Assiut university researches
Genetical Studies on Peanut (Arachis
Hypogaea L.) Tissue Culture
‫درا سات وراث ية ع لى زراعة االن سجة ف ى ال فول‬
‫ال سودان ى‬
Douha Salah Mohamed Mahmoud
‫ضحى ص الح محمد محمود‬
Adel Sayed Taghian, Hamdy Mohamed El-Aref, Bahaa El-Din ElSayed Abd-El-Fatah
‫ ب هاءال دي ن ال س يد ع بدال ف تاح‬،‫ حمدى محمد ال عارف‬،‫عادل س يد ت غ يان‬
Abstract:
The present investigation was carried out at the Department
of Genetics, Faculty of Agriculture, Assiut University, Assuit,
Egypt, during 2009 – 2013. The investigation aimed at
assessing the utility of peanut tissue culture for studying the
following characteristics: I- Study the tissue culture response
and plant regeneration in peanut (Arachis hypogaea L.). IIDetermine the genetic variability and polymorphism among
six peanut varieties by RAPD, ISSR and SRAP markers, and
to identify markers associated with tissue culture response.
III- Assessing the possibility of obtaining salt tolerant peanut
plants via tissue culture. VI- Studying the changes in gene
expression under salinity stress as revealed by protein
analysis. Six genotypes of peanut namely; Ismailia-1 (G1),
Giza-5 (G2), Agryl (G3), Gregory (G4), Giza-7 (G5) and Giza6 (G6) were used in the present investigation. The results
obtained could be summarized as follow: 1- Calluses and
shoots were formed from the emberyonated and deemberyonated cotyledon explants of all genotypes on all
tested media. The regeneration rate depended upon the
genotype, type of explant and medium. 2- The MS medium
supplemented with 25.0 mg/l BAP (SIM-P1 medium) was
superior in callus formation (78.50%) and number of shoots
per explant (10.28 shoots), which suggested that it was more
suitable for regeneration of peanut plants than the other
tested media. 3- The embryonated cotyledon was suitable
explant for peanut tissue culture which exceeded the deembryonated one in callus formation, shoot regeneration and
rooting of shoots. 4- The high efficiency of in vitro root
formation could be obtained by culturing of regenerated
shoots on MS medium supplemented with 1.0 mg/l NAA (Rzmedium). 5- The range of Euclidean distance among all
genotypes using tissue culture traits was relatively wide
(0.628 – 4.651), which indicated high amount of variation in
tissue culture traits among the genotypes. 6- The UPGMA
dendrogram based on tissue culture data separated the
peanut genotypes into two clusters; the first one contained
the highly responsive genotypes Giza-5, Gregory and Giza-7,
while the second included the low responsive genotypes
Ismailia-1, Agryl and Giza-6. 7- from the three molecular
markers (RAPD, ISSR and SRAP primers), a total of 202
DNA fragments were amplified by 18 primers from all
genotypes with an average 11.22 bands/primer. Out of these
fragments, 71 (35.15%) showed polymorphism and 131
(64.85%) were monomorphic. 8- Four molecular markers
generated by different primers [461bp (OPA06), 1480bp
(HB09), 205bp (HB12) and 465bp (Em1c – me4)] can be
considered as positive markers for tissue culture response in
peanut. 9- RAPD, ISSR and SRAP markers showed positive
correlation with tissue culture traits. 10- A total of 43 DNA
bands were obtained from the six peanut genotypes using six
RAPD primers and ranged in size from 772bp (OPA07) to
71bp (OPA01). 11- Only 37.21% of the DNA bands generated
by RAPD were polymorphic while 62.79% were common
between the tested genotypes. 12- Unique positive RAPD
markers were detected for the genotypes Giza-6 (1 marker)
and Giza-5 (2 markers) which could be used for genotype
identification and discrimination. 13- Negative RAPD markers
were detected for the genotypes Giza-7 (2 markers), Agryl (3
markers), Gregory (1 marker) and Giza-6 (1 marker). 14- A
total of 60 DNA bands were obtained from the six peanut
genotypes using ten ISSR primers and ranged in size from
1480bp (HB09) to 93bp (HB15). 15- Twenty-seven (45%) of
ISSR bands were polymorphic while 33 bands (55%) were
common between the tested genotypes. 16- Positive RAPD
markers were detected for the genotypes Ismailia-1 (4
markers) and Giza-6 (1 marker) which could be used for
genotype identification and discrimination. 17- A total of 99
DNA bands were obtained from the six peanut genotypes
using six SRAP primer combinations and ranged in size from
1461bp (Em1a – me4) to 51bp [(Em1a – me2), (Em1c – me4)
and (Em2 – me1b)]. 18- Twenty-eight (28.28%) of SRAP
bands were polymorphic while the rest of bands (72.72%)
were common between the tested genotypes. 19- One unique
positive marker in Giza-6 and four negative markers in
Gregory which were detected by SRAP primer combinations
and could be used for genotype identification and
discrimination. 20- Based on combined data of RAPD, ISSR
and SRAP, the highest genetic similarity (0.951) and shortest
genetic distance (0.049) were found between Giza-5 and
Gregory. While the lowest genetic similarity (0.87) and
longest genetic distance (0.130) was found between Gregory
and Giza-7. 21- The dendrogram generated based on
combined RAPD, ISSR and SRAP data separated the peanut
genotypes into two clusters; the first one contained the highly
responsive genotypes Giza-5, Gregory and Giza-7, while the
second included the low responsive genotypes Ismailia-1,
Agryl and Giza-6. 22- The development of callus and shoot
differentiation were decreased with the increment of NaCl
level from 50 to 150 mM. 23- At higher concentration of NaCl
(200mM), the cultured explants turned brown and the few
developed calli failed to regenerate shoots or regenerate
abnormal shoots with very low frequency in most genotypes.
24- A total of 1760 embryonated cotyledons from Giza-5,
Gregory, Giza-7 and Giza-6 were cultured under selection
conditions of salinity stress (150 mM NaCl). 25- The
regeneration rate was reduced under salinity stress to be
28.67% in callus and 15.15% in shoot formation of that on the
control treatment. 26- The selected and unselected plants in
comparison to their donor parents were tested for salinity
tolerance in vitro on 0.0 and 150mM NaCl. The selected
plants showed significant enhancement in their growth under
salinity treatment, as compared with the unselected plants
and their donor parents. 27- Salinity-stress induced the
synthesis of two new protein bands (RF = 0.931 and 0.816) in
all tested genotypes. While, one band at RF 0.978 in Giza-5
and other band at RF 0.532 in Giza-7 were also induced
under salinity stress. 28- Salinity stress also reduced the
expression of 5 proteins in G5, and 3 protein bands in G4 29Salinity stress also enhanced and/or decreased the
expression of other proteins in the tested genotypes of
peanut. 30- Salinity stress treatment lead to differential
expression of the genetic information in peanut, resulting from
changes in gene products, including induced/ enhanced the
synthesis of certain proteins and simultaneously
reduced/decreased the expression of other protein sets in the
tested genotypes.