III. MATERIALS AND METHODS
3.1 Time and Place
The research was conducted from November 12
th
, 2013 to May 12
th
, 2014 at Center of Agricultural Biotechnology CAB, Kasetsart University, Kamphaeng
Saen campus No 1, Village 6, Malai Maen road, Kamphaeng Saen Sub-district, Kamphaeng Saen District, Nakhon Pathom 73140, Thailand.
3.2 Preparation
3.2.1 Equipment Preparation
Basic instrument alike laminair air flow cabinet was turned on and sterilized with UV radiation prior using. Culture bottles, petri dish, tweezers, and
scalpel were washed and then sterilized by autoclaving at 121 C for 20 minutes.
3.2.2 Media Preparation
There were two culture media used namely callus induction media and shoot regeneration media. Callus induction media
was made from well-mixing of
MS Murashige and Skoog, 1962 basal medium, 20 gl sucrose, 10 coconut water, and 3 mgl 2,4-D. Shoot r
egeneration media was made from well-mixing of
basic MS medium supplemented with 20 gl sucrose and 10 coconut water. Both media were solidified with 7 gl agar and pH adjusment of 5.7 prior to autoclave.
The aliquot of 20 ml both medium was poured out into culture bottle. 3.2.3
Plant Material Preparation Plant material in form of certain Thailand sugarcane Saccharum
officinarum L. cultivars were obtained from Sugarcane Research Center,
Kasetsart University at Kamphaeng Saen Campus, Thailand. Four high yielding local cultivars were chosen as plant materials namely LK 95-127, LK 92-17, K
93-219, and K 93-347. Sugarcane was 7 months-old at the time of used. Top shoot
of canes were collected by using chopper knife. Top shoot of cane, composed of 2-4 alternating nodes and internodes, was further processed manually by using
15
chopper knife to produce smaller plant material in form of young leaf rolls with the lenght of 12 - 15 cm and 1 cm in diameter.
3.2.4 Explant Preparation
The surface sterilization was conducted in all plant material. Plant material were washed and were immersed in two concentration of 25 and 10
commercial bleach solution Haiter ™ ai: sodium hypoclorite, respectively. Each
step took 20 minutes with continuously placed on 120 rpm shaker. Then, explants were subjected to three successive washings with sterile RO water. All washing
protocol was done inside culture bootle and under laminary air flow cabinet. After sterilization, explant was transfered on petri dish and outer three whorls of
explants was removed by using tweezers. The inner spindle was cut by using scalpel into approximately 20 mm pieces as explant.
3.3 Callus Induction and Plant Regeneration Ability of Various Callus Ages