We got some transgenic rice seeds from an old batch. We don't know how were the seeds stored, but probably at room temperature. We tried to germinate them in water as usual, but the seeds failed to germinate. We also tried to use sucrose on an agar medium to stimulate germination but it didn't work as well.
Does someone have any suggestion to stimulate germination on these seeds?
Thanks
You can perhaps try excising the embryo (after imbibition) and see if they will germinate on a simple medium.
The GA3 treatment is a possibility, but you should select denser seeds by flotation. https://www.thcfarmer.com/community/threads/germinating-old-seeds-with-gibberellic-acid.8841/
Dear Dr. Tiago,
First you must apply a tetrazolium test on part of the sample. Thereafter, you can apply a serie of recommendation for rice seeds when they have dormancy. These recommendations are free for download in the Rules for Seed Testing in Brazil (2009)
Type: Regras para análise de Sementes, MAPA, 2009 Brasil.
Best regards,
Walter
I think you could check the viability first. Is very easy, you just need make a tetrazolium test.
You are welcome, Seed specific density altough primitive is high (1.2) in good seeds and failure totake up water after 24h is the first clue to follow in small batches. GA3 can remove dormancy if any.
The germination of rice increases if you treat them at 45-50 degree for ~4 days. This method is more effective for indica varieties than the japonica one. Do not seal the seeds otherwise they will never germinate.
At first viability test is very essential. then you go for other treatment with GA3 and KCL
Dear Taigo, incubate rice seeds in 2% H2O2 solution at room temperature for 12-24 hours, then rinse with tap water, hydrogen per oxide removes contamination and also acts as stimulant to germination in many cases. germinate at 30C immersed in water or as per ISTA between paper method, if still no success, apply GA3, transplant the germinated seeds to soil; wish you success.
First find viability of seeds using tetrazolium.Pre-treat viable seed with GA,Kinetin and KNO3 alone and in combination in different concentrations for different time durations.
You may also try gernination under defferent light spectra.
storage conditions are critical for seed storage. As seed ages, germination potential goes down. Probably your seed is dead. However, you may want to consider working backwards to find out the parents of the transgenic rice you are working with. Obtain the gene from the donor plant and introduce the gene to a live recipient plant cell using the appropriate technique. So, you have your transgenic rice plant. you can go ahead with your work....
I don't recommend that you test the embryos for viability, as those testing methods are destructive, and you already have precious few viable ones. But there are other things you can do to increase the probability of some precious remaining viable embryos to grow.
In normal rice germination, the imbibition of water triggers the embryo to produce gibberellin which diffuses to the aleurone layer where it stimulates de novo synthesis of alpha amylase and other starch-digesting enzymes that convert the starchy endosperm to sucrose to feed the growing embryo. Once the embryo begins developing, the gibberelins produced in the seed/embryo/seedling help the shoot to elongate, while cytokinins encourage root development.
The endogenous hormones and enzymes that support all of the above biochemistry become broken down or damaged over time, a p4roces that can be slowed with cooler temperatures. Storing seed in a refrigerator or freezer slows the breakdown process, and prolongs seed viability.
So - to germinate over-aged seed, you need to replace the missing components. Germinating the seed on sucrose-agar bypasses the need to digest the starch. Removing the embryo from the seed so that it can access the sucrose more directly can also help.
Germinating dissected embroyos like that is known as embryo rescue.
PUBLICSATION TO READ:
Application of Embryo Rescue in Recovering Rice (Oryza sativa L.) Germplasm
R. H. Dilday, Wengui Yan, F. N. Lee, R. S. Helms and F. H. Huang
Crop Science 1994 34: 6: 1636-1638
doi:10.2135/cropsci1994.0011183X003400060039x
Adding low levels of GA3 and cytokinin to the medium can also help a viable embryo develop faster.
All of this can help an aged embryo (of any species, not just rice) grow, but nothing can bring it back from the dead. There are no "Lazarus treatments".
i agree with Pinson, not to waste few seeds in TTZ test and go ahead with such test if you have ample amount of seeds.
I agree with the above. You might also try incubating on an osmoticum like mannitol to reduce the rate of imbibition. This would reduce solute leakage and would probably be beneficial for old seeds.
You have not mentioned age of the rice seeds. i.e. how old are they? The grass seeds are meant to germinate in next monsoon season. If not received favorable conditions, they lose seed viability significantly after one year of maturity. Especially, if not stored with proper precautions.
I experienced same problem with the seeds of halophytic grass Halopyrum mucronatum.
I tried alternate low-high temp treatments in various combinations. Alternate 10 and 25 degree temp (Night and Day respectively) with 12 hr photoperiod improved germination percentage from 7% (control) to 32% for the species. (UNPUBLISHED DATA). Such treatment may also improve germination in old rice seeds. Best of Luck.
One suggestion you may try.
Keep the seeds in 10% Lantana camara aqueous leaf extract solution for 7 days.
Article Allelopathy: Natural and an environment friendly-unique tool...
Pre-sowing seed treatment in different conc. of GA may give good germination as GA is effective in de novo synthesis of hydrolytic enzymes thus providing energy source for germinating seeds
First the check activities of root promoters.
After reassurance, you can seed priming with Ga3, Ga3+ CK or ascorbic acid.
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