Response of Some Egyptian and Introduced Wheat Hybrids to Androgenic Process
Aida A. Rizkalla,
Wheat anther culture produces homozygous doubled haploid plants, in a very
short time span as comparing with traditional breeding methods. It is affected
with several different factors, such as suitable spike stage as a donor for
anthers, anthers cold pretreatment duration, rate of embryogenic callus formation
and the time of anthers transferring from callus induction to regeneration medium.
The effects of these factors on androgenic process were studied in this work
the response of five F1 wheat hybrids, of ten varieties, differing
in their salt tolerance, was investigated in anther culture system for haploid
plantlets regeneration. Anthers were plated on P4S medium for callus induction,
anthers were taken from different spikes ages (as in a distance between the
top of the spike and flag leaf), different duration of cold pretreatment and
different time of transferring anthers from callus induction to regeneration
medium (190-2) were studied. Significant differences between genotypes on androgenic
process were detected. Genotype influence was significant on the suitable spike
length which has high percentage of proper stage of mid-uninucleate stage, consequently
on both of callus induction and plant regeneration (green, albino and total
plantlets). On the other hand, there was no significant influence in the embryogenic
callus formation per 100 anthers between the hybrids under same duration (14,
28 and 56 days). The suitable duration for staying on induction callus medium
was 28 days as the green regeneration plants increased. This is considered as
accurate parameter for evaluation efficiency of androgensis for anther culture.
January 27, 2012; Accepted: April 03, 2012;
Published: June 21, 2012
Anther culture is a competitive technique for traditional methods that plant
breeders use. This technique can produce haploids to improve breeding in both
mono and dicotyledonous plants to get new lines characterizing with both high
yield and disease resistance in a short time (Ramakrishnan
et al., 2005). Production of haploid plants with androgenesis in
cereals has been applied such as: wheat (Simmonds et
al., 1993), Barely (Luckett and Smithard, 1992),
Triticale (Charment and Bernard, 1984), Rice (Miah
et al., 1985), Maize (Petolino and Thompson,
1987) etc. Doubled haploid production technology, inducing homozygosity,
shortens breeding program and permitting variation between genotype within any
generation which attracts breeders attention (Maria
et al., 2006), reducing cost of cultivar development (Hu
and Yang, 1986; Hu, 1997; Liu
et al., 2002). In wheat breeding programs obtaining haploid plants
resultant from anthers of F1 hybrids through anther culture that
accelerates new cultivars development when reduces many years and permitting
easier and more efficient selection process (Camergo et
al., 1999; Ramos et al., 2000).
Before eighteenth century, developing crops by doubled haploid plants was limited.
Androgenesis process can be explained as formation of pseudo embryos (embryoids)
which can geminate into plants giving genetically true doubled haploid (DH)
plants. This is an important and attractive goal for plant breeders, geneticists
and biotechnology researches. DH progeny enables getting inherited traits of
different gene combinations (Konzak et al., 1987).
Anther culture technique gives a very important advantage of obtaining more
than thousand haploid plants/anther culture (Devaux, 1988).
In comparing to other methods, all of it are restricted to only one plant/floret
and consequently to a low number of responsive genotypes while others remain
without changing. Therefore, the urgent need for methods help to increase androgenesis
process of microspores in a great number of genotypes (Hu
et al., 1995).
In wheat, may be through crosses androgenic response can be passed to progeny
in great numbers as it is inherited (Henry and Buyser, 1981;
Lazar et al., 1984; Moieni
and Sarrafi, 1995). Thus, formation of haploids and their regeneration is
affected by three related traits; embryo formation rate, their regeneration
capability and ratio of green to albino plants (Henery and
Buyser, 1985; Ghaemi et al., 1995; Moieni
and Sarrafi, 1995). Regeneration frequency also is affected by the time
of transferring anthers from induction medium to regeneration conditions (Zheng
and Konzak, 1999; Redha et al., 2000).
Developing of microspore-anther culture methods was getting when stress treatment
was applied basing on the transferring from preprogrammed gametophytic to sporophytic
pathway for getting androgenesis (Touraev et al.,
1996; Touraev et al., 1997; Hu
and Kasha, 1999; Zhou and Konzak, 1997; Simonson
et al., 1997; Reynolds, 1997; Zheng
and Konzak, 1999; Ohnoutkoua et al., 2000).
In view of the above, present work was planned to study the androgenetic process
of anther cultures for F1 hybrids of several wheat parents differing
in salt tolerance level under different parameters i.e., (1) Proper stage of
donor spikes development (in centimeters) as a morphological character (2) Duration
of anthers cold pretreatment ( days) (3) Induction of embryogenic callus and
embryos formation and (4) The time of transferring anthers from callus induction
medium to regeneration medium.
MATERIALS AND METHODS
Plant materials and salt screening: Salt screening and field crossing were
done in 2009 and 2010, laboratory work was extending till Dec. 2011.
Ten varieties of bread wheat (Triticum aestivum L.), six were local
(Sids 4, Sakha 93, Gemmeiza 9, Sids 1, Sakha 8 and Giza 162) and four introduced
(Golan 2, Cham 6, Bow and Mexipak 69), were chosen to test the transactions
affected by levels of salinity. The experiment was conducted in Petri dishes
containing quarter strength of Hoagland solution (Hoagland
and Arnon, 1950) under five salinity levels: 0, 8,000, 10,000, 12,000 and
14,000 ppm of 2:1 (NaCl:KCl) as a source of salinity stress. Each treatment
was performed by 5 petri dishes (15 cm in diameter) in 3 replicates, each one
contained 100 seeds. The germination percentage was calculated after 2 weeks.
Seeds were scored as germinated when the primary roots were greater than 3 mm
and the shoots were longer than the seed itself. Seeds contaminated by fungi
during the test were ignored (Ye et al., 1987).
|| Crosses between selected parents for obtaining F1
hybrids as donor plants for anthers
On this base, six parents had been chosen, five of them were most resistant
to salinity, while the sixth variety (Gemmeiza 9 is sensitive to salinity but
high yielder) to set some crosses as shown in Table 1. The
materials of this study were kindly provided by Dr. Samy. A.A. Attia who performed
Laboratory work of anther culture, haploid production and regeneration was
performed in Genetics and Cytology Department, Genetic Engineering and Biotechnology
Division, National Research Center, Egypt.
Determining suitable spike age for anther culture: Anthers of F1
hybrid plants from primary and three secondary tillers were collected when microspores
from the anthers in the central region of the spikes were at the mid to late
uninucleate stage before the first pollen mitosis (Barnabas
et al., 1991; Navarro-Alvarez et al.,
1994; Redha et al., 1998). The developmental
stage of microspores was checked by acetocarmine staining of anthers from the
central region of the spike and subsequent light microscope observation. The
sampled spikes at different ages as a distance between the top of the spike
and flag leaf were O = 7.5, 10, 12.5 or 15 cm.
Cold pre treatment: Suitable spikes were placed in flasks containing
tap water and stored in dark at 4°C for 2-7 or 14 days as an anther stress
Induction of embryogenic callus and embryos formation: Spikes were surface
sterilized in 80% ethanol for 2 min intervals and the enclosed anthers were
excised with fine forceps (Zheng and Konzak, 1999).
Anthers were then placed aseptically in 50 mL glass jars containing 15 mL of
P4S medium (Ouang et al., 1983) to induce embryogenic
callus formation at 26±2°C in dark. The largest embryos (>2 mm)
produced were then counted and transferred into regeneration medium 190.2 (Zhuang
and Jia, 1983), green and albino plantlets were recorded.
The time of transferring anthers from callus induction medium to regeneration
medium: The following parameters were recorded to show the effects of the
time of transferring anthers from callus induction medium to regeneration medium:
embryogenic callus formation plant/100 anthers, regenerated green plants/100
anthers, regenerated albino plants/100 anthers and total regenerated plants/100
anthers after 14, 28 or 56 days on induction medium (P4S) to regeneration medium
(190-2). Cytological examination for haploids was done.
RESULTS AND DISCUSSION
Salt screening: As shown in Fig. 1, the ten different
wheat varieties had emerged the effect of different levels of salinity on their
germination rate were among the terms of higher and lower tolerance salinity.
It is noticed that Mexipak 69, Bow Giza 162, Sakha 8 and Cham 6 gave the highest
percentage of germination under all salinity levels.
||Seed germination percentages of the ten studied cultivars
under five salinity levels. 1: Sids 4, 2: Sakha 93, 3: Gemmeiza 9, 4: Sids
1, 5: Sakha 8, 6: Golan 2, 7: Cham 6, 8: Giza 162, 9: Bow, 10: Mexipak 69
|| Developmental stages of anther culture, (a) Suitable stage
of anther culture (mid-uninucleate stage), (b) Chromosome number of regenerated
haploid plantlets 1n = 3x = 21, (c) Callus formation from anther culture,
(d) Initiation of shoot tip, (e) Regenerated plantlets from embryogenic
Suitable spike age for anther culture: According to Ding-Gang
and Jun-Wen (1984) and Lantos et al. (2006)
the most critical steps of wheat microspore anther cultures is the choice of
ideal microspore development stage (Fig. 2a). It is the first
important step of androgenesis induction.
|| Percentage of microspores/spike at mid-uninucleate stage
induced at different spike stages of the five wheat hybrids (crosses)
|| Percentage of wheat embryogenic callus/100 anthers of the
five crosses in response to cold pretreatment durations
Data in Table 2 showed the percentage of microspores/spike
at mid-uninucleate stage in the central part of spikes of the 5 used hybrids
expressed in centimeter as distance between the top of the spike and the flag
leaf auricles. Table 2 revealed that spikes at <10 cm stage
for the five used F1 hybrids possessed the highest mean frequency
of microspore cells in the mid-uninucleate stage (52.88) as compared to the
other four spike ages, i.e., 0, 7.5, 12.5 or 15 cm where, their mean values
reached 8.78, 51.02, 50.18 and 31.16, respectively. It seems that genotype effect
has significant influence on the differences among the mean percentage of microspores/spike
in the proper stage of the 5 genotypes. These result is in agreement with Ding-Gang
and Jun-Wen (1984), Maluszynski et al. (2001)
and Lantos et al. (2006), who stated that percentage
of microspores in mid-uninucleate stage in a certain age of spike morphology
depend on the genotype. So, correlation between developmental stage of microspores
and morphological characters of spike such as distance between the top of the
spike (last floret) and flag leaf auricles is important to define the suitable
spike age for anther culture. These results also are supported by findings of
Mehmet et al. (2008), who concluded that the
spike length is significantly correlated with callus number and green plantlet.
Effect of cold pretreatment of spikes on callus induction: Low temperature
~4°C for several days, prior to normal culture procedure, was used under
speculation that it might has a dual function: interrupting normal gametophytic
development and the nursing effects of anther tissue on microspores (Fazar
et al., 1985; Amstrong et al., 1987;
Henry and Buyser, 1981). Table 3 showed
the response of the 5 used crosses to cold treatment durations as percentage
of callus produced from 100 anthers.
The results in Table 3 indicated that cold treatment of spikes
for duration shorter than 7 days could not have enough impact on starving microspores
to switch their normal pathway to form embryos. On the contrary, starving microspores
for 7 or 14 days was highly recommended for the used crosses. Table
3 presented that the five crosses significantly varied as the percentage
of cross H5 reached 58.4 and 56.6% under cold duration of 7 and 14 days, respectively.
|| Number and percentage of callus and embryoids from anthers
of the five crosses on inductions (P4S) medium
It was followed by cross A which reached 47 and 49.7%, respectively. While,
cross C showed the lowest response, its percentages were 30.9 and 28.4, respectively,
also the two crosses B and H1 produced intermediate levels of anther
response. The diverse of the five genotypes responses for cold pretreatment
were attributable to varying genotypes, pretreatment durations and the genotype
x pretreatment durations. Present results are in agreement with the findings
of Lazar et al. (1984), Amstrong
et al. (1987) and Henry and Buyser (1981).
In contrary, Khiabani et al. (2008) included
that cold pretreatment was found ineffective on androgenic ability of genotypes
where they showed low response to cold treatments.
Induction of embryogenic callus and embryos formation: Embryogenic callus
formation (Fig. 2c) of the five crosses on induction medium
(P4S), their percentages and No. of embryos are in Table 4
which showed that the percentages ranged from, the lowest percentage (2.8) for
cross C and the highest (55.5) for the cross A. While crosses H5,
B and H1 ranged in between the previous crosses where, their callus
formation percentages were 36.6, 30.8 and 7.3%, respectively. These differences
among crosses could be attributed to the effects of genetic make-up and or the
culture composition, where genotype affected anther culture response significantly
(Lu et al., 1991; Konieczny
et al., 2003; Bagheri and Jelodar, 2008;
Zheng et al., 2001).
Others suggest that these differences in androgenic response could be referred
to the presence of several genes located on the different wheat genome (Szakacs
et al., 1988; Sibikeeva and Sibikeev, 1996).
Others referred the difference in androgenic responses, of used genotypes, to
their differences in endogenous hormones levels , which in turn in different
requirements for the culture medium.
Effect of transferring wheat anthers (from induction to regeneration medium)
on the androgenic response, of the five studied crosses: Data in Table
5, showed the percentages of embryogenic callus, green regenerated plants
(Fig. 2d-e), albino regenerated plants and
total number of regenerated plants/100 anther after 14, 28 or 56 days on induction
medium (P4S) to regeneration medium (190-2). From Table 5,
we notice that, there were no significant differences in the embryogenic callus
formation per 100 anthers between the five studied crosses under the same duration
(14, 28 or 56 days). On the other hand, we find that 28 days duration is the
proper time of transferring for obtaining higher percentages of green regenerated
plants as well as for albino and total regenerated plants (haploid 1n = 3x =
21, Fig. 2b).
|| Percentages of embryogenic callus, green, albino and total
regenerated plantlets per 100 anthers
|Em.: Embryos, An: Anthers, GRP: Green regenerated plants,
ARP: Albino regenerated plants, TRP: Total regenerated plants
Data showed great increment percentage of green regenerated plants at 28 days
than 14 or 56 days, where for cross (a) the increment reached 2.8 and 2.9 fold,
respectively; while for cross (b) it reached 2.7 and 2.85 fold; for cross (c)
it was 1.71 and 2.16 fold, respectively; for cross (H1) it increased
2.6 and 2.72 fold; while for cross (H5) it was the highest increasing
2.82 and 3.07 fold.
In terms of regeneration, 28 days duration was the best as the percentage of
green regenerated plants is considered as accurate parameter for efficiency
evaluation of anther culture.
On the other hand, in comparing with 14 days, the percentages of the albino
regenerated plants increased greater with 28 days, while the greatest increment
was with 56 days for all the studied crosses which reflects as a problem for
the application of anther culture technique in wheat breeding programs as Saidi
et al. (1997) stated that, production of albino plants was affected
by genotype and medium composition. They added that albinism might occur by
mutation in chloroplast DNA. While Caredda et al.
(1999) referred albinism to the probability of the abnormalities of plastids
features during embryogenesis process.
The early transfer of anthers (that formed callus) from the induction medium
to regeneration medium caused a strong reduction in the production of embryogenic
callus, while the overall regeneration of plants/100 anthers showed an improvement
for delaying. These results are in agreement with previous findings on wheat
(Henery and Buyser, 1985; Redha
et al., 2000). The reduction of embryogenic callus production with
the early transfer, suggesting that, the earlier transfer interferes with the
development of the embryos. However, it had a positive effect on the quality
of the embryos and, as a result, the regeneration of the plants improved considerably.
When the embryos are left for longer time (56 days) on induction medium, the
quality of the embryos and their totipotency to regenerate may be lower (Redha
et al., 2000).
Anthers of five F1 wheat hybrids, their parents differ in salt tolerance
showed a variation in the productive response of callus formation and regeneration
capacity (green, albino and total plant numbers) under different studied factors.
It was concluded that androgenic response is affected mainly by genotype which
has significant influence on the differences among the mean percentage of microspores/spike
in the anther proper stage of the 5 used genotypes. While, embryogenic callus
formation was better in response to 7 days of anthers cold pretreatment durations.
Also the proper time of transferring calluses from callus induction medium to
regeneration medium affected androgenic process.
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