Effect of self-pollination with heat-treated pollen on parthenocarpy and homozygosity in cassava

Cassava\u2019s ( Manihot esculenta Crantz) high heterozygosity complicates its genetic improvement via selective breeding. Double haploid (DH) technology can be used to improve the crop\u2019s heterozygosity, thereby improving the capacity for genetic improvement. The objective of this study was to evaluate the effect of self-pollination using heated pollen on pollen tube penetration, fruit set, seed and haploid embryo development in cassava genotypes for the production of haploid cassava. Pollen from two cassava genotypes, NASE3 and NASE14, was heated at 40, 50 and 60 oC for 0.5, 1.0 and 2.0 hr each. The heated pollen was used in six rounds of self-pollinations. Pollen tube penetration was monitored by fluorescent microscopy, followed by early embryo rescue and ovule culture. Ploidy and zygosity were assessed using flow cytometry and single-nucleotide polymorphism analysis, respectively. Pollen germinated on the stigma, grew within the style through the nucellar beak, but did not reach the embryo sac, thus achieving no fertilisation in all the 5756 self-pollinated flowers. There was a reduction in pollen germination (in vitro and in vivo), pollen tube penetration and fruit set with increasing temperature. Heat-treated pollen stimulated division of the egg cell and induced development of parthenocarpic fruits. Up to 6 embryoids per ovule were observed and all regenerated plantlets were diploid, with up to 93.0% increased homozygosity. For the first time, plant regeneration from ovules, pollinated with fresh pollen at 14 days after pollination, was achieved indicating improved speed in plant regeneration. The data generated are important for the development of protocols for cassava DH plant production.La forte h\ue9t\ue9rozygotie du manioc ( Manihot esculenta Crantz) complique son am\ue9lioration g\ue9n\ue9tique par s\ue9lection s\ue9lective. La technologie d\u2018 haplo\uefde double (DH) peut \ueatre utilis\ue9e pour am\ue9liorer l\u2019h\ue9t\ue9rozygotie de la culture, am\ue9liorant ainsi la capacit\ue9 d\u2019am\ue9lioration g\ue9n\ue9tique. L\u2019objectif de cette \ue9tude \ue9tait d\u2019\ue9valuer l\u2019effet de l\u2019auto-pollinisation \ue0 l\u2019aide de pollen chauff\ue9 sur la p\ue9n\ue9tration du tube pollinique, la nouaison, le d\ue9veloppement des graines et des embryons haplo\uefdes dans les g\ue9notypes de manioc pour la production de manioc haplo\uefde. Le pollen de deux g\ue9notypes de manioc, NASE3 et NASE14, a \ue9t\ue9 chauff\ue9 \ue0 40, 50 et 60 oC pendant 0,5, 1,0 et 2,0 heure (s) chacun. Le pollen chauff\ue9 a \ue9t\ue9 utilis\ue9 dans six cycles d\u2019auto-pollinisation. La p\ue9n\ue9tration du tube pollinique a \ue9t\ue9 surveill\ue9e par microscopie fluorescente, suivie d\u2019un sauvetage pr\ue9coce des embryons et d\u2019une culture d\u2019ovules. La plo\uefdie et la zygosit\ue9 ont \ue9t\ue9 \ue9valu\ue9es \ue0 l\u2019aide de la cytom\ue9trie en flux et de l\u2019analyse du polymorphisme mononucl\ue9otidique, respectivement. Le pollen a germ\ue9 sur le stigmate, s\u2019est d\ue9velopp\ue9 dans le style \ue0 travers le bec nucellaire, mais n\u2019a pas atteint le sac embryonnaire, n\u2019obtenant ainsi aucune f\ue9condation dans toutes les 5756 fleurs autogames. Il y avait une r\ue9duction de la germination du pollen (in vitro et in vivo), de la p\ue9n\ue9tration du tube pollinique et de la nouaison avec l\u2019augmentation de la temp\ue9rature. Le pollen trait\ue9 thermiquement a stimul\ue9 la division de l\u2019ovule et induit le d\ue9veloppement de fruits parth\ue9nocarpiques. Les 6 embryo\uefdes par ovule ont \ue9t\ue9 observ\ue9s et toutes les plantules r\ue9g\ue9n\ue9r\ue9es \ue9taient diplo\uefdes, avec 93,0% d\u2018augmentation d\u2019homozygotie. Pour la premi\ue8re fois, la r\ue9g\ue9n\ue9ration des plantes \ue0 partir des ovules, pollinis\ue9es avec du pollen frais 14 jours apr\ue8s la pollinisation, a \ue9t\ue9 r\ue9alis\ue9e, indiquant une vitesse am\ue9lior\ue9e de r\ue9g\ue9n\ue9ration des plantes. Les donn\ue9es g\ue9n\ue9r\ue9es sont importantes pour l\u2019\ue9laboration de protocoles de production de plantes de manioc de DH