SCREENING FOR SEED FERTILITY THROUGH PLOIDY MANIPULATION AND INTER-SPECIFIC HYBRIDIZATION AMONG Musa ACCESSIONS.

SCREENING FOR SEED FERTILITY THROUGH PLOIDY MANIPULATION AND INTER-SPECIFIC HYBRIDIZATION AMONG Musa ACCESSIONS.

ABSTRACT

This work was aimed at assessing the variability of male and female fertility to enhance yield, quality and physiology in Musa breeding program. Musa germplasm at Ebonyi State University Abakaliki were characterized using morphological descriptors, SCoT markers and the count of chloroplasts in stomatal guard cells. The accessions were assessed for male and female fertility using artificial hand pollination, in an attempt to ascertain the possibility of generating new hybrids. In addition, micro-propagation of identified genotypes (‘Owom’ and ‘Efol’) was carried out using MS media supplemented with different concentrations of plant hormones (BAP at 0 mg/L, 5 mg/L and 10 mg/L; IAA at 0 mg/L, 2 mg/L and 4 mg/L) and cultured in two environments (screen house and growth chamber) in order to identify cost-effective ways of generating seedlings. Macro-propagation was also carried out to access the effect of different growth media (sawdust and spear grass) and acclimatization potting media (Poultry droppings with topsoil, Cow dung with topsoil and topsoil) on the generation of Musa seedlings (‘Atagafong’ and ‘Owom’). Results indicated morphological variation among the accessions studied for most of the traits studied. About 9 (≥ 90%) of the SCoT markers were highly informative (i.e., PIC > 0.50) in accessing genetic diversity of the Musa accessions, grouping them into five clusters. Results of the chloroplast indicated that the accessions had chloroplasts ranging from 9 to 18. Accessions were then grouped into different ploidy based on number of chloroplasts with Calcutta 4 and PITA 14 classified as tetraploids, ‘Efol red’ as a diploid, and the remaining accessions as triploids. There was no seed generated from crosses among the Musa accessions, indicating high level of sterility among the accessions in the Musa genebank. Prior to micropropagation, Treatment 6 sterilisation protocol produced very healthy and clean cultures with 100 % survival. There were significant differences in the performance of the Musa accessions on MS media supplemented with different hormone concentrations and kept under different environments. For ‘Owom’, BAP 10 mg/L concentration responded significantly in both environments while for ‘Efol’, BAP 5 mg/L and BAP 0 mg/L concentrations were significantly different in growth chamber and screen house respectively. At 5 weeks after initiation, enhanced development of 70 % plantlets was observed. IAA 0 mg/L and IAA 4 mg/L concentrations were significantly different for rooting of ‘Efol’ and ‘Owom’ respectively in growth chamber while in screen house, IAA 4 mg/L and IAA 2 mg/L were significantly different for ‘Efol’ and ‘Owom’, respectively. Screen house cultures produced higher number of shoots, but with less health performance even at acclimatization stage. Physicochemical properties showed that sawdust was a better macro-propagative media than spear grass, and it produced higher number of shoots per corm for ‘Atagafong’ (3.66) and ‘Owom’ (3.00). A 100 % mortality recorded across the genotypes weaned in medium of poultry droppings and top soil was attributed to its very high nitrogen content. The work showed that responses of accessions are influenced by environment.

1.1       Background of the Study

The situation in Nigeria is worrisome as food insecurity has become a critical element of Nigeria’s poverty profile (Oshewolo, 2011). Statistically, about 14 million people, including children, are malnourished in Nigeria (Owoo, 2021). With a projected rise in the prices of staple food items, according to Oderinde et al. (2022) food security in Nigeria is presently in dire straight owing to factors like skyrocketing energy price, climate change and terrorism. This could be attributed to unavailability of different food crops, with minimal supply of limited available food ones. The unfavourable weather condition, degraded soil, improper response of people towards farming, lack of planting materials, insect and disease attack among others could have high effect on agricultural productivity. To offset hunger among the population, there is high need to increase the agronomic value of available food crop species, among which is Musa spp.

Plantain and banana (Musa spp) are one of the most important staple food crops in the tropics and a source of income to millions of poor subsistence farmers. They constitute the fourth most important global food commodity (after rice, wheat and maize) grown in more than 100 countries over a harvested area of approximately 10 million hectares, with an annual production of 88 million tonnes (Frison and Sharrock, 1999). The all year round fruiting habit of the crop puts the crop in a superior position in bridging the hunger gap between crop harvests. It therefore contributes significantly to food and income security of people engaged in its production and trade, particularly in developing countries. In Africa, they provide more than 25% of the carbohydrate requirements for over 70 million people (IITA, 1998).

The genus Musa has 22 chromosomes in wild species and 22, 33 and 44 chromosomes in cultivated forms, with a basic haploid number of 11 chromosomes. Majority of cultivated varieties (Simmonds and Shepherd, 1955; Simmonds, 1995a) are triploid (3x=33), that are derived from intra-specific crosses within M. acuminata Colla (A genome) and inter-specific crosses between M. acuminata and M. balbisiana Colla (B genome).

Different species and cultivars of Musa possess different levels of competence in the production of microspores, which correlates positively with levels of pollen fertility. A study by Fortescue and Turner (2004), where pollen viability was examined using the Alexander’s pollen stain procedure, showed that diploid species M. acuminata and M. balbisiana had 3 times more viable pollen than the edible tetraploids (AAAB), and the tetraploids contained 3 times more viable pollen than the edible triploids AAA, AAB and ABB. This shows high sterility among the triploids. Most cultivated bananas are triploid and so, characterized by high male and female sterility (Nyine and Pillay, 2007), hence, propagated vegetatively through suckers. As a result, use of conventional breeding methods for improvement is difficult and cumbersome. Owing to these constraints, mutation breeding and genetic engineering techniques have been employed in its breeding. These methods of breeding are very expensive, may be more prone to unexpected outcomes because it creates profound disruption in the normal interactions of genes, require highly skilled labourers and laboratories with sophisticated technological equipment and finally, weed off wild species which are souces of genetic materials for breeding. Therefore, conventional breeding being less complicated and employing processes such as sexual and asexual reproduction which occur in nature is still being advised.

1.2       Problem Statement

Research has been carried out on breaking the sterility of Musa spp during which TMB2x 9128-3 was discovered at the International Institute of Tropical Agriculture (IITA), Ibadan. TMB2x 9128-3 is a diploid breeding stock which was developed at IITA from 1988 to 1990 and has good combining ability, fertility, and resistance to Black Sigatoka and Nematodes (Tenkouano et al. 2003). According to Tenkouano et al. (2003), breeding values in 2x – 2x and 4x – 2x crosses were assessed in subsequent years and favourable results were achieved. With the ever-increasing population and its consequent increasing demand for food, there is high need for extensive research on available Musa accessions in every genebanks and their possible hybridization as this will enhance provision of improved varieties of Musa spp, especially those with high fertility. This will promote its agricultural productivity, breeding progress and reduce hunger in the society, especially for children and pregnant woman, and also increase the food security and financial status of farm families. For this to be achieved, accessions have to be screened to assess level of male and female fertility and/or sterility for maximum exploitation in a breeding program. As explained by Poerba and Ahmad (2010), frequent somatic mutations and morphological changes resulting from environment have resulted in large number of cultivars.

Genetic relatedness among cultivars can be studied based on morphological characteristics among cultivars and varieties. However, the use of only morphological parameters could result in over or under estimation of the degree of relatedness among plantain cultivars. Therefore, employment of genomic approach in determination of relatedness among available accessions is needful to enhance understanding of the accessions’ diversity prior to its hybridization in the germplasm.

 1.3       General Aim

This work was aimed at assessing the variability in Musa germplasm for male and female fertility, sterility, through crossing the accessions to generate fertile hybrids which will be used for maximum exploitation of yield, quality and physiological enhancement in Musa breeding program, that targets consumer preferred characteristics. This will establish the prospects of using different groups of accessions as seed parents for different sexes, as well as developing hybrids from their crosses.

1.4       Specific Objectives

• To characterize Musa accessions within Ebonyi State University Abakaliki (EBSU) Field Musa Germplasm using morphological descriptors and genetic markers for their relatedness.
• To determine the ploidy status of Musa accessions in the field germplasm.
• To screen the Musa accessions for seed fertility using artificial hand pollination.
• To micro-propagate identified accessions, including embryo rescue of seeds generated from crosses.
• To efficiently macropropagate selected Musa accessions using cost effective procedures.

1.5       Study Hypothesis

1) There is no variation in genomic and morphological traits of Musa accessions in EBSU Field Musa Germplasm.

2) Musa accessions in EBSU Field Musa Germplasm are sterile.

3) hormones, growth environment and accessions have no effects in performance of Musa spp during tissue culture.

4) Accessions show the same response in different substrate during micropropagation.

1.6   Scope and Limitation of Research

This research was carried out using accessions from Musa germplasm in Ebonyi State University Abakaliki, which is located in Southeastern Nigeria with coordinate of latitude 6° 19′ N and longitude 8° 6′ E. Accessions in the germplasm were assembled from South-Eastern Nigeria and International Institute of Tropical Agriculture, Ibadan.

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