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).

       Overcoming the problem of inadequate hybrid seed developmentAbnormal embryo or endosperm development following a wide cross may be overcome by using proper parent selection and reciprocal crossing as previously described. In addition, the technique of embryo rescue is an effective and common technique. The embryo is aseptically extracted and nurtured into a full plant under tissue culture conditions. Overcoming lack of vigor of the hybridHybrids may lack the vigor to grow properly to flower and produce seed. Techniques such as proper parent selection, reciprocal crossing, and grafting the hybrid onto one of the parents may help.Overcoming hybrid sterilitySterility in hybrids often stems from meiotic complications due to lack of appropriate pairing partners. Sterility may be overcome by doubling the chromosome number of the hybrid to create pairing mates for all chromosomes, and hence produce viable gametes.