Application of pollen germination media on stigmas during pollination increases seed set in east African highland cooking bananas (Musa spp.)

Open Access Journal; Published online: 27 May 2021Seed set in East African Highland Cooking bananas (EAHBs) is extremely low and therefore hampers breeding. Pollen–pistil interaction is a key contributing factor. We assessed the effect of pollen germination media (PGM) on seed set in EAHBs. Five EAHB cultivars were pollinated with pollen from the wild banana ‘Calcutta 4’. Glucose-based PGM sprayed on freshly emerged stigmas significantly increased seed set per 100 fruits per bunch. Increases were 73.5% in ‘Enzirabahima’, 39.9% in ‘Mshale’, and 302.4% in ‘Nshonowa’. However, PGM did not increase seed set in the female sterile ‘Mlelembo’ and ‘Nakitembe’. As larger bunches were more fertile, good field management practices are also recommended to get more seed to improve breeding efficiency

Seed set patterns in east African highland cooking bananas are dependent on weather before, during and after pollination

Open Access Journal; Published online: 29 Jun 2021Seed set in banana is influenced by weather, yet the key weather attributes and the critical period of influence are unknown. We therefore investigated the influence of weather during floral development for a better perspective of seed set increase. Three East African highland cooking bananas (EAHBs) were pollinated with pollen fertile wild banana ‘Calcutta 4′. At full maturity, bunches were harvested, ripened, and seeds extracted from fruit pulp. Pearson’s correlation analysis was then conducted between seed set per 100 fruits per bunch and weather attributes at 15-day intervals from 105 days before pollination (DBP) to 120 days after pollination (DAP). Seed set was positively correlated with average temperature (P < 0.05–P < 0.001, r = 0.196–0.487) and negatively correlated with relative humidity (RH) (P < 0.05–P < 0.001, r = −0.158–−0.438) between 75 DBP and the time of pollination. After pollination, average temperature was negatively correlated with seed set in ‘Mshale’ and ‘Nshonowa’ from 45 to 120 DAP (P < 0.05–P < 0.001, r = −0.213–−0.340). Correlation coefficients were highest at 15 DBP for ‘Mshale’ and ‘Nshonowa’, whereas for ‘Enzirabahima’, the highest were at the time of pollination. Maximum temperature as revealed by principal component analysis at the time of pollination should be the main focus for seed set increase

Seed set patterns in east African highland cooking bananas show asymmetric distribution in bunches and fruits

Open Access Journal; Published online: 14 Apr 2021Low female fertility in bananas is the biggest hurdle for banana breeding. The aim of this study was to determine seed set patterns in East African Highland Cooking bananas (EAHBs) to inform future decisions on a more targeted approach of increasing seed set and subsequently banana-breeding efficiency. Matooke (AAA) and Mchare (AA) bananas are genetically distinct but belong to the same genetic complex, referred to as EAHBs. Seed set patterns in “Enzirabahima” (AAA), “Mshale” (AA), and “Nshonowa” (AA), all with residual fertility, were examined after hand pollination with a highly male fertile wild banana “Calcutta 4” (AA). Seed set in “Enzirabahima” is predominant in distal hands. Mchare cultivars have a slightly more even distribution of seeds in their hands compared to “Enzirabahima”. There is a gradual increase in seed set from proximal to distal hands with a slight drop in the last hand. This pattern is more definite in “Enzirabahima” and “Mshale”, while “Nshonowa” has a somewhat inconsistent pattern. There is also a drop in seed set per 100 fruits per hand from small to larger bunches. However, larger bunches have a higher pollination success compared to smaller bunches. They therefore set more seed on 100 fruits per hand and per bunch basis, if bunches without seed are accounted for. Pollination success rate increases from smaller to larger bunches of EAHBs. Seed set is biased toward the distal third part of fruits of examined EAHBs, as well as tetraploid Matooke hybrid “401K-1” (AAAA), and improved diploid “Zebrina” GF (AA) that were used for comparison. In comparison, in the highly female fertile “Calcutta 4”, seed set is along the entire length of the fruit. Seed set bias in the distal hands and distal end of fruits suggests a systematic mechanism rather than a random occurrence. It is expected that this information will provide a foundation for increased crossbreeding efficiency in bananas

Use of timelapse photography to determine flower opening time and pattern in banana (Musa spp.) for efficient hand pollination

Open Access Journal; Published online: 30 Sep 2021Sterility and low seed set in bananas is the main challenge to their conventional genetic improvement. The first step to seed set in a banana breeding program depends on pollination at the right time to ensure effective fertilization. This study aimed at determining bract opening time (BOT) to enhance efficient pollination and seed set in bananas. A Nikon D810 digital camera was set-up to take pictures of growing banana inflorescences at five-minute intervals and time-lapse movies were developed at a speed of 30 frames per second to allow real-time monitoring of BOT. Genotypes studied included wild banana (1), Mchare (2), Matooke (4), Matooke hybrid (1), and plantain (1). Events of bract opening initiated by bract lift for female flowers (P < 0.01) started at 16:32 h and at 18:54 h for male flowers. Start of bract rolling was at 18:51 h among female flowers (P < 0.001) and 20:48 h for male flowers. Bracts ended rolling at 02:33 h and 01:16 h for female and flowers respectively (P < 0.05). Total time of bract opening (from lift to end of rolling) for female flowers was significantly longer than that of male flowers (P < 0.001). On average, the number of bracts subtending female flowers opening increased from one on the first day, to between one and four on the fourth day. The number regressed to one bract on day eight before start of opening of bracts subtending male flowers. There was a longer opening interval between bracts subtending female and male flowers constituting spatial and temporal separation. Bract rolling increased from partial to complete rolling from proximal to the distal end of the inflorescence among female flower. On the other hand, bracts subtending male flowers completely rolled. Differences in BOT of genotypes with the same reference time of assessment may be partly responsible for variable fertility. Hand pollination time between 07:00 and 10:00 h is slightly late thus an early feasible time should be tried

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

Evolution of cassava brown streak disease-associated viruses

Cassava brown streak disease (CBSD) has occurred in the Indian Ocean coastal lowlands and some areas of Malawi in East Africa for decades, and makes the storage roots of cassava unsuitable for consumption. CBSD is associated with Cassava brown streak virus (CBSV) and the recently described Ugandan cassava brown streak virus (UCBSV) [picorna-like (+)ssRNA viruses; genus Ipomovirus; family Potyviridae]. This study reports the first comprehensive analysis on how evolution is shaping the populations of CBSV and UCBSV. The complete genomes of CBSV and UCBSV (four and eight isolates, respectively) were 69.0–70.3 and 73.6–74.4% identical at the nucleotide and polyprotein amino acid sequence levels, respectively. They contained predictable sites of homologous recombination, mostly in the 39-proximal part (NIb-HAM1h-CP-39-UTR) of the genome, but no evidence of recombination between the two viruses was found. The CP-encoding sequences of 22 and 45 isolates of CBSV and UCBSV analysed, respectively, were mainly under purifying selection; however, several sites in the central part of CBSV CP were subjected to positive selection. HAM1h (putative nucleoside triphosphate pyrophosphatase) was the least similar protein between CBSV and UCBSV (aa identity approx. 55 %). Both termini of HAM1h contained sites under positive selection in UCBSV. The data imply an on-going but somewhat different evolution of CBSV and UCBSV, which is congruent with the recent widespread outbreak of UCBSV in cassava crops in the highland areas (.1000 m above sea level) of East Africa where CBSD has not caused significant problems in the past