Can we use trifluralin (Preem) treated pollen from a diploid?

Rose Genetics Ploidy Level
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Can we treat pollen from a diploid and then add the treated pollen to a tetraploid mother to get tetraploid seedlings?

Title: Heterofertilization exhibited by trifluralin-induced bicellular pollen on diploid and tetraploid maize crosses

Author: Kato A

Author affiliation: Univ Missouri, 117 Tucker Hall, Columbia, MO 65211 USA

Published in: GENOME, volumn 44, pages 1114-1121, (2001).

Abstract: “The heterofertilization rates and fertility of trifluralin-induced bicellular pollen were investigated in maize (Zea mays L.). A diploid inbred line, Oh43 (r1/r1), and a tetraploid line, Q28-1 (r1/r1/r1/r1), were pollinated with a trifluralin treated diploid stock heterozygous for R1-scm2. The gene R1-scm2 conditions purple pigmentation in both the embryo and the aleurone layer. Heterofertilized kernels were detected as discordant kernels, i.e., yellow kernel with purple embryo or purple kernel with white embryo. The diploid-diploid crosses treated with 0.2-0.3% Trefanocide solution (0.09-0.13% trifluralin) resulted in incidences of discordant kernels (3.7-4.8%) that were significantly higher than the control (2.3%). Most of the seedlings (86%) of the discordant kernels in the 0.3% treatment were triploids or triploid-class aneuploids. In tetraploid-diploid crosses, trifluralin treatments increased the number of plump kernels on the tetraploid ears. In the 0.3% treatment, 5.2% of ovaries produced plump kernels on the ears and most of the seedlings (92%) were tetraploids or tetraploid-class aneuploids, whereas in the control, only 1.5% ovaries produced plump kernels and most of the seedlings (98%) were triploids or triploid-class aneuploids. A high rate of discordance was observed among the plump kernels both in the treated plots (36.1-48.0%) and in the control (33.3%). Consequently, almost all of the plump kernels from the tetraploid-diploid crosses were considered to be the results of heterofertilization.”

Title: Induction of bicellular pollen by trifluralin treatment and occurrence of triploids and aneuploids after fertilization in maize.

Author: Kato, Akio

Author Address: University of Missouri, 309 Tucker Hall, Columbia, MO, 65211-7020, USA.



Published in: Genome, volumn 42, pages 154-157, (1999).



Abstract: “By spraying tassels of maize (Zea mays L.) with a trifluralin solution before flowering, viable bicellular pollen grains (with one vegetative nucleus and one mitotically arrested diploid generative cell) were produced. Fertilization between a central cell (2n) of diploid plants and the mitotically arrested generative cell (2n) of the bicellular pollen induced by trifluralin treatment was detected by the presence of shriveled kernels on pollinated ears. A covered method (tassels covered with aluminum foil for 24 h after spraying) and a non-covered method were compared, and the non-covered treatment with 0.2-0.4% trefanocide solutions was the most effective treatment in producing viable bicellular pollen. About 40-50% of the kernels were shriveled on pollinated ears from the treatments. Chromosome counts on seedlings obtained from 0.3% non-covered treatment revealed 24% were triploid and 4% were aneuploid (2n = 19, 21, and 22).”

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This is a great question Henry. I’ve read these articles before and really enjoyed them. For corn, the final post meiotic mitosis happens before the pollen is mature (corn pollen is trinucleate at maturity, the pollen tube nucleus and two generative nuclei are all present). The final division of the one nucleus into the two generative nuclei is what Kato aimed to prevent by spraying the tassels with trifluralin. So, there was just one generative nuclei left with twice as many chromosomes. If it fertilized the egg and somehow another pollen grain supplied an appropriate generative nuclei for viable endosperm then he was able to get triploid (and such depending on his crossing scheme) offspring he describes. If the nucleus fertilizes the central cell for endosperm development and not the egg, sometimes haploids resulted. Also, if it fertilized the egg and nothing fertilized the central cell that one often aborted. Multiple pollen grains participating in the fertilization of an ovule is the heterofertilization component (“hetero”=different) and his papers is where I first learned that this can happen.

Since roses are binucleate at pollen maturity and the final division to form the two generative nuclei doesn’t happen generally until its way down the style, trifluralin applications to mature pollen sounds like a fabulous idea! I hope you try this Henry and others do too. I am excited to learn what happens.

Sincerely,

David

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But how to do this in a pratical home setting? Got my surflan, can make an approximate streangth solution. I imagine that one must harvest the flower with stem, remove the petals down to the anthers, and coat the the surflan solution on the anthers and keep it in a cool and draft free place until it dries within 2 or 3 days.

Or…

Collect the pollen in a container, and give a very light spritz of the solution, and dry it.

I’m going to dry this with R. foliolosa, and see how I could more or less re-create this type of conditions.

(Update: I’ve rooted the possible tetraploid rugosa Roseraie de l’H

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Enrique, your 2 suggestions are certainly worth trying. A variation on your second suggestion would be:

Add a very small amount of the solid chemical to the stigmatic fluid of the flower to be fertilized. This could be done before adding the pollen or right along with the pollen (having the solid chemical powder mixed with the pollen).

I may also try depositing the chromosome doubling agent on the stigmas by preparing an organic solution of the chemical in a fast evaporating non polar organic solvent. This method may give a more even distribution of the chemical.

I am concerned that using a water solution may “ruin” the pollen. Of course the answer lies in trying.

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Just thinking about it… Do you think that if the stem of the rose was put into a surflan solution, would it be absorbed into the the whole flower and also into the pollen? To better illustrate this, think about putting blue ink into water in order to get roses with blue petals.

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Enrique, a potential problem with using a soak is that the chemical may act on other process such as pollen germination. Since Dave has stated that: “roses are binucleate at pollen maturity and the final division to form the two generative nuclei doesn’t happen generally until its way down the style”, it would appear that whatever method we chose would have to allow normal germination of the pollen.

Since I have a microscope, I will be able to determine if caffine or triflorine inhibits pollen germination in sugar, boric acid, test solutions.

If germination is inhibited, we may have to first add the pollen to the stigmatic fluid; and then after a “to be determined” time delay, add the division preventing chemical to the stigmatic fluid.

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In doing a Google search I found an earlier related discussion:

Link: forums.gardenweb.com/forums/load/hybrid/msg111522196232.html?4

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I was interested in finding out what the time period is regarding germination of the pollen, the final mitosis, and fertilization.

I found the following abstract:

I obtained the full paper and it states that: “fertilization is achieved in the 12-24 h. after pollination”.

It also states: “Second pollinic mitosis take place during pollen tube germination as most of the Rosaceae”.

The caption of one of the figures states: "pollinic mitosis of the generative cell inside the pollen tube after 24h of germination. Two sperm nuclei are easily viewable.

Thus, it appears that there will not be much of a time window between the pollen tube formation and the final mitosis if it turns out that I must not apply the chemical until after the pollen germinates. I guess the next step is to purchase some florist roses and see whether these chemicals inhibit germination (by observing with my microscope).

Link: www.actahort.org/books/547/547_48.htm

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More research has been done on Nicotine than on roses. Hopefully the behavior of the pollen is similar.

Title: Requirements for division of the generative nucleus in cultured pollen tubes of Nicotiana.

Authors: Read, S. M.; Clarke, A. E.; Bacic, A.



Authors affiliation: Plant Cell Biol. Res. Cent., Sch. Botany, Univ. Melbourne, Parkville, Victoria 3052, Australia.



Title: Requirements for division of the generative nucleus in cultured pollen tubes of Nicotiana.



Published in: Protoplasma, volumn 174, pages 101-115, (1993).



Abstract: “Production of sperm cells by division of the generative cell occurs during growth of Nicotiana (tobacco) pollen tubes through the sporophytic tissue of the style, and is associated with transition to the second phase of pollen-tube growth. When Nicotiana pollen tubes are grown in liquid culture, the extent of generative-nucleus division and the timing of this division depend on the chemical composition of the medium. Addition of reduced forms of nitrogen, either as mixed amino-acids (0.03% w/v of an acid hydrolysate of casein) or as 1 mM ammonium chloride, induces division of the generative nucleus in over 90% of the tubes; 3 mM calcium nitrate does not stimulate division. Individual amino-acids differ in their ability to induce this division. Contaminants in some batches of poly(ethylene glycol), which is a major component of pollen-tube growth media, inhibit generative-nucleus division; this inhibition is greater in the absence of nitrogen, which increases the observed nitrogen-dependence of division. Reduced forms of nitrogen are also required for growth of pollen tubes after division, when callose plugs are deposited. In the absence of nitrogen, growth continues until the point where sperm cell production would normally occur, then ceases. Addition of amino-acids or ammonium chloride thus allows cultured pollen tubes of Nicotiana to progress to their second phase of growth. When Nicotiana pollen is germinated in a complete culture medium at 25-26 degree C, sperm nuclei are first observed in the growing tubes after about 10 h, and by about 16 h most of the tubes have undergone division; at lower temperatures, division is delayed. The timing of division also varies between species of Nicotiana, but division occurs similarly in self-compatible and self-incompatible species. Anaphase in an individual pollen tube is calculated to take less than 4 min. The resultant sperm nuclei usually trail behind the vegetative nucleus, but a variety of arrangements of the three nuclei are observed.”

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This may be worth trying:

Title: Pronamide disrupts mitosis in a unique manner.

Authors: Vaughan, Martin A.; Vaughn, Kevin C.

Authors affiliation: South. Weed Sci. Lab., ARS, Stoneville, MS, USA.

Published in: Pesticide Biochemistry and Physiology, volumn 28, pages 182-193, (1987).

Abstract: “Pronamide, the active ingredient in the herbicide Kerb, has traditionally been grouped with mitotic disrupters, such as colchicine or trifluralin, that inhibit polymn. of tubulin into microtubules. Many of the effects of pronamide (c-metaphases, polymorphic nuclei, isodiametric cells, abnormal xylem development, swollen root tips) are similar to these well-studied mitotic disrupters. However, immunofluorescence microscopy studies using anti-tubulin sera revealed that, unlike other mitotic disrupters, pronamide-treated cells have greatly shortened microtubules that are located only at the kinetochore region. These structures detected by immunofluorescence studies were detd. by electron microscopy to be microtubules. Because these short kinetochore microtubules did not allow for mitosis to proceed normally, the nuclear envelope reforms around the chromosomes leading to polymorphic nuclei. Frequently, other cycloplasmic organelles normally excluded in the zone occupied by the chromosomes and spindle during mitosis were retained within the reformed nuclei. These data indicate that although the net effect of pronamide is similar to those of colchicine and trifluralin, pronamide acts by a different mechanism.”

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Method 2 apears similar to what we are considering. My initial plan was to inject the 2,4-D some time after the injection of the chromosome doubling agent. This article suggests that that offset may not be necessary.

Title: Development of novel chromosome doubling strategies for wheat x maize system of wheat haploid production

Authors: Sood S, Dhawan R, Singh K, Bains NS

Authors affiliation:

Bains NS, Punjab Agr Univ, Dept Plant Breeding, Wheat Sect, Ludhiana 141004, Punjab, India

Punjab Agr Univ, Dept Plant Breeding, Wheat Sect, Ludhiana 141004, Punjab, India

Punjab Agr Univ, Dept Genet & Biotechnol, Ludhiana 141004, Punjab, India

Published in: PLANT BREEDING, volumn 122, 493-496, ( DEC 2003)

Abstract: "Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine-free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling.

In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4-D (100 ppm), were injected into the uppermost internode of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early-stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant."

Link: www.blackwell-synergy.com/links/doi/10.1111/j.1439-0523.2003.00894.x/abs/

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This is what I posted under what came out as a title of only “A”. I am putting it here also so that this thread is continuous.

Posted by Henry Kuska [email] on Wed, Jan 7, 2004

In my reading about intergeneric crosses, I came across what may be tricks to try in wide crosses within the rose family.

The first is based on the following paper:

Title: Pollen tube growth and early embryogenesis in wheat x maize crosses influenced by 2,4-D

Author: Wedzony, Maria; Van Lammeren, Andre A. M.

Authors affiliation: Dep. Plant Physiology, Polish Academy Sci., Slawkowska 17, 31-016 Krakow, Poland.

Published on: Annals of Botany (London), volumn 77, pages 639-647, (1996).

Abstract: " Pollen tube growth and embryogenesis were investigated in the intergeneric Triticum aestivum times Zea mays cross. Emasculated wheat florets were pollinated with maize pollen, and at one day after pollination, the wheat plants were injected with 2,4-dichlorophenoxyacetic acid (2,4-D). The influence of 2,4-D on pollen tube behaviour was determined applying callose staining in whole mount preparations of pistils. Changes in the embryo sac and early embryogenesis were analysed on sections after various histochemical stainings. Maize pollen tubes germinated within 30 min and grew much slower through the pollen tube pathway compared with selfings of both maize and wheat. Deviations in pollen tube growth occurred such as coiling, widening and forking, irrespective of treatment with 2,4-D. Pollen tubes reached the micropyle between 5 and 24 h after pollination. 2,4-D treatment increased the number of the pollen tubes that reached the micropyle, and additionally, multiplication of sperm cells was found in the maize pollen tubes. Embryo formation was analysed at 2 d after pollination. Only when pollen tubes were in the micropyle, zygotes and embryos were observed. This points to their hybrid origin. Moreover, the embryos are likely of zygotic origin since multicellular embryos exhibited micronuclei, a sign of chromosome elimination not occurring in parthenogenic embryos and leading to haploidy of the embryos. Treatment with 2,4-D increased successful intergeneric fertilization from 18.7 to 69.3%. Immature embryos were rescued by in vitro culture from 14% of pollinated florets when excised at 14 d after pollination."

My comments: a 1 ml syringe was used to inject 1 ml of a 100 mg 2,4-D per liter of solution into the stem cavity of the highest internode. My question is: will this introducing 2,4-D result in the plant eventually dying? They removed the embryos after 14 days in order to culture in a petri dish; so, I assume the plants were still living then. If that amount of 2,4-D would eventually kill the plant, is there a chemical that could used several days after the injection of the 2,4-D in order to save the plant (such as GA3) and allow the hips to form normally?

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The body of the above cited article states the following:

"Colchicine treatment via tiller injection involved the postpollination injection of various concentrations of colchicine in combination with 2,4-D (which is routinely administered in wheat