Euphytica 31 (1982) 613-619

POLLEN AND POLLINATION EXPERIMENTS. VII. THE EFFECT OF POLLEN TREATMENT AND

APPLICATION METHOD ON INCOMPATIBILITY AND INCONGRUITY IN LILIUM

J.M.VAN TUYL’, M.CLARA MARCUCC12 and T.VISSERt

’ Institute for Horticultural Plant Breeding (IVT), Wageningen, theNetherlands 21stituto Cultivazioni Arboree, Universita di Bologna, Italy

Received22 January 1982

INDEX WORDS

Lilium longiforum, Lilium ‘Enchantment’, overcoming incompatibility, incongruity, pollen treatment, tem- perature treatment, cut-style pollination, mentor pollen, pioneer pollen.

SUMMARY

Four methods were investigated for their capacity to overcome incompatibility and incongruity in Lilium longiflorum and the Lilium hybrid ‘Enchantment’. Temperature treatments of pollen at 40” to 60°C for one hour had no effect on germination capacity. None of the temperature treatments influenced incompati- bility or incongruity. Application of cut-style pollination had a positive effect on self-pollination of ‘En- chantment’, but did not overcome self-incompatibility in L. longiflorum. With the aid of genetically inacti- vated (by irradiation) compatible ‘mentor’ or ‘pioneer’ pollen self-incompatibility of L. longiflorum could be largely overcome. Both methods also gave promising results in realizing interspecific crosses.

INTRODUCTION

Incompatibility and incongruity (interspecific incompatibility) continuously cause the lily breeder complications in realizing his breeding programme. It is therefore not sur- prising that many studies deal with methods to overcome these barriers by application of growth regulators (EMSWELLER & STUART, 1948; MATSUBARA, 1973), heating of the style before pollination (ASCHER & PELOQUIN, 1966; HOPPER et al., 1967), cut-style pollination (MYODO, 1962; ASCHER & DREWLOW, 1975; CHENG & MATSON, 1972), application of flower organs and extracts (MATSUBARA, 1981) temperature pretreat- ment of pollen (MATSUBARA, 198 1) and application of a mixture of pollen (KUNISHIGE & HIRATA, 1978). From this literature and from previous (unpublished) experiments of the first author it appears that heating or cutting the style before pollination or pollination at relatively high greenhouse temperatures are quite effective in overcom- ing crossing barriers. The mentor pollen technique, succesfully applied to overcome incompatibility as reported by STETTLER (1968) in Populus, DAYTON (1974) in apple, DEN NIJS & OOST (1980) in Cucumis and the pioneer pollen method applied by VISSER (198 1) in apple and pear have not yet been used for Lilium. In this paper four methods designed to overcome self incompatibility or incongruity are compared: the tempera- ture treatment of pollen, used with success by MATSUBARA (1981), the mentor and pioneer pollen technique and the cut-style method.

613

J. M. VAN TUYL. M. CLARA MARCUCCI AND T. VISSER

MATERIAL AND METHODS

Plants. Lilium longzjlorum ‘White Europe’, a virus free selection of ‘Arai No. 5’ was kindly provided by G. van Veen, Moerbeke, Belgium. Lilium IongifZorum ‘White Amer- ican’ is a variety released by IVT, Wageningen. It originated from crosses made by Dr A. N. Roberts (Oregon State University) with ‘Ace’, ‘Nellie White’, ‘Gibson’ and ‘King Georgia’ as ancestors. The asiatic Lilium x hybrids ‘Enchantment’ and ‘Nut- megger’ are standard commercial varieties.

Pollen treatment. The pollen was freshly collected from glasshouse flowers and, prior to use, stored for a few days over silicagel in a desiccator at 2”-4°C. Some pollen was treated for one hour in a drying stove at constant temperatures of 40”, 50” and 60 “C. Other pollen was irradiated at ITAL with 100,250 and 500 krad gamma. Germi- nation was determined after three hours in a 1% agar medium with 10% sucrose and 20 ppm boric acid at a temperature of about 22°C. Prior to germination, the pollen was rehydrated at 100% RH. The germination percentage was based on three replicates per treatment; in each of the three samples five counts were made totalling about 100 grains.

Pollination trials. To prevent unintentional self-pollination, flowers were emasculated before opening. For pollination, the pollen was brought on the stigma or stylar surface using a brush. After pollination the stigmata were enveloped with a cap of aluminium foil. For cut-style pollination the style was cut O-2 mm above the ovary with a razor blade. The pollinations were made in the greenhouse from August 21 to September 2, 198 1. The mean daily temperature of the greenhouse during this period varied from 17 to 24°C. As the pollination period covered a fortnight, one or two flowers were pollinated at a given day for each of the treatments of a trial so as to ensure the same conditions for each set of pollinations. The treatments were randomized over the avail- able flowers.

Pods were harvested 8-30 October. The seeds were sorted into poor light and good heavy seeds by using an illuminated glass sheet. From incongruous combinations em- bryo culture was performed at about 45 days after pollination using the Murashige & Skoog medium, pH 5.0, supplemented with 20 g/l sucrose and lo-* mg/l NAA.

RESULTS

Germination of pollen grains (effect of treatments). As to the period of rehydration, preliminary trials with the three lily cultivars showed no differences between 20, 40 and 80 minutes of rehydration, mean germination (9 replicates/treatment) being 65.7, 65.3 and 65.7 respectively. Accordingly as a routine, all pollen was rehydrated for 30 minutes.

Table 1 shows that rehydration had little effect on non-heated pollen, which germin- ated quite well without it. On the other hand, rehydration had a markedly positive effect on the germination of pollen treated at 60°C for one or more hours. Tables 1 and 2 as well as Fig. 1. demonstrate that heating up to 60°C for one to four hours did not greatly decrease the germination of the pollen just after treatment. Longer

614 Euphytica 31 (1982)

POLLINATION EXPERIMENTSIN LILIUM

Table 1. Effect of pollen heating at 60°C for l-8 hours on the germination percentage of fresh Lilium pollen (‘White Europe’) without (-R) and with (+ R) rehydration.

Treatment -R +R

Control’ 77.0 86.0 I hour 60°C 26.4 61.1 2 hours 60°C 21.3 59.8 4 hours 60°C 20.3 59.4 8 hours 60°C 15.3 45.0

’ Non-heated pollen.

Table 2. Effect of pollen heating at 40”, 50” and 60°C for one hour on the germination percentage of rehydrated Lilium pollen determined directly afterwards (0) and after three weeks (3) of cold storage.

Cultivar Control’ 40°C 50°C 60°C

0 3 0 3 0 3 0 3

‘White Europe’ 80.5 69.5 87.4 60.0 80.5 46.4 61.1 50.6 ‘White American’ 83.0 82.8 84,4 41.3 86.9 39.9 65.9 40.7 ‘Enchantment’ 66.3 47.6 67.6 45.3 68.9 32.2 35.6 36.7

’ Non-heated pollen.

periods of heating and higher temperatures (Fig. 1) and also storage for a few weeks following treatment (Table 2) clearly depressed the germination capacity.

Regarding the effect of irradiation, it appeared (Fig 2) that 100 krad had no effect

v. lOOr

Fig. 1. The effect of heating Lilium pollen (‘White

\ Europe’) at different temperatures-and times on the relative germination (expressed as a percent-

I I I L 40’ 60’ 60* 70. 80’ 90’ age of the non-heated control = 100%); derived

Temperature from data of MARCUCCI et al. (1982).

on germination, 250 krad reduced germination by about one quarter, while 500 krad largely or entirely inhibited germination. Pollen tube growth was relatively more de- pressed by irradiation than the germination percentage, e.g. pollen tubes at 250 krad were very short compared with those at 100 krad. Pollinations. With respect to the effect of heat treatment of pollen, Table 3 illustrates that heating the pollen at 40”-60°C for one hour had had no noticeable effect on its capacity to produce seeds in compatible crosses. On the other hand, this treatment

Euphytica 31 (1982) 615

J. M. VAN TUYL, M. CLARA MARCUCCI AND T. VISSER

% 100 r

s S 60 - E

E 2 40 -

20 -

o-

Fig. 2. The effect of irradiation dose on the pollen germination percentage of four Lilium cultivars.

.- White Europe X---X Whate Amencan o----o Nutmegger n-.-a Enchantment

0 100 250 500 Irradiation dose in krad

did not contribute at all to overcome self incompatibility or incongruity. Not even fruits were formed, except with ‘Enchantment’ where selfmg produced fruits with a few seeds, irrespective of pollen treatment. Selting of this cultivar was improved by the cut-style method, but not by the mentor or the pioneer pollen method (Table 4). It is worthy of note however, that the interspecific cross with ‘White Europe’, which produced no fruits - and thus no seeds - unaided, did so to a small extent with the aid of mentor pollen (Table 4). This also happened in the reciprocal crosses of ‘White Europe’ and ‘White American’ with ‘Enchantment’ in which case also the pioneer pollen method produced a seed (Table 5). With these two cultivars the cut-style method was completely unsuccessful to achieve sehing. However, with the mentor pollen meth- od self-incompatibility was largely overcome in both. The pioneer pollen method was equally effective with ‘White Europe’, though not with ‘White American’ (Table 5). It is finally of interest to mention that pollen irradiated at 100 krad was much more effective in stimulating (a parthenocarpic) fruit set than pollen having received a dose of 250 krad.

CONCLUSION AND DISCUSSION

Heating pollen at 40” to 60°C for one hour had only a negligible effect on germination capacity. In agreement with this, such pollen was found to be as productive in terms of fruits and seeds as unheated pollen in compatible combinations. However, contrary to the results of MATSUBARA (1981), heat treatment of pollen completely failed to over- come self-incompatibility. It could be that this investigator did not carry out all his treatments under the same conditions. As Table 6 illustrates, self-incompatibility is largely overcome when the prevailing temperature is high enough.

MATSUBARA’S results would be understandable if selting with untreated pollen was done under much cooler conditions than selting with the heated pollen.

ASANO (1981) also tried heat treatment of pollen (1 hour at 4073, but obtained no capsules after self-pollination of 20 flowers of L. longzj7orum ‘Georgia’. He also failed to overcome incompatibility in L. longiflorum using the cut-style technique, which confirms our lack of success in this species (Table 5). However, in our trials the cut-style method was successful in overcoming self-incompatibility in Lilium ‘En- chantment’. As has been shown earlier by VISSER (1981), irradiated pollen is quite

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POLLINATION EXPERIMENTS IN LILIUM

Table 3. The effect of the heating of pollen at 40”, 50” and 60°C for one hour on the fruit and seed set of a complete diallel cross involving the Lilium longiflorum cultivars ‘White Europe’ and ‘White American’ and the Lilium hybrid ‘Enchantment’.

- Female parent ‘White Europe’:

number of Male parent and treatment flowers fruits seeds

‘White American’: number of

flowers fruits seeds

‘White Europe’ Fresh 5 0 - 2 2 252 40°C 5 0 2 2 278 50°C 5 0 - 2 2 231 60°C 5 0 - 2 1 198

‘White American’ Fresh 5 5 1256 2 0 - 4O’C 5 5 1338 2 0 - 50°C 5 5 1485 2 0 - 60°C 5 5 1230 2 0

‘Nutmegger” Fresh - - - - - ‘Enchantment’ Fresh 5 0 - 2 0

40°C 5 0 - 2 0 - 50°C 5 0 - 2 0 - 60°C 5 0 - 2 0 -

‘Enchantment’: number of

flowers fruits seeds

5 0 ~ 5 0 - 5 0 - 5 0 -

5 0 - 5 0 - 5 0 - 5 0 -

10 10 211 15 4 10 10 3 4 10 2 0 10 4 3

‘Compatible partner for ‘Enchantment’.

Table 4. Fruit and seed set of L. x ‘Enchantment’ after:: - pollination with compatible L. x ‘Nutmegger’ pollen: untreated (C) or irradiated at 100 krad (Crt) or 250 krad (CQ); - selling (S) only or with the help of the cut-style method (S(CSM)), the mentor pollen method (C,t + S, C,z + S) and the pioneer pollen method (C,t/ S, C,z/ S, C,t/ S/S); - pollination with incongruous L. longzjlorum ‘White Europe’ only (WE) or with mentor pollen (Crt + WE).

Treat- Number of Seeds Treatment’ Number of Seeds ment per per

flowers fruits seeds fruit flowers fruits seeds fruit

C 10 10 211 21.1 CdW h) 10 10 0 - Gl 20 10 0 - G2/W3 h) 10 6 1 0.2 cr2 10 0 - - CrI/S(24 h) 5 5 1 0.2

Cr2/S(24 h) 5 1 0 - 45 13

Z(CSM) 10 10 :;I 1.3 C,t/S/S(S and 24)2 5 5 3 0.6 3.6

c,1+s 10 IO 2 0.2 WE 20 0 0 cr2+s 10 2 0 -. C,I + WE 5 4 23 0.5

‘Within brackets interval between pollinations in hours, 2Sehing carried out twice: 8 and 24 hours after the first pollination. 3Seeds grown in embryo culture.

capable to stimulate a parthenocarpic fruit set, provided its germination capacity is good. In both the previous trials with apple pollen and the present ones with lily pollen this was still the case after a dose of 100 krad, but not after 250 krad. The latter dose

Euphytica 31 (1982) 617

J. M. VAN TUYL, M. CLARA MARCUCCI AND T. VISSER

Table 5. Fruit and seed set of the L. longiflorum cultivars ‘White Europe’ and ‘White American’ after: -crossed with each other with untreated (C) or irradiated (100 krad) pollen (C,); - selfed only (S), selfed with the cut-style method (S(CSM)), selfed with the aid of irradiated mentor (C, + S) or pioneer pollen (C/S) at 8 hours interval;

- crossed with L. x ‘Enchantment’ only (E) or with the aid of irradiated mentor (C, + E) or pioneer pollen (CJE).

Treat- ment

C 20 S 20 S(CSM) 5 G 4 c,+s 4 G/S 4 E 20 C, + E 4 G/E 4

‘White Europe’ ‘White American’

number of seeds

per flowers fruits seeds fruit

20 5309 265 0 0 0 - - 2 259 130 3 516 172 0 -

3 0 - 2 2 I

number of

flowers fruits

8 7 8 0 3 0 2 2 2 2 2 2

20 0 2 1 2 2

seeds

seeds

per fruit

959 137

0 205 103

3 1.5

6 6t 1 I

t Seeds grown in embryo culture.

Table 6. The effect of the temperature in the phytotron glasshouses on fruit and seed set of L&m cultivars after cross and selfpollination.

Treat- ment

Crossed’ Selfed

number of seeds number of seeds per per

flowers fruits seeds fruit flowers fruits seeds fruit

L. x ‘Enchantment’ 10°C 10 0 IO 0 - - 14°C 10 9 3 0.3 10 0 - - 17°C 10 8 13 1.6 10 0 -

20°C 10 10 190 19 10 9 9 1 23°C 10 10 172 17 10 8 13 8 26°C 10 10 412 41 10 9 225 25

L. 1ongiJlorunl ‘White Europe’ 10°C 4 0 5 0 14°C 3 2 0 - 5 0 - - 17°C 5 3 292 77 4 0 - 20°C 8 7 255 36 7 2 0 - 23°C 10 9 601 60 8 2 16 8 26°C 8 8 808 101 7 2 95 48

t ‘Enchantment’ was crossed with ‘Connecticut King’, ‘White Europe’ with ‘Ace’.

considerably inhibited the growth of the pollen tubes and consequently stimulated fruit growth much less.

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POLLlNATlON EXPERIMENTS IN LILIIJM

The application of irradiated mentor or pioneer pollen did not help to overcome self-incompatibility in ‘Enchantment’. In remarkable contrast to the complete failure of the cut-style method, both methods aided selfing of ‘White Europe’ to a great extent; the mentor pollen method was also very successful with ‘White American’. Although in view of the few flowers per treatment the trials must be called preliminary, the results with respect to the usefulness of the two methods appear to be promising. This conclusion is supported too by the fact that the two methods also produced some viable seeds in a very few crosses between incongruous cultivars. To achieve even this relatively poor result unaided, would have required the pollination of at least l&20 times as many flowers otherwise.

Further and more extensive experiments will be carried out to ascertain whether there is a difference between the mentor pollen and pioneer pollen method, taking into account environmental temperature and interval between pollinations.

ACKNOWLEDGEMENTS

The participation of Miss Marcucci was made possible by a grant of the National Council of Research (NR), Italy. Project: Biology of reproduction.

REFERENCES

ASANO, Y., 1981. Pers comm. ASCHER, P. D. & L. W. DREWLOW, 1975. The effect of prepollination injection with stigmatic exudate

on interspecific pollen tube growth in Lilium longiflorum THUNB. styles. Plant Sci. Letters 4: 401-405. A.SCHER, P. D. & S. J. PELOQUIN, 1966. Effect of floral aging on the growth of compatible and incompatible

pollen tubes in Lilium longiforum. Amer. J. Bot. 53 (1): 99-102. CHENG, J. H. & R. H. MATTSON, 1972. Effect of intrastylar pollination methods on seedset of Lilium x

‘midcentury’ hybrid lilies. J. Amer. Sot. Hort. Sci. 97 (5): 591-592. DAYTON, D. F., 1974. Overcoming self-incompatibility in apple with killed compatible pollen. J. Amer.

Sot. Hort. Sci. 99: 19&192. DEN NIIS, A. P. M. & E. H. Oosl, 1980. Effect of mentorpollen on pistil-pollen incongruities among species

of Cucumis L. Euphytica 29: 261-27 1. EMSWELLER, S. L. &N. W. STUART, 1948. Use of growth regulating substances to overcome incompatibilities

in Lilium. Proc. Amer. Sot. Hort. Sci. 51: 581-589. HOPPER, J. E., P. D. ASCHER &S. J. PELOQUIN, 1967. Inactivation of self-incompatibility following tempera-

ture pretreatments of styles in Lilium longiforum. Euphytica 16: 215-220. KUNISHIGE, M. & Y. HIRATA, 1978. Studies on crosses of lilies: On the mixed-pollen method. (Jap.). Bull.

Hort. Res. Sta. (Kurume), 1978: 80-85. MATSUBARA, S., 1973. Overcoming self-incompatibility by cytokinins treatment in Lilium longiflorum. Bot.

Mag. Tokyo 86: 43-46. MATSUBARA, S., 1981. Overcoming the self-incompatibility of Lilium longif2orum THUNB. by application

of flower-organ extract or temperature treatment of pollen. Euphytica 31: 97-103. MYODO, H., 1962. Experimental studies on the sterility of some Lilium species. Journ. Facul. Agr. Hokk.

Univ., Sapporo vol. 52 (pt 1): 70-122. VISSER, T., 1981. Pollen and pollination experiments. IV. ‘Mentorpollen’ and ‘pioneerpollen’ techniques

regarding incompatibility and incongruity in apple and pear. Euphytica 30: 363-369. MARCUCCI, M., T. V~SER & J. M. VAN TUYL, 1982. Pollen and pollination experiments. VI. Heat resistance

of pollen. Euphytica 31: 287-290.

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