Relationship between cytological and cultural indices of corn anther
culture ability
--Satarova, TN; Chernousova, NM
It is known that success in maize anther culture strongly depends on genotype with many genotypes being unresponsive. The early and rapid diagnosis of anther culture ability could help to find responsive genotypes among a great number. As the transition of a microspore to the sporophytic pathway of development is necessary for embryoid and callus formation, cytological determination of the amount of microspores having divided in an unusual manner, with embryoid and/or callus initiation, could characterize the androgenic ability of a genotype. Such characteristics, however, may be true only in case of strong correlation between the percentage of induced pollen grains in the initial period of cultivation (%IPG) on the one hand, and the percentage of responding anthers (%Resp.A), and the number of embryoids produced per 100 anthers plated (E/100A), on the other.
In this investigation the correlation between %Resp.A, E/100A, and %IPG was determined. Anthers for culture in vitro were taken from tassels of field donor plants in summer 1995 and after the cold pretreatment during 14 days at 8 C were planted on the medium UP (Genovesi and Collins, Crop Sci. 22:1137-1144, 1982). The first experiment was carried out for the responsive hybrid Wf9xLH148. Its anthers were plated at the stages of the uninucleate vacuolated pollen grain and the young binucleate pollen grain, before the generative cell migration. The second experiment included 9 genotypes, whose anthers were plated only at the young binucleate stage. %IPG was determined on the 8th and 15th days of culture by counting the number of promorphogenic pollen grains and different structures which could be the result of the induction of the sporophytic pathway. The following structures were found in our material: bicellular pollen grains with abnormal position of cells (equal division in microspore), pollen grains in which one or several mitoses in the generative and/or vegetative cell occurred, multicellular pollen grains, coenocytic structures, young embryoids, young calli. The count of the number of the induced pollen grains was made for 6 anthers per tassel for Wf9xLH148. 1000 pollen grains for each anther were examined, then the average of six determinations was found. For the experiment with 9 genotypes, 6 anthers from 2-3 tassels for each genotype, 1000 pollen grains for each anther, were examined, then the average was found. %Resp.A and E/100A were determined on the 42nd day of culture.
The results of the first experiment for Wf9xLH148 are shown in Table 1. Correlation coefficients between %Resp.A and %IPG and between E/100A and %IPG (the 15th day of culture) for the uninucleate stage and for the young binucleate stage (the 8th day) were not significant.
For the young binucleate stage (the 15th day of culture) coefficients of correlation between the variables mentioned above were significant and high (0.824; 0.822), so the strong correlation is confirmed. Perhaps, it is very difficult to distinguish visually promorphogenic pollen grains on the 8th day of culture, some of them may not have started their development yet. For the stage of uninucleate vacuolated pollen grains, when the process of induction is unstable as a whole and is being realized with lower efficiency (Satarova, Izv. Akad. Nauk Ser. Biol. 5:771-778, 1994), visual estimate of induced pollen grains may be also difficult.
Taking into account all the reasons above it is necessary for future estimation of the androgenic ability of a genotype to determine the percentage of induced pollen grains on the 15th day of cultivation with the anthers having been plated at the stage of the young binucleate pollen grains.
Table 1. Correlation between the percentage of responding anthers (%Resp.A),
the number of embryoids produced per 100 anthers plated (E/100A) and the
percentage of induced pollen grains (% IPG) for the corn cross Wf9xLH148.
| Plant number | % Resp.A | E/100A | %IPG | Coefficients of correlation |
| The stage of the uninucleate vacuolated pollen grains,15th day |
| 1 | 1.43 | 1.91 | 0.20 | |
| 2 | 1.00 | 1.00 | 0.59 | |
| 3 | 0 | 0 | 0.13 | |
| 4 | 0 | 0 | 0.02 | r 12 = 0.988* |
| 5 | 0.40 | 0.40 | 0 | r 13 = 0.580 |
| 6 | 0 | 0 | 0.18 | r 23 = 0.483 |
| 7 | 0 | 0 | 0 | |
| Average | 0.40 | 0.47 | 0.16 | |
| The stage of the young binucleate pollen grain, 8th day |
| 1 | 0 | 0 | 0.1 | |
| 2 | 7.39 | 9.85 | 0.15 | |
| 3 | 1.43 | 1.43 | 2.61 | r 12 = 0.985* |
| 4 | 7.23 | 12.05 | 0.70 | r 13 = - 0.014 |
| 5 | 1.23 | 1.23 | 0.03 | r 23 = - 0.047 |
| 6 | 5.48 | 7.31 | 2.03 | |
| Average | 3.79 | 5.31 | 0.94 | |
| The stage of the young binucleate pollen grain, 15th day |
| 1 | 7.23 | 12.05 | 4.38 | |
| 2 | 7.39 | 9.85 | 4.01 | |
| 3 | 1.54 | 1.54 | 2.92 | |
| 4 | 0.76 | 0.76 | 1.00 | r 12 = 0.990* |
| 5 | 3.33 | 3.33 | 1.20 | r 13 = 0.824* |
| 6 | 0.59 | 0.59 | 1.54 | r 23 = 0.822* |
| 7 | 1.23 | 1.23 | 2.78 | |
| 8 | 10.24 | 15.61 | 4.04 | |
| 9 | 0 | 0 | 0.02 | |
| 10 | 5.48 | 7.31 | 2.63 | |
| 11 | 0 | 0 | 0.17 | |
| Average | 3.44 | 4.75 | 2.25 |
*The coefficient of correlation is significant, P<0.05
In the second experiment we determined correlation between %Resp.A and %IPG and E/100A and %IPG for different corn genotypes (Table 2). Correlation coefficients were significant and high (0.827; 0.776) and this also proves the strong correlation between cytological (%IPG) and cultural (%Resp.A, E/100A) indices of androgenic ability. Genotypes which had %IPG being more than zero, were responsive in anther culture.
Table 2. Correlation between the percentage of responding anthers (%Resp.A),
the number of embryoids produced per 100 anthers plated (E/100A) and the
percentage of induced pollen grains (%IGP) for different corn genotypes
(the stage of the young binucleate pollen grains).
| Genotype | %Resp.A | E/100A | %IPG | Coefficient of correlation |
| 15th day | ||||
| Wf9xLH148 | 2.46 | 3.39 | 2.35 | |
| And 44 | 12.14 | 29.57 | 2.71 | |
| H99xWf9 | 1.32 | 1.70 | 0.21 | |
| Wf9xH99 | 3.78 | 6.33 | 1.03 | |
| 3633xAnd44 | 0 | 0 | 0 | r 12 = 0.992* |
| LH59xAnd44 | 0 | 0 | 0 | r 13 = 0.827* |
| M377 | 0 | 0 | 0 | r 23 = 0.776* |
| 502xH99 | 0 | 0 | 0 | |
| And44xLH148 | 2.78 | 6.85 | 0.71 |
*The coefficient of correlation is significant, P<0.05
Thus, the suggested approach allows taking in culture and analysis a minimum number of anthers during one vegetation to differentiate the large circle of corn genotypes and to pick out those with a potential for androgenesis.
The induced pollen grains in a great amount are contained in cultured anthers at the end of their cultivation, on the 42nd day of culture, when all the formed embryoids and calli are removed from an anther. Table 3 demonstrates that up to 5.42% of pollen grains in an anther (for B14xNf9, and in some lower rate for other genotypes) have entered the sporophytic pathway of development and formed the high unrealized androgenic potential of the anther.
Table 3. The percentage of induced pollen grains (%IPG) on the 42nd
day of anther culture.
| Genotype | %IPG |
| Wf9xLH148 | 3.70 |
| H99xWf9 | 0.65 |
| Wf9xH99 | 0.13 |
| B14xWf9 | 5.42 |
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