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

Fruit set and plant regeneration in cassava following interspecific pollination with castor bean

The increasing demand for cassava ( Manihot esculenta Crantz) for food and non-food uses in the tropics necessitates that its breeding for increased root productivity be made faster. The characteristic long breeding cycle and heterozygous nature of this crop, pose a major obstacle to its rapid genetic improvement. This study aimed at inter-pollinating cassava with castor bean ( Ricinus communis ), with a purpose of inducing and regenerating cassava doubled haploids (DHs). A total of 3,349 flowers from twelve elite cassava varieties were inter-pollinated with caster bean. A total of 803 fruits were harvested for early embryo rescue and/or ovule culture. Of these, three were dissected to obtain seven unique embryos, while 800 were dissected to obtain 1312 young ovules, all of which were cultured in vitro. Overall, 82 (6.25%) of the cultured ovules formed callus that originated from the embryosac region, which is haploid. Four out of seven rescued embryos (57.1%) regenerated into plantlets. Ploidy analyses of 24 samples using flow cytometry revealed that 23 of the analysed samples were diploid. However, one callus sample was anueploid. Only one sample had an exceptionally high level of homozygosity ( 84.2%). These findings lay a foundation for future research aimed at induction of haploids in cassava.La demande croissante de manioc (\ua0 Manihot esculenta \ua0Crantz\ua0) \ue0 usage alimentaire et non alimentaire dans les tropiques\ua0n\ue9cessite que sa reproduction pour une productivit\ue9 accrue des racines soit faite plus rapidement.\ua0Le long cycle de reproduction et le caract\ue8re h\ue9t\ue9rozygote de cette plante constituent un obstacle majeur dans la rapidit\ue9 de son am\ue9lioration g\ue9n\ue9tique.\ua0Cette \ue9tude visait \ue0 inter-polliniser le manioc avec le haricot (\ua0 Ricinus communis \ua0), dans le but d\u2019induire et de r\ue9g\ue9n\ue9rer le manioc d\u2019haplo\uefdes doubl\ue9 (HD).\ua0Un total de 3 349 fleurs de douze \ue9lites vari\ue9t\ue9s de manioc ont \ue9t\ue9 inter-pollinis\ue9es avec le haricot.\ua0Un total de 803 fruits ont \ue9t\ue9 r\ue9colt\ue9s pour les embryons pr\ue9matur\ue9s qui etaient sauves et\ua0/ ou la\ua0culture d\u2019ovules\ua0.\ua0Parmi ceux-ci,\ua0trois ont \ue9t\ue9 diss\ue9qu\ue9s pour obtenir sept embryons uniques\ua0,\ua0tandis que 800 ont \ue9t\ue9 diss\ue9qu\ue9s pour obtenir 1312 jeunes ovules, qui ont tous \ue9t\ue9 cultiv\ue9s\ua0in vitro\ua0.\ua0Un total de 82 (6,25%) des ovules en culture ont form\ue9 des cals provenant de la\ua0r\ue9gion\ua0embryonnaire\ua0, qui est haplo\uefde. Quatre parmi sept embryons sauv\ue9s (57,1%) se sont r\ue9g\ue9n\ue9r\ue9s en plantules.\ua0Les analyses de plo\uefdie de 24 \ue9chantillons par cytom\ue9trie en flux ont r\ue9v\ue9l\ue9 que 23 des \ue9chantillons analys\ue9s \ue9taient diplo\uefdes.Cependant, un \ue9chantillon de cals \ue9tait anueplo\uefde.\ua0Un seul \ue9chantillon pr\ue9sentait un niveau d\u2019homozygotie exceptionnellement \ue9lev\ue9\ua0(84,2\ua0%).\ua0Ces r\ue9sultats sont les bases des recherches dans le futur sur la cause des haplo\uefdes dans le manioc

Fruit set and plant regeneration in cassava following interspecific pollination with castor bean

The increasing demand for cassava (Manihot esculenta Crantz) for food and non-food uses in the tropics necessitates that its breeding for increased root productivity be made faster. The characteristic long breeding cycle and heterozygous nature of this crop, pose a major obstacle to its rapid genetic improvement. This study aimed at inter-pollinating cassava with castor bean (Ricinus communis), with a purpose of inducing and regenerating cassava doubled haploids (DHs). A total of 3,349 flowers from twelve elite cassava varieties were inter-pollinated with caster bean. A total of 803 fruits were harvested for early embryo rescue and/or ovule culture. Of these, three were dissected to obtain seven unique embryos, while 800 were dissected to obtain 1312 young ovules, all of which were cultured in vitro. Overall, 82 (6.25%) of the cultured ovules formed callus that originated from the embryosac region, which is haploid. Four out of seven rescued embryos (57.1%) regenerated into plantlets. Ploidy analyses of 24 samples using flow cytometry revealed that 23 of the analysed samples were diploid. However, one callus sample was anueploid. Only one sample had an exceptionally high level of homozygosity ( 84.2%). These findings lay a foundation for future research aimed at induction of haploids in cassava