In Vitro Seed Germination, Seedling Development, Multiple Shoot Induction and Rooting of Actinidia chinensis

Abstract

Worldwide, the yellow-fleshed kiwifruit (Actinidia chinensis) is an important crop that possesses great economic significance due to its nutritional, medicinal and ornamental values. The call for the expansion of the kiwifruit industry in South Africa, due to rising local and international market demand, resulted in the introduction of new plant species in sub-mountainous areas, where soil and climate conditions are more suitable for intensive kiwifruit production than in lowland areas. Consequently, a need to develop suitable commercial protocols for mass propagation of A. chinensis emerged. This study introduces an optimized micropropagation protocol for A. chinensis, facilitating seed germination, seedling development and multiple shoot induction. For seed germination, the effect of cold stratification (CS) and gibberellic acid (GA3) alone and in combination on in vitro germination of A. chinensis seeds was studied. Sterile seeds were stratified at 4 °C for 28 and 42 days. Batches of stratified and non-stratified (control) seeds were germinated on plant growth regulator-free Murashige and Skoog (MS) media and also on sterile filter paper bridges moistened with dH2O and GA3 concentrations of 500, 1000, 1500, 2000 and 2500 ppm. Seeds from the control and the CS treatments alone did not germinate on MS medium. However, on filter paper bridges, seeds cold stratified for 28 days yielded only a 20% germination percentage (GP), whereas CS for 42 days did not promote germination. A maximum GP of 64% and a mean germination time (MGT) of 27.52 days were achieved at a 2000 ppm GA3 concentration. Cold stratification (28 days) followed by GA3 treatments yielded an optimum GP of 80% and optimum MGT of 18.94 days at GA3 concentrations of 500 ppm. In contrast, CS (42 days) followed by GA3 yielded a maximum GP of 72% and MGT of 18.80 days at a GA3 of 500 ppm. Conclusively, CS alone had little effect on germination, whereas CS (28 and 42 days) followed by GA3 significantly (p ≤ 0.05) improved GP. Germinated seeds on moist filter paper can produce seedlings when sub-cultured on MS medium for seedling development. For multiple shoot induction, in vitro shoot culture of A. chinensis was carried out using apical and basal shoot explants from the above in vitro-produced seedlings. These explants were cultured on MS supplemented with 2.2 µM and 4.4 µM 6-Benzylaminopurine (BAP) for shoot multiplication. Axillary shoot proliferation was not observed on apical shoot explants after 4 weeks of culture on MS medium with 2.2 µM BAP. In contrast, the basal shoot explants produced 2–3 axillary shoots, tendrils and calluses at the base on the same medium. The highest number (3–4) of multiple shoots was attained from these basal shoot explants after subculture (10–12 weeks) in the same culture medium. In contrast, only elongation and rooting of apical shoot explants, without axillary shoot induction, occurred after the subculture. Regenerated plantlets derived from both apical and basal shoot explants were successfully acclimatised under a controlled environment at 24 ± 2 °C and 16 h photoperiod of 150–200 µmol m−2 s−1 light intensity. A similar response was observed for both types of explants of A. chinensis when cultured on MS with 4.4 µM BAP, although the higher concentration of BAP affected the morphological appearance of the regenerated plantlets that had shorter stems and smaller and narrower leaves compared to plantlets derived from 2.2 µM BAP.