5. KESIMPULAN DAN SARAN
5.2. Saran
Saran dalam penelitian ini adalah:
1. Perlu dilakukan analisis stabilitas genetik dan stabilitas fitokimia pada mutan
yang mengandung kadar andrographolide tinggi pada beberapa generasi sehingga didapatkan mutan yang benar-benar solid.
2. Perlu dilakukan analisa kimia lanjutan untuk elusidasi struktur kimia beberapa puncak baru yang muncul dari beberapa mutan yang berbeda dengan kontrol sehingga dapat diketahui struktur kimia senyawa tersebut yang berbeda dengan kontrol.
Abd El-Twab MH, Zahran FA. 2010. RAPS, ISSR and RFLP analysis of phylogenetic relationhips among congeneric species (Anthemideae,
Asteraceae) in Egypt. Int J Bot. 6(1): 1-10.
Abdel-Hady MS, Okasha EM, Soliman SA, Talaat M. 2008. Effect of gamma radiation and gibberellic acid on germination and alkaloid production in
Atropa belladonna L. Aust J Basic Appl Sci. 2(3): 401-405.
Adeogun AI, Adeogun MB. 2009. Effect of irradiation and extractive solvents on
the Thevetia seed oil. Afr J Biotechnol. 8(5): 841-843.
Aharoni A, Jongsma MA, Kim TY, Ri MB, Giri AP, Verstappen FWA, Schwab W, Bouwmeester HJ. 2006. Metabolic engineering of terpenoid biosynthesis
in plants. Phytochemistry Reviews. DOI 10.1007/s11101-005-3747-3.
Ahloowalia BS, Maluszynski M. 2001. Induced mutations – A new paradigm in
plant breeding. Euphytica. 118: 167-173.
Aisyah SI, Aswidinnoor H, Saefuddin A, Marwoto B, Sastrosumarjo S. 2009.
Induksi mutasi pada stek pucuk anyelir (Dianthus caryophyllus Linn.)
melalui iradiasi sinar gamma. J Agron. Indones. 37(1): 62-70.
Akbar S. 2011. Andrographis paniculata: a review of pharmacological activities
and clinical effect. Monogr Alt Med Rev. 16(1): 66-77.
Akowuah GA, Zhari I, Norhayati I, Mariam A. 2006. HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata. J Food Comp Anal. 19: 118–126.
Anonim. 2005. Pasar domestik dan ekspor produk tanaman obat (biofarmaka).
Pusat Studi Biofarmaka. IPB. Bogor.
Anwar FS, Ahmad N, Gulzar A, Shidiq AM, Rahman S, Ahmad I, Rauf A. 2004. Effect of gamma radiation on growth and yield of barley under different
nitrogen levels. Pak J Biol Sci. 7(6): 981-983.
Aromdee C, Wichitchote P, Jantakun, N. 2005.Spectrophotometric determination
of total lactones in Andrographis paniculata Nees. Songklanakarin J Sci
Technol. 27(6): 1227-1231.
Aryanti. 2011. Peningkatan kandungan artemisinin melalui mutasi tunas in vitro
tanaman obat Artemisia cina. Majalah Farmasi Indon. 22(1): 60-64.
Atak C, Sema A, Leyla A, Yasemin C. 2004. Induced of plastid mutations in
soyben plant (Glycine max L. Merril) with gamma radiation and
Atienzar FA, Awadhesh NJ. 2006. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and
carcinogenesis studies: a critical review. Mutation Res. 613: 76-102.
Azzam EI, Jay-Gerin JP, Pain D. 2012. Ionizing radiation-induced metabolic
oxidative stress and prolonged cell injury. Cancer Lett.
doi:10.1016/j.canlet.2011.12.012. 13 pages.
Babaei A. 2010. Genetic diversity analysis of nemat rice mutant (Oryza sativa L.)
via RAPD marker. Am-Eur J Agric & Environ Sci. 8(4): 452-456.
Bahar YH. 2012. Kebijakan pengembangan tanaman obat mendukung saintifikasi
jamu. Prosiding Seminar Nasional Pokjanas TOI XLII. 16 Mei 2012.
Bandung.
Beltagi MS, Ismail MA, Mohamed FH. 2006. Induced salt tolerance in common
bean (Phaseolus vulgaris L.) by gamma irradiation. Pak J Biol Sci. 9(6):
1143-1148.
Bhan MK, Dhar AK, Khan S, Lattoo SK, Gupta KK, Choudhary DK. 2006.
Screening and optimization of Andrographis paniculata (Burm.f.) Nees for
total andrographolide content, yield and its components. Scientia
Horticulturae. 107: 386–391.
Borzouei A, Kafi M, Khazae H, Naseriyan B, Majdabadi A. 2010. Effects of gamma radiation on germination and physiological aspects of wheat (Triticum Aestivum L.) seedlings. Pak J Bot. 42(4): 2281-2290.
BPS.2012. http://www.bps.go.id/tab_sub/view.php?kat=3&tabel=1&daftar=1&id _subyek=55¬ab=6. diunduh pada tanggal 08 Juni 2012 pukul 15.00 WIB.
Camargo EES, Telascrea M, Vilegas W. 2008. Effect of the decontamination
using gamma irradiation on the essential oil of Turnera diffusa Wild.
Brazilian J Pharmacog. 18(3): 356-359.
Chao WW and Lin BF. 2012. Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian). Chinese Med. 5:1-15.
Chmielewski AG, Migdal W. 2005. Radiation decontamination of herbs and
spices. Nukleonika. 50(4): 179-184.
Cho Y, Song KB. 2000. Effect of ɣ-irradiation on molecular properties of BSA
and β-lactoglobulin. J Biochem Mol Biol. 33: 133-137.
Chobkarjing U. 2004. Chemical investigation of irradiated medicinal Plant (Andrographis paniculata). Master Thesis. Mahidol University. Bangkok. 1- 73.
Chopra V L. 2005. Mutagenesis: Investigating the process and processing the
outcome for crop improvement. Curr Sci. 89(2): 353-359.
Chowdhury A, Biswas SK, Raihan SZ, Das J, Paul S. 2012. Pharmacological
potensials of Andrographis paniculata: an overview. Int J Pharm. 8(1): 6-9.
Chung BY, Leeb YB, Baeka MH, Kima JH, Wia SG, Kim JS. 2006. Effects of low-dose gamma-irradiation on production of shikonin derivatives in callus
cultures of Lithospermum erythrorhizon S. Rad Phys Chem. 75: 1018–1023.
Cretu R, Gille E, Danila D, Floria F. 2009. Effects induced by singular and combined treatments with gamma radiation and flavonoid compounds on
Triticum aestivum L. Analele Ştiinţifice ale Universităţii, Alexandru Ioan
Cuza”, Secţiunea Genetică şi Biologie Moleculară, TOM X. 55-62.
Datta, Kumar S, Chakrabarty D, Verma AK, Banerji BK. 2011. Gamma ray induced chromosomal aberrations and enzyme related defense mechanism in
Allium cepa L. Caryologia. 64(4): 388-397.
Dhakshanamoorthy D, Selvaraj R, Chidambaram A. 2010. Physical and chemical
mutagenesis in Jatropha curcas L to induce variability in seed germination,
growth and yield traits. Rom J Biol–Plant Biol. 55(2) 113–125.
Dhakshanamoorthy D, Selvaraj R, Chidambaram ALA. 2011. Induced
mutagenesis in Jatropha curcas L. using gamma rays and detection of DNA
polymorphism through RAPD marker. C. R. Biologies. 334: 24–30.
Ditjen POM. 2001. Kebijakan Nasional Pengembangan Obat Tradisional. Departemen Kesehatan RI. Jakarta. 20 hlm.
El-Sayed OE, Rizkalla AA, SRS Sabri. 2007. In vitro mutagenesis for genetic
improvement of salinity tolerance in wheat. Res J Agric Biol Sci. 4(5): 377-
383.
Erdem G, Oldacay S. 2004. Employment of RAPD technique to assess the genetic
stability of Helianthus annuus treated with different mutagenic agents. J
Appl Sci. 4(2): 277-281.
Fanaro GB, Silveira APM, Nunes TCF, Costa HSF, Purgatto E, Villavicencio
ALCH. 2009. Effects of radiation on white tea volatiles. Int Nuclear
Atlantic Conf - INAC 2009. Rio de Janeiro, Brazil, September 27 to October 2 2009.
Hadi HA, El-Mokadem HE, El-Tayeb HF. 2008. Relationship of Four Ficus
Species Using Random Amplified Polymorphic DNA (RAPD) and Inter-
Hegazi AZ, Hamideldin N. 2010. The effect of gamma irradiation on
enhancement of growth and seed yield of okra [Abelmoschus esculentus (L.)
Monech] and associated molecular changes. J Hort Forestry. 2(3): 038-051.
Hoang TML, Filippis LFD, Le XT. 2009. Salt tolerance and screening for genetic change in rice mutants after gamma radiation using RAPD and
microsatellite (RAMP) markers. Open Hort J. 2: 62-69.
Hussein TS, Tawfik AA, Khalifa MA. 2008. Molecular identification and genetic
relationships of six strawberry varieties using ISSR markers. Int J Agri Biol.
10: 677–80.
Ibrahim, R. 2000. Radiation Technology for the improvement of colour in ornamental plants. p.131-153. Proc. of Seminar on Methodology for Plant Mutation Breeding: Screening for Quality. Jakarta.
Jarukamjorn K, Nemoto N. 2008. Pharmacological Aspects of Andrographis
paniculata on Health and its Major Diterpenoid Constituent
Andrographolide. J Health Sci. 54(4): 370-381.
Jha Z, Sharam SN, Sharma DK. 2011. Differential expression of 3-hydroxy-3-
methylglutaryl-coenzyme A reductase of Andrographis paniculata in
andrographolide accumulation. J. Chem. Pharm. Res. 3(3):499-504.
Joshi K, Chavan P, Warude D, Patwardhan B. 2004. Molecular markers in herbal
drug technology. Curr Sci. 87(2): 159-165.
Khan SJ, MA Khan. 2010. Application of in-vitro mutation techniques for
sugarcane improvement. J. Agric. Res. 48 (4): 429-435.
Khodary SEA. 2004. Effect of NaCl salinity on improvement of nitrogen metabolismand some ions uptake in Lupine plants subjected to gamma
irradiation. Int J Agri Biol. 6(1): 1-4.
Kintoko, 2006. Prospek pengembangan tanaman obat. Prosiding Persidangan
Antarabangsa Pembangunan Aceh. 26-27 Disember 2006, UKM Bangi. 178-188.
Kiong ALP, Lai AG, Hussein S, Harun AR. 2008. Physiological responses of
Orthosiphon stamineus plantles to gamma irradiation.Am-Eur J Sust Agric.
2(2): 135-149.
Koobkokkruad T, Chochai A, Kirdamanee C, De-Eknamkul W. 2008. Effects of low-dose gamma irradiation on artemisinin content and amorpha-4,11-diene
Lamseejan S, Jompuk P, Wongpiyasatid A, Deedeepan S, Kwanthammachart P. 2000. Gamma-rays induced morphological changes in chrysanthemum (Chrysanthemum morifolium). Kasetsart (Nat Sci). 34: 417-422.
Lattoo SK, Dhar RS, Khan S, Bamotra S, Bhan MK, Dhar AK, Gupta KK. 2008. Comparative analysis of genetic diversity using molekuler and
morphometric markers in Andrographis paniculata (Burm.f) Nees. Genet
Resour Crop Evol. 55: 33-43.
Lattoo SK, Dharr RS, Khan S, Bamotra S, Bhan MK, Dhar AK, Gupta KK. 2009. Molecular and morphometric diversity ini relation to breeding system in
Andrographis paniculata (Burm. f.) Nees. In: Plant tissue culture and molecular marker. Ashwani Kumar, H. C. Arya (Eds). I. K. International Pvt Ltd. 445-518.
Lattoo SK, Khan S, Dhar AK, Choudhary DK, Gupta KK, Sharma PR. 2006.
Genetics and mechanism of induced male sterility in Andrographis
paniculata (Burm. f.) Nees and its significance. Curr Sci. 91(4): 515-519. Leaman D. 2001. Conservation, trade, sustainability and exploration of medicinal
plant species. In: Development of Plant-Based Medicines: Conservation,
Efficacy and Safety. Editor: P.K. Saxena. Kluwer Academic Publishers. 1-5. Lee S, Lee M, Song K. 2005. Effect of gamma irradiation on the physicochemical
properties of gluten films. Food Chem. 92:621-925.
Lu S, Wang Z, Niu Y, Chen Y, Chen H, Fan Z, Lin J, Yan K, Guo Z, Li H. 2009. Gamma-ray radiation induced dwarf mutants of turf-type bermudagrass.
Plant Breeding. 128: 205-209.
Maison T, Volkaert H, Boonprakob U, Paisooksantivatana Y. 2005. Genetic
diversity of Andrographis paniculata Wall. Ex Nees as revealed by
morphological characters and molecular markers. Kasetsart J (Nat Sci). 39:
388-399.
Mamatha A, Patil KS, Ashok P, Kushal, Soujanya, Gokul. 2010. Effect of gamma
irradiation on pharmacological activity of Andrographis paniculata. J
Pharm Res. 3(11): 2638-2639.
Mamatha A. 2011. Quantitative HPTLC Analysis of Andrographolide in
Andrographis paniculata obtained from different geographical sources
(India). Int J Pharm Pharm Sci. 3(2): 42-44.
Minn M, Khai AA, Lwin KM. 2008. Study on the fffect of gamma radiation on
rice Sin Thwe Latt (IR53936). GMSARN International Conference on
Sustainable Development: Issues and Prospects for the GMS. 12-14 Nov. 2008.
Mishra SK, Sangwan NS, Sangwan RS. 2007. Phcog rev.: plant review
Andrographis paniculata (Kalmegh): A review. Pharmacol Rev. 1(2): 283- 298.
Moghaddam SS, Jaafar H, Ibrahim R, Rahmat A, Aziz MA, Philip E. 2011. Effects of acute gamma irradiation on physiological traits and flavonoid
accumulation of Centella asiatica. Molecules. 16: 4994-5007.
Nassar AH, Hashim MF, Abo-zaid H. 2004. Effect of gamma irradiation and phosphorus on growth and oil production of Chamomile (Chamomilla
recutita L. Rauschert). Int J Agri Biol. 6(5): 776-780.
Ngan DH. 2005. Bioactivities and chemical constituents of Vietnamese medicinal
plant Jasminum subtriplinerve Blume (Che vang). Master Thesis. Roskilde
University Roskilde. Denmark. 1-70.
Novak FJ, Brunner H. 1992. Plant breeding: Induced mutation technology for
crop improvement. IAEA BULLETIN. 4: 25-33.
Otake T, Mori MHLT, Hattori M, Namba T. 1995. Screening of Indonesian plant extracts for anti-human immuno deficiency virus-type I (HIV-I) activity.
Phytother Res. 9: 6-10.
Pandey AK, Mandal AK. 2010. Variation in morphological characteristics and
andrographolide content in Andrographis paniculata (Burm.f.) Nees of
Central India. Iran J Energy & Environ. 1(2): 165-169.
Parasher R, Upadhyay A, Khan NA, Dwivedi SK. 2011. Biochemical estimation and quantitative determination of medicinally important andrographolide in
Andrographis paniculata at different growth stages. EJEAFChe. 10(7):
2479-2486.
Patarapanich, Laungcholatan S, Mahaverawat N, Chaichantipayuth C, Pummangura S. 2007. HPLC determination of active diterpene lactones
from Andrographis paniculata Nees planted in various seasons and regions
in Thailand. Thai J. Pharm. Sci. 31: 91-99.
Phianphak W, Rengpipat S, Cherdshewasart W. 2007. Gamma irradiation versus
microbial contamination of Thai medicinal herbs. Songklanakarin J Sci
Technol. 29(1): 157-166.
Raina AP, Kumar A, SK Pareek. 2007. HPTLC analysis of hepatoprotetive
diterpenoid andrographolide from Andrographis paniculata Nees
(Kalmegh). Indian J Pharm Sci. 69(3): 473-475.
Ramesh HL, Murthy VNY, Munirajappa. 2012. Effect of gamma radiation on
morphological and growth parameters of mulberry variety M5. Int J Sci Nat.
Sabu KK, Padmesh P, Seeni S. 2001. Intraspecific variation in active principle
content and isozymes of Andrographis paniculata Nees (Kalmegh): a
traditional hepatoprotective medicinal herb of India. J Med Aromatic Plant
Sci. 23: 637–647.
Sakuanrungsirikul S, Jetana A, Buddanoi P, Dithachaiyawong J. 2008.
Intraspesific variability assesment of Andrographis paniculata collections
using molecular markers. Proc. International Workshop on Medicinal and
Aromatic Plants. Eds.: N. Chomchalow and V. Chantrasmi. ISHS Acta Horticulturae 786: 283-286.
Selvi BS, V Ponnuswami, T Sumathi. 2007. Identification of DNA polymorphism
induced by gamma irradiation in amla (Emblica officinalis Gaertn.) grafts of
V1M1 and V2M1 generation. J Apll Sci Res. 3(12): 1933-1935.
Sema Alikamanoglu & Orkun Yaycili & Ayse Sen. 2011. Effect of Gamma Radiation on Growth Factors, Biochemical Parameters, and Accumulation of Trace Elements in Soybean Plants (Glycine max L. Merrill). Biol Trace Elem Res.141:283–293.
Sharma A, Krishan L, Handa SS. 1992. Standardization of the Indian crude drug Kalmegh by high pressure liquid chromatographic determination of
andrographolide. Phytochem. Anal. 3: 129-31.
Sharma SN, Sinha RK, Sharma DK, Jha Z. 2009. Assessment of intra-specific variability at morphological, molecular and biochemical level of
Andrographis paniculata (Kalmegh). Curr Sci. 96(3): 402-408.
Srivastava N, Akhila A. 2010. Biosynthesis of andrographolide in Andrographis
paniculata. Phytochem. 71: 1298–1304.
Su H, Wang L, Liu L, Chi X, Zhang Y. 2008. Use of inter-simple sequence repeat
markers to develop strain-specific SCAR markers for Flammulina velutipes.
J Appl Genet. 49(3). 233–235.
Subramanian R, Asmawi MZ, Sadikun A. 2012. A bitter plant with a sweet future? A comprehensive review of an oriental medicinal plant:
Andrographis paniculata. Phytochem Rev. 11:39-75.
Suganda AG. 2002. Standardisasi simplisia, ekstrak dan produk obat bahan alam.
Prosiding Simposium Standardisasi Jamu dan Fitofarmaka. ITB. Bandung, 26 September 2002.
Sunardi. 2008. Teknik pembibitan sambiloto untuk menghasilkan bibit yang
Suprasana P, Sidha M, Ganapathi TR. 2008. Characterization of radiation induced and tissue culture derived dwarft types in banana by using a SCAR marker.
Aust J Crop Sci. 1(2): 47-52.
Tah PR. 2008. Studies of leaflet mutants in Mungbean (Vigna radiate (L)
Wilczek). Int J Plant Breeding Gen. 2(2): 75-84.
Thohirah LA, Fakhrurazi S, Nazir BM. 2009. Changes in Flower Development,
Chlorophyll Mutation and Alteration in Plant Morphology of Curcuma
alismatifolia by Gamma Irradiation. Am J Appl Sci. 6 (7): 1434-1437. Timprasert S, Boonruad T, Nouuhpramool K, Setakanna P, Rattanasiri J,
Pannanususorn A, Kaewchoung P, Orankanok W. 2005. Effect of irradiation on chemical changes and contaminated microorganisms in turmeric and
creat powder. International Symposium “New frontier of irradiated food
and non food products. KMUTT Bangkok Thailand. 22-23 September 2005. Van Harten AM. 1998. Mutation breeding. Cambridge Univ Press. 1-353.
Vandenhove H, Vanhoudt N, Cuypers A, Van Hees M, Wannijn J, Horemans N.
2010. Life-cycle chronic gamma exposure of Arabidopsis thaliana induces
growth effects but no discernable effects on oxidative stress pathways. Plant
Phys Biochem. 48: 778-786.
Viccini LF, de Carvalho CR. 2002. Meiotic chromosomal variation resulting from
irradiation of pollen in maize. J Appl Genet. 43(4): 463-469.
Wahyuni, S, Xu DH, Bermawie N, Tsuretmasu H, Ban T. 2004. Skrinning ISSR primer studi pendahuluan kekerabatan antar jahe merah, jahe emprit dan
jahe besar. Buletin BALITTRO. 15(1): 33-42.
Wijarat P, Keeratinijakal V, Toojinda T, Vanavichi A, Tragoonrung S. 2011.
Genetic diversity and inbreeder specie of Andrographis paniculata (Burm.
f.) Nees by randomly amplified polymorphic deoxyribonucleic acid (RAPD)
and floral architecture analysis. J Plant Breeding Crop Sci. 3(12): 327-334.
Wijarat P, Keeratinijakal V, Toojinda T, Vanavichit A, Tragoonrung, S. 2012.
Genetic evaluation of Andrographis paniculata (Burm. f.) Nees revealed by
SSR, AFLP and RAPD markers. J Med Plants Res. 6(14): 2777-2788.
Xiang TH, Yang JB, Zhu QS, Li L, Ni DH, Wang XF, Hang DN. 2002. Molecular biological effect of (CO)-C-60 gamma-ray irradiation on rice genome DNA.
Progress in Biochem Biophys. 29:754–759.
Xiaorui Z.1999. Role of the WHO monographs on selected medicinal plants. Vol
2.World Health Organization. Geneva. 12-24.
Xiaorui Z. 2003. WHO guidelines on good agricultural and collection practices
Yang M, Wang J, Kong L. Quantitative analysis of four major diterpenoids in
Andrographis paniculata by H NMR and its application for quality control
of commercial preparations. 2012. J. Pharm. Biomed. Anal.
http://dx.doi.org/10.1016/j.jpba.2012.05.037. 7 pages.
Zeid IM, Gharib FA, El-Ghate HMA. 2001. Response of fennel (Foeniculum
vulgare) to gamma irradiation and gibberelic acid treatments. Pak J Biol Sci. 4(7): 805-808.
Zhang WJ, Björn LO. 2009. The effect of ultraviolet radiation on the
accumulation of medicinal compounds in plants. Fitoterapia.
doi:10.1016/j.fitote.2009.02.006. 12 pages.
Zhou Y, Gao W, Duan H, Wang F, Gu F. 2007. Assesment of Genetic Diversity of Rehmannia glutinosa Libosch Based on ISSR Markers. Analele Ştiinţifice
ale Universităţii. Alexandru Ioan Cuza. TOM VIII. 2007.
Zietkiewicz E, Rafalski A, Labuda D. 1994. Genomic fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification.
LAMPIRAN
Lampiran 1. Sequence primer ISSR yang digunakan
Tabel Sequence primer ISSR yang digunakan
No Primer Sequence primer Tm
1 SBLT 2 (AG)8T 52
2 SBLT 3 (AG)8C 50
3 SBLT 5 (GA)8C 53
4 SBLT 8 (CT)8G 52
6 SBLT 14 (GACA)4 50
7 SBLT 15 (GA)8 50
8 SBLT 17 (TG)8G 52
9 SBLT 18 (CCCT)4 51
10 SBLT 19 (GATA)2(GACA)2 51
Lampiran 2. Tahapan isolasi DNA genom daun tanaman sambiloto
Tahapan isolasi DNA genom tanaman sambiloto adalah: daun dibersihkan dari kotoran yang menempel dan ditimbang sebanyak 200 mg. Daun yang telah ditimbang dimasukkan dalam mortal dan dipotong kecil-kecil kemudian ditambahkan 1% PVPP sebanyak 100 µL, larutan 2% merkaptoetanol hangat sebanyak 100 µL dan bufer ekstraksi sebanyak 250 µ L. Daun tersebut ditumbuk
sampai halus menggunakan pestle sampai membentuk pasta. Kemudian pasta daun dimasukkan dalam tabung mikrotube 1,5 mL dan ditambahkan bufer
ekstraksi sebanyak 250 µL. Larutan DNA divortex dan diinkubasi selama 30
menit dalam waterbath suhu 60oC. Larutan ditambahkan larutan CIAA dengan
perbandingan 24:1 sebanyak 500 µ L. Tabung dibolak balik selama 15 menit dan kemudian disentrifugasi pada kecepatan 800 rpm selama 10 menit pada suhu
25oC. Supernatan yang didapatan dipindahkan pada tabung mikrotube baru dan
ditambahkan larutan 5M NaCl sebanyak 250 µL sambil dibolak balik perlahan- lahan. Supernatan ditambahkan larutan isopropanol dingin sebanyak 0,6 volume supernatan dan dibolak balik perlahan-lahan. Larutan tersebut kemudian diinkubasi pada suhu ruang selama 60 menit. Setelah itu larutan disentrifugasi
dengan kecepatan 10000 rpm selama 10 menit pada suhu 25oC. Supernatan
dibuang dan pelet yang didapatkan dicuci dengan menggunakan 80% etanol dingin sebanyak 200 µL dan dibolak balik perlahan. Supernatan dibuang dan pelet DNA dikeringkan dengan membalikkan tutup mikotube pada kertas tissu sampai tidak tercium bau etanol. Pelet ditambahkan larutan Tris-EDTA (TE) sebanyak 100 µ L kemudian dihomogenkan. Larutan DNA ditambahkan 1 µL RNAse
kemudian diinkubasi dalam waterbath suhu 37oC selama 30 menit. Larutan
ditambahkan larutan CIAA (24:1/v:v) dengan volume yang sama dan dibolak balik tabung perlahan-lahan. Larutan disentrifugasi 8000 rpm selama 10 menit
pada suhu 25oC. Supernatan dipindah pada tabung mikrotube baru dan
ditambahkan isopropanol dingin dengan volume yang sama kemudian diinkubasi
pada suhu -20oC selama semalam (overnight). Larutan disentrifugasi 10000 rpm
selama 10 menit pada suhu 25oC. Supernatan dibuang dan pelet DNA yang
didapat dikeringkan kemudian dilarutkan dalam larutan TE sebanyak 20 µ L. Lampiran 3. Analisa variasi (ANOVA) pada mutan tanaman sambiloto generasi
M1V0-M1V3
1. ANOVA mutan generasi M1V0
Analysis of Variance for tinggi, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
Dosis 18 2960.58 2960.58 164.48 2.09 0.007 Error 257 20253.46 20253.46 78.81
Analysis of Variance for daun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
Dosis 18 607.15 607.15 33.73 2.33 0.002 Error 257 3713.61 3713.61 14.45
Total 275 4320.77
Analysis of Variance for pdaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
Dosis 18 59.8806 59.8806 3.3267 4.39 0.000 Error 257 194.5512 194.5512 0.7570
Total 275 254.4318
Analysis of Variance for ldaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
Dosis 18 5.27920 5.27920 0.29329 3.39 0.000 Error 257 22.23708 22.23708 0.08653
Total 275 27.51628
Analysis of Variance for Nodus, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
Dosis 18 146.162 146.162 8.120 2.23 0.003 Error 257 937.356 937.356 3.647
Total 275 1083.518
2. ANOVA mutan generasi M1V1
Analysis of Variance for tinggi, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 151.195 151.195 8.400 1.53 0.082 Error 217 1191.936 1191.936 5.493
Total 235 1343.130
Source DF Seq SS Adj SS Adj MS F P dosis 18 12322.7 12322.7 684.6 3.04 0.000 Error 217 48882.0 48882.0 225.3
Total 235 61204.7
Analysis of Variance for pdaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 114.681 114.681 6.371 2.90 0.000 Error 217 477.134 477.134 2.199
Total 235 591.814
Analysis of Variance for ldaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 5.0152 5.0152 0.2786 2.08 0.007 Error 217 28.9986 28.9986 0.1336
Total 235 34.0137
Analysis of Variance for Nodus, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 4375.48 4375.48 243.08 3.83 0.000 Error 217 13761.51 13761.51 63.42
Total 235 18137.00
Analysis of Variance for cabang, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 279.194 279.194 15.511 2.32 0.002 Error 217 1449.700 1449.700 6.681
Total 235 1728.894
3. ANOVA mutan generasi M1V2
Analysis of Variance for tinggi, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 620.42 620.42 34.47 1.26 0.215 Error 204 5563.59 5563.59 27.27
Total 222 6184.01
Source DF Seq SS Adj SS Adj MS F P dosis 18 13701.2 13701.2 761.2 1.15 0.308 Error 204 135202.2 135202.2 662.8
Total 222 148903.4
Analysis of Variance for pdaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 93.861 93.861 5.214 1.72 0.038 Error 204 618.926 618.926 3.034
Total 222 712.787
Analysis of Variance for ldaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 6.6270 6.6270 0.3682 3.31 0.000 Error 204 22.6685 22.6685 0.1111
Total 222 29.2955
Analysis of Variance for Nodus, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 13271.6 13271.6 737.3 2.19 0.005 Error 204 68527.2 68527.2 335.9
Total 222 81798.8
Analysis of Variance for cabang, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 906.82 906.82 50.38 1.89 0.018 Error 204 5429.04 5429.04 26.61
Total 222 6335.86
4. ANOVA mutan generasi M1V3
Analysis of Variance for tinggi, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 1832.20 1832.20 101.79 3.40 0.000 Error 178 5328.04 5328.04 29.93
Total 196 7160.24
Source DF Seq SS Adj SS Adj MS F P dosis 18 6006.8 6006.8 333.7 1.25 0.228 Error 178 47579.7 47579.7 267.3
Total 196 53586.5
Analysis of Variance for pdaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 31.762 31.762 1.765 1.21 0.260 Error 178 260.326 260.326 1.463
Total 196 292.088
Analysis of Variance for ldaun, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 2.6430 2.6430 0.1468 0.54 0.938 Error 178 48.8179 48.8179 0.2743
Total 196 51.4610
Analysis of Variance for Nodus, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 2306.2 2306.2 128.1 0.35 0.993 Error 178 64446.6 64446.6 362.1
Total 196 66752.7
Analysis of Variance for jcabang, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P
dosis 18 289.59 289.59 16.09 0.37 0.991 Error 178 7678.48 7678.48 43.14
Total 196 7968.07