1. EFFECTS OF SEED STERILIZATION TREATMENTS ON SEEDLING VIGOR AND IN VITRO CALLUS INDUCTION OF FOUR MAIZE INBRED LINES Anita Dutta1, Juan Carlos Martinez2 and Kan Wang2 1 Scavo Campus, Des Moines Public Schools, 2Center for Plant Transformation, Agronomy Department, Plant Science Institute, Iowa State University
BACKGROUND AND GOAL
Maize is one of the most important crops for plant biologists not only for its tremendous value for agriculture but also for its abundant genetic information for biology. One of the most important tools for crop improvement and basic biological study is genetic transformation. To perform genetic transformation some requirements have to be fulfilled. The first requirement is to establish a robust tissue culture system and this depends upon of having a reliable system to sterilize the starting material. An efficient sterilization method for highly seed-borne pathogen seeds was developed (Martinez, 2006) and used routinely, however it seems that it has a strong impact in seedling vigor and development and possibly in callus initiation as well. The objective of this project was to assess seedling vigor and callus induction of four maize inbred lines using two sterilization treatments. This project is part of the research project entitled “Establishment of transformation systems using inbred maize mature seeds”
RESULTS AND DISCUSSION
Contamination rate
Contamination rate was evaluated only in T2 and T3 every day, until the eighth day and number of contaminated seeds were recorded. Inbred lines B73 and W22 showed no contamination at all for T2 and a relatively higher contamination for T3. Inbred B104 has a lower contamination rate with no significant difference between treatments. Inbred H99 showed a higher contamination rate in T2. Comparison among genotypes showed that H99 has a higher contamination rate followed by B73 and W22, B104 showed lower contamination rate. (Table1, fig.1)
This may be an indication about the seed borne pathogen content. In general T2 seems to be the best treatment to sterilize seeds, however even if T3 showed some contamination, it doesn’t go beyond 11% which is acceptable in tissue culture.
Seedling vigor
Seedling growth rate (SGR) was assessed in all three treatments for each genotype. For B73 among treatments there is no significant difference between T2 and T3 but T1 (control) is significantly different. Genotype B104, T1 is significantly different from T2 and T3. For W22, there is no significant difference between T2 and T3 (data from T1 were lost due to contamination). For genotype H99, all treatments are significantly different. Among inbred lines, SGR is significantly different in H99, showing the highest vigor. In general all genotypes except H99 showed a similar pattern in vigor rate. Number of dead and abnormal seedlings was also recorded (not considered for SGR) (Table 2, fig. 2).
For genotype B73, T1 showed no abnormal seedlings and the highest number of dead seedlings because the contamination was not controlled. For T2 and T3 the number of abnormal seedling is higher as compared to the number of dead seedlings, maybe this is because of the stress caused by the sterilization methods. For genotype B104, T1 showed fewer dead embryos as compared to T2 and T3, number of abnormal seedlings for T1 and T2 are slightly higher than T3, maybe this is due to the genetic background of the genotype itself, since it has been seen that B104 is not well responsive in tissue culture (data not showed). For genotype W22, T1 (control) was completely lost because of the higher amount of seed borne pathogens, T2 and T3 showed no dead seedlings and few abnormal seedlings. Genotype H99 exhibited the best performance in vigor and lowest number of dead and abnormal seedlings.
Callus induction
Up to now (30 days after starting experiments) the only genotype that has showed callus initiation is H99, first callus induction in this genotype was observed after 21 days (Table3, fig.3). At this point it has been seen that T2 has higher callus induction as compared to T3 which is contrary to our expectations since T2 is stronger seed sterilization method than T3, however we cannot conclude at this time since there are still some lines with potential to generate callus that may require few more days. There is a positive correlation between H99 genotype vigor and callus initiation.
Table1. fig.1. Contamination rate per inbred line and per treatment among inbred lines
MATERIALS AND METHODS
Four inbred lines harvested from field were used for this study: B73, B104, W22 and H99.
Seedling vigor
Three treatments (T) were compared.
T1: Control. No sterilization, no in vitro conditions. Seeds were softened 24 hours in moisturized sterile paper towels, then embryos were dissected and placed into sterile soil in magenta boxes.
T2: Routinely used seed sterilization method. Three minutes Ethanol 80 %, two periods of 15 minutes of 50 % commercial bleach, then seeds were softened for 24 hours in moisturized sterile paper towels. Seeds were re-sterilized 3 minutes with ethanol 80 % and 12 minutes 50% commercial bleach. Embryos were removed from seeds and sterilized a third time, 10 minutes with 15% commercial bleach then embryos were placed into MSVS34 media (Sidorov et al, 2005).
T3: Three minutes Ethanol 80% followed by two periods of 15 minutes of 50 % commercial bleach, then seeds were softened for 24 hours in moisturized sterile paper towels. Seed were re-sterilized, 2 minutes ethanol 80%. Embryos were removed from seeds and sterilized a third time, 5 minutes with 10% commercial bleach, then embryos were placed into MSVS34 media.
After 10 days, the meristematic part of each plantlet (located in the base of the coleoptile and the upper part of the mesocotyl) was removed (around 1 cm length) and placed into MSW57 media (Sidorov et al, 2005) for callus induction. Leaves were used to assess plant vigor using the Seedling Growth Rate (SGR) Test (ISTA, 1999).
Callus induction
Callus induction was evaluated as the first callus appearance (number of days) and the number of callus-deriving lines (every seedling is considered as a line). Assessment of plant vigor was performed on all treatments however contamination rate and callus induction were only evaluated on treatment two and three. The experimental design was a complete randomized design (CRD) with three replications per treatment. Seedling growth rate was analyzed using the analysis of variance (GLM procedure, SAS institute). Treatment mean comparison was estimated using Duncan’s test.
** Significant at the 0.05 probability level. (P≤ 0.01)
Table3, fig.3. Callus induction correlated with SGR
CONCLUSIONS
Genotype H99 shows the highest seedling vigor rate and it was also the first one showing callus initiation.
Treatment two and three are both efficient for seed sterilization and there is no significant difference in SGR for both treatments among genotypes.
The difference in number of abnormal seedlings between inbred lines is possibly due to the genotype background.
There is a positive correlation between H99 genotype vigor and callus initiation.
Table2, fig.2. Seedling growth rate, dead and abnormal seedlings
ACKNOWLEDGEMENT
Authors are thankful to Bronwyn Frame, Diane Luth and all the personnel from the Plant Transformation Center as well as NSF, Adah Leshem-Ackerman, Jay Staker and Eric Hall.