George Acquaah - Principles of Plant Genetics and Breeding

Hybridization is often conducted routinely without any problems when individuals from the same species are involved, provided there are no fertility regulating mechanisms operating. Even when such mechanisms exist, hybridization can be successfully conducted by providing appropriate pollen sources. Sometimes, plant breeders are compelled to introduce desired genes from distant relatives or other more or less related species. Crossing plants from two different species or sometimes even plants from two genera is challenging and has limited success. Often, the breeder needs to use additional techniques (e.g. embryo rescue) to intervene at some point in the process in order to obtain a mature hybrid plant. Reproductive isolation barriers may be classified into three categories ( Table 6.2). These barriers maintain the genetic integrity of the species by excluding gene transfer from outside species. Some barriers occur before fertilization, some after fertilization. These barriers vary in degree of difficulty to overcome through breeding manipulations.

Spatial isolationSpatial isolation mechanisms are usually easy to overcome. Plants that have been geographically isolated may differ only in photoperiod response. In this case, the breeder can cross the plants under a controlled environment (e.g. greenhouse) by manipulating the growing environment to provide the proper duration of day length needed to induce flowering.

Pre‐fertilization reproductive barrierThese barriers occur between parents in a cross. Crops such as wheat have different types that are ecologically isolated. There are spring wheat types and winter wheat types. Flowering can be synchronized between the two groups by, for example, vernalization (a cold temperature treatment that exposes plants to about 3–4 °C) of the winter wheat to induce flowering (normally accomplished by exposure to the winter conditions). Mechanical isolation may take the form of differences in floral morphology that prohibit the same pollinating agent (insect) from pollinating different species. A more serious barrier to gene transfer is gametic incompatibility, whereby fertilization is prevented. This mechanism is a kind of self‐incompatibility (see Chapter 4). The mechanism is controlled by a complex of multiple allelic system of S‐genes that prohibit gametic union. The breeder has no control over this barrier.

Post‐fertilization reproductive barriersThese barriers occur between hybrids. After fertilization, various hindrances to proper development of the embryo (hybrid) may arise, sometimes resulting in abortion of the embryo, or even formation of a haploid (rather than a diploid). The breeder may use embryo rescue techniques to remove the embryo and culture it to a full plant. Should the embryo develop naturally, the resulting plant may be unusable as a parent in future breeding endeavors because of a condition called hybrid weakness. This condition is caused by factors such as disharmony between the united genomes. Some hybrid plants may fail to flower because of hybrid sterility (F1 sterility) resulting from meiotic abnormalities. On some occasions, the hybrid weakness and infertility manifest in the F2 and later generations (called hybrid breakdown).

Table 6.2A summary of the reproductive isolation barriers in plants as first described by G.L. Stebbins.

The reproductive barriers previously discussed confront plant breeders who attempt gene transfer between distant genotypes via hybridization. The primary challenge of wide crosses is obtaining fertile F 1hybrids, because of the mechanisms that promote, especially, gametic incompatibility. As previously indicated, this mechanism acts to prevent (i) the pollen from reaching the stigma of the other species; (ii) germination of the pollen and inhibition of growth of the pollen tube down the style, or the union of male gamete and the egg if the pollen tube reaches the ovary; and (iii) the development of the zygote into a seed and the seed into a mature plant. Gametic incompatibility ends where fertilization occurs. However, thereafter, there are additional obstacles to overcome. Gametic incompatibility and hybrid breakdown are considered to be barriers to hybridization that are outside the control of the breeder.

Several techniques have been developed to increase the chance of recovering viable seed and plants from a wide cross. These techniques are based on the nature of the barrier. All techniques are not applicable to all species.

Overcoming barriers to fertilizationConduct reciprocal crossesGenerally, it is recommended to use the parent with the larger chromosome number as female in a wide cross for higher success. This is because some crosses are successful only in one direction. Hence, where there is no previous information about crossing behavior, it is best to cross in both directions.Shorten the length of the styleThe pollen tube of a short‐styled species may not be able to grow through a long style to reach the ovary. Thus, shortening a long style may improve the chance of a short pollen tube reaching the ovary. This technique has been successfully tried in corn and in lily.Apply growth regulatorsChemical treatment of the pistil with growth‐promoting substances (e.g. naphthalene acetic acid [NAA], gibberellic acid [GA]) tends to promote rapid pollen tube growth or extend the period over which the pistil remains viable.Modify ploidy levelA diploid species may be converted to a tetraploid to be crossed to another species. For example, narrow leaf trefoil, (Lotus tenuis, 2n = 12) was successfully crossed with broadleaf bird's foot trefoil (L. corniculatus, 2n = 24) after chromosome doubling of the L. tenuis accession.Use mixed pollenMixing pollen from a compatible species with pollen from an incompatible parent makes it possible to avoid the unfavorable interaction associated with cross‐incompatibility.Remove stigmaIn potato, wide crosses were accomplished by removing the stigma before pollination and substituting it with a small block of agar fortified with sugar and gelatin.GraftingGrafting the female parent to the male species has been reported in some crops to promote pollen tube growth and subsequent fertilization.Protoplast fusionA protoplast is all the cellular component of a cell excluding the cell wall. Protoplasts may be isolated by either mechanical or enzymatic procedures. Mechanical isolation involves slicing or chopping of the plant tissue to allow the protoplast to slip out through a cut in the cell wall. This method yields low numbers of protoplasts. The preferred method is the use of hydrolytic enzymes to degrade the cell wall. A combination of three enzymes – cellulase, hemicellulase, and pectinase – is used in the hydrolysis. The tissue used should be from a source that would provide stable and metabolically active protoplasts. This calls for monitoring plant nutrition, humidity, day length, and other growth factors. Often, protoplasts are extracted from leaf mesophyll or plants grown in cell culture. The isolated protoplast is then purified, usually by the method of flotation. This method entails first centrifuging the mixture from hydrolysis at about 50× g, and then resuspending the protoplasts in high concentration of fructose. Clean, intact protoplasts float and can be retrieved by pipetting. Protoplasts can also be used to create hybrids in vitro (as opposed to crossing mature plants in conventional plant breeding) ( Figure 6.3).