Hybridizers who have an understanding of the specific fertility problems they face can make breeding decisions that may help them overcome or circumvent those problems. There are many different causes of infertility as discussed in an earlier article (Factors affecting Fertility in Daylilies, Daylily Journal, Vol. 3, No. 4). Determining the exact causes of specific fertility problems requires equipment and skill that are beyond the level of the average daylily hybridizer. However, with some careful observations hybridizers should be able to make inferences and educated guesses about the causes of the fertility problems they face.
Daylilies can be more or less divided into three groups based on fertility:
Those that are infertile or very reluctant as both pod and pollen
Those that are more or less fertile to very fertile as both pod and pollen parent.
Those that are fertile as either pod or pollen parent, but not both.
PRELIMINARY PROCEDURES FOR DETERMINING RELATIVE FERTILITY.
By making a large number of different crosses, hybridizers can determine the relative fertility or infertility of particular parents. However, there are a few simple things hybridizers can do to get a quick assessment of the fertility of a new cultivar or seedling.
When working with a new cultivar or seedling a hybridizer should first self-pollinate it, then pollinate it with a mixture of pollen from parents that are known to be highly fertile, and then use its pollen on a number of different pod parents that are known to be fertile. These crosses should be made solely based on the high fertility of the other parents, even if they have absolutely no breeding value.
If a daylily will self-pollinate, we then know it is both pod and pollen fertile and that it either lacks a self-incompatibility system or has a self-fertility gene present. Daylilies that are pod- and pollen-fertile but do not self-pollinate probably have a self-incompatibility gene system present. Problems with either pod or pollen fertility should be relatively evident from the results, although it may take time to find good test parents. These preliminary results may not be completely reliable but should give hybridizers an indication of potential fertility problems.
THE INFERTILE OR VERY RELUCTANT PARENT.
Genome incompatibility: Of the various causes of infertility, genome incompatibility is probably the most significant. Daylilies that are infertile or very reluctant as both pod and pollen parents most likely have a problem with genome incompatibility, although chromosome abnormalities or aneuploidy can also result in infertility or low fertility. Infertile or difficult progenies from two highly fertile parents is most likely due to genome incompatibility. On the contrary, two difficult parents can produce progenies that are highly fertile because the progenies have compatible genomes. Daylilies are a complex mixture of many different genomes so it is very difficult to predict how fertile or infertile the resulting progenies will be from particular crosses.
There is very little that hybridizers can do with daylilies that are infertile or very reluctant as a result of genome incompatibility, because these daylilies fail to produce viable gametes, both on the pod and pollen side. However, diploid daylilies that are completely infertile often make good candidates to convert to tetraploids because at the tetraploid level each chromosome will then have a compatible chromosome to pair with, and thus be fertile.
Hybridizers who insist on using very difficult parents will probably have better luck using them as pod parents and using a mixture of fertile pollen. Also, the authenticity of the parentage is much easier to establish when the difficult parent is the pod parent. Daylilies that have impaired meiosis due to genome incompatibility sometimes do produce a rare viable gamete. However, it may take a long time before any seeds are produced.
Chromosome abnormalities and aneuploidy: Chromosome abnormalities and aneuploidy also result in low fertility, and can be distinguished from genome incompatibility by examining the microspores (immature pollen) during meiosis. This is generally beyond the ability of most amateur hybridizers. It is doubtful, however, that chromosome abnormalities and aneuploidy are significant causes of infertility in daylilies and for practical purposes can be lumped together with genome incompatibility. In general, daylilies that are aneuploids or have chromosome abnormalities have better pod fertility than pollen fertility and are best used as pod parents.
Misclassified Ploidy: An important factor that hybridizers should consider when daylilies appear to be infertile or are very reluctant parents is whether or not the daylilies they are using are the ploidy level they are registered as being. Many hybridizers have been using converted tetraploids or their progenies without actually checking their ploidy level. As a result we may have many diploids masquerading as tetraploids. Hybridizers using these misclassified diploids with true tetraploids are most likely producing triploid progenies, if they are getting any viable seeds. Triploids are generally infertile or have very low fertility. These triploids are most likely being registered as tetraploids. (see end note)
The problem of misclassified ploidy level will probably result in much confusion for a long time. Hybridizers who have infertile "tetraploids" should try using them with diploid parents in case they have a diploid masquerading as a tetraploid.
FERTILE BOTH WAYS - SOMETIMES.
Daylilies that are fertile as both pod and pollen parent can be divided into various groups based on their relative degree of fertility, how fastidious they are as parents, and how consistent they are as parents.
Low-range fertility: Fertility can range from low to high. Daylilies that have a fairly consistent low level of fertility, regardless of the other parents, probably have an underlying genome incompatibility not severe enough to prevent a reasonable number of gametes from being produced. Since there is far more pollen produced than egg cells, these daylilies are often better used as pollen parents because of the greater number of viable gametes on the male side. Oftentimes these daylilies will exhibit better fertility in particular crosses. When using these daylilies as pod parents, use different mixtures of pollen over a period of time until a particular mix seems to work. Then the individual pollens can be tried until the viable ones are found.
High-range fertility: Fastidious daylilies that are highly fertile, at least in certain combinations, but infertile or with low fertility in other crosses most likely have a problem with self- or cross-incompatibility, embryo failure or endosperm failure. Self- and cross-incompatibility are difficult to separate because of the complex germplasm mix of the modern daylily hybrids. From a practical point of view it is better to consider self- and cross-incompatibility together as pollen-style incompatibility. Hybridizers making reluctant crosses between otherwise fertile parents will need to repeat the cross as often as possible, reciprocally, until a sufficient number of seeds are produced. Sometimes crosses will be productive in one direction but not the other.
TECHNIQUES FOR PROMOTING RELUCTANT MATCHES.
There are a number of breeding techniques and schemes that can be used to overcome or circumvent infertile crosses between otherwise fertile parents. These include cut-style pollination, delayed and early pollination, embryo culture, the use of bridge crosses and the substitution of self-pollinated or backcross progenies for the desired parents.
Cut-style pollination. The cut-style pollination technique is accomplished by cutting the style about 5-6 mm above the ovary. A slit is then made in the style. Pollen is applied to the stigma and a mix of pollen and stigmatic tissue is then applied to the cut style. The cut style is then covered with aluminum foil to reduce drying of the stigmatic tissue pollen mix. This technique requires considerable time and effort and the results are often poor.
Delayed and Early pollinations: Delayed and early pollinations have been used to overcome self- and cross-incompatibility in other plants. The self- and cross-incompatibility reactions are not fully developed prior to anthesis (opening of the flower) and weaken as the flower ages. How well early or bud pollinations and delayed pollinations work in daylilies is not known, but may be worth a try if there appears to be a pollen-style incompatibility. Bud pollinations are made by forcing open the bud and pollinating 1 or 2 days prior to anthesis. Delayed pollination is done as late as possible but yet giving the pollen enough time to grow down into the ovary. Delayed pollination probably has limited value in daylilies because the daylily flower senesces so quickly.
Bridge crossing: Particular infertile crosses can often be circumvented by using a bridge cross, that is, by crossing each daylily to a third parent and then intermating the best half sibs. For example, if the cross A x B is infertile, then the cross (A x C) x (B x C) may be just as useful as the intended cross. Bridge crosses are far easier and more effective than using cut-style pollination.
Substituting progenies: Infertile crosses can sometimes be circumvented by crossing self-pollinated progenies from the desired parents. Unfortunately, self-pollinated progenies from fertile parents are not necessarily themselves self-fertile or even fertile with other parents. Self-pollinated progenies can sometimes be just as good or even better than the original parent. It may be necessary to use more than one self-pollinated progeny as a substitute for the original parent. If the desired parents do not self-pollinate, hybridizers can than use backcross progenies as a substitute for the desired parents; for example, ((A x C) x A) x ((B x C) x B) or ((A x C) x A) x ((B x D) x B).
Embryo culture: Fertility problems caused by embryo or endosperm failure are usually easy to detect. Fertilization of the egg cell and endosperm nuclei initiates the production of auxins that prevent the pod from aborting. All the other causes of infertility do not result in fertilization and the pod will abort 5-7 days after anthesis without any pod development. If fertilization occurs the pod will start to develop but failure of the endosperm leads to embryo failure and eventual pod abortion. However, pod abortion will occur after some pod development occurs, anytime from 7 to 45 days after fertilization. Opening the pod will reveal slightly swollen ovules that have not yet developed into seeds. Occasionally, enough seeds will develop in a pod to continue normal pod development even though most embryos abort and many of the resulting seeds will be empty. Lightly squeezing the seeds after they have dried will easily reveal the empty seeds.
Infertility caused by embryo abortion resulting from endosperm failure is often easy to overcome by embryo culture. Embryo culture can be done with a relatively simple nutrient-sugar medium. Aseptic techniques are required but are not difficult to learn. The use of bridge crosses and the substitution of self-pollinated or backcross progenies for the desired parents can also be used to circumvent infertility resulting from endosperm-embryo failure.
Some daylilies are inconsistent in their ability to set pods, either in general or sometime only in particular crosses. On some days a high percentage of pollinations will result in pod set while on other days there will be little or no pod set. Environmental factors may play a role that is not well understood. Hybridizers should repeat the cross as often as necessary to get a sufficient number of seed. Anything that will reduce stress may help seed set. Keep blooming plants well watered, healthy and free of insect attack. Cool temperatures also seem to be helpful. Daylilies can be potted up and kept in a cooler shady location or forced to bloom before hot weather sets in. In hot climates pollinate as early in the morning as possible.
FERTILE ONE WAY - AND WHAT TO DO ABOUT IT.
Daylilies that are only fertile as either pod or pollen parents, but not both, can have all the causes of infertility as already discussed in addition to male or female sterility genes. The pod or pollen fertility can range from low, if there is an underlying genome incompatibility, to high, at least in certain crosses.
Pod and pollen sterility genes are generally recessive and can be carried along in the heterozygous state. Since there are a large number of genes involved in the formation of the male and female gametes and the development of the ovule into a seed, it is possible that a large number of undesirable fertility alleles are present in the modern daylily germplasm pool. Some of the genes may result only in reduced fertility and as such may mimic pollen-style incompatibility, and on the female side may mimic endosperm-embryo abortion if the gene results in the failure of the seed to develop properly. These genes are probably a significant factor causing fertility problems.
The pod The presence of pod sterility genes is not easy to detect in pod infertile but pollen fertility parents. Such daylilies should be pollinated with a mixture of different fertile pollen over a long period of time to ascertain if the plant is truly pod sterile or if it is very fastidious about what pollen it will accept. Some daylilies that are pod infertile or have low fertility will produce a reasonable number of seeds with certain pollen parents.
The pollen: Male sterility genes are quite common and are often easy to detect as pollen either fails to develop, has a very abnormal appearance or the anthers fail to develop properly resulting in functional male sterility. Examining an anther with a hand lens should easily detect abnormal anthers lacking pollen or pollen that has a very lumpy, sticky or waxy appearance. Pollen should have a powdery, fluffy appearance and should look more or less dry. However, good looking pollen may not germinate or may only grow a short pollen tube. Fortunately, pollen gemination is easy to check, even without a microscope.
Pollen germination can be checked by placing pollen in a 10-15% sucrose solution with about 100 ppm boric acid and examining after 1 and 3 hours. The sugar concentration is fairly critical and the optimal concentration varies with different pollens for in vitro germination. It is generally a good idea to use two or three different sugar concentrations of about 10, 12.5 and 15%. One ounce (by weight) of sugar in one cup of water is approximately a 10% solution. For 10, 12.5 and 15% sucrose, use 1, 1.25 and 1.5 ounce of sugar per cup, respectively. Add about 1 drop of 1% boric acid (eye wash). It will probably be necessary to experiment to get the optimal proportions.
An inexpensive microscope is quite adequate to check pollen germination or a slide projector can be used in place of a microscope. Place a drop of the pollen-sugar mix on the sticky side of clear cellophane tape and affix to a slide made from developed but unexposed B&W film. After 1 hour the pollen tubes should have emerged and by 3 hours there should be a tangled mess of pollen tubes.
Hybridizers wishing to cross two parents that are pod infertile but pollen fertile, or pod fertile but pollen infertile can circumvent the difficulty by using a bridge cross or using the respective backcross progenies as parents. Daylilies with male sterility will sometimes produce viable pollen under certain conditions.
COMPLEMENTARY STYLES - BOTANY FASHION.
Crosses between parents that vary considerably in style length should be made with the short style plant as the pod parent, although the reciprocal cross should also be made. Pollen from species with short styles may not have the genetic determination to grow pollen tubes long enough to reach the ovary of parents with long styles. This seems to be a problem with many of the spider types which have very long styles. Crosses of miniature and small flowered types with large flowered types may also manifest reciprocal differences in crossability.
Hybridizers confronted with serious fertility problems need to evaluate the parents or specific crosses that are causing problems. In some cases it may be desirable to work around the fertility problem. However, there are many good parents that are fertile so that it may be better to discard those cultivars or seedlings that are very difficult. Decisions about if and when to discard breeding lines, cultivars or seedlings, because of fertility problems, are personal decisions that need to be based on many factors.
Notes added 9 Jan 1995.
This digital version of this article differs slightly from the published manuscript in that I replace the word "sterile" with the more correct word "infertile" in a few places.
Since originally writing this article I have made some crosses onto spider daylilies and other daylilies using pollen from plants with short styles and didn't have any problems in getting seed set. This doesn't mean that style length couldn't be a problem in other crosses. However, I don't think this is a serious problem in daylilies.
As with many other hybridizers, I have encountered many fertility problems with certain parents or with certain crosses. I am becoming less enthusiastic about trying to use parents that give me problems. There are just too many other good daylilies, both named cultivars and my own seedlings that are easily fertile and that have the same germplasm potential.
Since first writing this article for publication I've discovered the occurance of unreduced gametes in daylilies and the occurance of triploid block in daylilies that mostly prevent triploid progenies from forming in diploid-tetraploid crosses. This is detailed in the "Polypolidy and Unreduced Gametes" article.