Euphytica 57: 10~107, 1991. 0 1991 Kluwer Academic Publishers. Printed in the Netherlanris.

A new method of producing 4x hybrid true potato seed

Rodomiro Ortiz & Stanley J. Peloquin Departments of Horticulture & Genetics, University of Wisconsin, Madison, WI 53706, USA

Received 28 June 1991; accepted 11 September 1991

Key words: 2n gametes, 2x X 2x crosses. bilateral sexual polyploidization, haploid-species hybrid

Summary

New haploid-wild species hybrids (H-S) with desirable tuber type and 2n gamete production have been identified. They provide the opportunity for the production of inexpensive, 4x hybrid TPS from 2x x 2x crosses. A proposed system to utilize plants producing either 2n eggs or 2n pollen is described. One H-S hybrid (female parent) would be selected for male fertility, self-incompatibility, high frequency of 2n eggs and no 2n pollen; an unrelated H-S (male parent) for male fertility and high 2n pollen production. In addition both parents would be selected for profuse flowering, attractiveness to bumblebees and other desirable characteristics. The male and female parents would be planted in alternate hills with bumblebees doing the pollinations. The elimination of emasculation, pollen collection, and hand pollination could reduce 4x hybrid TPS costs by more than 50 percent.

Introduction

The use of true potato seed (TPS), in production of potatoes by farmers in developing countries, has several advantages. Two of the most important are: a) healthy planting material, i.e. PVY and PLRV are not seed transmitted in potato: and b) reduced cost of planting material, in locations where virus free seed potato tubers are not available and where it is very expensive to import them (Malagamba & Monares, 1988). In these locations the use of TPS is an excellent alternative for potato production, since cost of seed potato tubers can represent 20- 60% of total production costs.

The occurrence of 2n gametes (gametes with the sporophytic chromosome number) in potato allows for the production of TPS using different ploidy manipulations (Peloquin et al., 1984). In potato 2n gametes are formed as a result of either a first division restitution mechanism (FDR) or a second division restitution mechanism (SDR) (Peloquin et

al., 1989). Genetically, the FDR mechanism is su- perior to the SDR mechanism in transmitting het- erozygosity and epistasis, and results in increased yield in the resultant progeny (Mendiburu & Pelo- quin, 1977; Mendoza & Haynes, 1974). The most common mechanism of 2n pollen formation, paral- lel spindles in the second meiotic division (Mok & Peloquin, 1975a), is controlled by a recessive meiotic mutantps (Mok & Peloquin, 1975b) and is a FDR mechanism. In contrast, 2n egg formation results mainly from omission of the second meiotic division. This is a SDR mechanism and is con- trolled by a recessive meiotic mutant OS (Werner & Peloquin, 1990).

The production of hybrid TPS requires emascu- lation, to avoid selfing if male fertile clones are used as female parents, collection of pollen, and hand pollination. The International Potato Center, CIP (1987; 1988; 1989) indicates that 7.5 rn: of land and 24 man days are needed to produce 1 kg of TPS. The average total cost of production was esti-

104

Female Haploid-Species 2x hybrid (H-S) male fertile self-incompatible high frequency of 2n eggs and no 2n pollen attractive to bumblebees profuse flowering

\

M& unrelated H-S male fertile

high frequency of 2n pollen attractive to bumblebees profuse flowering

/ bumblebees as pollinator

PRODUCT: 4x hybrid TPS Advantages

inexpensive seed production near maximum genetic diversity

good tuber yields and uniformity

Fig. 1. 4x hybrid TPS production using meiotic mutants.

mated to be $246, and ranged from $128 (most efficient combination in fruit setting) to $307. Plower collection, pollen extraction and hand polli- nation represents approximately 40% of total costs, and emasculation 12%. Therefore a new in- expensive method for hybrid TPS production is highly desirable.

The objectives of this paper are to: a) outline a new, inexpensive method of producing 4x hybrid TPS from 2x x 2x crosses through bumblebee pol- linations, and b) indicate the problems that need to be solved to exploit this new method.

Outline of the method

New haploid-wild species 2x hybrids (H-S) with desirable tuber type and 2n gamete production have been identified. They provide the opportunity to explore a new inexpensive method of 4x hybrid TPS production from crosses between 2x parents. Normally, 2x potatoes are self-incompatible due to a gametophytic system (Cipar et al., 1964). There- fore, selfed progeny are only obtained when a 2x parent produces both 2n eggs and 2n pollen.

The main elements of the method are presented in Fig. 1. One H-S hybrid (female parent) is select- ed for male fertility, self-incompatibility, high fre- quency of 2n eggs and no 2n pollen; an unrelated H-S (male parent) is selected for male fertility and

high 2n pollen production. In addition both parents would be selected for profuse flowering, attractive- ness to bumblebees and other desirable character- istics. The male and female parents would be plant- ed in alternate hills. Bumblebees would collect pol- len and do the pollinations necessary to obtain the hybrid 4x progeny. In this way, flower emascula- tion, pollen extraction, and hand pollination are eliminated. Fruit from the female plants would be harvested and seed extracted. The 4x progeny from this seed would have plants with good tuber uni- formity and high yields. The elimination of emas- culation, pollen collection, and hand pollination could reduce 4x hybrid TPS costs by more than 50 percent.

Theoretical assumptions

It is important to consider two concepts in maxi- mizing tuber yield in potatoes: a) transmission of heterozygosity using FDR and SDR 2n gametes, and b) allelic interactions; intralocus interactions (heterozygosity) and interlocus interactions (epis- tasis) as the result of crosses between unrelated 2n gamete, 2x parents.

The 4x progeny, from crosses between 2x par- ents, involve the possible complementation of FDR and SDR 2n gametes, i.e., 100% of the heter- ozygosity from the centromere to the first cross- over is transmitted to the 4x progeny with FDR, and 100% of the parental heterozygosity from the first to the second crossover is transmitted to the 4x progeny by SDR 2n gametes. However, only 50% of the heterozygosity from the centromere to the first crossover is transmitted to the 4x progeny by SDR 2n gametes. Both transmit a large fraction of the epistasis of the 2x parents to the 4x progeny.

Tetra- and tri-allelic loci are expected in 4x prog- eny from both FDR (2n eggs) X FDR (2n pollen) and SDR (2n eggs) x FDR (2n pollen) crosses. However, no diallelic loci occur in the 4x progeny of the latter if both parents are unrelated. For example, assume a) two loci ‘a’ and ‘b’ in which locus ‘a’ is between the centromere and the first crossover and locus ‘b’ is between the first and second crossover, and b) a cross between unrelated

10.5

2x hybrid clones: a,/a, b,/bz (2n pollen) X a3/a4 b,/b, (2n egg). Then, the resultant 4x progeny will have 25% diallelic loci (bIblblb,, blb,b4b4, bzbtb,b,, b2bIb4b4) for the chromosome region between the first and second crossover (locus b) if both parents produced FDR 2n gametes. In contrast there will be no diallelic loci from SDR x FDR crosses for this chromosome region. In effect the genotypes for the locus b could be 25% b,bIb3b4, 50% b,b2bjba, and 25% b2b2b3b4. Assuming tri- and te- tra-allelic loci are equal, and both are superior to mono- and di-allelic loci, SDR x FDR would be superior to FDR x FDR. This is very important in regard to the concept of maximum heterozygosity (more than two alleles per locus), to maximize yield in potatoes (Mendiburu & Peloquin, 1977a; Mendoza & Haynes, 1974). Therefore, 4x progeny from crosses between SDR x FDR parents could be more desirable for yield.

Practical aspects

Tuber yield and other tuber traits of 4.x progeny from 2x X 2x crosses. Werner & Peloquin (1991) produced 4x families from crosses between SDR x

T&e I. Percentage of 4x progeny after 2x x 2x crosses accord- ing to the frequency of 2n gametes in female and male parents

Parent Frequency of 2n gametes

2n eggs’ 2n pollen

Percentage 4xifamily

Females (2n eggs): l-248 T706 TX0 Males (2n pollen) P 100.1 P 94.1 P 23062 P 126.3 CIP 38.13

10.1 s/f 4.5 (5)3 28.7 s/f 16.0 (5) 40.1 s/f 18.2 (2)

l- 5% 0.0’ l- 5% 3.32 l- 5% 6.02 6-10% 12.92

1 l-20% 21.2”

1 based on seed set (s/f) after 2x x 4x crosses. 1 of average crosses with T 248 and T 706. ‘number of male parents crossed with 2n egg producers in brackets.

FDR parents. They reported that the average tuber yield of 19, 4x families from 2x x 2x crosses ex- ceeded the 4x cultivars by 28% at Hancock (irrigat- ed conditions) and 30% at Rhinelander (non-irri- gated conditions).

However, the 2x parents of these 4x families had rough tubers and high tuber set per hill. The 4x progeny from 2x x 2x crosses also had these unde- sirable characteristics and for these traits were sig- nificantly inferior to the 4x cultivars. The solution to this problem is to obtain H-S hybrid with good tuber type and set (Or&, 1991). New FDR 2n pollen and SDR 2n egg producers with improved tuber appearance have been identified in a 2x H-S population. Therefore, the production of uniform high yielding families with good tuber type and adequate number and size of tubers for utilization as TPS progenies through 2x x 2x crosses is now feasible.

Percentage of 4xprogeny folIowing2x X 2x crosses. The percentage of 4x progeny after 2x x 2x crosses ranged from 3% to almost 100%) with an average of 10.4% in our trials. The production of 4x proge- ny was influenced by the frequency of 2n gametes in both parents. For example W-5295.7 (I), a Phu- reja x Tuberosum haploid 2x hybrid with a high percentage of FDR 2n pollen (20-30%) produced mainly 4x progeny after crosses with 2n egg clones with a medium to high frequency of SDR 2n eggs. Moreover, other clones with high frequencies of FDR 2n pollen produced almost entirely 4x proge- ny in crosses with SDR 2n egg producers (Werner & Peloquin, 1991).

An experiment (under controlled conditions in the greenhouse) was carried out to determine the relationship between 4x progeny after 2x x 2x crosses and frequency of 2n gametes in the 2x par- ents. The results (Table 1) indicated that the signif- icant differences detected in percentage of 4x prog- eny in each family after 2x X 2x crosses depends on the percentage of 2n gametes produced by both parents. Highly significant correlations were found between the percentage of 4x progeny, after con- trolled 2x x 2x crosses, and the frequencies of SDR 2n eggs (r = 0.987; p = 0.003) and FDR 2n pollen (r = 0.952; p = 0.002) in the 2x parents.

106

An increase in both the frequency and stable production of 2n gametes, and subsequently 4x progeny after intermating 2x parents, can be achieved efficiently through recurrent selection (Or&, 1991).

The percentage of 4x progeny after intermating 2x parents also depends on fertility of the parents, e.g. pollen shedding and viability.

Detection of tetraploids. The detection of 4x proge- ny from 2x x 2x crosses can be done at early stages in the nursery if transplants are going to be used in the field for production of potato from TPS, or at later stages in the nursery if seedlings tubers will be harvested to be used as seed in the next generation. The 4x can be identified by darker green color, smaller length-breadth ratio of terminal leaflet, coarser appearance, and increased vigor as com- pared to their 2x ‘full-sibs’.

Choice of parents. Haploid X species hybrids are the most desirable parents in 2x x 2x crosses to produce 4x TPS progenies. The advantages are related to their good vigor, abundant flowering, and high fruit and seed set. More importantly, the use of H-S parents increases the genetic diversity which leads to maximum yield in potatoes.

Problems to be solved to exploit this method

More research must be done to: a) Determine the location of the field for 4x TPS production. Should the required TPS production field be an isolated plot or should it be a part of a large potato produc- tion plot? Is it feasible to produce TPS with the methodology proposed by using screened enclo- sures? The answers to these questions should take into consideration the attractiveness of the flowers to bumblebees as well as the behavior of bumble- bee populations. In this regard, different methods have been described to induce Bombus species to start and maintain colonies in captivity (Plowright & Jay, 1969; Pomeroy & Plowright, 1980). There- fore, the possibility of managing bumblebee pop- ulations seems feasible; b) Identify male and fe- male parents which flower over a similar time peri-

od. This is essential for success in TPS production using natural pollination by bumblebees; c) Con- tinue the selection of clones with high frequency of 2n gametes (especially SDR 2n eggs) in order to obtain a high frequency of 4x progeny from 2x x 2x crosses. Recurrent selection for both FDR 2n pol- len and SDR 2n eggs seems appropriate to achieve this goal (Ortiz, 1991); d) Develop the agronomic practices most suitable for seed production. They involve, among other factors, spacing between par- ents, and the proportion of male and female paren- tal plants.

Preliminary results

In summer 1990, 16 SDR 2n egg producers were planted and each surrounded with four FDR 2n pollen producers in replicated plots at Rhinelan- der, Wisconsin. Ten SDR 2n eggs producers had more than 15 seeds/fruit (average of 39 seeds/fruit). Most of these 2x clones had low fruit set (average was 6 fruits/plant). However, E-649 (a SDR 2n egg producing parent) had 55 seeds/fruit and 54 fruits/ plant. Thus, E-649 produced almost 3000 hybrid TPS in 1.5m2 using the methodology proposed. SDR-2n egg producers such as E-958 and E-1041 which are male sterile did not produce fruits. This corroborates that pollination by bumblebees in po- tatoes depends on high levels of male fertility (Amdt et al., 1990).

The preliminary result with one clone (E-649) is encouraging, but higher TPS production per plant is required. A reasonable goal would be to identify clones that produce 10,000 hybrid seeds/plant (100 fruits with 100 seeds/fruit).

Acknowledgements

Paper No. 3213 from the Laboratory of Genetics. Research supported by the College of Agricultural & Life Sciences; International Potato Center; US- DA-CRGO-88-37234 3619, and Frito-Lay, Inc.

107

References

Amdt, G.C., J.L. Rueda, H.M. Kidane-Mariam & S.J. Pelo- quin, 1990. Pollen fertility in relation to open pollinated true seed production in potatoes. American Potato .I. 67: 499-505.

Cipar, M.S., S.J. Peloquin & R.W. Hougas, 1964. Inheritance of incompatibility in hybrids between Solanum tuberosum ha- ploids and diploid species. Euphytica 13: 163-172.

International Potato Center, 1987. Annual Report CIP 1986- 1987.

International Potato Center, 1988. Annual Report CIP 1988. International Potato Center, 1989. Annual Report CIP 1989. Malagamba, P. & A. Monares, 1988. True Potato Seed: Past

and Present Uses. International Potato Center. 40 pp. Mendiburu, A.O. & S.J. Peloquin, 1977. The significance of 2n

gametes in potato breeding. Theor. Appl. Gener. 49: S%61. Mendoza, H.A. & F.L. Haynes. 1974. Genetic basis ofheterosis

for yield in the autotetraploid potato. Theor. Appl. Genet. 4s: 21-2s.

Mok, D.W.S. 81 S.J. Peloquin, 1975a. Three mechanisms of 2n pollen formation in diploid potatoes. Can. J. Genet. Cytol. 17: 217-225.

Mok, D.W.S. &S.J. Peloquin, 1975b. The inheritance ofdiplan- droid (2n pollen) formation in diploid potatoes. Heredity 35: 295-302.

Ortiz, Rodomiro, 1991. Efficiency of potato breeding using 2n gametes; Male sterility and 2n pollen production in 4x potato. PhD Thesis. University of Wisconsin-Madison. 291 pp.

Peloquin, S.J., G.C. Arndt & H.M. Kidane-Mariam, 1984. Utilization of Ploidy Manipulations for True Potato Seed. In Report of CIP Planning Conference of Innovative methods for propagating potatoes. International Potato Center. Lima, Peru. pp. 17-24.

Peloquin, S.J., G.L. Yerk & J.E. Werner, 1989. Ploidy Manip- ulations in Potato. In: K.W. Adolph (Ed.) Chromosomes: Eukaryotic, Prokaryotic, and Viral. Vol. II. CRC Press. Bo- ca Raton, Florida. pp. 167-178.

Plowright, R.C. & S.C. Jay, 1969. Rearing bumble beecolonies in captivity. J. of Agricultural Research 5: 155-165.

Pomeroy. N. & R.C. Plowright. 1980. Maintenance of bumble bee colonies in observation hives (Hymenopthera: Aphidae). Can. Em. 112: 321-326.

Werner, J.E. L S.J. Peloquin, 1990. Inheritance of two mecha- nisms of 2n egg formation in 2x potatoes J. Hered. 81: 371-374.

Werner. J.E. & S.J. Peloquin. 1991. Yieldand tuber character- ization of 4x progeny from 2x X 2x crosses. Potato Research (In press).