as food additives
(Pestryakova et al. 1978).
Sweetness showed the highest correlation with sugar:acid ratio (III, Table 3). The
sugar:acid ratio generally peaks after the berries ripened (II, Fig. 6). This implies that a
better flavor is expected after the berries have fully ripened. The same tendency has
also been reported for many other fruits, such as apples (Poll 1981), strawberries
(Burton 1982) and arctic bramble (Häkkinen et al. 1995). A sucrose concentration of
around 10 % in soft drinks was preferred by a majority of subjects (Pangborn 1980,
Tuorila-Ollikainen et al. 1984). The study of Poll (1981) suggested that a sugar:acid
ratio of 15-16 gave the optimal balance of sweetness-sourness in apple juice. The
present study showed much lower sugar concentrations and sugar:acid ratios for sea
buckthorn original juices. However, a sea buckthorn genotype with a sugar
concentration over 20% was observed in the raw juice of Chinese genotype by Zhang
et al. (1989) and Kallio et al. (1999). This could represent a target for breeders seeking
to improve the qualities of sea buckthorn berries. Moreover, the higher pleasantness
ratings of hybrids over non-hybrids was observed. It would appear that hybridization
combines the two different sets of flavors from their respective origins, so that the
hybrids appeal to a wider range of people than the original bushes.
The intensity of aroma did not differ significantly among genotypes, and consequently
affected the overall pleasantness rating very weakly. Subjects preferred the red color
of the juice, but color only slightly affected overall preference. The weak effect of
intensity of aroma and appearance on overall hedonic ratings has also been illustrated
in the studies of Tuorila-Ollikainen et al. (1984).
,QKHULWDQFHRIIORZHULQJDQGPDWXULQJDQGWKHLUFRUUHODWLRQVZLWK\LHOG,9
4.2.1 Crosses between origins
Among three parental control origins, Chi and Fin flower simultaneously, significantly
earlier than Dan, while Chi and Dan matured simultaneously, later than Fin (IV, Table
1). Yao and Tigerstedt (1995) observed that Chi showed the earliest budbreak, and
Dan the latest. The same tendency, reflected in the start of flowering, suggests that
earlier budbreak correlates with earlier flowering.
The mixed progenies, Fin x Dan and Dan x Fin, fell between the mean values of the
parentals for all phenological events examined. This indicates that characters related
to flowering and maturity were quantitatively inherited in within-subspecies crosses.
However, one can not exclude the possibility of matroclinal effect, since the progenies
Fin x Dan and Dan x Fin were not compared separately.
29
The inter-subspecific hybrids, Fin x Chi and Dan x Chi, did not differ significantly
from the respective Dan or Fin parentals for any of the characters studied. This
suggests that Dan and Fin were dominant to Chi in characters related to flowering and
maturity, at least when their hybrids are grown in a high latitude region. This
tendency, which also extended to most characters of other inter-subspecific hybrids
involving Dan or Fin, highlighted that flowering and maturity related characters could
be predicted satisfactorily in inter-subspecific hybridizations from parental means.
However, the dominance may also be due to cytoplasmic inheritance.
4.2.2 Heritabilities
The flowering related characters showed a range of heritabilities from 0.20 to 0.44
(IV, Table 2). Their relatively low heritability coupled with moderate nonadditive
variance implies that flowering related events are highly subjected to environmental
factors. The same tendency is also reflected in corresponding characters in almond
[3UXQXVGXOFLV (Mill) D.A. Webb] (Dicenta et al. 1993).
The maturity parameters, start of maturity and mid-maturity, displayed strong
heritabilities with a predominantly matroclinal inheritance. This finding suggests that
inheritance of the both maturity parameters may be sex-linked or cytoplasmic, and that
selection of female bushes for these two traits would be quite efficient. However,
heritability for date of full maturity showed a combined value of only 0.41 for two
years. The relative lower heritability for date of full maturity than those of date for
other two maturity parameters is probably due to the lower and more fluctuating
temperatures at the time when the berries are approaching full ripeness, since duration
of maturity (h2 = 0.18) and whole reproductive period (h2 = 0.44) also show very low
heritabilities. The characters analogous to date of full maturity and whole reproductive
period in peach and almond were highly heritable (de Souza et al. 1998, Dicenta et al.
1993).
4.2.3 Genetic and phenotypic correlations
Late flowering and early maturity are considered important in a northern climate for
securing a high and stable yield. The date for full maturity correlated moderately
strongly, both genetically and phenotypically, with the onset of flowering, indicating
that late flowering is associated with late ripening (IV, Table 3). This trend makes it
difficult to combine late flowering and early maturation in sea buckthorn. However,
The start of flowering correlated negatively with the duration of flowering (UG = -0.48)
and reproductive time (UG = -0.12), indicating that later flowering tended to associate
with a short duration of flowering and short reproductive time. These results are in
agreement with the observation that the lengths of different developmental periods are
highly correlated with their corresponding temperature in peach (Boonprakob et al.
1992). Early flowering individuals develop slowly at the relatively cool temperatures
after flowering, compared to late flowering individuals. However, the efficacy of the
start of flowering in predicting reproductive time is largely offset by the cooler
temperatures experienced by the late ripening individuals as compared with the early
ripening ones in sea buckthorn. The strong correlation between reproductive time and
full maturity (UG = 0.73 and UP = 0.76) indicates that full maturity is a major factor
determining the period of reproductive time.
30
Full maturity correlated, both genetically and phenotypically, very strongly with the
start of maturity and mid-maturity. This suggests that these three maturity-related
characters were controlled by the same genes. This finding further confirms the idea
that the heritability of full maturity was weakened by environmental effects.
4.2.4 Correlations of yield with flowering and maturity events
Yield showed a moderate to strong genetic correlation with flowering and maturity
related events. (IV, Table 3). These results imply that earlier flowering, especially
earlier maturing, result in higher yields, as demonstrated by the strong negative
genetic correlations between yield and the maturity related character: full maturity (ra=
-0.73), maturity duration (ra = -0.72) and whole reproductive period (ra = -0.68). This
finding is reasonable in high latitude regions, since late ripening individuals may not
have enough energy, after attaining berry maturation, to form sufficient flower
primordia for the following yield.
Phenotypically the correlations between yield and these phenological characters were
much weaker than their corresponding genetic correlations. These trends are in
agreement with the observation that yield was strongly affected by environmental
effects, as shown by its low heritability (IV, Table 2). Meanwhile, it suggests that
individual phenotypic selection for yield may not be so efficient as family selection.
*HQHWLFDODQGELRFKHPLFDOEDVHVRIFROGKDUGLQHVVDQGLWVUHODWLRQZLWK\LHOG
4.3.1 Genetical basis of winter hardiness and yield (IV)
Sea buckthorn plants introduced from China (Chi) suffer serious winter injury under
Finnish climatic conditions. Intersubspecific hybrids both Fin x Chi and Dan x Chi are
significantly hardier than Chi, and are as hardy as, if not more than, Fin and Dan,
respectively. However intersubspecific hybrids of the type Sib x Fin are cold sensitive
(IV, Fig. 1). This suggests that a cytoplasmic inheritance mechanism for cold
hardiness may be operative in sea buckthorn. A similar tendency has also been
observed in apple (Wilner 1965) and 5KRGRGHQGURQ (Uosukainen and Tigerstedt
1988). Unfortunately, there are no reciprocal crosses between Chi and Fin or between
Fin and Sib which would have given more definite indications of matroclinal
inheritance.
Slight winter injuries were observed in the present study on native sea buckthorn
plants, a finding confirmed in the study of Pietilä and Karvonen (1999). Danish
origins showed significantly lower winter hardiness than Fin. However, Fin x Dan
(including Dan x Fin) are even hardier, albeit non-significantly, than Fin, implying
that there is heterosis for winter hardiness between crosses of different origins.
Heterosis, or hybrid vigor, is generally due to genetic homeostasis acting in
heterozygotes which have an advantage over their parents for specific biochemical
pathways across different environments and are thereby better buffered against
challenging environments (Lerner 1954, Barlow 1981, Clare and Luckinbill 1985,
Tigerstedt 1985). Consistently high levels of winter hardiness of Fin x Dan progenies
31
results in low differentiation among half sibs or full sibs, explaining the low value of
heritability for winter hardiness (h2 = 0.02).
Yield also showed a weak combined heritability (h2 = 0.23) over the two years, and
was attributed mainly to the paternal effects (Table 2). The low maternal heritability
implies that selection based on yield performance of female bushes will be inefficient
in sea buckthorn breeding. However, the low heritability could also be due to selection
of female parents, which reduces maternal variance, and consequently the covariance
between their offspring (Dicenta et al. 1993, Li and Wu 1997).
The phenotypic correlation between fruit yield and winter hardiness (U = 0.95) is
strong and highly significant (3 = 0.001) on the basis of means of original and hybrid
groups. This implies that gain in yield could be achieved through improvement of
winter hardiness where it constitutes a limiting factor. However, in regions where the
crops is well adapted, e.g., in the center of its distribution range, fitness or fruit yield
can generally be improved at the expense of some adaptation (Austin 1993). Generally
speaking, the greater the winter hardiness obtained in fruit species, the lower the yield
or quality of the fruits (Shen et al. 1994, Jakubowski and Monet 1998). Therefore the
attainment of adaptability to various environments and fitness should only be
compromised to the extent that sustainable yield can be secured.
4.3.2 Freezing tolerance after controlled freezing (V)
Three genotypes, representing Chi, Fin and Fin x Chi, were subjected to controlled
freezing tests. After plant acclimation at low temperatures for 8 days, a subsequent
freezing temperature of –5°C for one day in spring or –4°C for three days in autumn
(V, Fig. 1 - CA2) caused serious injury to leaves of Chi but not to those of Fin or Fin
x Chi. In contrast, the cold acclimated leaves of Chi were significantly cold hardier
than those of the other two genotypes, as revealed by ion leakage tests over a wide
range of temperatures (V, Table 1). The contradictory results were explicable if Chi
requires more time to be cold acclimated than two other genotypes. The rate of
hardening is an important factor for plants avoiding winter injuries (Stushnoff 1972),
especially in fluctuating climatic conditions. An insufficient rate of hardening may
account for the winter injuries observed in Chinese sea buckthorn (Yao and Tigerstedt
1995, Tang and Tigerstedt 2001), as well as in Russian sea buckthorn growing in
Finland (Pietilä and karvonen 1999).
Older leaves were observed to be more susceptible to freezing injury than younger
ones in 5XEXV plants (Warmund et al. 1989). This trend was confirmed in the present
study as demonstrated by the fact that most leaves turned yellow or wilted, except for
a few new leaves near the shoot tips in Chi.
4.3.3 Changes in biochemical components change during cold acclimation (V)
The abundance of dehydrin mRNAs increases during cold acclimation and decreases
during de-acclimation (V, Fig. 3, Fig. 4), confirming a general trend illustrated in
many other plants (Hajela et al. 1990, Thomashow 1993, Arora et al. 1997). Sugar
components also followed a general trend that low temperatures facilitate
carbohydrate accumulation in various tissues or plants (Levitt 1980, Palonen et al.
2000). However, sugars varied significantly among genotypes during cold
32
acclimation. A peak for sucrose and glucose occurred around a temperature of 0ºC in
all genotypes, but also for fructose in Fin and Fin x Chi. Sucrose percentages were
significantly higher than those of glucose, and glucose was higher than fructose at the
same temperature for each genotype (V, Fig. 5).
The levels of sugars appeared to associate positively with plant cold hardiness among
the three genotypes, since Fin and Fin x Chi were considered as relatively cold-hardy
and Chi not. However, the associations depended mainly on sucrose. Not only was
sucrose present in higher concentrations than the other two monosaccharides in the
present study, but it also shows more effective cryoprotectant properties than the other
sugars (Anchordoguy et al. 1987, Crowe et al. 1987). Moreover, sucrose percentages
paralleled low temperatures better than the other two sugars in the genotypes we
studied. These results confirm the more crucial role of sucrose than that of the other
two sugar components in protecting sea buckthorn tissues from freezing injury.
Dehydrin mRNAs are capable of responding to low temperature stimuli much more
rapidly and flexibly than sugars (Fig 3, Fig. 5). The initial rapid accumulation of
dehydrins plays an important role in stabilising proteins and membranes (Close 1996,
Palva and Heino 1998). This rapid response to low temperature or ability to harden
has been considered an important factor allowing plants to avoid freezing injuries
(Stushnoff, 1972). The rapid release of dehydrins at –4ºC provided further
cryoprotection of cells, even though sugars did not increase in response to this
freezing temperature (V, Fig. 5).
Of the three genotypes, Fin responded most rapidly to the low temperature stimulus,
as demonstrated by its highest accumulation of dehydrins at the initial stage, whereas
Fin x Chi showed the highest levels of transcripts and Chi the least at freezing
temperatures during spring and autumn (V, Fig. 3). Differences in dehydrin mRNA
levels were not reflected in any difference in cold hardiness between Fin and Fin x
Chi, but different dehydrin levels did account for differences in cold hardiness
between Chi and the others under freezing temperatures, especially in autumn (V, Fig.
3).
Dehydrin mRNA accumulation varied seasonally, although the conditions of cold
acclimation imposed were almost the same. In spring, levels of dehydrin mRNA in
three genotypes investigated all increased steadily with the falling temperatures (V,
Fig. 3A). In autumn, the original genotypes Fin and Chi assumed different patterns of
development of dehydrin mRNA levels which initially rose with low temperatures to
high levels, thereafter declining, and subsequently become restored in response to
temperatures below freezing for Fin, though not in Chi (V).
According to Sakai and Larcher (1987), cold acclimation can be characterized by two
steps in temperate zone woody perennials. The first stage is due to progressively
shorter daylengths in late summer and the second stage is caused by low temperatures.
In the present study, the plant material used in autumn had been exposed to
progressively shorter daylengths following a summer daylength of over 20 hours, so
that the shorter photoperiod had presumably induced the first stage of the cold
acclimation process. This stage of cold acclimation was not, however, reflected in any
33
accumulation of dehydrin mRNA in non-cold acclimated controls. Acclimation
appears initially to affect transcript patterns in the supposedly second stage of
acclimation associated with low temperatures (V, Fig. 3B). Since conditions for the
first stage of cold acclimation were already met by autumn, leaves of Fin and Chi may
have been fully acclimated with a shorter period at low temperatures than in spring (V,
Fig. 3B).
After plants have acclimated, the depressed dehydrin mRNA levels may be associated
with a balance between the effects of dehydrins and other metabolites like sugars in
ameliorating dehydrative stress in Fin and Chi. The ameliorative effects of sugars,
whose accumulation paralleled falling temperatures (V, Fig. 5), decreased the
protective requirement from dehydrins and resulted in decreased levels of transcripts
(V, Fig. 3B). This speculation was further confirmed by the observation that Chi also
showed decreased accumulation of dehydrin mRNA following initial higher
accumulation under constant low temperatures in spring (V, Fig. 4). The decrease of
dehydrin mRNA levels after full acclimation was also observed in blueberry in floral
buds of cultivar ‘Tifblue’, while transcripts increased in the hybrid cultivar
‘Gulfcoast’ (Arora et al. 1997). The same trend was also observed in the hybrid Fin x
Chi in the present study (V), implying effects of gene interaction.
34
&RQFOXVLRQVDQGLPSOLFDWLRQVIRUEUHHGLQJ
The present study confirmed that considerable genetic variations exist among berry
quality related traits such as berry size, sugar components, vitamin C and titratable
acidity among subspecies and hybrid groups. Different origins also varied for rhythm
of berry maturation. The degree of berry maturity can be predicted most easily from
the berry color. During berry maturation, concentrations of sugar components,
especially glucose and ratio of sugar to acidity, increase while concentration of
vitamin C, titratable acidity and berry hardiness decline. Chemical components were
less affected by the physical environments and years than were physical traits.
In crosses between subspecies, berry size, vitamin C and titratable acidity showed
intermediate inheritance. A high concentration of glucose is dominant, whereas a high
concentration of fructose is recessive. Flowering related traits had low heritability,
while maturity related traits displayed relatively greater heritabilities. Heritabilities of
flowering or maturity related traits were considerably depressed by low temperatures
in spring and autumn, respectively. This may explain why the date of onset of berry
maturation and full maturity were supposed to be controlled by the same genes but the
former showed a much higher heritability.
Cold hardiness, flowering and maturing related events tend to be cytoplasmically
inherited in crosses between subspecies, implying that improving quality related traits
by introducing pollen batches from disparate origins will not compromise freezing
tolerance or growth rhythm in the progenies. Earliness of flowering and maturity
correlate negatively with yield, indicating that selection for early maturing cultivars
should result in a gain in yield.
Different tissues or organs vary with regard to cold hardiness or cold acclimation
mechanisms. Freezing tolerance assessments derived from leaves may not be valid for
the other tissues or organs. However, the underlying biochemical bases associated
with cold hardiness among genotypes of various origins provide clues for further
research into cold hardiness in other tissues or organs of sea buckthorn. Owing to cold
acclimation, concentrations of sugars and levels of dehydrin increase, which, in turn,
results in elevated freezing tolerance. Both levels of dehydrin mRNA and sugar
components appear to correlate with cold hardiness among genotypes in certain
circumstances.
Owing to the limitations of the materials used in the present study, some of the results
and conclusions pertaining to inheritance of quality traits need further confirmation.
However, the information obtained from the present study does allow one to indicate
certain strategies for a programme aimed at improving certain traits efficiently for sea
buckthorn in Finland. The improvement of yield related characters and adaptation
should be based upon subsp. UKDPQRLGHV. The quality related characters vitamin C,
sugars and titratable acidity would be improved effectually through crosses between
subspecies. In particular, subsp. VLQHQVLV offers great potential for enhancing quality
characteristics. Since sea buckthorn is basically dioecious, identification of superior
paternal parents is a prerequisite for achieving the breeding goals. The paternal parent
can be determined first by identifying which populations display superiority in the
35
desired traits, after which candidates could be selected by their general combining
ability in crosses within the population. Dominant traits such as glucose concentration
can easily be improved via crosses in which the paternal parent has high
concentration, while recessive traits like fructose may need further backcrosses with
the paternal parents. The latter can also apply to additive traits like vitamin C and
titratable acidity.
When breeding outcrossing plants, one must avoid close relatives mating, which are
likely to lead to inbreeding depression. In the case of sea buckthorn, the dioecious
habit, which prohibits selfing, indicates that the species is likely to suffer severe
symptoms of inbreeding depression. This should be taken into account during the
design of matings, and any breeding programme must begin with a broad genetic base.
For long-term breeding prospects, it would be advantageous to incorporate a
hermaphrodite mode of reproduction into parental stocks, so that selection for berry
traits need not occur solely through the maternal parent. This change has already
occurred during the domestication of the grape vine, 9LWLV YLQLIHUD and there are
observations on monoecy also in our material of sea buckthorn. This would also
obviate the need to use males in commercial plantations. However, in the near future,
DNA marker assisted identification of sexes of sea buckthorn at the seedling stage
may well become feasible (Persson and Nybom 1998) which may be an alternative to
the construction of monoecious plants.
36
$FNQRZOHGJHPHQWV
This study has been mainly carried out at the Department of Applied Biology,
University of Helsinki. My thanks are due to many people who have contributed to
this research work.
I am mostly grateful to my supervisor Prof. (emer.) PMA Tigerstedt during this study
for his kind guidance and firm support which continued unabatedly after his retired. I
would like to express my sincere thanks to Dr. Pertti Pulkkinen from the Finnish
Forest Research Institute for his supervision and encouragement of my work during
the period when he was as acting professor of plant and tree breeding. My warmest
thanks are due to Prof. Hely Tuorila for her supervision and offering me the
opportunity to carry out the research work at the Department of Food Technology. Dr.
Ari Pappinen is highly appreciated for his guidance in molecular work.
My sincere thanks are due to Mr. Peter Joy for his encouragement and help in study
and many other aspects. His friendship has smoothed my life during my period of
study in Finland.
My warmest thanks are also due to Terttu Parkkari who took care of the greenhouse
and field materials. Mrs Marjo Kilpinen is thanked for preparing tissue-cultured
plants. Thanks are extended to Dr. Yingmou Yao for his contribution on the
establishment of the plantation of sea buckthorn.
I wish sincerely to express my gratitude to all my colleagues in the Department of
Applied Biology for their support and friendship, especially Anna-Maija Niskanen,
Yeshitila Degefu, Jirong Lu, Nora Garcia, Matti Kangaspunta, Qibin Yu, Aki Höltken.
Dr. Anu Väinölä and Dr. Sunil Kumar Kundu.
I deeply thank Niina Kälviäinen for the rewarding collaboration towards our coauthored paper included in this thesis. I would like to express my gratitude to Dr.
Matti Haapanen from the Finnish Forest Research Institute for his review of
manuscript for paper III.
Finally, I would like to thank my parents, my brothers and sisters and all other family
members for their encouragement. I thank my daughter Min for her understanding and
love. My dearest thanks I owe to my wife, Jianyun, for her whole-heart support and
taking care of our daughter and the house work.
The financial support was provided by the Foundation for Research of Natural
Resources in Finland and the Rector of the University of Helsinki during the PhD
study. The scholarship towards my earlier studies as a visiting scholar in Finland,
37
provided by the Chinese Ministry of Education and the Center of International
Mobility (Finland), are gratefully acknowledged.
38
5HIHUHQFHV
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flowering in mungbean (9LJQDUDGLDWD (L.) Wilczeck). Euphytica 26: 207-219.
Aitken, Y. 1974. Flowering time, climate and genotype. The adaptation of agricultural
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Aksel, R. and Johnson, L.P.V. 1961. Genetic studies on sowing-to-heading and
heading-to-ripening periods in barley and their relation to yield and yield
components. Canadian Journal of Genetics and Cytology 3:242-259.
Anchordoguy, T.J., Rudolph, A.S., Carpenter, J.F. and Crowe, J.H. 1987. Modes of
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