IN-VITRO GERMINATION AND MICROPROPAGATION OF ALFALFA (Medicago sativa L.) AS CLOROPHIL SOURCES Perkecambahan In Vitro dan Mikropropagasi Alfalfa (Medicago sativa L.) Sebagai Sumber Klorofil

  

IN-VITRO GERMINATION AND MICROPROPAGATION OF ALFALFA

(

Medicago sativa L.) AS CLOROPHIL SOURCES

Perkecambahan In Vitro dan Mikropropagasi Alfalfa (Medicago sativa L.)

  

Sebagai Sumber Klorofil

Fitrahtunnisa*, Sajimin** and Selvia Dewi Anomsari***

• *Assessment Institute for Agricultural Technology (AIAT) West Nusa Tenggara

  Jln . Paninjauan Narmada – 83371

• **Indonesian Center for Animal Research and Development (ICARD)

• ***Assessment Institute for Agricultural Technology (AIAT) Central Java

  E-mail : fit_biotek@yahoo.co.id

  

ABSTRAK

Alfalfa (Medicago sativa L.) dikenal sebagai tanaman penghasil klorofil yang berkhasiat menyembuhkan berbagai

penyakit. Biji Alfalfa yang diproduksi di Indonesia tidak memiliki embrio dan tidak bisa berkecambah, sehingga

biji masih harus diimpor. Salah satu cara yang dapat digunakan untuk menghasilkan benih alfalfa dalam jumlah

besar dan relatif cepat adalah dengan menggunakan teknik mikropropagasi. Penelitian dilakukan untuk mengem-

bangkan teknik germinasi (perkecambahan) dan mikropropagasi alfalfa secara in-vitro. Perkecambahan dilakukan

dengan menggunakan dua teknik yang berbeda. Pertama, benih steril direndam dalam 50 mg/l GA3, dan kedua

tanpa direndam GA3. Benih dikecambahkan pada media MS yang mengandung 0 atau 0,5 mg/l BA dikombinasikan

dengan 0,1, 0,5 atau 1 mg/l GA3. Inisiasi dan mutiplikasi tunas dilakukan pada media MS yang mengandung 0, 0,5

atau 1 mg/l BA dikombinasikan dengan 0, 0,1 dan 0,5 mg/l Th menggunakan eksplan epikotil. Variabel yang diukur

adalah tinggi tanaman, jumlah daun dan jumlah tunas. Sedangkan inisiasi kalus dilakukan pada media MS yang

mengandung 0, 1 atau 3 mg/l 2.4D dan dikombinasikan dengan 0, 3 atau 5 mg/l Pic menggunakan eksplan hipoko-

til. Variabel yang diukur adalah panjang, lebar dan tebal kalus. Hasil penelitian menunjukkan bahwa rata-rata

perkecambahan biji yang direndam dengan GA3 pada media yang berbeda berkisar antara 60-87,5%, sedangkan

tanpa direndam GA3 berkisar antara 4-75%. Rata-rata perkecambahan benih paling tinggi diperoleh dari biji yang

direndam dengan GA3 pada media MS tanpa BA dan GA3 (kontrol). Media terbaik untuk induksi dan multiplikasi

pucuk eksplan epikotil adalah MS + 1 mg/l BA. Sedangkan media terbaik untuk induksi kalus adalah MS + 3 mg/l

Pic.

  Kata kunci: Medicago sativa L., perkecambahan, mikropropagasi.

  

ABSTRACT

Alfalfa (

  Medicago sativa L) known as chlorophyll producing plants are efficacious cure for various diseases. Alfalfa

seeds produced in Indonesia do not have embryos and could not germinate. So seeds are still to be imported. One

way that can be used to produce alfalfa seeds in large quantities and relatively fast is the micropropagation tech-

niques. An experiment was carried out to develop technique for in-vitro germination and micropropagation of al-

falfa. The in-vitro germination was conducted using two different techniques. Firstly, sterile seeds were soaked in

50 mg/l GA3, and secondly seeds were germinated without soak in GA3. Seeds were germinated on MS medium

containing 0 or 0.5 mg/l BA and combined with 0.1, 0.5 or 1 mg/l GA3. Shoot initiations and mutiplications were

done on MS medium containing 0, 0.5 or 1 mg/l BA combined with 0, 0.1 and 0.5 mg/l Th using epycotyl explants.

Variables measured were plant height, number of leaves and shoots number. While callus initiations were done

on 0, 1 or 3 mg/l 2.4D combined with 0, 3 or 5 mg/l Picloram using hypocotyl explants. Variables measured were

length, width and thick of callus.The results showed that the seed germination rate with GA soaked on different

media ranged from 60 to 87.5%, while without GA3 soaked ranged from 4 to 75%.

  

The seed germination rate with GA3 soaked on MS medium without BA and GA3 (control) was the highest. The

best medium for induction and shoot multiplication of epycotyl is MS+1 mg/l BA. While the best medium for callus induction was MS+3 mg/l Pic.

  ., 2005). According to Mariska and Sukm- adjaja (2003) propagation by in vitro culture technique is much faster than the conventional way. Additionally, this technology also ensures uniformity, disease-free and cheaper freight costs.

  Alfalfa seed were cultured in two tech- niques. First, the seeds were soaked in GA3 50mg/l; second, the seeds were not soaked in GA3. Then, seeds were germinated on MS medi- um containing 0 or 0.5 mg/l BA and combined with 0.1, 0.5 or 1 mg/l GA3. Observations were made of the percentage of seed germination. ^{Fitrahtunnisa*, Sajimin** and Selvia Dewi Anomsari***}

  In vitro germination

  Alfalfa seeds were sterilized using 70% alco- hol for 5 minutes, 0.02 ppm HgCl ₂ for 2 min, 30% clorox for 10 minutes and 20% clorox for 5 min, andsteriledistilled water and antiseptic solution as a rinse.

  The study was conducted at the Tissue Culture Laboratory of Research Group of Biol- ogy Cell and Tissue, Indonesian Agency for Ag- ricultural Research and Development, Ministry of Agriculture from February to October 2009.

  ., 2002; Liz and Levith, 1997). The purpose of this study is to obtain a method of in vitro ger- mination of alfalfa seed, and the formulation of appropriate media for shoot induction and multiplication.

  al

  The success of in vitro propagation of plants through shoot multiplication, organo- genesis, and somatic embryogenesis is strongly influenced by genotype and explant, basic me- dia types, as well as the type and concentration of used growth regulator hormones (Hutami et

  et al

  Key words: Medicago sativa L., germination, micropropagation.

  Each shoots produced can be used as a source for further multiplication, so obtain a lot of shoots in a relatively short time (Kosmiatin

  Propagation through in vitro culture can be done in three ways, namely the formation of adventitious shoots, lateral shoots prolifera- tion and somatic embryogenesis. Proliferation costs in order to obtain fast multiplication of shoots.

  are less developed in the tropics due to pests, diseases, and weeds (Ashigh et al ., 2009). To prevent and address pest problems, diseases and weeds, cultivation of alfalfa should be very concerned about pesticides being used. This is related to the quality and safety of alfalfa plants that will be consumed by humans as a drug or a source of chlorophyll, or as animal feed. If these issues are not addressed, it will reduce the quality of the protein of alfalfa to 9%.

  lifera, as apollinator. In addition, alfalfa plants

  . (2004), in order to produce alfalfa seeds, pollination of flowers need the help of insects such as the honey bee, Apis mel-

  Alfalfa propagation is by seed. In tropi- cal regions such as Indonesia, alfalfa plants do not produce seeds. According to Mercer (1943) and Hirnyck et al

  Alfalfa( Medicago sativa L.) is legume that is efficacious to treat cancer, diabetes, lu- pus, and hepatitis. Alfalfa known as a producer of chlorophyll is also used as a dietary supple- ment. Chlorophyll is an organic molecule in plants. Its structure such as hemoglobin at hu- mans, can increase the body resistance. Alfal- fa also contains carotenoids, acidic acids, fla- vonoids, phyto estrogen, and saponins. Other benefits of alfalfa are as feed of cattle and oil producer (Horne, 2010). Alfalfa has high pro- tein, energy, vitamins, minerals, and effective fiber. Its composition of amino acids is bal- anced (Gonzalez et al., 2001).

  INTRODUCTION

MATERIALS AND METHOD

  Shoot induction and multiplication

  The plant material used as explants in this study was epycotyl and hypocotyl. Epycot- yl and hypocotyl of normal seedling were cut along 1 cm and cultured on shoot regenera- tion medium. Epycotyl of normal seedling was cultured on MS medium containing 0 , 0.5 or 1 mg/l BA combined with 0, 0.1 or 0.5 mg/l Th. Variables measured were plant height, leaves and shoots numbers. While the initiation of callus was done on MS medium combined with 0, 1 or 3 mg/l 2.4D and 0, 3, or 5 mg/l Pic. The variables measured were length, width, and thick of callus.

  MS medium (Murashige and Skoog, 1962) used equipped with 3% sucrose (w/v), and made solid by adding 0.2% (Phytagel Gel- rite). pH of media was made 5.8 by adding 1 N NaOH or 1 N HCl before autoclaved at temper- ature of 121oC for 15 minutes. Cultures were incubated at 25 ± 2°C under fluorescent light of 1.000-2.000 lux for 16 hours.

  In vitro germination

  Seed germination by in vitro can be per- formed on the appropriate medium for germi- nation.

  This technique is often used for plants or seeds that is difficult to germinate at starting with a high risk of miscarriage of embryo.

  The first series of seeds germination were soaked with GA3, while the second series seeds was without soaked on GA3. Seeds then were germinated on MS medium added with 0 or 0.5 mg/l BA and combined with 0.1 , 0.5 or 1 mg/l GA3. The results showed that the addition of BA and GA3 on the medium in which both of seeds soaked or not soaked GA3 did not result better germination than controls (Table 1). Ko- smiatin et al

  . (2005) showed that the addition of GA3 in the medium only slightly increased the percentage of germination of agarwood. The addition of cytokinin with strong activity (BA) could also improve germination, although it was only 25%. Media without vitamins could improve germination and reduce the formation of abnormal sprouts (Kosmiatin et al., 2005). The media was also successful for germinating vanilla beans (Mariska et al

  ., 1998).

RESULT AND DISCUSSION

  

Table 1. Average of alfalfa seeds germination on MS with and without GA3 soaked

Treatment Percentage of alfalfa germination

  BA 0 GA BA 0 GA BA 0 GA BA 0,5 BA 0,5 BA 0,5 Control 0,1 0,5

  IN-VITRO GERMINATION AND MICROPROPAGATION OF ALFALFA (Medicago sativa L.) AS CLOROPHIL SOURCES Perkecambahan In Vitro dan Mikropropagasi Alfalfa (Medicago sativa L.) Sebagai Sumber Klorofil

1 GA 0,1 GA 0,5 GA 1

  8

  4

  20

  16

  75 soaked With GA3

  60

  60

  76

  72

  68 68 87,5 soaked

  Shoot induction and multiplication

  Multiplication of alfalfa using seeds in vitro propagation can use various explants. Explants used in this study was epycotyl and hypocotyl. Results of induction and multipli- cation of shoots using epycotyl explants were listed in Table 2.

  16

  Without GA3

  48 Fitrahtunnisa*, Sajimin** and Selvia Dewi Anomsari***

Table 2. Plant height, leaves and shoot number of induction and multiplication of shoots using epycotyl explants.

  Treatment Plant height (cm) Number of leaves Number of shoots BA0 Th 0,1 11,3

  5

  3 BA 0 Th 0,5 9,5 3,7 2,9 BA 0,5 Th 0 9,6 5,1 3,6 BA 0,5 Th 0,1 10,2 3,3 2,6 BA 0,5 Th 0,5 10,7 8,3 3,2 BA 1 Th 0 13,4 10,4 5,4 BA 1 Th 0,1 12,7 7,8 6,5 BA 1 Th 0,5 13 5,3 3,9

  The addition of BA and Th did not give a Table 3 shows that length, width and significant difference on plant height and num- thickness of callus were not significantly dif- ber of shoots produced. However, the number ferent. But after combined, size of the largest of leaves produced by the addition of BA 1 mg/l callus was obtained from the addition Pic3 resulted in plants with more leaves in the in- mg/l into MS medium(Figure 1). The greater duction and shoot multiplication. Kosmiatin et concentration of 2,4D + Pic into the media, the

  al

  . (2005) stated that the addition of BA 1 mg/l less callus was formed. In vitro Callus induc- resulted the highest number of shoots with tion in wheat showed that the concentration the fastest time of shoot induction. Research and type of hormones used greatly affected in Malaysia showed that MS medium plus BA the ability of explants to form callus or tissue gave the best shoot multiplication (Normah (Rashid et al., 2002;Satyavathi et al., 2004).

  et al

  ., 1995). However Goh et al. (1990), men- tioned that the best shoot multiplication was obtained from medium WPM + BA 5 mg/l. The results of the study in Solok showed that spray- ing the source of explants (shoot) with BA 0.5 ppm + 1 ppm GA3 before cultured, gave the highest number of growing explants (42%), whereas explants from cotyledons provided more leaves than shoot buds (Triatminingsih

  et al ., 2001).

  Shoot induction using hypocotyl did not give better results than using epycotyl ex- plants. Shoots induction using hypocotyl pro-

  Figure 1. Volume of callus using hypocotyl eksplant

  duced callus at first so that the shoots induc- tion process was longer. Shoot induction using hypocotyl explants were listed in Table 3.

  

Table 3. Induction of shoots by using hypocotyl explant.

Treatment

  Callus size Length (mm) Height (mm) Width (mm) 12,9 4,6

  6 2,4D0 Pic5

  

13,1 3,6 5,1

2,4D1 Pic3

  

13,1 4,2 5,2

2,4D1 Pic5

  

13,5 3,7 4,3

2,4D3 Pic0

  

11,7 2,7 3,6

2,4D3 Pic3

  

12,2 2,4 3,3

2,4D3 Pic5

  

11,8 2,2 3,3