Micropropagation of Some Dwarf and Early Mature Walnut Genotypes
In current study, the proliferation and rooting ability
of some low vigor and early mature seedlings of Persian walnut were compared
with those of semi- and high vigor seedlings in in vitro condition.
To do this, from each vigor group, nodal explants of newly grown shoots
of 5- year-old seedlings were cultured on DKW medium. These explants were
subcultured every mount and up to 13 times to increase the number of microshoots.
Results showed that the number of axillary shoots arising from the microshoots
was the highest in the dwarf and semi-dwarf genotypes compared to the
high vigor ones (3.3 vs. 2.3). The low vigor genotypes also showed the
highest number of nodes per a given size of shoot, smaller shoot size
(2.6 vs. 4.5 cm), lower callus formation and higher rooting percentage
(63.5 vs. 37.1%). Moreover, these genotypes showed in vitro flowering
on micro-shoots, which are consistent with the field observations. These
results proved the consistency of low vigor, precocity, basitonic growth
tendency and easy rooting of dwarf and precocious genotypes under in
vitro condition. In conclusion, a simultaneous recurrent selection
program is recommended for both dwarfing and rooting ability (selection
of dwarf/semi dwarf as well as easy to root clones) to utilize their advantages
in high-density orchard systems.
Persian walnut (Juglans regia L.) as its name suggests, originated from
Persia (present Iran), where walnut populations exhibit great genetic diversity
in terms of tree size and pomological traits (Vahdati and
Khalighi, 2001). From there, it spread to west through eastern and central
Europe ( Greece and Rome) and to east toward Afghanistan, Kyrgyzstan, India
and China (Forde and McGranahan, 1996; Vahdati,
2000; Breton et al., 2004).
Walnut trees grow very large, making them inconvenient to prune, spray and
harvest (Forde and McGranahan, 1996). One of the most important
characteristics with an interesting application in walnut breeding is the presence
of precocious (early mature) and low vigor walnut genotypes, which are frequently
found in some seed sources in Iran (Rezaee et al.,
2006) or in Kyrgyzstan (Germain et al., 1997;
Breton et al., 2004). The low vigor genotypes
could provide the genetic material for tree size control. To date, tree size
reduction using genetically dwarf rootstocks has been a key component of high
density orchard systems (Cousins, 2005) and many walnut
growers are now interested in shifting to high density planting systems (McGranahan
et al., 1985; Olson et al., 2001;
Ramos et al., 2001).
The main limiting factor in exploring this valuable germplasm is the lack of
an efficient vegetative propagation method as result of difficult-to-root nature
of Persian walnut species. A number of attempts have been made to propagate
walnut by conventional cutting and/or layering (Kuniyuki and
Forde, 1985; Gunes, 1999; Vahdati
et al., 2004). The results of these studies were so disappointing
that the conventional methods of vegetative propagation are no longer considered
practical (Kuniyuki and Forde, 1985). Meanwhile, a high
variability in rooting ability was reported among different seed sources (genotypes)
by layering (Vahdati and Khalighi, 2001).
Cultivar or genotype dependent variability in rooting is also reported in peach
(Tsipouridis et al., 2003), grapevine (Peros
et al., 1998) and other woody plants (Foster,
1990), suggesting that genotype plays a central role in rooting. Vahdati
et al. (2008) recently compared the rooting ability of low vigor
and precocious walnut seedlings of 3-year-old with semi- and high vigor ones
in response to modified stool layering method. They concluded that low vigor
seedlings display an improved rooting ability in terms of quantity and quality
of adventitious root formation, probably because of their lower degree of lignifications,
wood density and/or rigidity of their sclerenchyma ring.
While modified layering method provides a simple and effective way of vegetative
propagation for conventional nurseries, micropropagation provides more efficient
technique for mass and large scale multiplication (Kuniyuki
and Forde, 1985; McGranahan et al., 1988;
Vahdati et al., 2004). In addition, one can use
tissue culture technique to investigate the behavior of low vigor genotypes
in completely homogenous condition rather than heterogeneous field condition.
However, it has been proven that walnut is also hard to propagate through micropropagation.
Various attempts have been made using different types of explants, media, culture
condition and rooting techniques, with promising results (Driver
and Kuniyuki, 1984; McGranahan et al., 1988;
Scaltsoyiannes et al., 1997; Saadat
and Hennerty, 2002; Vahdati et al., 2002,
2004). Poor proliferation and rooting rate is one of
the main obstacles that limit the micropropagation efficiency in some walnut
varieties and further researches are needed to allow efficient plant propagation
for both scientific and commercial purposes.
As genotype plays a major role in vegetative propagation, in particular for
micropropagation (Lupez, 2004; Scaltsoyiannes
et al., 1997), the present study aimed to compare the response of
low vigor and early mature walnut genotypes versus high vigor ones to micropropagation
condition, in terms of proliferation and rooting rate, to study their growth
consistency using a standard in vitro propagation procedure.
MATERIALS AND METHODS
Plant material: Details of the plant material were described by Rezaee
et al. (2006). In brief, through a nursery selection program (2001-2002),
a number of 125 early mature and dwarf seedlings were selected from local nurseries
and maintained at the Kahriz Agricultural Research Station in Western Azerbaijan
Province, Northwest of Iran (45°10` E; 37° 53` N; 1,325 m a.s.l.). Five years
after growing under uniform orchard condition (2005), different morphological
traits were recorded and data were subjected to the cluster analysis, which
revealed three clusters of high vigor (HV), semi vigor (SV) and low vigor (LV)
trees, each with significantly distinctive characteristics. From each of three
clusters, a number of four mature trees were chosen as a representative for
Explants preparation: Newly grown shoots, approx. 15-20 cm long,
were collected in early May 2006 and leaves were removed, leaving only
1 to 2 cm of the petiole. Shoots were cut into nodal segments, 3 to 5
cm long and then surface sterilized in 0.5% sodium hypochlorite plus one
or two drops of Tween 20 L-1 for 15 min and after repeated
washing with sterile distilled water were cultured on sterile media.
Media and culture conditions: Driver-Kuniyuki-Walnut (DKW) medium supplemented
with 2.1 g L-1 Phytagel (Sigma Chemical Co.), 30 g L-1
sucrose, 4.4 μM 6-benzyladenine (BA) and 0.05 μM indole -3- butyric acid (IBA)
(McGranahan et al., 1988) was used for shoot induction
and multiplication. The pH of medium was adjusted to 5.5 before adding of Phytagel
and autoclaving. Micropropagated shoots were maintained in canning jars (0.3
l), four shoots per jar, at 25±2°C under a 16 h photoperiod and light intensity
of 40-60 μmol m-2 sec-1 supplied by cool white
Philips fluorescent lamps (Vahdati et al., 2004).
All the cultures (20 explants per genotype) were transferred to fresh medium
of the same composition every four weeks. Contaminated explants and those with
sustained growth (no shoot proliferation) were eliminated during the 13 months
period. At the end of the multiplication phase, the number of the newly grown
auxiliary shoots arisen from each explant, as well as shoot length, number of
nodes, presence of flower on shoots, callus and leaf color (based on a scale
of 1 to 3) were recorded or visually scored.
In root induction phase, regenerated shoots, 3 to 5 cm in length, were excised
and transferred onto full-strength MS salts and vitamins (Murashige
and Skoog, 1962), supplemented with 15 μM IBA, 30 g L-1 sucrose
and 9 g L-1 agar (Serva Co.) and were kept in darkness for 7 days
at 24±1°C. In root development phase, shoots (20 to 28 explants per genotype)
were transferred into glass canning jars (1 l) containing a mix of 1:1.25 (v/v)
quarter-strength DKW: Vermiculite (free of hormone) and maintained as described
for multiplication (Vahdati et al., 2004). Rooting
percentage, root length, number of roots, diameter of longest root, callus size
and number of nodes per shoot were recorded after two weeks.
The experimental design was a completely randomized design with four replications.
The experiment was started with 12 genotypes, but only six were finally remained
(other genotypes were eliminated because of contamination or ceased growth).
The data were transformed as necessary and were analyzed using analysis of variance
(ANOVA), followed by Duncan`s mean comparisons test (SPSS,
RESULTS AND DISCUSSION
Establishment: In this study, 12 genotypes (four genotypes from each
of three clusters of vigor) were initially introduced into culture. The number
of successfully established genotypes were three (75%), one (25) and one (25%)
out of four, in low vigor, semi vigor and high vigor clusters, respectively,
indicating better adaptation of low vigor genotypes under in vitro condition.
The effect of genotypes on the establishment of explants has been previously
reported for Persian walnut (McGranhan et al., 1988; Lopez, 2004) as
well as other tree species (Scaltsoyiannes et al.,
Multiplication: Significant differences in multiplication rate,
shoot elongation, size of callus and leaf color were observed among the
six genotypes (Table 1). Number of newly grown shoots
per explant was the highest (3 to 3.3) for low vigor genotypes (G4, G12
and G16) compared to 2.3 in high vigor genotype (G8). The highest (4.5
cm) and the lowest (2.6) elongation of shoots were observed in G8 (high
vigor) and G12 (low vigor) genotypes, respectively.
The average number of nodes per explants ranged from 6.6 to 7.5 which were
not significantly different among the studied genotypes, but considering the
short length of shoots, the number of nodes per a given length of shoot was
comparably higher in low vigor genotypes. Moreover, the low vigor genotypes
showed less basal callus formation as well as dark green leaves compared to
the excessive basal callus formation and lighter color of leaves in high vigor
genotype. Higher rate of multiplication, lower shoot elongation and other morphological
observations clearly documented the basitonic tendency and limited growth rate
of low vigor genotypes which is consistent with their field behavior as explained
in earlier publication (Rezaee et al., 2006).
An interesting phenomenon was the formation of female flowers on the microshoots
of two genotypes (G4 and G12) (Fig. 1A). The flowered microshoots
were shorter and bushier than the non-flowered ones. These observation represent
the early mature behavior (short juvenile period) in low vigor walnut genotypes,
even under in vitro condition and are in agreement with the reports of
Breton et al. (2004).
Rooting: Except for root length and quality, significant differences
in terms of rooting percentage, number of roots per explant and root diameter,
were observed among six genotypes of different vigor (Table 2).
The highest (63.5%) and lowest (37.1%) rooting percentages were observed on
low vigor (G12) and high vigor (G8) genotypes, respectively. The low vigor genotypes
(Fig. 1B) also expressed the highest number of roots per
shoot (2.8 to 3.7) compared to the lowest root number (1.7) on the high vigor
genotype, implying substantial structural and or hormonal differences between
low vigor and high vigor genotypes. The effect of genotypes on rooting has been
also reported by other researchers in different plant species (Scaltsoyiannes
et al., 1997; Peros et al., 1998; Vahdati
et al., 2004), reflecting different levels of endogenous hormonal
balances among different genotypes as suggested by Grochowska
et al. (1984).
||Multiplication rate of the established genotypes of
Persian walnut after 13th subculture
|AMeans followed by the same letter(s) are
not significantly different (p≤0.01).
1 to 3 score: 1 = less, 2 = medium and 3 = excessive basal callus
formation on microshoots. CBased on 1 to 3 score: 1 = light,
2 = medium and 3 = dark color of leaves
||Rooting ability of established genotypes microshoots
|AMeans followed by the same letter(s)
are not significantly different (p≤0.01).
on 1 to 3 score:1 = fair 2 = medium and 3 = good distribution of roots
||In vitro flowering (A) and rooting (B) of low
vigor genotype of walnut (G4). bar = 10.0 mm
The improved rooting ability of low vigor genotypes could be attributed to
the lower amount of endogenous gibberellins and lower degrees of lignifications,
wood density and rigidity of sclerenchyma ring, which facilitate rooting of
micro cuttings. This differential ability of low vigor genotypes was more evident
in the case of layering method at field condition (Vahdati
et al., 2008).
Improved plantlet regeneration as well as rooting ability and in-vitro
flowering of low vigor genotypes in current study provides more supports
on our initial assumptions on the consistency of low vigor behavior as
well as their easy-to-root capacity in both layering (field) and micropropagation
(in vitro) condition. Further researches are needed to develop
size reducing clonal rootstocks and or cultivars by selection of easy-to-root
and low vigor genotypes both under field and in vitro condition.
We thank the Seed and Plant Improvement Institute (SPII), University
of Tehran and Iranian National Science Foundation (INSF) for providing
support for this research.
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