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

- Makful, Sudarmadi Purnomo, - Sunyoto, R Iswanto, T.I.R. Utami

Abstract


Penelitian ini dilaksanakan dari bulan Januari 2002 sampai Februari 2003, bertujuan untuk mendapatkan kalus
transforman gen 1-aminosiklopropan-1-asam karboksilat oksidase yang mampu hidup dan dapat berdiferensiasi,
wahana untuk membuat pepaya transgenik tahan simpan, telah dilakukan di Laboratorium Biologi Molekuler, Balai
Besar 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 yang 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 bombardemen
partikel. 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 1-aminosiklopropan-1-asam karboksilat 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 aminocyclopropane carboxylic
acid (ACC) ox i dize gene, there are vi a 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 of pa paya qual ity was de ter mined by fruit fresh ness. The pro -
cess of pa paya ma tu rity was very fast. This prob lem made dif fi cult for hap pened pa paya trans por ta tion. The pro cess of
ma tu rity was con trolled by in creas ing con cen tra tion of eth yl ene hor mone and it was syn the sized from
1-aminocyclopropane-1-carboxylic acid. One of ef fort pressed eth yl ene pro duc tion was long dis tance block ing
ethylene biosynthesis path way. Mech a nism of block ing eth yl ene biosynthesis path way was made antisens of gene
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 was de scrip tive quan ti ta tive with 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, and
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 sults in di cated the op ti miz ing of both pa paya on 150 mg/l
kanamycin. This con cen tra tion made all explant of results. 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 cotyledone em bryo) in dampit but in sarirona was not growth.
Transforman growth on se lec tive me dium in di cated pGA643 SM4 have in serted to zy gotic em bryo be cause plasmid
con tain ing se lec tion gene of kanamycin (NPT II gene).


Keywords


Carica pa paya; 1-aminocyclopropane-1-carboxylic acid ox i dize; Gene trans fer; Rip en ing re tar da tion

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DOI: http://dx.doi.org/10.21082/jhort.v14n2.2004.p76-83

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