Monday, November 4, 2013

Chemical Emasculation in Plant Breeding

This is the page of the “Chemical Emasculation in Plant Breeding: Chemical Gametocide: Hybridizing chemicals (HYC): Chemical Hybridization Agents” where I welcome you.

I prepared the review paper as the term paper in the Ph.D. Plant Breeding 2nd Semester (R2011-PLB-01 P) in Tribhuvan University, IAAS, Chitwan, Nepal. 

Chemical Emasculation in Plant Breeding

Nav Raj ADHIKARI, Associate Professor, Plant Breeding Department, Rampur Campus
Institute of Agriculture and Animal Science
Tribhuvan University, Rampur
Chitwan, Nepal

For citation:
Adhikari, Nav Raj, 2012. Chemical Emasculation in Plant Breeding.

Principally, artificial crossing involves removal of anther or sterilization of pollen and transfer of pollens of male parent from different breeding line to the emasculated plant population. In different times, crop scientists have been exploring different ways to facilitate artificial crossing for commercial hybrid seed production. Chemicals have also been used to emasculate female flower of one parent line for breeding research as well as hybrid seed production.

Plant Breeders have been exploring effective chemicals and effective application method to emasculate female parent pollens for hybrid seed production. In fact, plant breeders or big innovative farmers must perform some preliminary works to start producing hybrid seeds of any crops species even they have to use chemical for emasculation.

First the breeders or farmers must identify or select suitable female and male parent. After selection, they must determine a particular chemical that sterilize pollen without lowering fertility of egg cell and seed setting potentiality of the seed parent. Once they know or determine the exact chemical and its dose for the crop of their interest, they have to apply the chemicals very precisely and correctly on the plant population of female parent only.

In the third stage, the farmers must need pollinator if it is cross pollinating crop species or the crop requires honeybees or other insects for pollination. In some crops, wind also works as a pollinator since it carries pollens from male parent population to the stigma of the female parent population. In the manipulated environment, the bees or pollinators are left.  After seed set or fruit maturity, the fruits or matured seeds are harvested very carefully without any seed contamination.

Hybridizing chemicals must work very selectively to sterilize male gametes without injuring female reproductive organ as well as seed setting potentiality of the female seed parent. The hybridizing chemicals must be economic, easy and flexible in use and time of application. The chemicals must not be mutagenic and hazardous to the environment, safe to use, free from the forming seeds. The hybridizing chemicals must be more effective to cause more than 95% male sterility without injuring the seed set of the female parent population.

What to call to the emasculating chemicals?
Hybridizing chemicals or Male gametocides
There are some terminologies to designate the chemicals that sterilize male gametes or make the male gametes or male gamete producing germ cells or floral organs non-functional to fertilize egg cell. I prefer to say hybridizing chemicals since artificial crossing or artificial hybridization is facilitated after emasculation of male floral parts of the plant population of the crop species of our interest. Some prefer to say male gametocides since the chemicals kill male gametes or pollen or pollen mother cells.

Male sterilants or Selective male sterilants
Some prefer to say the hybridizing chemicals ‘male sterilants’ since the chemical sterilizes the male gametes or destroys the male reproductive organ to sterilize the pollen or male gametes. Some prefer to say the hybridizing chemicals ‘selective male sterilants’ since the chemicals selectively sterilizes the pollen or male gametes only without influencing the female gametes or female gamete  reproducing organ or seed setting potentiality of the female plant.

Pollen suppressants or pollenocide or androcide
Some prefer to say ‘pollen suppressants’ since they suppress the fertilizing potentiality of the pollen or male gametes. Some prefer to say the hybridizing chemicals as the ‘pollenocide’ since the chemicals kills the pollen or make the pollen or male gamete non-functional to fertilize the egg cell. Some prefer to say the hybridizing chemical ‘androcide’ since the chemical kills the tissue of androcium such as anther, or pollen mother cells or pollens or male gametes.

Here, emasculation means removal of anthers from the female parent plants. Hand emasculation and manual transfer of pollen for crossing are very expensive and cumbersome. There is maximum chance of working mistakes in the hand emasculation. Besides, emasculation is highly skilled labor requiring technique for commercial hybrid seed production. So plant breeders have been continuously working to develop male sterile lines.

Plant breeders have also been working to find out effective chemicals for emasculation, emasculation method, and their appropriate concentration to emasculate female parent to facilitate commercial hybrid seed production of our crop species since development of male sterile lines is very long, complex and expensive. But; chemical sterilization is comparatively easier, less expensive and less time consuming.
Moore and Naylor independently reported some possibility of the use of some chemicals to sterilize pollen in plant species. They independently worked with a chemical Maleic hydrazide (MH) in the crop of maize to induce male sterility in the year of 1950. Thereafter serially scientist started to report on the hybridizing chemicals in plant species.

Maleic hydrazide (MH)
Maleic hydrazide is one of the hybridizing chemicals used in the induction of male sterility in the crop species of maize, wheat, cotton, onions. Maleic hydrazide is a class of chemical under antiauxin. The hybridizing chemical is toxic to the plants inducing abscission of flowers. The sterilizing property of the maleic hydrazide was discovered by Moore (1950) and Naylor (1950). But, Warren and Dimmock (1954) reported that maleic hydrazide cannot be an effective male gamete sterilizing chemical. Natrova (1973) further reported that maleic hydrazide can be used to sterilize pollen in the hybrid seed engineering of rye (Secale cereal L.).
Concentration of 500 ppm of maleic hydrazide can work as chemical emasculation on the pollen of tomato plant inflorescence. It also works in the crops of wheat and cotton for experimental purpose only.

Triiodobenzoic acid (TIBA)
TIBA is under antiauxin group. It is also known as male sterility inducing chemical. Hensz and Mohr (1959) reported that TIBA induced male sterility in watermelon. Kiermayer (1959) reported that TIBA could induce male sterility in sunflower. Similarly, Iyer and Randawa (1965) reported that TIBA can induce male sterility in grape (Vitis vinifera L.). TIBA has not been studied extensively since there is not more report on the use of the TIBA as male sterility inducing chemical.The dose of 25 to 30 ppm (parts per million) of triiodobenzoic acid can induce sterility of pollen of tomato flower buds without damage to egg cell fertility.

Auxin Group chemicals
Rehm (1952) reported that 2,4-D (2,4-dichlorophenoxyacetic acid) induced partial male sterility in watermelon with slight improvement in female fertility. There is some report that NAA (naphthalene acetic acid) could induce male sterility in tomato. Some other auxins have also been tested for induction of male sterility in several crops.

Halogenated Aliphatic acids:
FW 450
Eaton (1957) reported that FW 450 (sodium alpha, beta-dichloroisobuyrate, Mendok) could induce male sterility in cotton with no significant loss in female fertility. But; later, it was found that the FW 450 caused phytotoxic effect in crop plants. The chemical: FW is not selective to induce sterility in male and female gamete.

Dalapon is closely related to the FW 450 since it is under halogenated aliphatic acid and its action to induce sterility in male and female gamete. Previously, it was found that dalapon could be used as an effective chemical hybridizing chemical in the crops of cotton, tomato, pea and maize. But, its action was found similar to the FW 450. It has similar drawbacks as the weakness of the FW 450.

Sodium dichloroacetate as hybridizing chemical
The chemical has produced  a positive effect on the sterilization of the pollen of Antirrhinum majus nanum L. Spray of the 0.5% solution of the sodium dichloro acetate in water at the first flower bud opening stage sterilized the pollen. The stage was about two weeks before the flowering. The treatment was done in glass house at 17 degree Celsius. The experiment was conducted by Kho and Bruyn (1962) at the laboratory of the plant breeding in Wageningen Agricultural University, the Netherland.

Gibberellic acid (GA3)
Nelson and Rossman (1958) reported that potassium gibberellate induced 100% male sterility in maize without any damage to the female fertility. The induction of the male sterility was very specific to the stage of the maize. This event initiated plant scientists to use gibberellins as male sterilizing agent. Hansen et al. (1976) also found similar effect of GA.

Successful male sterility was found in sunflower with GA shown by Anascenko (1967). Schuster (1969) and Piguemal (1970) also reported result of the effect of GA. Gibberellins have been used in several crops to induce male sterility in several crops. Similar effect was shown in crop of onion (Allium cepa L.) as shown by Badino (1981) and van der Meer and van Bennekom (1982), but its commercial use is very expensive as the breeding tool of chemical hybridization. Similar encouraging effectiveness were obtained in the crops of lettuce, Brussel’s sprouts, cabbage, cauliflower and kale as shown by van der Meer and van Dam (1979).

Application of GA3 is very effective to cause male sterility in plant species before the start of cell division of pollen mother cell but does not work after the pollen mother cell inters to meiotic cell division phenomenon. Desired effect of the application of GA3 is very stage specific. Research has shown that application of the GA3 is very effective in maize, barley, wheat, rice and sunflower.

GA3 application on crop plants elongates the inter-nodal region of the crop plants and lowers the hybrid seed set percent in the crop population of sunflower. Different genotypes of the same crop species behave differentially to the applied GA3. So, research should be carried out to examine the response of female parent of different breeding lines for hybridization.

Stage of plant and environmental factors also influence on the emasculation effect of the GA3. GA3 is not used commercially in the hybrid seed engineering. GA3 is expensive. How GA3 works in the male sterility induction is also interesting. Systemic entry of GA3 into the plant system does not let plant assimilates ‘food nutrients’ flow to the developing pollen generating anthers. As the result, the pollen forms abnormally and then becomes non-competent to fertilize egg cells.

Ethephon or Ethrel:
Ethephone or ethrel (2-chloroethyl phosphonic acid) is the substance which produces organic gas ‘ethylene’. Initial discovery of the use of the ethephon as the male sterilizing chemical was done in the crop of wheat (Triticum aestivum L.) by Rowel and Miller (1971). The wheat has been the primary crop to respond ethephon as male sterilizing chemical. Other crops also include in it such as Hordeum vulgare L. (barley), Avena sativa L. (oats) , Triticosecale (Wittmack) (triticale), Pennisetum spp (proso), Eragrostis tef (eggplant), Linum usitatissimum L. (flax), Solanum melongena L (eggplant), maize, rye, lettuce and sugar beet.

The action of male sterilizing of ethephon is very effective in stage specific. In the wheat crop, all tillers are not of the same age. So the ethephon does not work 100% in commercial hybrid seed production in bread wheat. But, high seed rate of wheat crop for minimum late tillering can minimize the less effect of male sterilizing of the ethephon. The use of ethephon checks the culm elongation of the wheat plants.

The applied ethylene induces male sterility in crop plant species. The concentration and amount required for the induction of male sterility varies from crop species to crop species. Furthermore, different genotypes of the same crop species behave differentially too to the applied ethephone.

Another important fact is that the response of the plants to ethrel is also dependent on stage of the plants. Besides, ethrel application causes physiological imbalance of the crop plants. The application of ethrel delays vegetative growth and reduces the plant height including the inflorescence length of the plants of the crop species too. The application of the ethrel also lowers the fertility of the egg cell and seed set.

The application of the ethrel is if done before the megaspore mother cell undergoes first meiotic prophase, it causes the meiotic cell division of megaspore mother cell unbalanced and produces aberrant egg cell and lowers the female gamete too.

The application granular form of ethrel into soils can also be done for emasculation of female parent of the crop species but huge amount of the ethrel is required for 100% male sterility. But soil application does not cause physiological imbalance of the crop plants whereas foliar application causes physiological imbalance of the crop plants.

DPX 3778
There is another hybridizing chemical known as DPX 3778 which is also effective in emasculation of some crop species such as rice and wheat. The chemical is applied at late tiller initiation, early and late boot stages. But the crop requires higher dose of DPX 3778.

Zinc methyl arsenate MG1
First, zinc methyl arsenate (ZMA: MG1) was used as a fungicide in the crop of rice. Similarly, the arsenic compound: monosodium methane arsenate (MSMA: also called MG2) was used as a herbicide in the crop of rice. Then, possibility of using the chemicals were examined as emasculating or male gamete sterilizing chemicals in the crop species. The chemicals showed that the chemicals can induce male sterility in plant species.

Monosodium methane arsenate MG2
MG2 has been found very effective to cause 99% male sterility in the crop of rice. So the MG2 has been used for hybrid rice production successfully. Concentration of MG2 is 150 mg per liter water which is the appropriate dose for its application. The application of MG2 should be 5 days before heading of the rice to find less toxic effects. The time of application can be 15 days earlier to the heading for the effective emasculation. MG2 has been used in hybrid rice production in China.

Androcium parts of floral organ are very sensitive to the arsenicals such as zinc methyl arsenate and sodium methyl arsenate produce but pistilate parts of the floral organ. It has been determined that the arsenicals inhibit anther cells to respire through some enzymes such as succinate dehydrogenase and cytochrome oxidase.

If the arsenic chemical is applied just before the start of first meiosis of pollen mother cells, it does not let the pollen mother cells undergo meiotic reductional phenomenon normally. As the result of which, unbalanced pollens forms with aberrant chromosome structures. Consequently the phenomena do not let the male gametes inter the embryo sac to fuse the egg cell for zygotic seed formation.

RH 531
RH 531 is a patented compound. Chemically RH 531 is known as sodium 1 (p-chlorophenyl) 1, 2-dihydro 4, 6-dimethyl-2-oxonicotinate. Yih et al. (1971) reported that RH 531 induced male sterility in barley. Wang and Lund (1975) reported that the use of RH 531 in inducing male sterility without loss in female fertility. Some studies showed that RH 531 did not behaved selectively in barley to cause male sterility without disturbing female sterility (Hockett and Feltner 1978; Batch 1978.

RH 532
RH 532 is also patented compound. It is chemically named as sodium 1-(3, 4 dichlorophenyl)-1, 2-dihydro 4,6-dimethyl-2-oxonicotinate. The use of the RH 532 is not under public’s hand. It has also been used as chemical hybridizing agents in breeding some crops. Its effect is similar to RH 532. The RH 532 is not so selective in inducing male sterility without loss in female fertility. Three kilograms of the RH 532 is enough to cause 100% male sterility in the crop of the wheat for hybridization.

LY 195259
It is one of the best hybridizing chemicals which does not produce any harmful effects on hybrid seeds setting and quality of the seeds obtained. From the perspective of commercial hybrid seed production, the hybridizing chemical is one of the best which causes more than 95% male sterility in wheat if sprayed in foliar form at the rate of 1.12 kg active ingredient of the LY 195259 per hectare.

The correct stage of the application is when the female parent population of the wheat crop plant have very young spike of 5 mm long in the abdomen. It is the stage at which no pollen mother cells have formed in any of the florets. Soil surface application of the hybridizing chemical is also possible.

High dose of HYC may prevent anther development or produce abnormal anthers causing more than 90% male sterility in most of the bread wheat varieties. The hybrid seed set recorded up to date with the HYC is up to 80% in wheat crop.

RH 0007 (Hybrex)
The hybridizing chemical: RH 0007 has been in use for engineering hybrid seed of wheat in the USA since 1980. Ninety to 100% male sterility have been recorded with the application of the RH0007 application. Most of the bread wheat genotypes respond the hybridizing chemical very well with respect to cause male sterility induction.

Red spring wheat requires 0.2 kg whereas red winter wheat requires 2 kg for emasculation of each hectare of the wheat crop field. The hybridizing chemical causes male sterility uniformly on all the tillers, but does not injure egg cell fertility and seed set. The hybridizing chemical is also sold in the trade name of Hybrex in the USA. Hybrex inhibits pollen development injuring the meiotic cell division in pollen mother cells and male gamete formation.
RH 5148
RH 5148 is also similar to Hybrex with respect to the induction of male sterility and hybrid seed setting. The RH 5148 is partially soluble in water which is the main drawback of the application of the chemical.

Shell’s W 84811 
The crops of wheat, barley, maize and oats respond well with Shell up to 100% for male sterility induction. Male sterility can be obtained in the wheat crop species from the hybridizing chemical. The stage of the plants for its application is from preboot to boot stage or during the origin of pollen mother cells. The appropriate dose recorded for its application is 1.2kg per hectare. But the hybridizing chemical reduces the receptivity of the stigma of the female plant population to the pollen from male parent population.

Hybridizing chemicals in plant breeding
Ample sunshine favors the spontaneous crossing of the female parent population from male parent population. In colorful flowers, honeybee population should be used as the pollinator. In cereal crop species such as rice, wheat, sorghum, maize; mild wind blow favors the spontaneous crossing for the artificial chemical hybridization.

We must be careful in the selection of the breeding lines of male and female parent for synchronous flowering. Planting date manipulation and nutrient supplementation can be done to make synchronous flowering of male and female parent populations.

Precise hybridizing chemicals, precise stage of plant population and precise concentration of the chemicals are the most important factors to be considered for successful chemical hybridization.  We must be careful while applying hybridizing chemicals to female parent population in order to protect male parent population rows from the chemicals.

  • Anascenko, A. V. 1967. Chemical castration of sunflower (in Russian). Dok. Vsesojuznoj Ordena Lenina (Moskva) 2: 17-18.
  • Badino, M. 1981. Gametocidal effects of gibberellic acid (GA3 + GA4+7) on common onion (Allium cepa L.). Acta Hort. 111:79-87.
  • Batch, J.J. 1978. Chemical gametocides for wheat and barley in relation to F1 hybrid breeding programmes. P.33-43. In: Proc. Joint BCPC and BPGRG Symp. Opportunities for Chemical Plant Growth Reulation.
  • Hansen, D. J., S.K. Bellman, and R.M. Sacher. 1976. Gebberellic acid-controlled sex expression of corn tassels. Crop Sci. 16:371-374.
  • Hensz, R.A. and H.C. Mohr. 1959. Functional male sterility in watermelon induced by chemical treatment. Proc. Amer. Soc. Hort. Sci. 74:552-554.
  • Hockkett, E.A. and K.C. Feltner. 1978. Sterility induced in barley by a synthetic pyridine. Crop Sci. 18:296-301.
  • Iyer, C.P.A. and G. S. Randhawa. 1965. Chemical induction of pollen sterility in grapes. Curr. Sci. 34:411-412.
  • Kho Y. O. and J . W. De Bruyn (1962). Gametocidal action of Dichloroacetic acid. Euphytica 11: 287-292.
  • Kiermayer D.  1959. Induction of male sterility in flowers of Helianthus annus with 2,3,5-triiodobenzoic acid. Naturwissenschaften 46:457.
  • Moore, R.H. 1950. Several effects of maleic hydrazide on plants. Science 112: 52-53.
  • Natrova Z. 1973. Potential use of maleic hydrazide and dalapon for inducing pollen sterility in rye.  Genet. Slecht. 9:163-172.
  • Naylor, A.W. 1950. Observations on the effects of maleic hydrazide on flowering of tobacco, maize and cocklebur. Proc. Natl. Acad. Sci. 36:230-232.
  • Nelson, P.M. and E.C. Rossman. 1958. Chemical induction of male sterility in inbred maize by use of gibberellins. Science 127:1500-1501.
  • Piguemal, G. 1970. How to produce hybrid sunflower seeds by inducing male sterility with gibberellic acid. P. 127-135. In: Proc. Fourth International Sunflower Conf.
  • Rehm, S. 1952. Male sterile plants by chemical treatment. Nature 179:38-39.
  • Rowell, P.L. and D.G. Miller. 1971. Induction ofmale sterility in wheat with 2-chloreethylphosphonic acid (Ethrel). Crop Science: 11:629-631.
  • Schuster, W. 1969. Experiments on male-sterility of sunflowers caused by genetical, physiological and applied chemical factors. Theor. App. Genet. 39:261-273.
  • Van der Meer, Q.P. and J.L. van Bennekom. 1982. Gibberellins as gametocides for the common onion (Allium cepa L.). III. GA4+7 and pollen viability. Euphytica 31:503-506.
  • Van der Meer, Q.P. and R. van Dam. 1979. Gebberellic acid as a gametocide for cole crops. Euphytica 28: 717-722.
  • Wang, R. C. and S. Lund. 1975. Studies on male sterility in barley induced by sodium 1-(p-chlorophenyl)-1,2-dihydro-4,6-dimethyl-2-oxonicotinate. Crop Science. 15:550-553.
  • Warren, F.S. and F. Dimmock. 1954. The use of chemicals and of pollen male sterility to control pollen production in corn. Can. J. Agri. Sci. 34:48-52.
  • Yih, R. Y., P.J.McNulty, M.C. Seidel and K.L. Viste. 1971.Plant growth regulating properties of 3-carboxy-2-pyridones. HortScience 6: 460-461.
 Thanks for visiting the webpage of the “Chemical Emasculation in Plant Breeding: Chemical Gametocides: Hybridizing chemicals.”


  1. Great blog nice n useful information , it is very helpful for me , I realy appreciate thanks for sharing. I would like to read more information thanks.

    Antirrhinum majus nanum

  2. dear sir my name is sami .im from jordan in medlleeast. i need to now the best chemical to make male stalrty in tomato in greenhouses . please can u help me