Gamma Irradiation Effect on Embryogenic Callus Growth of Citrus reticulata cv. Limau Madu

(Kesan Sinaran Gamma ke atas Pertumbuhan Kalus Embriogenik Citrus reticulata cv. Limau Madu)

Sains Malaysiana 45(3)(2016): 329–337

Dita Agisimanto, Normah Mohd Noor, Rusli Ibrahim & Azhar Mohamad

ABSTRACT

Induced mutagenesis using gamma ray has been proven applicable to improve varieties of many genotypes of crop species. The effects of 60Co gamma ray dosage on growth and callus induction of nucellus segments of Citrus reticulata cv. limau madu were investigated. The nucelli were exposed to gamma rays at doses of 10, 20, 40, 60, 80, 100 and 120 Gy, followed by embryogenic callus (EC) induction on Murashige and Skoog medium supplemented with 500 mg L-1 malt extract (ME), 146 mM sucrose, 0.8% (w/v) agar and 13.3 μM benzyl amino purine (BAP). Survival, callus type and colour, degree of callus formation, time of callus formation and total fresh weight of callus varied among the treatments. All untreated explants (controls) survived and produced friable EC in the 2nd or 3rd week of culture, whereas the irradiated nucelli showed delayed response. EC derived from the nucelli irradiated at 10, 20 and 40 Gy appeared in the 3rd week of culture, whereas EC from the 60 and 80 Gy doses appeared in the 4th week. Exposure to higher doses (100 and 120 Gy) completely suppressed callus formation. After 35 days of culture, an average of 697 and 660 mg of EC were harvested from the nucelli irradiated at 10 and 20 Gy, respectively, which was higher than those at 40 Gy (441 mg), 60 Gy (436 mg) and 80 Gy (380 mg). EC were initiated and proliferated and subsequently regenerated into plantlets. DNA of plantlets from the 20, 40 and 60 Gy exposure were individually amplified and compared to the control for early detection of mutagenesis using retrotransposon, inter simple sequence repeat and markers related to seedlessness. No variants were observed from the plantlets produced.

Keywords: Embryogenic callus; gamma-rays; LD50; limau madu; nucellus

ABSTRAK

Mutagenesis aruhan menggunakan sinaran gamma dibuktikan berupaya untuk menambah baik pelbagai genotip tanaman. Kesan dos sinar 60Co gamma pada pertumbuhan dan induksi kalus pada segmen nuselus Citrus reticulata cv. limau madu dikaji. Nuselus didedahkan kepada sinar gamma pada dos 10, 20, 40, 60, 80, 100 dan 120 Gy , diikuti dengan aruhan kalus embriogenik (EC) di atas medium Murashige dan Skoog dengan penambahan 500 mg L-1 ekstrak malt (ME), 146 mM sukrosa, 0.8% (w/v) agar dan 13.3 μM benzil amino purin (BAP). Kemandirian, jenis dan warna kalus, tahap pembentukan kalus; masa pembentukan kalus dan berat segar kalus berbeza antara rawatan. Semua eksplan tidak dirawat (kawalan) hidup dan menghasilkan EC rapuh dalam masa 2 hingga 3 minggu kultur, manakala nuselus radiasi menunjukkan tindak balas yang lambat. EC daripada nuselus diradiasi pada 10, 20 dan 40 Gy muncul pada minggu ke-3 kultur, manakala EC daripada dos Gy 60 dan 80 muncul pada minggu ke-4. Pendedahan kepada dos lebih tinggi (100 dan 120 Gy) telah menghalang pembentukan kalus sepenuhnya. Selepas 35 hari dikultur, purata 697 dan 660 mg EC dicatatkan daripada nuselus yang diradiasi masing-masing pada 10 dan 20 Gy, iaitu lebih tinggi daripada 40 Gy (441 mg) , 60 Gy (436 mg) dan 80 Gy (380 mg). EC dimulakan, berkembang biak dan seterusnya dijana semula menjadi anak pokok. DNA anak pokok daripada pendedahan 20, 40 dan 60 Gy telah diamplifikasi secara individu dan dibandingkan dengan kawalan untuk pengesanan awal mutagenesis menggunakan retrotransposon, antara ulangan jujukan ringkas dan penanda berkaitan ketidakhadiran biji benih. Tiada variasi diperhatikan daripada anak pokok yang dihasilkan.

Kata kunci: Kalus embriogenik; LD50; limau madu; nuselus; sinar gamma

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