Techniques and methods to overcome self-incompatibility barriers

In several crops, self incompatibility (SI), in which self pollen or pollen tubes

are inhibited on the stigma surface, is a useful tool for the production of commercial F1

hybrid seed. However, the existence of SI constitutes a problem in obtaining parental

inbred lines. In many situations, e.g., during the production of inbreds for use as parents

in hybrid seed production, it is essential that temporary self-fertility is achieved in

manner so that self-incompatibility is fully functional in the selfed progeny. Such self-

fertility is known as pseudofertility and is achieved by temporarily suppressing the

incompatibility reaction using one of the following techniques.

(A)Mixed pollination:

A mixture of live compatible pollen and killed (by chemicals or irradiation)

compatible pollen, which is called as “Mentor” or recognition pollen is employed for

pollination. The treatments used to kill the pollen do not disturb the wall held proteins.

When the killed compatible pollen along with incompatible pollen is used for pollination,

the proteins emitted from recognition pollen mask the inhibition reaction at the surface of

the stigma. Chatterjee and More (1991) employed technique of mentor pollen, in addition

to use of benzyl adenine (1%) to overcome pre-fertilization barrier during inter-specific

hybridization of C. melo with C. figari, C. meeusii, etc.

(B) Bud pollination:

In some plant species pollination at early bud stage has proved very effective

in overcoming the incompatibility. Bud pollination means application of mature pollen to

immature non-receptive stigma, generally 2-4 days prior to anthesis. This is most

practicable and successful method both in the gametophytic and sporophytic system.

Obviously, SI system does not become active until shortly before anthesis. In some cases,

lack of stigma receptivity may be a problem e.g., in Lilium longiflorum. Seed set varies

considerably in different inbred lines. Bud pollination is adequate for the production of

inbred lines, but for their multiplication, other methods need to be employed. In some

cases, application of the fluid from mature stigmas may improve the success of bud

pollination.

In Petunia axillaries delayed pollination or pollination with stored pollen

could not shatter the self-incompatibility barrier, but self pollination of buds, two days

before anthesis resulted in seed set (Shivanna and Rangaswamy, 1969). At the bud stage

stigma probably lacks the exudate, which appears only at anthesis. Probably protein

secretion covers the stigmatic surface, which acts as a barrier to penetration of stigma by

germinating pollen grains. Protein secretion is probably laid down just before anthesis. If

buds are opened and pollen is placed on stigmatic surface i.e., before protein barriers

formed, seed set may be obtained. For inbred line production of cabbage, bud pollination

has been recommended. With self-incompatible plants of cauliflower, bud pollination has

been found to be effective in self-fertilization.

(C)Surgical Techniques:

Removal of stigmatic surface, the whole of stigma or a part or whole of the

style may permit an otherwise incompatible mating. Removal of the stigma is very useful

in the sporophytic system, e.g., Brassica oleracea, but it does not work in B. compestris.

In B. napus stigma removal may be more effective than bud pollination, while removal of

the style is helpful in some cases of gametophytic self-incompatibility e.g., Petunia. In

Petunia the whole of the style may be removed and the pollen grains may be directly

dropped on to the ovules in ovarian cavity.

(D)Stub pollination:

In those cases where incompatibility reaction is confined to stigma or in those

cases where the length of style of the female parent is relatively longer than the pollen

parent, removal of stigma and part of the style has been helpful to overcome the

incompatibility barriers. In Ipomea trichocarpa the primary site of the self

incompatibility is the stigmatic surface, which inhibits pollen germination. Removal of

the stigmatic lobes or trimming the terminal half of the style and placing the pollen on cut

surface of style, results is good-pollen germination and pollen tube growth down upto the

ovary, without any inhibition.

(E) Intra-ovarian pollination:

Where the zone of incompatibility resides on stigma or in the style, direct

introduction of pollen suspension into the ovary may be helpful to attain self-pollination.

In this technique the ovary surface is sterilized with ethanol. Two holes are made on the

ovary wall – one for the introduction of pollen suspension and another for escape of the

air present in ovarian cavity (Bhojwani and Bhatnagar, 1988).

(F) Test-Tube Pollination:

In this method the stigmatic, stylar and ovary wall tissues are completely

removed from the path of pollen tube and the exposed ovule is directly dusted with pollen

grains from same plant. The pollinated ovules are then cultivated in a suitable nutrient

medium, which favours pollen germination as well as development of fertilized ovule

into the seed.

(G)Late season or End-of-season Pollination:

In some species, the degree of incompatibility is reduced towards the end of

flowering season or in mature plants. A tobacco plant self-incompatible throughout the

season was self-compatible at end of the season (Allard, 1960). But there are

controversial reports on the usefulness of this technique.

(H)Repeated pollination:

Repeated pollination with the incompatible pollen can sometimes lead to

selfed seed formation. Repeated pollinations in self-incompatible chicory led to some

selfed seeds (Kalloo, 1988). Further CO2 treatment was found to be effective to overcome

self-incompatibility barrier in Brassica.

(I) Double Pollination:

In some species, self-incompatible matings become possible when

incompatible pollen in applied as a mixture with a compatible pollen, or it is applied after

pollination with compatible pollen.

(J) High temperature or heat treatment:

In some species, e.g., Trifolium,Lycopersicon, Brassica, Oenothera. etc.,

exposure of pistils to high temperatures upto 600C induces pseudo-fertility. In some lines,

temperatures of 300C or more induce seed set. But in many cases, it is genotype-

dependent response. For example, in Trifolium hybridum a single dominant gene

produces self-incompatibility at 320C. In B. compestris, polygenes reported to influence

this response. In addition, high temperature may reduce the strength of self-

incompatibilty.

Heat treatment of pistil upto 55-600C reduces the self-incompatibility in

Lycopersicon peruvianum. High temperatures reduced the incompatibility in cole crops

also (Kalloo, 1988).

(K)Increased CO2 Concentration:

Increased CO2 concentration is reported to overcome self-incompatibility in

the sporophytic system. This approach has promise for use on large scale for

multiplication of inbred lines. Plants of Brassica napus and B. oleracea (Kale and

Brussels sprouts) were grown in polythene tunnels (2 m high) with CO2 pellets; a small

fan was used for air circulation. Pollination was effected by either hand or blowflies, and

a good seed set was obtained.

(L) High humidity:

High humidity can be created simply by enclosing the inflorescence within a

suitable bag, e.g., polythene bags. High seed set in Brussels sprouts was obtained by

covering inflorescence with cellophane bags and introducing within them blowflies as

pollinators. This could have been due to increased CO2 levels due to blowfly activity or,

possibly due to increased humidity. Covering of the inflorescence with polythene bags

resulted in rapid increase in humidity inside the bags. This also resulted in seed set from

similar lines of Brassica sp. A combination of high humidity and high CO2 concentration

is very effective in promoting high seed in cauliflower and B. napus; this type of gas

mixture is obtained by breathing out. Good seed set was obtained in B. napus when the

inflorescence was enclosed within a polythene bag and its air was displaced with the

breathed-out gas mixture simply by blowing into it.

(M) Salt (NaCl) Sprays:

Chinese workers have developed a salt spray technique to overcome self-

incompatibility in B. napus. In this technique, the flowers are sprayed with a 5-10%

sodium chloride solution for 3-5 days. The method is easy, economical and as effective as

bud pollination. It has also been shown to work with B. compestris and B. oleracea.

However, it is not known if it will work with gametophytic system as well. This approach

in any case has the promise to develop into a simple and reliable methods of field

multiplication of self-incompatible inbred lines and of hybrid seed production,

particularly in case of complex hybrids.

(N) Irradiation:

In the single-locus gametophytic system, e.g., in Solanaceae, acute irradiation

with X-rays or gamma-rays induces a loss of self-incompatibility. It induces mutation in

the incompatibility alleles. Gametophytic incompatibility system has been much easier to

change through the mutation than the sporophytic system. By application of X-rays, P32

and chemical mutagenes to pollen mother cells, Lewis (1954) was able to mutations of S

alleles to self fertile types (sf) in Prunus avium. Mutations were easy to identify on selfed

plants because the mutated pollen grains accomplished fertilization and led to seed

production. According to Kalloo (1988) irradiation of pollen is effective in overcoming

the self-incompatibility reaction of pollen.

(O) Grafting:

Grafting of a branch onto another branch of same plant or of another plant is

reported to reduce the degree of self-incompatibility in Trifolium pretense. There is only

one report on this phenomenon and the mechanism of this reduction is not known.

(P) Polyploidy:

It was noticed that the self-incompatibility system observed in diploids of a

species was often weakened, or disappeared, at the polyploidy level (Briggs and

Knowles, 1967). This is particularly true for gametophytic system. However, where

incompatibility is determined sporophytically, it is not altered greatly due to doubling of

chromosome number. Pear was self incompatible when diploid but was compatible as

autotetraploid. Briggs and Knowles (1967), were of opinion that when pollen grain

carries two different alleles in a tetraploid, they might interact to neutralize the

incompatibility reaction. Thus pollen grain carrying two different alleles may be function

on stigma whose style carries same two alleles for self-incompatibility. Thus self

pollination could be possible. But when pollen grain carries same two alleles, interaction

is not possible and the pollen may not be functional on stigma with style carrying same

two alleles as in the pollen.

(Q) Other Techniques:

A number of other techniques have been tried with varying degrees of

success, but they are not commonly used. These techniques are:

treatments of flowers with carbon-monoxide

injecting styles with immunosuppressant

application of electrical potential difference of about 100 V between

the stigma and pollen grains

treatments of pistil with phytoharmones

treatments with protein synthesis inhibitors

steel brush pollination

Examples:

(1) Tetsu Nakanishi and Kokichi Hinata (1975), studied self-seed production by

CO2 gas treatment in self-incompatible cabbage. An adequate number of self-

seeds was obtained in self-incompatible cabbage (Brassica oleracea L. var.

capitata) when CO2 gas was supplied to flowers after self-pollination. A strain,

which set 0.2 self-seeds per flower in the ambient air condition, set over 10 self-

seeds per flower when treated with 3.6-5.9% CO2 for 5 h. In a strain with weaker

self-incompatibility, a 4 h treatment with 1.4% CO2 was still effective. Seed set in

bud-pollination was also enhanced by applying COz. This method is so simple

that it may be used for practical self-seed production in Brassica vegetables.

(2) Sachiko Matsubara(1981) done study on overcoming the self-incompatibility of

Lilium longiflorum THUNB . by application of flower-organ extract or

temperature treatment of pollen The application of flower organ extracts to

stigmas and the temperature treatment of pollen were tried to overcome self-

incompatibility of Lilium longii forum cv . Georgia. Substances in stigma, style,

ovary and anther were extracted with ethanol and fractionated with ethyl acetate

into the acidic, basic and aqueous fraction. The extracts melted in a small volume

of distilled water were applied to stigmas prior to self-pollination. Hinomoto

stigma extract, self-pollinated and cross pollinated Georgia stigma extracts of

high concentrations and Georgia anther extract of high concentration were

effective in overcoming the self-incompatibility and resulted in a high percentage

of fruit set and many normal seeds . Extracts from Hinomoto ovary, style and

anther were ineffective, except a basic fraction, which was very slightly effective.

Pollen was treated with 400C for 60 or 90 minutes and 500C for 30 or 60 minutes,

and a half of each lot was followed by -20°C for 24 h, prior to self-pollination. All

treatments were effective, especially at 40°C for 60 minutes or 500C for 30

minutes, and 400C for 90 minutes or 500C for 60 minutes followed by -200C for

24h.

(3) Overcoming self-incompatibility by application of three kinds of plant

hormones, sucrose, 3 kinds of amino acids and 2 kinds of vitamines was tested in

cvs. Honbashi-taibyo Minowase (H-Mino) and Minowase (Mino) of Raphanus

sativus by Sachiko Matsubara (1984). Effects differed between the cultivars. In

`H-Mino', BA (100 mg/I) and glutamic acid, folic acid and nicotinic acid (500

mg/l) resulted in higher fruit set and higher number of seeds per pollinated flower.

In 'Mino', BA and NAA (100 mg/1) and glutamic acid and glycine (500 mg/1)

induced a high number of seeds per pollinated flower. These chemicals, however,

induced parthenocarpic fruit set, especially GA3. From the observation of pollen

on stigmas washed with glutamic acid, it appeared that the pollen-tube penetrated

into a papilla cell after 1 hour and openings of papillae and detached pollen grains

and tubes were found after 2 hours as the result of successful pollen tube

penetration of papillae. Pollen was heated at 50°C for 30, 45 or 60 minutes, at

60°C for 15, 30 or 45 minutes and at 70°C for 10, 20 or 30 minutes prior to self-

pollination . In 'H-Mino', 60 and 70°C were effective, and expecially 600C for 15

or 30 minutes resulted a higher percentage fruit set and more seeds per fruit. In

`Mino', although 50-700C were effective, the mean number of seeds per pollinated

flower was lower than in 'H-Mino'.

(4) K. Okazaki and K. Hinata studied the effect of short-term high temperature

on the expression of self-incompatibility in detached flowers of Brassica

oleracea, B. campestris and Raphanus sativus. The expression of self-

incompatibility was repressed by treatment of pistils at 400C for 15 minutes.

Treatment at 500C repressed self-incompatibility but it also disturbed pollen tube

elongation into stylar tissue. S-glycoproteins did not show any quantitative

changes during the intact pistil treatment under 500C Callose was occasionally

found in the treated papilla where the self pollen tube penetrated. The repressing

effect of the 400C treatment was found to be reversible, and this reversibility

depended upon the environmental temperature of plant. Plants grown at 15/50C

(day/night temperature) completely recovered self-incompatibility 2 h after

treatment, while those grown at 20/100C, 25/150C did not. The reversibility of the

expression of self-incompatibility correlated with the distortion of plasma

membrane in the papilla. It is considered that high temperature affects the pollen

tube penetration system in pistils rather than the recognition system between

pistils and pollen. The treatment of dehiscing anthers at 400C killed the pollen.

(5) Palloix and et al., (1985) studied the effect of carbon dioxide and relative

humidity on self incompatibility in cauliflower, Brassica oleracea. The events of

the progamic phase of fertilization have been monitored by in vitro experiments in

self compatible (SC), partial self-incompatible (PSI) and self incompatible (SI)

lines. The duration of the progamic phase is about 30 h. Treatment with low

concentrations of CO2 (3 to 5%) at high relative humidity (rH, 100%) had the

following effects: pollen quality, which declines normally during flower ageing,

was prematurely reduced; pollen adhesion and germination, both low in SI

matings, were increased; the stigma callose response in SI matings was reduced to

the low level of SC matings; and the number of pollen tubes in the style after SI

matings significantly increased. CO2 concentrations of 4 to 6% applied for 8, 16

or 24 h at 100% rH proved to be the most effective treatment for blocking the SI

response in cauliflower. (6) D. G. Voyiatzis showed overcoming self-incompatibility and increasing fruit set

in olive trees with benzyladenine. In olive (Olea europaea L.) orchards, fruit set

may be very low when a self-incompatible cultivar is grown without pollinizers,

or the existing pollinizers differ in the time of blooming. Since most greek olive

cultivars are partly self-incompatible (Porlingis and Therios, 1973), a problem of

low productivity may arise when no appropriate pollinizers are interplanted with

the main cultivar. Early tests with various chemicals for increasing fruit set in

olive had no effect (Hartmann, 1950). Later, it was shown that self-

incompatibility could be overcome and fruit set increased in the partly self-

incompatible olive cv. “Chalkidikis” with benzyladenine (BA), (Porlingis and

Voyiatzis, 1976). Cytokinins have also been found to increase fruit set in

muskmelon (Jones, 1965), and to help overcome self-incompatibility in Lilium

(Matsubara, 1973; Henny and Ascher, 1975). The purpose of this work was to

investigate further the role of BA in increasing fruit set in three partly self-

incompatible olive cultivars, and to study some factors, as the surfuctant Tween-

20 and the solvent dimethylsulfoxide (DMSO), that might influence this effect.

They also found the positive results in overcoming self-incompatibility with

benzyladenine application.

(7) Neelam Sharma and K. R. Shivanna (1986) studied the effect of treatment of

the stigma with an extract of a compatible pistil overcomes self-incompatibility in

Petunia. This treatment just before pollination, was very effective in overcoming

self-incompatibility in Petunia hybrida. Progressive delay either in the application

of the extract or in pollination marketedly reduced its efficacy in overcoming self-

incompatibility. The treatments seems to mask the self-incompatibility

recognition molecules of the pistils.

(8) Monteiro, A. A. studied use of sodium chloride solution to overcome self-

incompatibility in Brassica campestris. Sodium chloride solution applied to the

stigmas of self-incompatible plants of the rapid-cycling stock CrGC1 induced self

seed setting. Maximum seed set/siliqua occurred on flowers treated 10 to 15

minutes before self pollination with sodium chloride solution applied either by

means of a micropipette (8.2 seeds/siliqua) or with a moistened cotton wool swab

(7.2 seeds/siliqua). The salt treatments increased pollen adhesion and pollen

germination on the stigmas and reduced callose formation on the papillae.

(9) Neelam Sharma and et al., 1986 studied the use of Lectins and Sugars in

Petunia and Eruca to overcome self-incompatibility. Treatment of stigma with a

lectin (Con A/PHA) before pollination was effective in overcoming self-

incompatibility in Petunia hybrida, a gametophytic self-incompatible system, and

Eruca sativa, a sporophytic self-incompatible system. Treatment of pollen with

glucose/N-acetyl-D-galactosamine (tested only with Petunia) was also effective.

These results suggest the involvement of pollen lectins and specific sugar

components of the pistil in self-incompatibility recognition.

(1) S. Niikura and S. Matsuura (2000) shown that in radishes, self-incompatibility

(SI) is governed by the S-locus, which consists of a series of multiple alleles. This

SI can be overcome by CO2 gas treatment, a characteristic that is very useful in

obtaining large amounts of parental seeds for F1 commercial seeds. They have

used 4% CO2 to overcome self-incompatible and found that there are genetic

variations in the reaction level of self-incompatibility (RLSI) to a 4% CO2 gas

treatment in the radish.

(1) Wancang Sun and et al., (2005) studied the overcoming self-incompatibility in

Eruca sativa by chemical treatment of stigmas. As a member of the tribe

Brassiceae, Eruca sativa, although a minor crop worldwide, is considered a

valuable genetic resource for cabbage, rapeseed and other Brassica crops. Self-

incompatibility (SI) in Brassica has been extensively studied, but information on

SI in E. sativa is limited. Of six chemicals used to treat the stigmas to overcome

SI in five E. sativa lines, gibberellin was the most effective. As gibberellin is

well known for its ability to break dormancy and to promote cell elongation, its

effectiveness may help to understand the mechanisms of SI. Urea and ammonium

sulphate were also effective. These two chemicals are known to affect protein

stability, which may help explain their effects on SI. Although table salt has been

reported as being effective in overcoming SI in B. rapa, B. oleracea and B. napu,

it was not effective in E. sativa. Sucrose and alcohol also had negligible effect.

There was significant variation among the genotypes in SI intensity and response

to chemicals, but the genotype–chemical interaction was not significant. The data

presented in this paper add to their understanding of SI in E. sativa and may lead

to a better use of this genetic resource.

(2) Nasrallah and et al., (2007) studied epigenetic mechanisms for breakdown of

self-incompatibility in interspecific hybrids. They suggested that as a major agent

of rapid speciation, interspecific hybridization has played an important role in

plant evolution. When hybridization involves species that exhibit self-

incompatibility (SI), this prezygotic barrier to self-fertilization must be overcome

or lost to allow selfing. How SI, a normally dominant trait, is lost in nascent

hybrids is not known, however. They demonstrate that hybrid self-fertility can

result from epigenetic changes in expression of the S-locus genes that determine

specificity in the SI response. They analyzed loss of SI in synthetic hybrids

produced by crossing self-fertile and self-incompatible species in each of two

crucifer genera. It showed that SI is lost in the stigmas of A. thaliana–lyrata

hybrids and their neo-allotetraploid derivatives and in the pollen of C. rubella–

grandiflora hybrids and their homoploid progenies. Aberrant processing of S-

locus receptor kinase gene transcripts as detected in Arabidopsis hybrids and

suppression of the S-locus cysteine-rich protein gene as observed in Capsella

hybrids are two reversible mechanisms by which SI might break down upon

interspecific hybridization to generate self-fertile hybrids in nature.

(3) Jovanka D. and et al., (2007), isolated pistils of distylous buckwheat

(Fagopyrum esculentum Moench) were treated with protease inhibitors (PMSF,

pepstatin A, and antipain). Pistils were cross- or self- pollinated, and growth of

pollen tubes was observed under a fluorescence microscope. Treatments with all

inhibitors suppressed inhibition of self-pollen tube growth, suggesting that

activity of proteases is involved in rejection of self-pollen during the SI response.

All these studies indicated that there are several different ways to

overcome self-incompatibility which helps in developing inbred lines for hybrid seed

production in self compatible crops.