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Production of Haploids and Polyembryos in Wheat (triticum Aestivum L.) Through Wheat × Maize System

U. Asad
Oilseed Research Centre, Regional Agricultural Research Station (RARS), Ishurdi, Pabna, Bangladesh

A. K. M. Shamsuddin
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

S. Alam
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M. M. Rahman
Pulses Research Centre, Regional Agricultural Research Station (RARS), Ishurdi, Pabna, Bangladesh

Abstract/ Executive Summary:

Six varieties of spring wheat such as Mayoor, Sebia, Prodip, Bulbul, Bijoy, Sowgat were crossed with a single maize variety Pop corn as pollinator. Fertilization of wheat eggs, formation, retention and development of haploid embryos were facilitated by the application of 2, 4-D at 100ppm.The haploid embryos were cultured on MS medium, which regenerated haploid plants. Highly significant variations were observed for the characters related to haploid production such as development of caryopsis, recovery of embryos and regeneration of haploid plants over total pollinated florets. The highest caryopsis setting over pollinated florets was noticed in the variety Sebia. Recovery of embryos over caryopsis was the highest in the variety Bijoy. Germination of embryos over total embryos and regeneration of haploid plants over total pollinated florets were highest in the variety Mayoor. At the time of embryo rescue, occurrence of more than one embryo in a single caryopsis were noticed which were considered as polyembryos. Generation of multishoot from attached polyembryos (appeared as single embryo) was also considered as polyembryos. Highly significant variations were observed among the six varieties of wheat for formation of polyembryos over caryopsis, germination of polyembryos over total embryos and formation of polyembryos accounted to multishoots over total embryos. The highest number of polyembryos formation and germination of polyembryos were observed in the variety Mayoor. The varieties Sebia and Mayoor gave the highest polyembryos formation accounted to multishoots which were more than 8.00%.

Keywords: Wheat (Triticum aestivum L.), Haploids, Polyembryos

Location: Green house and tissue culture laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

Start Date: 00-10-2005

End Date: 00-06-2006

Program Area: Variety and Species

Commodity: Wheat

Objectives:

The present research program was undertaken to develop a protocol for the production of haploids and to assess the genotypic variation of wheat varieties for the production of haploids and polyembryos through wheat × maize system.

Methodology:

The experiment was carried out in the green house and tissue culture laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, during the period from October, 2005 to June, 2006. Six varieties of spring wheat namely Mayoor, Sebia, Bulbul, Bijoy, Prodip and Sowgat were used as female parents and one maize variety, Pop corn was used as male parent in wheat × maize cross. These varieties were sown in the earthen pots in different dates to synchronize flowering of wheat varieties with that of maize. At booting stage both wheat and maize plants were transferred in the green house where the temperature was maintained at 25⁰C/18⁰C day/night with 16h/8h day/ night photoperiod. Pollination was done consecutively two days for better setting of caryopsis. Post pollination treatment was done by application of 2, 4-D at 100 ppm concentration. The hormone 2, 4-D was taken in a hypodermal syringe and an amount of 0.5 ml was injected into the uppermost internode of the tiller bearing crossed spike on the second day of pollination. On the same day a drop of the hormone was poured on the ovary of each pollinated floret. On the next day one drop of 2, 4-D was again poured on the pollinated florets. After 16-18 days of pollination the spikes with developed caryopsis were harvested. These caryopses were surface sterilized by treating with 70% ethyl alcohol for five minutes. The treatment with alcohol was followed by treatment with commercial bleach (chlorox containing 4.7% chlorine) supplemented with a few drops of Tween-20 for 15 minutes. The caryopses were then washed 4 to 5 times with doubled distilled water for five minutes. To extract embryos, the caryopsis were placed under the focal view of stereomicroscope and dissected by using fine pointed forceps. When the embryos were found floating in the fluid of dissected caryopsis, they were picked up and put on the solid MS medium contained in the culture petridish. All of the operations were done in sterile condition under a laminar airflow cabinet. For the germination of embryos, the vials were kept in the dark room at 25⁰C temperature for 3 days. After germination of embryos, when the coleoptiles were about 1-2 cm long, they were transferred in the culture room at 25⁰C with 16 h photoperiod provided by using fluorescent lamps. After 15-20 days of germination of embryos, the regenerated plants developed roots and shoots and some of them showed multishooted plants from a single embryo. The regenerated plants were transferred in cubicles containing sterile soil with sufficient organic matter. The cubicles were covered by transparent polythene bags and kept in growth chamber for hardening. In the growth chamber temperature was maintained at 16-18⁰C with 4500 Lux light intensity. After 8-9 days of keeping the regenerated plants in growth chamber, bags were removed and plants were transferred in green house. When the plants attained 10-12 cm in height, they were transferred in larger earthen pot with normal soil in the green house. In the green house, temperature was maintained at 25⁰C/18⁰C days/ night with 16h/8h photoperiod. Data were taken on a number of florets crossed, caryopsis developed, and number of embryos obtained (number of single embryos and polyembryos), number of embryos germinated (single shoot regenerating embryos and multi shoots regenerating embryos) and number of plants regenerated were recorded in percentage. These data were transformed by arcsine transformation and analyses of variance for different parameters were performed following the principles of randomized complete block design (RCBD). The difference between the treatments means were compared by the least significant difference (Lsd) test. The regenerated plants were confirmed to be haploids by counting their somatic chromosome number and also by checking their pollen sterility.

Source of Information: Bangladesh J. Genet Pl. Breed: 20(2): 43-50, 2007

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Production of polyembryo in the wheat varieties through crossing with maize gives an additional benefit to obtain higher number of haploid plants. In the present study, the variety Mayoor performed better for polyembryo over caryopsis and germination of polyembryo.This variety also ranked first in the regeneration of haploid plants over the total pollinated florets. This result suggests that the variety Mayoor could be used in crossing program for haploid breeding in wheat. In addition the variety Sebia also performed better for production of haploids in wheat. So, the varieties Mayoor and Sebia may be selected as parents for haploid breeding program in wheat. The variety Mayoor appeared as the best among the six varieties of spring wheat for the formation of polyembryony. Higher number of polyembryos in the variety Mayoor resulted increased number regenerated haploid plants in this variety and it is followed by Sebia and Bulbul. Therefore, polyembryos obtained here considered as an added advantage for regeneration of higher haploid plants through wheat × maize crosses.

Knowledge Management: Journal