Proe. Indian Ac, ad. Sci. (Plant Sci.), Voi. 91, Number 3; Junr 1982, pp. 227-234. O Printed in India

Effect of CO2 in ovecroming self-incompatibility bar¡ in Brassica campestris L. var. toria

A S DHALIWAL* and C P MALIK Department of Botany, Punjab Agricultural University, Ludhlana 141004, India *Seed Teehnology Section, Department of Plant Breeding, Punjab Agricultural University, Ludhiana 141 004, India

MS reeeived I May 1981 ; r•vised 14 May 1982

Abstraer. CO2 mediated induction of pollen adhesion, germination, tub• pene- tration, tube growth through the stylar tissue and seed set was studied in Brassica campestris L. var. toria following incompatible pollination. This report stressr the effect of CO2 not at a single step but at various levels which normally prevent and/or hinder successfui fertilization and seed set. The possible role of CO2 in overcoming these barriers is discussedo

Keywords. Brassica campestris L. var. toda; incompatibility; CO2; seed set.

1. Introduction

The incompatibility reaction in Brassica is sporophytically controlled with multiple S-alleles (Bateman 1955 ; Th£ 1957). Double cross hybrids raised from self-incompatible inbred lines would give low yielding crop because of high level of cross-incompatibility with each other. In recent years, two developments have taken place to overcome such problems, One is based on the manipulation o f genetic attributes (Thompson 1978) and the other involves putting the self-incom- patible plants in an atmosphere of 3 to 5% COz after selfing the flowers (Nakanishi et al 1969, 1975). The latter technique is now successfully employed in obtaining self-seeds.

Following incompatible pollination various check points exist at the level of pollen germination, adhesion, germination, tube penetration, tube growth through the stylar tissue and ultimately seed set. The present study describes the effect of CO2 on the aforementioned levels following illegitimate pollination.

2. Material and methods

Seeds o f Brassica campestris L. var. toria were obtained f rom the Department o f Plant Breeding, Punjab Agricultural University, Ludhiana and the plants were

227

228 A S Dhalq and C P Malik

raised in the Botanical Garden. Experiments were performed in full ¡ season to avoid early or late season effect.

Excised pistil technique was employed to study the effect of CO2 on pollen adhesion, germination, pollen tube penetration and growth through the stylar tissue. Flower buds were emasculated in the evening before opening. Next morning the flowers were removed and the pedicels with pistils were embedded in 1% agar medium in small glass vials. Self- and cross-pollinations were done with a fine brush to obtain a thin and even coating of pollen on the stigmatic surface. The vials containing self- and cross-pollinated pistils were put in a glass container which was sealed with parafilm. COz was injected with the help of an air tight syringe to obtain 4% concentration. After incubation for 6 hr at 22+2~ pollen adhesion, germination and tube penetration into stigmatic papillae were studied by staining pistils with cotton blue (0.1%) in lactophenol.

Growth of pollen tubes in cross-pollinated pistils without CO2 and self-pollinated pistils with 4% CO2 was followed at times ranging between 6 to 30 hr after polli- nation. The number of pollen tubes in 0.5 mm region alongwith the length of the pistil were counted in the two situations (three replicates of six flowers each).

The emasculated flowers were pollinated with self- and cross-pollen and enclosed within known volumeofpolythene chamber and 4% CO9 was injected with an air tight gas syringe. After exposing the flowers for 6 hr the polythene chamber was replaced by emasculating bags. The number of seeds formed per siliqua was deter- mined 50-60 days after pollination.

3. Results

Due to full operation of self-incompatibility, adhesion of self pollen to the stigmas was low. Those which made contact with the papillae showed poor germination. Figure l(a, b, c) depicts the adhesion of pollen grains in three situations. Figure la shows incompatible pollination with very few pollen grains adhering to the stigmatic sufface and without any germination. Figure lc is that of compatible pollination where most of the pollen grains are adhering to the papillae. Pollen grains show germination and pollen tubes grow in the stigmatic and stylar tissues. The firm attachment of pollen grains, following self-pollination, was observed when they were enclosed in an atmosphere of CO2 (4%) (Figure lb).

The data given in table 1 show that no pollen tube was recorded in the pistil following self-pollination. In majority of the styles, the longest pollen tube reached almost the same position in response to both self-pollination -I- CO2 and cross-pollination styles. The data on percentage of pollen tubes present at different levels of the pistil after 24 hrs of pollination are set in figure 2. A gradual reduction in the number of pollen tubes from the top of the pistil down into the ovary was noticed. A decrease in cross pollen tubes as well as self pollen tubes (in the presence of CO2 ) is evident. This decrease was more pronounced in the cross pollen tubes as compared with the self pollen tubes with CO2.

Table 2 shows the number of seeds produced per siliqua after different pollination treatments. The number of seeds per siliqua following bud pollination was nearly

Self-incompatibility barriers in Brassica campestris L. 229

C T

Figure 1. Pollen adhesion, germination and tubr penetration after 6 hr pollination. The stigmas alongwith styles were stained gith 0.1% cotton blue in lactophenol. (a) Noticed very few pollen grains adhering to the stigmatic surface following self-pollination (b) The number of pollen grains sticking to the stigmatic surface is more as comparcd with figure (a) following CO2 trealment Some pollen tubes are pcnetrating the stigmatic papillae. (c) Most of the pollen grains germinate and penetrate following cross-pollination (pollen tubes arrowed).

Self-incompatihility barriers in Brassica campestris L. 231

Table 1. Growth of pollen tubes in the pistil at different times after pollination in cross-, ~lf- and self q- CO2 laollinations. Data are the mean of six flowers.

Time after Pollination Maximum Rate of pollen tube growth pollination treatment oollen tube mm/hr

(hr) length (mm) Max. rate Mean rate

6 CP 0.66 0.11 0.05 SP + CO,2 0.45 0.08 0.04 SP 0.03 0.01 0.00

11 CP 1.40 0.13 0.07 SP q- CO2 1.35 0.12 0 05

18 CP 3.00 0.17 0.09 SP -t- CO~ 2.70 0.15 0.05

24 CP 3.50 0.15 0.08 SP -F CO~ 3,00 0.13 0.07

30 CP 4.00 0 13 0.07 SP q- CO~ 3.60 0.12 0.05

Table 2. Effect of CO2 (4%)on seed production following self- and cross-pollination.

Pollination Self seeds Cross seeds treatment Number of Number of Number of Number of

fruits seeds per fruits ~eeds per fruit fruit

Bud 36 17.3 30 18.2 Open flower

--CO2 0 0.0 38 19.6 +COs 34 8.2 30 18.8

same in the two s i tua t ions i. e. self- and c ross -po l l ina t ions . N o seed set was recorded when the f reshly opened flowers were se l f -po l l ina ted . H o w e v e r , a b o u t 8 seeds per s i l iqua were fo rmed when se l f -pol l ina ted flowers were kep t in 4 % COs a tmosphe re for a b o u t 6 hr. CO2 d i d not affect seed set af ter c ros s -po l l i na t ion .

4. Diseussion

The s t rong se l f - incompa t ib i l i t y ba r r i e r exh ib i ted by Brassica campestris var. t o r i a depends on the recogni t ion and the re jec t ion reac t ion . The events g r o u p e d in these two stages cons t i tu te the var ious c h e c k ' p o i a t s as men t ioned ear l ier . The re jec t ion response is c h a r a c t e r i z e d by cal lose depos i t ion .

The adhes ion o f the po l l en gra in to the s t igmat ic surface sectas to be the in i t ia l bu t mos t i rnpor tan t event to ensure po l l en ge rmina t i on and tube pene t r a t i on . CO2 p lays an i m p o r t a n t ro le in the adhes ion o f the po l l en gra ins to the self s t igma surface as is ev ident f rom the presence o f more pol len gra ins on the self s t igma fo l lowing CO2 t r ea tmen t than con t ro l (wi thout CO2) even 6 hr a f te r po l l i na t i on ,

232 A S Dhalq and C P Malik

too

. o

80

J g 8O

4O +6 + ~ 2o

e - - e C t~$ e - - - o S e l f -i- CO 2

, , A

OiStsnc~ t r o m lop ol st|gres(mm)

Figure 2. Percentage of pollen tubes at differ~nt levels from top of the stigma follovdng 24 hr of pollination.

If pollination is compatible the tryphine is induced to 'gel' and promotes both adhesion and hydration of the pollen grain (Dickinson et al 1980). The number of pollen grains adhering to the stigma surface following cross-pollination is higher than self-pollination. Stead et al (1980) proposed that differences in the degree/ extent of adhesion may result from physical or chemical alteration(s) taking place in the components that bind the pollen to the stigma surface. They suggested the involvement of stigma surface proteins in the pollen grain adhesion which have a rapid turnover rate. C(:2 has been shown to affect changes in the kinetic behaviour of allosteric proteins (Mitz 1979). CO2 may be affectingpollen adhesion through the above mentioned processes. CO2 has been reported to affect protein synthesis in maize roots (Splittstoesser 1966), Chlorella (Miyachi and Hogestu 1970) and germinating Amaryllis vittata P011en grains (Sharma et al 1981).

Dhalq and Malik (1980) and Dhalq et al. (1981) have suggested the role of CO2 in pollen hydration and recognition rejection response which are essential for the activation of enzymes and acceptance of pollen grain, respectively. Non- spec]fic esterases have been suggested to be involved in the active cutinase complex essential for the breakdown of cuticle. C02 stimulates the leaching of these esterases on to the stigma surface following illegitimate pollination (Dhalq and Malik 1982).

CO2 increased both pollen germination and tube penetration following incom- patible pollination. CO2 action was correlated with the metabolic changes of pollen tubes and/or papilla cells at the time of their attachment (Nakanishi et al 1969). The growth rates of pollen tubes in self-pollination + CO2 and cross- pollination styles indicate that the fastest self pollen tube (in presence of CO2) grew nearly as fast as the fastest cross pollen tube. The gradual reduction in the number ofpollen tubes from the top of the pistil down into the ovary indicate much variation in the tate of pollen tube growth. Thus, the most rapidly growing tubes registered two to three times faster rate than the average tubes. This deerease in the number is more pronounced in the cross pollen tubes as compared to selfpollen tubes in the presence of CO2. The variatioa in the growth tate may be attributed to competition between the pollen tubes for the nutrients. Clearly, single pollen

Self- incompatibi l i ty barriers in Brassica campestris L . 233

i:~)i[en iands on the stlgma

Adhesion

Hydration

Germination

C~tinase~act ivation

Penetration

erowlh through the stytar tlssue

1

. m . - - J p ~

. m . - - 4 b - -

�91

ee,- - , .b-

Figure 3. Effect of COz (4%) on various check points that lead to self-incompatibility in Brassica camoestris L.

tube growing th rough the style will have less compet i t ion with each other than several o f them growing at the top o f the stigma. The seed set increased when the freshly opened flowers were self-pollinated and were kept in 4 % CO2 atmos- phere for about 6 hrs. CO: did not affect seed set after cross pol l inat ion.

Taking the present discussion in conjunct ion with our previous inferences on the tole o f C O , on various check points is indicated (figure 3). We believe that CO2 act at several stages promot ing successful fert i l ization and seed set in incompatible pol l inat ion.

Acknowledgements

Financia l assistance in the form o f Junior Research Fellowship to ASD from Gove rnmen t o f India , Depar tment o f Atomic Energy is gratefully acknowledged.

References

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fresh weight of Brassica campestris L. polIen in vitro ; Indian J. Exp. Biol. 18 1522-1523 Dhalq A S and Malik C P 1982 Release of esterases from incompatible Brassica

campestris L. pollen in vitro and in vivo ; Indian J Exp. Biol. 20 95-96 Dhalq A S, Malik C P and Singh M B 1981 Overcoming incompatibility in Brassicacampestris

L. by carbon dioxide and dark fixation of the gas by self- and cross-pollinated pistils; Ann. Bot. 48 227-233

Dickinson H G, Roberts I N and Stead A D 1980 The role of exine held substances in the development and behaviour of the pollen tube in Brassica : 5th International Palyr~ological Conference. Cambridge, Abs.

Mitz M A 1979 COa biodynamics: a new concr of cellular control; 3". Theor. BioL 80 537-551

234 A S Dhalq and C P Malik

Miyachi S and Hogestu O 1970 Effect of pre-illumination with light of diffcrcnt wavelengths on subscquent CO~ fixation in Chlorella cells ; Can. J. Bot. 48 1203-1207

Nakanishi T, EsashiY and Hinata K 19s Control of sr by CO2 gas io Brassica; Plant Cell Physiol. 10 925-927

Nakanishi T and Hinata K 1975 Self seed produetion by CO~ gas trr in self-incompatiblr cabbage; Euphytica 24 117-120

Sharma S, Singh M B and Malik C P 1981 Dark CO2 fixation dufing germination of Amaryllis vittata pollen in suspension eultures ; lndian. J. Exp. BioL 19 710-714

Splittstor W E 1966 Dark CO~ flxation and its role in the growth of plant tissue; Plant PhysioL 41 755-759

Str A D, Roberts I N and Dickinson H G 1980 Pollen-pistil intr in Brassica oleracea; Events prior to pollen germination; Planta 146 211-216

Thomvson K F 1957 Self-incompatibility in narrow-stem kalr oleracea var acephala L Demonstration of a sporophytie system; J. Genet. 55 45-60

Thompson K 1978 Application of recessive self-incompatibility to production of hybrid rapeseed ; 5th International Rapesr conferenc~. Abstract 12.6-16.6 1978 Malmo, Sweden.