Thesis (M.Sc.)-Cairo University, 2022.

Bibliography: pages 110-118.

As a consequence of the ongoing global climate changes, low water
availability and salinization are expected to affect up to 50% of all arable lands
by the year 2050. Wheat is one of the most consumed crops all over the world,
especially in Egypt. Therefore, improving the tolerance of wheat to drought
and salinity stresses which are correlated (abiotic stress) is vital to ensure food
security. Lately, the CRISPR/Cas9 (Clustered Regularly Interspaced Short
Palindromic Repeats/CRISPR associated nuclease 9) system has been used as
a powerful tool for improving traits with high efficiency. So, this study aimed
for using the CRISPR/Cas9 system to knock out the SALI gene (which down-
regulation increases drought tolerance in plants other than wheat) in a bread
wheat cultivar to increase its drought and salinity tolerance. Marker-assisted
selection using 9SSR primer pairs, 43 SCoT primers, and 42 combinations of
SRAP primers (7 Me and 6 Em) along with biochemical analysis of K+/Na+
ratio on 10 of the most cultivated bread wheat cultivars in Egypt (Giza 171,
Giza 168, Misr 1, Misr 2, Sids 11, Sids 12, Gemaiza 9, Gemaiza 11, Gemaiza
12, and Shandaweel 1) revealed that Giza 171, Gemaiza 9, and Gemaiza 11
are genetically similar sharing the same ancestor and also Giza 171 and
Gemaiza 11 showed the lowest K+/Na+ ratio and high Na+ content, so they
are considered non-tolerant under high salt concentrations. Construct “2000”
containing 3gRNAs targeting 3 different exons on all copies of wheat’s SALI
gene were used to induce mutations in Giza 171 to induce stomatal closure,
thus increasing abiotic stress tolerance. Screening of transgenic T0 plants
regenerated from both mature and immature embryos showed an average
transformation % of about 4.612%. Screening of T1 mutants using the CAPS
PCR method within 20 transformed plants showed successful mutagenesis for
all lines selected, especially lines 2, 3, 8,12,13,14 which resulted in uncut
bands. Sanger sequencing and alignment analysis of the mutated lines 2 and
12 (MLs-2 and12) along with wild-type Giza 171 (WT-G171) ensured
complete knockout in both lines. Stomatal measurements under an electron
microscope revealed significant reductions in both stomatal dimensions.
IV
Drought tolerance evaluation of both lines under the effect of PEG showed the
inability of WT-G171 to tolerate PEG concentrations >10% while MLs-2 and
12 showed high significant improvement under 15 and 20% PEG. Salinity
tolerance evaluation conducted on MLs-2, 3, 4, 8, 12, 13, 14, and 18 using 0,
150, 200, and 300 mM of NaCl revealed different behavioral patterns of the
lines either showed non-significant reductions or significant distinction when
compared to themselves through all treatments thus considered as susceptible
salinity tolerant genotypes (ex. ML-2 and ML-13) or showed significant
reductions in the studied parameters (Eg. ML-4 and ML-18). Therefore, ML-
2 can be considered as a susceptible abiotic stress-tolerant genotype and it is
important to dive deeper into the study of the other susceptible tolerant
mutated lines’ mutations, that gave it its excellency in most of the studied traits,
both on the molecular level and field level.

يمثل تغير المناخ العالمي تحديًا كبيرًا لإ نتاج الزراعي ، لا سيما في المناطق شبه الجافة والجافة ، حيث تتسبب ندرة
المياه في خسائر فادحة في إنتاجية المحاصيل وذلك نتيجة للتغيرات المناخية العالمية المستمرة ،ومن المتوقع أن يؤثر انخفاض
توافر المياه والتملح على ما يصل إلى50٪ من جميع الاراضى الصالحة للزراعة بحلول عام2050. يعتبر القمح أحد أكثر
المحاصيل استهالكًا في جميع أنحاء العالم ، وخاصة في مصر ، و يمثل أكثر من20٪ من السعرات الحرارية اليومية. لذلك ،
فإن تحسين تحمل القمح للجفاف وضغوط الملوحةالعالية(الاجهادات الغير حيوية) أمر ضرورى لضمان الامن الغذائي في
الاونة الاخيرة ، تم استخدام نظامCRISPR / Cas9(Clustered Regularly Interspaced Short Palindromic
Repeats/CRISPR associated nuclease 9) كأداة قوية لتحسين السمات بكفاءة عالية. لذلك ،تهدفهذه الدراسة إلى
استخدام نظامCRISPR / Cas9لتثبيط عملجينSALI(مما أثر فى زيادة تحمل الجفاف في بعض النباتات بخلاف القمح)

Issues also as CD.

Text in English and abstract in Arabic & English.