Culturing Interspecific Hybrids

Recently Chuck Leslie and his crew at the Walnut Improvement Program modified the method for extracting interspecific walnut embryos for culture to improve its success and efficiency. Earlier work extracting the embryos of interspecific hybrids had been challenging, requiring a team of two to extract embryos. In addition to the modified extraction method, modifications were also made to the disinfestation process, such as rinsing the fruit in a soapy solution prior to immersion in a bleach solution. Embryos are also transferred every 24 hours due to phenolic exchange with the media.

Improved method to extract embryos for culture.

First some background on the material and reasons for improving the embryo extraction method. The main interspecific hybrids being embryo cultured are F1 hybrids of J. microcarpa x J. regia and J. cathayensis x J. regia, the small fruit/nut and larger fruit/nut in the photos, respectively. The fruits are collected directly from the mother trees just prior to full maturity. The fruit of J. microcarpa and J. cathayensis do not split open at maturity like J. regia, although J. microcarpa fruit often develop some cracks. It was previously determined that the shells of the nuts from both mother trees hardened at earlier stages of maturity and it would make little difference to collect them later thus allowing the embryos additional maturation time. Therefore the shells are fully hardened and both mother trees produce nuts with very thick shells, unlike the much thinner shells of J. regia nuts. Additionally, the J. microcarpa fruit are small and round with an approximate diameter of 1/2-3/4”, whereas J. cathayensis fruit are oblong, approximately 1 1/4-1 1/2” wide and 2-2 1/2” long, and smaller than J. regia nuts. Lastly, J. microcarpa embryos are very small even at full maturity.

J. microcarpa x J. regia

The fruit is placed into a sterile paper towel to prevent reinfestation from placement in the vice. The paper towel is then rolled into a tube containing the fruit and placed into a vice, preferrably with the blossom end up, but it is hard to track because of the round shape and small size of J. microcarpa nuts. The vice is then tightened until the shell cracks, enough to hear a pop but not so much as to crush the nut and embryo, and then removed from the vice. After cracking and removal from the vice, the paper towel is unrolled and used as a cutting surface during the embryo extraction. The cracked nut is opened further to reveal the embryo located at blossom end of the nut, the point of the ‘heart’ in the photos. The embryo is then placed on culture media to germinate and once large enough is further multiplied, rooted, and send to the different pathology and nematology groups. In some cases the nut cracks poorly and it is difficult to determine the embryo location. When this occurs the assorted pieces are all placed on media and observed until the section with the embryo is evident.

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J. cathayensis x J. regia

This method is similar to the one used for the J. microcarpa x J. regia hybrids. However, in the photos it is shown without the paper towel and with a line drawn on the fruit following the suture, but this was done for demonstration purposes only. In reality a sterile paper towel is still used, the fruit is placed in the vice with the suture against the sides, and the blossom end pointing up. Again, the vice is tightened until the shell cracks open the fruit is removed from the vice, and the embryo extracted and placed in culture media to germinate.

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