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Influence of age of the embryo and method of hormone application on haploid embryo formation in wheat x maize crosses

Navneeta Kaushik, Mukta Sirohi and V.K. Khanna

Department of Genetics and Plant Breeding, G.B.Pant University of Agriculture and Technology-263145 (Uttaranchal) India. navneeta_kaushik@yahoo.co.in

Abstract

Wheat polyhaploids were regenerated from embryos resulting from crosses with maize variety “Navin”. The frequency of the recovery of embryos was low when an aqueous solution of 2,4-D was injected to the uppermost internodes of crossed spikes or when 2,4-D was applied via spray and dipping methods. Higher embryo recovery was obtained using spikelet culture method due to the availability of favorable cultural conditions. The best age of the hybrid embryos to be rescued for in vitro condition was 17-19 days after pollination, which produced healthy looking seedlings. The embryos also varied in shape, size and developmental stage.

Media summary

Spikelet culture is a useful alternative to increase the efficiency of wheat x maize haploid production system. Best time for embryo rescue is 17-19 days after pollination.

Key words

Triticum aestivum, Zea mays, chromosome elimination, auxin treatment, embryo stimulation, wide hybridization.

Introduction

Double haploid production by means of hybridization between hexaploid wheat (Triticum aestivum) and maize (Zea mays) is potentially of great value to wheat breeding programme because it may reduce the time required to achieve homozygosity in breeding lines. The production of embryos through wheat x maize crosses was first reported by Zenkteler and Nitzsche (1984). Laurie and Bennett (1986) cytologically examined embryos produced via. this system and found maize chromosomes to be preferentially eliminated during the first three cell divisions, leaving a haploid complement of wheat chromosomes. Maize is not sensitive to the action of dominant genes Kr1 and Kr2, located on the long arms of chromosome 5B and 5A, respectively. These two genes drastically reduce the frequency of fertilization in crosses between hexaploid wheat and Secale cereale. In wheat x maize crosses, the embryo soon aborts; however exogenous treatment with the synthetic auxin 2,4-D promotes seed and embryo development until the embryo can be excised and plated onto a synthetic medium for continued growth and plant regeneration.

Haploid production efficiency is also influenced by the proportion of haploid embryos which germinate and develop into plantlets. One factor which is likely to influence the germination success of haploid embryos is the time of embryo rescue. In most recent reports, haploid embryos have been rescued from 2 to 3 weeks after pollination. Therefore the present investigation was undertaken to study the effect of various methods of hormone application on haploid embryo production and to investigate the effect of timing of embryo rescue on embryo germination.

Material and Method

The experimental material consisted of four wheat F1’s ie.UP 2113 x UP 2338 (W1), PBW 396 x UP 2338 (W2), PBW 65 x PBW 343 (W3), PBW 175 x PBW 343 (W4) and a maize variety “Navin” with good pollen shedding ability (Table 1). One to two days prior to anthesis the wheat spikes were emasculated and covered with butter paper bags. Two days after emasculation the spikes were pollinated with freshly collected maize pollen with the help of a brush.

Experiment 1: Method of 2, 4-D application

1. 2,4-D spray method- Spikes were sprayed with 100 ppm 2,4-D, one two and three days after pollination.

2. 2, 4-D tiller injection method- One day after pollination, 1ml of 2,4-D@100 ppm was injected to the uppermost internodes of wheat spike. This was done for three consecutive days.

3. 2, 4-D dipping method- Pollinated spikes were dipped in aqueous solution of 2, 4-D for three consecutive days.

4. Spikelet culture method-Two days after pollination spikes were removed, surface sterilized and dried on filter paper. The rachis was cut into individual spikelets and placed upright on MS media containing 30mg/l sucrose and .2mg/l 2,4-D. The cultures were incubated for three weeks in continues light at 20 C with a 16h day length.

Experiment 2: Time of embryo rescue

In order to investigate the effect of time of embryo rescue on embryo germination, the immature embryos obtained from young wheat spikelets were collected 11-20 days after pollination and cultured on modified MS media (Murashige and Skoog,1962) .The cultures were maintained at 4 C in the dark. After 5-6 days, cultures were transferred to 25 C and 16/8 hours light/dark cycle.

Result and Discussion

Experiment 1

The role of 2,4-D in promoting seed set and embryo formation has been concluded as critical (Inagaki and Tahir, 1990, Laurie and Bennett,1988). Exogenous application with 2,4-D appears to enhance wheat haploid embryo viability resulting from wheat x maize crosses, although the mechanism is not clear.

Table 1: Number of florets pollinated, embryo recovered, and plantlets regenerated from wheat x maize crossing using three methods of hormone application

Crosses

W1xNavin

W2x Navin

W3 X Navin

W4 x Navin

Total

Spray

Florets Pollinated

60

60

60

60

240

Embryos recovered

2

1

3

2

8

Plants regenerated

1

-

1

-

3

Dipping

Florets Pollinated

60

60

60

60

240

Embryos recovered

2

1

3

3

9

Plants regenerated

1

1

2

1

5

Tiller Injection

Florets Pollinated

60

60

60

60

240

Embryos recovered

4

3

5

2

14

Plants regenerated

2

1

2

1

6

Spikelet Culture

Florets Pollinated

60

60

60

60

240

Embryos recovered

9

6

8

4

27

Plants regenerated

5

3

4

2

14

In the present study, low embryo recovery was observed when 2,4-D was applied using spray, dipping and tiller injection method, whereas high embryo recovery were obtained when 2,4-D was applied using spikelet culture method (Table 1). The extreme environmental conditions of the field might have negatively affected cross fertilization and seed development in spikes which were sprayed, dipped and tiller injected with 2, 4-D. Spikelet culture method was estimated to be more efficient for the recovery of embryos and haploid plants since the culture conditions were quite different from those prevalent in the field, so spikelet culture method seems to be superior and more versatile procedure than the rest of the methods.

Experiment 2

The success of embryo rescue in wide crosses is influenced by the age of the embryo at the time of its culture. Older, healthy embryos are easier to culture than young ones. The culture of young embryos is also more difficult due to their small size, damage due to desiccation and sensitivity to osmotic shock. However, such embryos can be rescued by the endosperm nursing technique (Williams, 1978), in which a milky to soft dough stage endosperm, extracted from a normal caryopsis was used as supplemental nourishment in conjugation with the MS medium. Milky endosperm was placed on the medium and the embryo was placed on the endosperm. Thus, the embryo grew, receiving nourishment from the endosperm and perhaps also the medium.

In the present study, the optimum age for the embryo was 17-19 days at which the embryos could grow and produce healthy seedlings when cultured on the media. Seeds dissected 11-13 days after pollination were watery and embryo detection was not possible. 14-16 days embryos were very small, showed swelling on media but no further growth. Embryos cultured after 19 days could not give better results as the abortion process had already set in by then. Similar reports have been proposed by several workers in wheat x maize crosses. Suenaga (1994) proposed that the time of pollination and embryo age were critical for efficiency of doubled haploid production. Chen et al. (1996) cultured 10 to14 days old embryos and found that most of the embryos larger than 400m germinated and produced normal plantlets but smaller ones failed to germinate.

Thus, from this investigation it can be concluded that the right age for culturing the embryos varies from genotype to genotype and need to be standardized in each case separately. In the present case, the optimum time of rescue was 17-19 days after pollination.

Conclusion

The cross incompatibility barrier in wheat has been successfully overcome by using maize pollen. The wheat x maize technique is currently being used as an alternative to the bulbosum technique and anther culture for wheat haploid production. In haploid production through wheat x maize crossing, application of 24-D is crucial and without it there may be no seed set and embryo formation. Of the various methods tested, the use of spikelet culture offers a practical and versatile alternative for wheat polyhaploid production using wheat x maize sexual crossing.

References

Chen XM, Lai GX, Chen Xiao, Zhou JF, Sun FH, Liu JX (1996) Differences in haploid production in crosses between different wheat F1’s and maize. Acta Agronomica Sinica 22 (4):437-441

Inagaki M, Tahir M (1990) Comparison of haploid production frequencies in wheat varieties crossed with Hordeum bulbosum L. and maize. Jap. J. Breed. 40: 165-174

Laurie DA, Bennett MD (1986) Wheat x Maize hybridization. Can J Genet Cytol 28:313-316

Laurie DA, Bennett MD (1988) The production of haploid wheat plants from wheat maize crosses. Theor. Appl. Gent. 76(3): 393-397

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco. Physiol. Plant 15: 473-497

Suenaga K (1994) Doubled haploid system using the inter-generic crosses between wheat (Triticum aestivum L.) and maize (Zea mays L.). Bull. Natt. Inst. Agrobiol. Resour. 9:83-139

Williams E (1978) A hybrid between Trifolium repens and T.ambiguum obtained with the aid of embryo culture. N. Z. J. bot. 16:499-506

Zenkteler M, Nitzsche W (1984) Wide hybridization experiments in cereals. Theor Appl Genet 68:311-315

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