Rajendra Agricultural University

Embryoid formation from cultured anthers of two inbreds and their hybrid
--Arti Kumari, Harsh Kumar, S. K. T. Nasar and M. Kumar

Low frequency of response has limited the use of anther culture technique for the production of haploid plants. With a view to improving results and gaining insight into genetical factors for tissue culture response, anther culture was done on two inbreds, PKMS and MSIDR, and their hybrid PKMS x MSIDR. Young tassels from selected healthy plants were cut from their bases, surface sterilized with 10% calcium hypochlorite solution, rinsed thrice with sterile distilled water and dissected. Anthers were placed on MS and N6 media supplemented with casein hydrolysate (500 mg/l), kinetin (1 mg/l) and 2,4-dichlorphenoxy acetic acid (2 mg/l). Sucrose was added at the level of 12% and 3% for N6 and MS media respectively. Active charcoal at the level of 0.5% was also added to the two media. An anther from each spikelet was kept and analysed for the pollen developmental stage.

On MS medium more anthers showed swelling but on N6 medium more anthers formed embryoids. A comparison of the anther size, pollen size and pollen developmental stage to the frequency of embryoid formation from the cultured anther was made. Results have shown that, contrary to earlier reports of middle uninucleate pollen stage being the most responsive, the pollen division (mitosis) stage and early binucleate stage showed better response (Table 1). Among the two inbreds and their hybrid, MSIDR showed better response than PKMS and the hybrid PKMS x MSIDR showed intermediate response for the frequency of embryoid formation from the cultured anthers (Table 2).

Table 1. Effect of pollen developmental stage on the frequency of embryoid formation from cultured anthers of MSIDR on N6 medium.
Sl no. Anther size (length-breadth), mm Pollen size (dia.), mm Pollen development stage Frequency of embryoid formation, %
1 1.71 - 0.28 0.049 Tetrad 0.86
2 1.99 - 0.32 0.062 Early uninucleate 1.37
3 2.28 - 0.37 0.068 Late uninucleate 3.87
4 2.57 - 0.42 0.074 Pollen mitosis 4.53
5 2.78 - 0.57 0.087 Early binucleate 4.35
6 2.99 - 0.71 0.101 Late binucleate 0.54

Table 2. Effect of genotype on frequency of embryoid formation from cultured anthers on N6 medium (approximate size 2.5x0.5 mm).
Sl no.  Genotype Frequency of embryoid formation (%)
1 MSIDR 4.31
2 PKMS 3.06

Cytological analysis of cultured anthers has shown that equal division of uninucleate pollen, development of the vegetative cell, and development of the generative cell were the three pathways for pollen embryoid formation. 

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