Reasons things do not take and possiable solutions

A meeting place for rose breeders.
Adam Eckstein

Reasons things do not take and possiable solutions

Post: # 28663Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

The original question from Lydia

Therese Bugnet makes hips with William's Double Yellow, and R. roxburghii, but not with R. primula. R. rox makes hips & winter hardy (in pots yet) seedlings with primula pollen. So what's the difference here? These are observations on two TB plants geographically separated. Naturally, no blooms on actual seedlings.

What do the scientists have to say. Any thoughts, theories?

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28664Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

So their s a number of reasons for failed crosses. I will list the ones I know and possible solutions if I know them. I will need help on this however. By the way this would be a great newsletter article. I might start writing one for the future.

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28665Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

1. One of the first ways seed set is prevented is because the chromosomes of plant A and the chromosomes of plant B have little in common. What happens here is that they can not match up. It is as though they are speaking two different languages. This is most likely what is happening in the case above.

Possible solutions that I Know of.

Use a link plant. In the case above. Therese Bugnet could have some chromosomes that match up to R. roxburghii but very little or anything that matches up to R. primula. However R. roxburghii has enough chromosomes that match up to R. primula that it makes possible a cross in that direction. So the game plan is to first cross R. roxburghii to R. primula. This offspring you then cross to the plant you originally wanted. How this works is hopefully you maintain enough similarity that makes the cross between Therese Bugnet and Roxburghii possiable but also add in the genes you want from R. primula.

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28667Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

2. Another possible thing that causes crosses not to take is the length of he pollen tube versus how far he pollen can grow. This is commonly seen in dayliles and lilies. I do not know how prevelent this is in roses but Ralph Moore did make a brief mention in a article at one time. The problem here is the pistal is too long for the pollen. The pollen when you pollinate a flower has to go from the stigma surface all the way down the style to use with the egg. (But this is probably a minnor situation with taking in all the miniature pollen that is used on full size roses ever year)

Possible solutions

A. Make the reciprocal of the cross if you can. By the reversing the cross the pollen from the former seed parent should have more than enough tube to successfully go down and fertilize the egg.

B. If the reciprocal is not possible you can cut the pistil down so it is not too long. I have not tried this method yet with roses, though I plain to in the future. But you cut the pistil to make it shorter. You can not pollinate right away because the sap from the pistil starts to flow up ward preventing things from going the other way. But after this flow has subsided you can try the initial cross. But some of your take percentage will be lower because of the damage you are causing. By the way when you cut the pistil you have to use something really sharp like a surgical knife or a razor blade because if you crush the tube the pollen grows down that's the end of the cross.

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28668Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

3. Another case can be seen in extreme miniaturization. In this case the seed parent is such a miniature in stature that it either lacks vigor for seed production or that by doing the cross pollination you damage the fragile parts of reproduction organs.

Possible solution

In this case a possible solution raised in early newsletters was grafting the miniature on a rootstock. There is a small sample of evidence to suggest this works. Again this is something I have not tested yet, but it does make sense. Many micro miniatures are not all that vigorous and you do make flowers larger in size when they are grafted. It is a weird concept when you consider that own root roses tend to be the better seed setters.


Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28669Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

4. The sap that glistens on top of the stigma that tells use it is the perfect time to pollinate can also be the reason we fail to make crosses. This sap is not just sugar water but it contains other substances that can facilitate pollen grains in germinating and can also cause them to just sit there forever in a comatose state. There is thousand a upon thousands of grains of pollen in the air naturally and nature needs a way to make sure only the right pollen fertilizes a plant. Grass pollen should not pollinate rose pollen a vice versus. So the sap helps the plant be selective of the right pollen. (Anyone who can explain this better I would appreciate it)

Possible solutions

A. Mix the pollen you want to take with pollen you know will take. By doing this you can sometimes pass the security guard at the top. However it is a good idea to choose a pollen parent that a very different from each other so that you can have a good idea which seedlings are which.

B. Clean of the sap from the pistil of plant you plain to use as the seed parent. Then replace it with a mixer of sugar water, or rub the sap from a plant that will except the pollen you want onto the pistil of the chosen seed parent. From the information I have read both have some success.

C. Cutting the pistil seems to exclude some of the barrier. These barriers are some how link to the stigma surface and not the sap. (again I need help on this one)

D. I read an article ounce where some one successfully started the pollen germination in a petri dish in the lab and then transferred it to the plant some how. This does not seem very feasible for us to do.

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28670Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

5. Chromosome numbers may not match.

Possible solutions

A. Increase the number of chromosmes in one of the plants through the use of Colchicine or another of other techniques. However a lot of times these techniques also reduce fertility in the plant that is induced.

B. The number of chromosomes can be decreased By either taking the pollen and doing a culture almost like tissue culture. It can also be decrease by making a cross with pollen that has been sterilized (usually by something like a microwave) onto the original plant. Then using this seedlings or reduced clones to cross with the other plant.

C. Finding a link that does not need to have chromosomes that match. In melon breeding their is one melon I do not remember the scientific name off the top of my head but it crosses with almost everything in that family no matter the ploidy if I remember right. In roses it seems that R. multiflora, Orangeade, anytime, and many of Ralph Moore's miniatures act in this way. Interesting Anytime and many of Ralph's miniatures are triploid. I wonder what the common link is between some of his miniatures.

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28671Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

6. Another reason may be auxin production and reduction. I do not know all of this so I will some up what I do know and some one will have to fill in the rest. Some how auxins is important to seed production. Sunlight breaks down auxin in the plant parts. In wide crosses the auxins may not be high enough for seed set.

Possible solution

Do wide crosses or unlikely crosses using shaded flowers on lower half of the plant. This supposedly works because these parts are shaded and the auxins are less broken down.


Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28672Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

That is all that I can think of off the top of my head. I am sure others can add to the list and also add to the ones already here. I am also sure that David, Henry and others have papers that describe in detail different methods and extra related to this question. Hope fully this has help Lydia, as you can see from what I posted I have not got all of these figured out myself and I know some where in my head are more. But right now the jelly beans in my head are rattling around to much to think of any more.

Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28673Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

Oh on problem #4 the sap scenario. Another solution would also be to make the reciprocal cross. In this case the thing that is preventing it in the sap or the stigma surface might not be a problem if you reverse the parents.


Adam Eckstein

Re: Reasons things do not take and possiable solutions

Post: # 28675Post Adam Eckstein
Fri Jun 11, 2010 1:00 am

I knew I would think of one more at least.

7. The temperature range that the pollen germinates at and the temperature range the seed parent accepts pollen at are different or they only overlap at a small interval. (this one is one I heard word of mouth so I do not know how accurate it is. I have not seen a study on it)

possible solutions

A. Probably not a big issue in roses because roses seem to have a large window when they are receptive. Roses also seem to have a fairly wide range of temperatures that they are receptive in. (I wonder if the petri method mentioned earlier would work here)

I hope I do not come off as knowing everything because I definitely don't. I will look forward to other peoples ideas and solutions.

Lydia S (5b ONT)

Re: Reasons things do not take and possiable solutions

Post: # 28683Post Lydia S (5b ONT)
Fri Jun 11, 2010 1:00 am

Thanks Adam. That certainly is just about an article. Now I'm thinking of putting primula pollen on multiflora seedlings - next year. Too much doing this year. I just had a look outside. There is one green & growing hip from R. primula on Hume's Blush. The rest have aborted. Maybe I could put Hume's Blush on Therese Bugnet & see how that goes.

You've given me some new ideas on mixing hardy with tender diploids.

Jadae (zone 8b)

Re: Reasons things do not take and possiable solutions

Post: # 28687Post Jadae (zone 8b)
Fri Jun 11, 2010 1:00 am

If I was using Rosa primula again, which I'm not because it was a total pain to work with logistically and it really doesnt play well with most other roses, I would definitely aim it at diploids. Unfortunately, there are not too many diploid briar types with traits I'd want to use other the rugosa =/ I'd definitely be tempted to try it on something like Coral Drift or Trier though. I tired it on Baby Faurax but it didnt yield anything.

Henry Kuska

Re: Reasons things do not take and possiable solutions

Post: # 28698Post Henry Kuska
Sat Jun 12, 2010 1:00 am

The link below gives a chapter from a book on this subject - methods of overcoming sterile barriers.

Link: http://www.liliumbreeding.nl/pollenbitech.PDF

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In the above document the following is stated:

"In interspecific crosses of Populus, treatment of the stigma with organic solvents such as hexane

and ethyl acetate before pollination has been reported to be effective in overcoming prefertilization

barriers (Willing and Pryor 1976)."

For a number of years I have been "playing" with the use of organic solvents and oils to store rose pollen. I also have tried to pollinate from a slurry of pollen and organic solvent.

Here are some results:

1) toluene, xylene, naphtha, and terpentine soak into the bud tissue and "kill" the bud. (this does not mean that toluene and xylene cannot be used to store the pollen, just that they would have to be removed before pollinating. An acetone wash can remove these.)

2) acetone, methylethylketone, (RV and Marine type antifreeze) propylene glycol, and DMSO do not kill the bud.

I also used a propylene glycol based car antifreeze (Autoguard). (The DMSO results sound surprising, but that is what my notebook says.)

In one successful pollination I mixed propylene glycol plus boric acid plus sucrose with William Baffin pollen and did get hips that set and resulting seedlings from wide crosses.

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The following references have to do with using organic solvents with pollen.

Title: Collection and storage of pollen from Salix (Salicaceae).

Authors: Kopp, Richard F.; Maynard, Charles A.; Rocha de Niella, Patricia; Smart, Lawrence B.; Abrahamson, Lawrence P.

Author Address: College of Environmental Science and Forestry, Faculty of Environmental and Forest Biology, State University of New York, Syracuse, NY, 13210, USACollege of Environmental Science and Forestry, Faculty of Environmental and Forest Biology, State University of New York, Syracuse, NY, 13210.

Published in: American Journal of Botany, volumn 89, pages 248-252,(2002).

Abstract: " Genetic improvement of willows through traditional breeding can be facilitated by pollen collection and storage so that female flower receptivity need not be synchronized with pollen shed for breeding. Two experiments were completed to test the effectiveness of various organic solvents for willow pollen collection. In the first experiment, seven pollen collection treatments and an untreated control were tested with two willow clones. The other experiment tested three treatments that showed promise in the initial experiment and an untreated control with eight willow clones. Toluene and carbon tetrachloride were effective for pollen extraction, with average pollen germination percentages that were >15%, but both chemicals reduced pollen viability by 10-20% compared with an untreated control based on in vitro germination tests. Pollen extracted with carbon tetrachloride or toluene was successfully used in controlled pollination, and >100 new families were produced with this technique. Pollen viability remained high after 18 mo of storage at -20degreeC. Based on our results, toluene is the preferred solvent for future willow pollen extractions because it is as effective as carbon tetrachloride, is not a known carcinogen, and is less expensive."

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Title: POLLEN COLLECTING METHOD FOR ARTIFICIAL KIWIFRUIT POLLINATION 1. WASHING METHOD BY ORGANIC SOLVENTS

Authors: WAKI K; TAKEUCHI K

Authors Address: FAC. AGRIC., TAMAGAWA UNIV., TAMAGAWAGAKUEN 6-1-1, MACHIDA-SHI, TOKYO 194, JPN.

Published in: Bulletin of the Faculty of Agriculture Tamagawa University, volumn 30, pages 59-72, (1990).

Abstract: " Pollen collecting method for artificial kiwifruit pollination: 1. Washing method by organic solvents.To develop a labor-saving method for collecting pollen from Kiwifruit flowers, several procedures were examined; the following results were obtained. Pollen from pollen loads collected by honeybees showed a germination rate of 85%, but it was decreased to 40% after 4 days storage at a room temperature. When pollen loads were washed with successively decreasing concentrations of sucrose and finally with water, the germination rate was 78%. When pollen loads and pollen collected directly from flowers were washed with either 50%, 30% or 15% sucrose solution and then with water, their germination rates after drying were about 20%. This result suggests that these washing and drying methods are not suitable. The following 11 organic solvents out of 26 solvents used for washing showed high germination rates (over 50% of non-washed pollen): cyclohexane, n-pentane, isooctane, toluene, benzene, xylene, methyl acetate, ethyl acetate, diethyl ether, n-butyl alcohol and isoamyl alcohol. Pollen from early-stage male flowers did not germinate at all. But when it was washed with organic solvents after drying for 24 hours, its germination rate was as high as 77%. After 2 years of storing benzene-washed pollen at -17.degree. C, the germination rate was as high as 89%. Even when pollen was stored for 4 years below -17.degree. C, the germination ability was maintained to some extent (41% .apprx. 69%) when the pollen was washed with several solvents just after collection. Washing flowers with toluene, sucrose solution or water yielded more pollen than sifting although the latter two solvents reduced the germination rate considerably after drying. When toluene or benzene-washed pollen was used for hand pollination in the field, the percentage of fruit that set was high with no significant difference compared to non-washed pollen. The fruits obtained did not show any deterioration. Toluene and benzene are useful for obtaining pollen for artificial pollination."

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Title: STORAGE OF POLLEN GRAINS IN ORGANIC SOLVENTS EFFECTS OF SOLVENTS ON POLLEN VIABILITY AND MEMBRANE INTEGRITY

Authors: JAIN A; SHIVANNA K R

Authors Address: DEP. BOTANY, UNIV. DELHI, DELHI 110007, INDIA.

Published in: Journal of Plant Physiology, volumn 132, page 499-501,(1988).

Abstract: "Storage of pollen grains in organic solvents: Effects of solvents on pollen viability and membrane integrity.In vitro germinability was studied in pollen grains of Crotalaria saltiana Andr. stored in various organic solvents and correlated with pollen membrane integrity (as assessed by the fluorochromatic reaction test) and its permeability (as assessec by leaching of water soluble UV-absorbing materials into organic solvents during storage). Pollen grains stored in organic solvents with low dielectric constants (a measure of their non-polar nature) such as n-hexane, cyclohexane and diethylether showed high scores for germinability and membrane integrity, and very little leaching of UV-absorbing materials. Pollen grains stored in solvents with high dielectric constants (a measure of their polar nature) showed low scores for germinability and membrane integrity, and extensive leaching of UV-absorbing materials. The results indicate that viability of pollen grains stored in organic solvents is primarily determined by the effect of organic solvents on the integrity of pollen membrane."

----------------------------------------------------

Title: Effect of storage in organic solvents on the germination of grapevine pollen

Published in: Journal of Horticultural Science, volumn 58, pages 389-392, (1983).

Author: P. K. AGARWAL

Author affiliation: Indian Institute of Horticultural Research, 255, Upper Palace Orchards, Bangalore-560080, India

Abstract: "Effect of storage in organic solvents on the germination of grapevine pollen. Grapevine pollen stored in amyl alcohol and benzene showed maximum germination even after 6 months storage, pollen tube growth being positively correlated with germination percentage." The following is part of the introduction: "FOR grapevine breeding pollen storage is an important consideration, since different cultivars flower at different times and some may produce pollen when others have ceased flowering. Studies on the effects of varying humidity and temperature on pollen viability have been conducted (Gollmick, 1942; Nagarajan, et al., 1965; Randhawa et al., 1982) but there is no available information on the effect of organic solvents on pollen viability in any fruit crop. Preliminary studies on this technique of pollen storage in Camelia, Lilium and Impatiens spp. by Iwanami (1972) and Iwanami and Nakamura (1972) have suggested its usefulness for fruit crops. This paper describes the effects of storage in ten organic solvents on pollen germinability in Vitis vinifera cv Anab-e-Shahi." The following are some of the data that were given in Table 1. For each solvent three numbers are given. The first is the percent of germinated pollen, the second is the mean pollen tube length in microns, and the third is the correlation coefficient between the percent germination and the mean tube length. "Caption of Table 1. Effect of 6 months storage in organic solvents on cv Anab-e-Shahi pollen germination and pollen tube growth Control 5.33; 3.93; +0.84 Acetone 5.02; 10.03; +0,77 Amyl alcohol 57.59; 72.51; +0.48 Benzene 50.71; 59.92; +0.85 n-Butanol 42.80; 40.07; +0.91 Carbon tetrachloride 41.96; 25.37; +0.84 Ethyl acetate 38.54; 34.73; +0.74 Isoamyl acetate 52.36; 42.94; +0 57 Petroleum ether 41.92; 39.64; +0.79 Toluene (S-free) 37.37; 16.97; +0.67 Xylene 43.86; 49.93; +0.76"

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Title: Studies on the artificial pollination of watermelon. 1. On the storage of pollens of watermelon by the utilization of organic solvents.

Authors: Shimizu, Tatsuo. Tottori

Authors affiliation: Veg. Ornamental Crops Exp. Stn., Tottori, Japan.

Published in: Tottori-ken Yasai Shikenjo Kenkyu Hokoku, volumn 4, pages 9-17, (1983).

Abstract: "Utilization of org. solvents for the storage of watermelon pollens was examd. with ref. to the extensions of their pollen storage period. Among 6 org. solvents tested for pollen storage, ethyl acetate [141-78-6] and ethyl ether [60-29-7] were suitable. The effective storage period for pollens in Et2O was extended by drying the pollen after ether extn. from anthers. Na2SO4 addn. to Et2O and low temp. during storage were effective for extension of the pollen storage period. After removal of Et2O or EtOAc, .apprx.50% of the pollen lost its germination ability within 2 h at room temp. Thus, pollination with stored pollens must be completed immediately after the removal of the solvents. Satisfactory fructification and fruit-thickening were obtained by actual crossing with the pollen following storage in Et2O and EtOAc for 2 wk. For the practical application, the crossing at lower temp. period should be examd."

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The following 2 articles may be useful to increase seed set:

http://home.roadrunner.com/~kuska/sprayingthehip.htm

http://home.roadrunner.com/~kuska/how_m ... ollina.htm

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Putrascene is reported to help pollen germination.

http://www.google.com/search?hl=en&rlz= ... n.&spell=1


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