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In Vitro Micropropagation of Strawberry (fragaria Ananassa)

S.M.L. RAHMAN
Citrus Research Station, Bangladesh Agricultural Research Institute, Jaintapur, Sylhet, Bangladesh

M.M. HOSSAIN
Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

M.M. RAHMAN
Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

M.A.K. MIAN
Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

T. HOSSAIN
Department of Crop Botany, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

Abstract/ Executive Summary:

The experiment was conducted at the Tissue Culture Laboratory, Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur during the period from February 2008 to December 2009 to investigate the effect of different concentrations of BAP on virus free plant regeneration, shoot multiplication and different concentrations of IBA on in vitro root formation of strawberry accession FA002. Number of shoots/explant was the highest of 29.0 for runner tip with 10.0 mg/l BAP and the controls were the least (3.0 for runner tip and 2.6 for shoot tip. The maximum leaves/culture (10.2) was produced for 10.0 mg/l BAP with runner tip while the minimum (3.6) for 0.0 mg/l BAP with shoot tip. The highest length (5.0 cm) was produced for 1.0 mg/l BAP with runner tip which was statistically identical to 0.5, 1.5, 2.0 and 2.5 mg/l BAP with runner tip (4.2 cm, 4.4 cm, 4.6 cm and 4.2 cm respectively) and also statistically identical to 1.0, 1.5 and 2.0 mg/l BAP with shoot tip (4.2, 4.04 and 4.24 cm, respectively). For root initiation half strength MS medium supplemented with different levels of IBA (0, 0.5, 1.0, 1.5 and 2.0 mg/l) were used. Root numbers varied with different concentrations of IBA and type of explants. Number of roots/explant was found maximum (19.0) at 1mg/l IBA with shoot tip which was statistically identical to 1.0 mg/l with runner tip explants.

Keywords: in vitro regeneration, Pineapple, Strawberry and IBA

Location: Tissue Culture Laboratory, Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur

Start Date: 00-02-2008

End Date: 00-12-2009

Program Area: Variety and Species

Commodity: Strawberry

Objectives:

1. To study the effect of BAP growth regulator in MS media on in vitro shoot proliferation of strawberry accession FA002

2. To determine the effect of IBA growth regulator in half strength MS media for in vitro root development of strawberry; and

3. To develop protocol for in vitro rapid propagation of strawberry.

Methodology:

The present study was carried out in the Tissue Culture Laboratory of the Department of Horticulture, Bangabandhu Sheikh Mujibur Brahman Agricultural University, Gazipur during the period from February, 2008 to December, 2009. The planting materials of strawberry accession FA002 was selected from a Ph. D. study experiment titled "Growth and yield performance of six strawberry genotypes in Bangladesh" (Rahman 2007). Runner tips and shoot tips were collected from the field experiment stated above and was brought to the preparation room. The two types of tips were washed throroughly under running tap water for about 30 minutes. The outer tissues of the tips were removed with the help of a sharp knife until the tips measured about 2-2.5 cm in length. Both the tips used for establishment of culture were prepared through dissection under the microscope. Then the initial explants were prepared under stereomicroscope by removal of outer tissues with the help of sterile scalpel, which was about 1-1.5 cm in length. Two treatments were conducted to assess the effect of different concentrations of BAP and IBA on shoot proliferation and subsequent rooting of the multiplied shoot. In the first experiment, runner tip and shoot tip of the strawberry accession FA002 were used as sources of ex plants to investigate the effect of BAP at different concentrations on shoot proliferation. Ten levels of BAP (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, and 10.0 mg/l) were used as treatments. In the second experiment, there were 5 levels of IBA (0.0, 0.5, 1.0, 1.5, and 2.0 mg/l) were used as treatments. The experiments were arranged in factorial design with 4 replications. Each treatment consisted of 10 culture tubes per replication. Data were recorded on the effect of different treatments on shoot proliferation and rooting. Murashige and Skoog medium supplemented with different phytohormones as per treatments were used as culture medium for shoot induction, shoot multiplication and maintenance and regeneration of roots from multiplied shoots. Hormones were added separately to different media according to the requirements. For the preparation of media, stock solutions were prepared at the beginning and stored at 9±1°C temperature. The respective medium was prepared from the stock solutions. The culture tubes with media were then autoclaved at 1.06 kg/cm2 pressure at 121°C for 25 minutes. The medium was then cooled at room temperature before use.

Both the two ex plants (1-1.5 cm length) were taken separately in two beakers. After surface sterilization under laminar Airflow Cabinet with 70% ethyl alcohol, the explants were again surface sterilized with 0.1% mercuric chloride and a few drops of Tween 20 for 15 minutes. Finally, the explants were then rinsed three to four times with sterile distilled water.
The isolated and surfaced sterilized explants collected carefully under the stereomicroscope through maintaining aseptic condition inside the laminar air flow cabinet to use those as ex plants. The individual explants were directly inoculated to each of the culture tube containing 20 ml of MS medium supplemented with different concentrations of hormones as per treatment covered with aluminium foil.

The culture tubes were transferred to growth room and allowed to grow in controlled environment. The temperature of the growth room was maintained with 25±10°C by an air conditioner. A 16 -hour light period was maintained with light intensity of 2000 lux for the growth and development of culture.
Some explants become black in colour within 5-6 days after inoculation. To control blackening after one week, the blackish tissues on the ex plants were removed and the meristematic tissues were transferred to fresh medium. It was repeated 10 days interval for about one month to minimize further blackening of the tissues.
Initial subculturing was done when the ex plants produced some shoots. For sub-culturing, the entire samples of in vitro shoot were cut into small pieces so that each piece would contain about one shoot. Each piece was inoculated into a similar fresh medium. It was practiced at the interval of every one month.
When the shoots grew about 3-5 cm in length with 3-6 well developed leaves, they were rescued aseptically from the culture tubes and were separated from each other and again cultured on freshly prepared medium containing different combinations of hormonal supplements for root induction.
Potting mixture contain grerden soil and cowdung in the ratio of 1:1 was mixed thoroughly and were placed into a 10 x 15 cm polyethylene bag for growing in vitro grown plantlets under ex vitro condition.

Source of Information: Int. J. Expt. Agric. 4(1):11-16, (2014)

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

For in-vitro shoot multiplication strawberry, runner tip explants was found better compared to shoot tip with 1 mg/l BAP whereas for root development, earlier roots could be found from both the explants with 0.5 mg/l IBA. For maximum roots/culture, 1.00 mg/l IBA with both runner tip and shoot tip was found better. Furthermore, the results could be used to produce large scale production of healthy and disease free planting materials commercially.

Knowledge Management: Journal