Male Kiwifruit Research Articles

Preliminary selection of wild male kiwifruit (Actinidia callosa var. strigillosa C. F. Liang) accessions in North Sikkim, India, for future breeding programmes

ABSTRACT The flower morphology and phenology of 10 healthy, profusely flowering wild male kiwifruit (Actinidia callosa var. strigillosa C. F. Liang) accessions growing in North Sikkim district in Sikkim state, Northeast India, were analysed in 2021–2022. There was distinct variation among the accessions in the number of flowers per inflorescence, number of sepals and petals, colour of the sepals, arrangement of the petals, and average number of stamens, as well as the timing and duration of flowering. Accessions SKNFF-04 and SKNLC-21 were superior in terms of pollinating traits, with the highest average numbers of flowers and stamens per vine and the longest duration of flowering. Principal component analysis (PCA) revealed that the first and second principal components (PC1 and PC2) had eigenvalues greater than 1 and together explained 95.13% of the variation. Hierarchical cluster analysis categorised the accessions into two major clusters: Cluster I with eight accessions and Cluster II with the two superior accessions. The presence of the pleiochasium inflorescence type among all the observed accessions is of particular significance, as it can increase the efficacy of male selection in kiwifruit breeding programmes. This research sets the stage for further investigations into the potential advantages and unique characteristics of wild kiwifruit germplasm.

RNA-seq analysis of synchronized developing pollen isolated from a single anther.

Pollen development, from unicellular microspores to anthesis, is a complex process involving the coordinated specification, differentiation and functions of different cell types. Key to understanding this development is identifying the genes expressed at precise stages of development. However, transcriptomic studies on pollen prior to anthesis are complicated by the inaccessible nature of pollen developing in the anther and the resistant pollen wall. To assist with understanding gene expression during pollen development we have developed a protocol to perform RNA-Seq on pollen isolated from a single anther (SA RNA-Seq). The protocol involves removing pollen from a single anther for analysis and viewing the remaining pollen to determine the developmental stage. The isolated pollen is chemically lysed and mRNA isolated from the lysate using an oligo-dT column before library preparation. Here, we report on the development and testing of our method and the generation of a transcriptome for three stages of pollen development from Arabidopsis (Arabidopsis thaliana) and two stages from male kiwifruit (Actinidia chinensis). This protocol enables the transcriptome of precise developmental stages of pollen to be analyzed, and uses a small number of plants, potentially facilitating studies that require a range of treatments or the analysis of the first generation of transgenic plants.

Shy Girl, a kiwifruit suppressor of feminization, restricts gynoecium development via regulation of cytokinin metabolism and signalling.

Kiwifruit (Actinidia chinensis) is a dioecious, long-living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate alternative farming for better food security in the face of climate change. Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male-specific Shy Girl (SyGl) gene with homology to type-C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis 'Bruce'. Biallelic mutations of CEN and CEN4 generated rapid-flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid-flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self-pollinate and produce fast-flowering offspring. These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.

The Leaf Bacterial Microbiota of Female and Male Kiwifruit Plants in Distinct Seasons: Assessing the Impact ofPseudomonas syringaepv.actinidiae

The Pseudomonas syringae pv. actinidiae pandemic has been compromising the production of the kiwifruit industry in major producing countries. Abiotic factors and plant gender are known to influence the disease outcome. To better understand their impact, we have determined the diversity of the leaf bacterial communities using the V5-V6 region of the 16S ribosomal RNA gene amplicon on the Illumina MiSeq sequencing platform. Healthy and diseased female and male kiwifruit plants were analyzed in two consecutive seasons: spring and autumn. This work describes whether the season, plant gender, and presence of P. syringae pv. actinidiae can affect the leaf bacterial community. Fifty bacterial operational taxonomic units were identified and assigned to five phyla distributed by 14 different families and 23 genera. The leaves of healthy female and male kiwi plants share most of the identified bacterial populations, which undergo major seasonal changes. In both cases, a substantial increase of the relative abundance of genus Methylobacterium is observed in autumn. The presence of P. syringae pv. actinidiae induced profound changes on leaf bacterial community structures, translated into a reduction in the relative abundance of previously dominant genera that had been found in healthy plants; namely, Hymenobacter, Sphingomonas, and Massilia spp. The impact of P. syringae pv. actinidiae was less pronounced in the bacterial community structure of male plants in both seasons. Some of the naturally occurring genera have the potential to act as antagonists or as enhancers of the defense mechanisms, paving the way for environmentally friendly and sustainable disease control.

Identification and sequence characterisation of molecular markers polymorphic between male kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) accessions exhibiting different flowering time

Fruit set in kiwifruit is strongly dependent on pollination, which is limited by the lack of efficient male pollen donors, among other factors. We searched for molecular markers that could be polymorphic in relation to flowering time in order to classify male kiwifruit plants to discard those that are not likely to perform as efficient pollen donors. Random amplified polymorphic DNA (RAPD) and modified amplified fragment length polymorphism (AFLP) markers were generated using 41 male kiwifruit plants in two flowering groups, early- and late-flowering males (with respect to the female cultivar ‘Hayward’). One RAPD and nine modified-AFLP markers polymorphic between male plants exhibiting different flowering time were identified, sequenced and analysed in databases. Unweighted pair group method with arithmetic average (UPGMA) clustering and multidimensional scaling showed that these markers could be used to classify the male plants into flowering groups. Analysis of molecular variance (AMOVA) agreed with this classification, showing that most of the genetic variation is found between flowering groups. Sequence analysis based on a database search revealed that the polymorphism PolM contains a 7-nucleotide long element involved in the repression of the phytochrome A gene, that Pol4 is a partial sequence of a phytochrome B gene, and that sequences Pol3, Pol5, Pol7, and Pol9 show high identity with ESTs from kiwifruit buds treated with hydrogen cyanamide. Clustering analysis supported the previous classification of males into flowering groups, making it feasible to predict male plants’ flowering times with respect to the cultivar ‘Hayward’ based upon these molecular markers.

Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation

The present work reports on a study of plant regeneration carried out with callus from the leaf blades and petioles of field-grown male adult kiwifruit plants (Actinidia deliciosa (Chev.) Liang and Ferguson). The cultivars used were ‘Tomuri’ and clone A, a selected male plant grown in north western Spain. The best shoot induction conditions were obtained in ‘Tomuri’ leaf blades cultured in K(h) medium in the presence of 23 µM Zeatin and 0.1 µM NAA. Under these conditions, more than 80% of organogenic callus induction was observed, with an average of 14 new shoots in the second subculture. The initial length of the shoots affected shoot elongation, which was accomplished by culturing isolated shoots in K(h) medium with half-strength salts, supplemented with 0.4 µM Zeatin and 0.1 µM NAA. A possible detrimental long-term effect of cytokinins on shoot elongation can account for the results, since elongation was not observed until 1 month of culture in elongation medium. For rooting, shoots (1 cm in length) were basally immersed in a 5 mM IBA solution for 15 s, and transferred to half-strength K(h) basal medium. Regenerated plants were acclimated in a sterile peat:perlite substrate for 10 days, and then transferred to soil. AFLP analysis was accomplished with 15 primer combinations from which 13 showed reproducible and well-resolved bands, producing a total of 1321 fragments from which 1281 were polymorphic (97%). A dendrogram was constructed using both monomorphic and polymorphic bands, showing genetic variation among field-grown plants and tissue culture-derived regenerants.

Molecular Characterization of Three Comercial Cultivars and a New Pollinator in Kiwifruit

We investigated the characterization of genotypes of Actinidia deliciosa (Chev.) Liang and Ferguson var. deliciosa by using isozymatic and molecular techniques [randomly amplified polymorphic DNA (RAPD), amplified fragment-length polymorphism (AFLP), standard AFLP, and modified AFLP]. Four genotypes were tested, the female cultivar `Hayward', the traditional New Zealand pollinizers `Matua' and `Tomuri', and a new pollinizer named clone A selected in a breeding program in Spain. PGI and PGM were the only isozymes that allowed us to distinguish the kiwifruit genotypes, although the accessions of `Matua' presented two different banding patterns for both isozymes. All three molecular markers differentiated between the genotypes of kiwifruit tested, although RAPD markers did not allow us to establish differences between accessions of `Matua', while both standard and modified AFLP did. These results, along with those of isozymes, support the hypothesis that the male kiwifruit genotypes present in Europe belong to different clones. None of the markers used showed differences between accessions of `Hayward', which would suggest that it is a uniform cultivar. On the other hand, clone A was a seedling derived from `Hayward' and an unknown pollinizer. The results obtained using AFLP markers strongly suggest that `Tomuri' may have been the male parent of clone A. A specific protocol for kiwifruit characterization based on a modified AFLP technique is also presented, that gave rise to the highest percentage of polymorphism while scoring the lowest number of bands. This, together with the technical features of modified AFLP markers, make them very useful for identifying propagated kiwifruit plant material in commercial nurseries.

Micropropagation of Two Selected Male Kiwifruit and Analysis of Genetic Variation with AFLP Markers

A simple and reliable protocol for micropropagation during 12 subcultures of two field growth male plants of kiwifruit [Actinidia deliciosa (A.Chev.) Liang and Ferguson] is described. The best results of shoot multiplication and elongation were obtained in Cheng's K(h) medium in the presence of 0.5 μm NAA, 22 μm BA and 1.4 μm GA3 for `Tomuri' explants, and of 0.1 μm NAA, 4.4 μm BA, and 0.3 μm GA3 for clone A explants. In addition, the cytokinin compounds TDZ and mT were also tested allowing improving the multiplication rate in `Tomuri' explants. For rooting, `Tomuri' and clone A developed shoots were treated by basal immersion in a 5 mm IBA solution for 15 seconds. Treated shoots were then cultured in half-strength K(h) medium without growth regulators showing 100% rooting after 30 days. Regenerated plantlets were successfully transplanted to soil (90% survival) and they are actively growing in the field. Somaclonal variation analysis by AFLP was carried out using 15 primer combinations, yielding reproducible and well-resolved bands with a 57% of polymorphism. AFLP markers showed to be effective to discriminate genetic variation in this species, being greater in clone A than `Tomuri'. Chemical names used: N6-benzyladenine (BA); gibberellic acid (GA3); indole-3-butyric acid (IBA); meta-topolin (mT); naphthaleneacetic acid (NAA); thidiazuron (TDZ).

ISOZYMES FOR CULTIVAR IDENTIFICATION IN KIWIFRUIT

The usefulness of isozyme banding patterns as genetic markers in kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) was investigated using starch gel electrophoresis. Fifty-four entries putatively belonging to seven female and two male kiwifruit cultivars were examined for 13 enzyme systems (AAT, ACO, GDH, G6PDH, IDH, MDH, ME, MNR, NDH, 6PGD, PGI, PGM, and SKDH). Four enzyme systems, ACO, MDH, NDH, and SKDH, showed identical banding patterns in all clones surveyed. Of the remaining enzymes, AAT, PGI, and PGM had the best discriminating power. Six enzyme systems (GDH, G6PDH, IDH, ME, MNR, and 6PGD), though showing polymorphic banding patterns, were poorly resolved. All the New Zealand cultivars were uniquely identified by the simultaneous comparison of the AAT, PGI, and PGM zymograms. Some enzyme systems were also polymorphic among plants within the same cultivar, thus proving the heterogeneity of kiwifruit material intro- duced into Europe in the early 1970s. -.

Sex expression in fruiting male vines of kiwifruit

In three canes on each of five fruiting male kiwifruit vines (A. deliciosa var. deliciosa (A. Chev) Liang and Ferguson), staminate and bisexual flowers were interspersed, so that staminate, bisexual and mixed inflorescences were found. Further variation in inflorescence composition resulted from the abortion of flower buds prior to anthesis. These two sources of variation, in combination, resulted in 19 inflorescence patterns, which varied substantially in frequency among and within vines. Some tendencies were determined. In particular, most of the inflorescences had retained their terminal flower, whereas just over half had retained one or both primary lateral flowers, and in two-thirds of the inflorescences retaining a terminal flower, this was bisexual. The five vines ranged in bisexual flower frequency from 40% to 70%. Phenotypic Gender (P) estimates are given for each vine, based on the frequencies of staminate and bisexual flowers from three canes per vine. Flower development, lability in sex expression and the use of P estimates as part of a selection index in breeding for hermaphrodite kiwifruit cultivars are discussed.

Isozymes for Cultivar Identification in Kiwifruit

The usefulness of isozyme banding patterns as genetic markers in kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson] was investigated using starch gel electrophoresis. Fifty-four entries putatively belonging to seven female and two male kiwifruit cultivars were examined for 13 enzyme systems (AAT, ACO, GDH, G6PDH, IDH, MDH, ME, MNR, NDH, 6PGD, PGI, PGM, and SKDH). Four enzyme systems, ACO, MDH, NDH, and SKDH, showed identical banding patterns in all clones surveyed. Of the remaining enzymes, AAT, PGI, and PGM had the best discriminating power. Six enzyme systems (GDH, G6PDH, IDH, ME, MNR, and 6PGD), though showing polymorphic banding patterns, were poorly resolved. All the New Zealand cultivars were uniquely identified by the simultaneous comparison of the AAT, PGI, and PGM zymograms. Some enzyme systems were also polymorphic among plants within the same cultivar, thus proving the heterogeneity of kiwifruit material introduced into Europe in the early 1970s.