Transformasi cDNA Gen 1-Aminosiklopropan-1-Asam Karboksilat Oksidase untuk Penundaan Kematangan pada Pepaya Dampit dan Sarirona

- Makful, S Subandiyah, I D Priyambada, W T Artama

Abstract


Penelitian ini dilaksanakan dari bulan Januari 2002 sampai Februari 2003. Penelitian bertujuan untuk mendapatkan
kalus transforman gen 1-aminosiklopropan-1-asam karboksilat (ACC) oksidase yang mampu hidup dan dapat
berdiferensiasi, wahana untuk membuat pepaya transgenik tahan simpan, telah dilakukan di Laboratorium Biologi
Molekuler, Balai Penelitian Bioteknologi dan Sumber Daya Genetik Pertanian, Bogor. Mutu buah pepaya salah
satunya ditentukan oleh kesegaran buah saat dikonsumsi. Proses pemasakan buah pepaya berlangsung sangat cepat,
hal ini menyulitkan dalam transportasi pepaya, terutama untuk menjangkau tempat yang jauh. Proses pemasakan buah
dikontrol oleh meningkatnya konsentrasi hormon etilen dan disintesis dari 1-aminosiklopropan-1-asam karboksilat.
Produksi etilen dapat ditekan dengan memblok jalur biosintesis etilen. Mekanismenya adalah membuat antisens gen
reg u la tor biosintesis etilen. Transformasi pepaya varietas dampit dan sarirona dilakukan menggunakan par ti cle bom -
bard ment. Rancangan percobaan adalah deskriptif kuantitatif dengan rancangan acak lengkap. Sumber eksplan
pepaya berupa embrio zigotik yang digunakan untuk optimasi taraf kematian terhadap kanamisin, uji gus
menggunakan plasmid pRQ6 (gen gus, NPH, promotor 35S, dan ter mi na tor NOS) dan introduksi gen interes dalam
plasmid pGA643 SM4 (gen antisens ACC oksidase, NPT II, promotor 35S, dan ter mi na tor NOS) pada me dia seleksi.
Hasil penelitian menunjukkan bahwa kedua eksplan pepaya op ti mal pada kanamisin 150 mg/l, di mana pada
konsentrasi ini eksplan mati seluruhnya. Pengujian gus terbanyak pada varietas sarirona 25% (25 spot biru) jarak 9
cm, sedangkan varietas dampit 10% (9 spot biru) jarak 5 cm. Spot biru menandakan gen yang disisipi telah terintegrasi
pada gen tanaman. Efisiensi gen antisens ACC oksidase pada me dia seleksi kanamisin 150 mg/l menunjukkan 16%
(14 embrio kotiledon) pada varietas dampit, sedangkan varietas sarirona tidak tumbuh. Tumbuhnya transforman pada
me dia seleksi menunjukkan eksplan tersebut sudah tersisipi pGA643 SM4 yang mengandung gen tahan terhadap
kanamisin (gen NPT II ).

Trans for ma tion of cDNA
ACC ox i dize gene for de lay rip en ing on pa paya dampit and sarirona. This re search was con ducted from Jan u ary
2002 to Feb ru ary 2003. The aim of this re search was to find out trans formed cal lus by ACC ox i dize gene, there are via -
ble and be able to dif fer en ti a tion, the means to de velop pa paya trans gen ic for de lay rip en ing. The re search was con -
ducted at the Lab o ra tory of Mo lec u lar Bi ol ogy of Re search In sti tute for Bio tech nol ogy and Ag ri cul tural Ge netic
Re sources, Bogor. One part of pa paya qual ity was de ter mined by fruit re fresh ment. The pro cess of pa paya ma tu rity
was went on very fast. This prob lem made dif fi cult for pa paya tranportation, ex cel lence to reach out long place. The
pro cess of ma tu rity was controled by incresing con cen tra tion of eth yl ene hor mone and it was syn the sized from ACC
(1-aminocyclopropane-1-carboxylic acid). One of ef fort pressed eth yl ene pro duc tion with to block eth yl ene
biosynthesis path way. Mech a nism of block ing eth yl ene biosynthesis path way was made antisens of gen reg u la tor.
The trans for ma tion of pa paya dampit and sarirona va ri ety have been de rived by bom bard ment par ti cle. The ex per i -
men tal de sign is de scrip tive quan ti ta tive and ran dom ized com plete de sign. The zy gotic em bryo as explant source
were used for op ti miz ing kanamycin lev els, gus as say with pRQ6 plasmid (gus gene, NPH, 35S pro moter, NOS ter mi -
na tor) and in tro duc tion in ter est gene with pGA643 SM4 plasmid (antisens ACC ox i dize gene, NPT II, 35S pro moter,
NOS ter mi na tor) on the se lec tive me dium. The re sult in di cated the op ti miz ing of both pa paya on 150 mg/l kanamycin.
This concentration made explant die all of it. Gus as say pref er ence of sarirona 25% (25 blue spot) in dis tance 9 cm and
dampit 10% (9 blue spot) in dis tance 5 cm. Ef fi ciency of antisens ACC ox i dize gene on se lec tive me dium con tain ing
150 mg/l kanamycin in di cated 16% (14 cotyledonae em bryo) in dampit but in sarirona do not growth. Transforman
growth on se lec tive me dium in di cated pGA643 SM4 have in sert to zy gotic em bryo be cause plasmid con tain ing se lec -
tion gene of kanamycin (NPT II gene).


Keywords


Carica pa paya; ACC Ox i dize; eth yl ene; Trans for ma tion; antisens; Bom bard ment par ti cle

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DOI: http://dx.doi.org/10.21082/jhort.v14n1.2004.p1-7

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