Post Autoclaved Auxin Application in Rooting Medium Preparation

Short Communication

Post Autoclaved Auxin Application in Rooting Medium Preparation by using Microwave oven as heat source to liquefy the Semisolid medium – medium use to induce root initiation in Eucalyptus Clone WK 013 Eh

Freddy Pangaribuan, SP - Plant Tissue Culture Research

Tree Improvement -  PT Wirakarya Sakti - Sinarmas Forestry

Jambi Region

Abstract

In horticulture, agriculture and forestry, vegetative propagation is used extensively to produce elite clones from breeding program and natural selection and rooting methods is one way to achieve this objectives.  NAA and IBA is the most common auxin being used to perform the rooting purpose in vitro.  Rooting in vitro use medium which is supplemented with auxin to the ½ MS basal medium, FeEDTA, micro nutrient and vitamin and sugar and sterilized using autoclaved.  The rooting medium  without auxin being autoclaved shows high rooting rate.  All treatment using from ½ MS NAA 0.6 ppm and IBA 0.6 ppm to 1/2MS NAA 2.4 and IBA 2.4 shows rooting rate from 82% to 92%.  This information gives a possible solution to difficult to root clones using the medium with auxin being autoclaved.

Introduction

             F.W. Went (1935) use auxin for the first time to develop his theory using Avena sp shoot tip to identify and quantify the biological activity of growth hormone-auxin.  Auxin was identified as  Indole-3-Acetic Acid (IAA) in 1934 and making plant tissue culture possible (Arditti, J., Yam, T.W.  2009)

            Practical application of auxin for root initiation becomes possible when the discovery of a fact that auxin also able to promote root initiation when added to the cut surfaces of cutting (De Klerk et al, 1999).  IBA is the most widely used commercially, followed by NAA and IAA, Rhizopon produces 80% IBA and the rest is the other auxin.   

            IAA continuous to be used in plant tissue culture inspite of evidence  suggesting that it is unstable.  The use of auxin, such as IBA, Naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy acetic acid, has increased because they often appear to be more effective than IAA for

Induction of morphogenic responses.  This enhanced effectiveness of auxin other than IAA is presumed to be due to their stability.  IBA is preferred for adventitious root induction in woody species   grown in vitro, since it is more effective (Nissan and Sutter, 1990).

            Clones Wk 013 Eh is a hybrid between E grandis x E pellita which has low rooting percentage (less than 50%) using medium B2RE (Makro B2RE + NAA 1 + IBA 0.5).  Medium B2RE are sterilized using autoclaved for 20 minutes and used on daily basis for rooting activities. 

            An approach have been conducted to improve the rooting percentage in rooted shoot of this eucalyptus clone using this short communication reported i.e. ‘post autoclaved hormone application’.  Hormone NAA and IBA are added to hormone free ½ MS medium in several concentration to see the best concentration of hormone that will bring the highest rooting percentage.

Materials and Methods

            Hormone free ½ MS medium were prepared using ½ MS Salt, micro MS, FeEDTA, Vit MS, Myo inositol, sugar 20 g/l and agar.  Medium are adjusted to pH 5.8 using NaOH 1 N.  One day later the semisolid medium were prepared for the  application of hormones.  NAA and IBA stock (1000 ppm) are being prepared by dissolving 0.1 g NAA or IBA using a few drops of NaOH I N with stirring gently  until dissolved completely and add deionized water to reach 100 ml volume.

            Treatment for hormone is being done using sterilized pipette (autoclavable pipette and fin tips) according to the treatment (Table 1.) by putting the hormone stock on the semisolid media surfaces.  The media were later heated up using microwave oven for 1’30’’ (1 minute 30 second) to liquefy the semisolid medium so the hormone can mixed through, and rest them to solidify again.  One day later the medium was used for rooting micro shoot.  Each vessel contain 10 micro shoot, average shoot selected is 2-3 cm.  All the rooted shoots were incubated in growth room with temperature 20-26oC and light intensities 2800 lux using white fluorescence lamp

            Assessment fro rooting rate was conducted 14 days later by calculating the number of shoot that produces shoot with root and count the percentage.  The data were processed using simple statistic (Sigma Zone).  The rooted shoot was later transplanted to the nursery.

Results

Table 2.  Rooting Rate (%) of Post autoclave auxins treatment to initiate rooting shoot of Wk 013 Eh 

No	Hormone Composition		Replication					Average (%)
	NAA	NAA	1**	2	3	4	5
A	0.6	0.6	100	100	100	80	80	92
B	1.2	1.2	80	90	80	90	80	84
C	1.8	1.8	90	80	90	80	90	86
D	2.4	2.4	80	80	90	90	80	84
E	0	0.6	90	90	90	80	90	88
F	0	1.2	50	100	80	80	80	78
G	0	2.4	100	100	70	70	80	84
H	2.4	0	80	60	50	60	50	60
I	0	3	80	80	80	70	60	74