Establishment of an endosperm culture and plant regeneration system in Actinidia arguta

【Objective】The endosperm of angiosperms is a product of double fertilization, belonging to triploid tissue, containing genetic material from both parents, and differs from ordinary hybrids. It contains two copies of maternal genetic material and one copy of paternal genetic material. Due to the rapid development of biotechnology, triploid plants can be obtained through endosperm culture, and new nucellus- free germplasm can be acquired through hybridization, which holds significant breeding value for fruit trees that primarily rely on vegetative propagation. The study aimed to establish a complete technical system for endosperm culture of Actinidia arguta, and obtained polyploid plants.【Methods】 In the early stage of the study, the callus induction screening experiments of different hormone ratios were carried out. It was found that the combination of 6-BA and NAA was more suitable for the callus induction culture of kiwifruit endosperm, and the callus induction rate was high and the differentiation of callus was promoted; Three kinds of artificially domesticated wild A. arguta and‘Longcheng No.2’ with different reproductive stages were used as experimental materials to study the effects of different sampling times and culture media on the formation and differentiation of endosperm culture organs, and determine the best sampling period and culture medium formula; The differentiated plantlets were cultured in succession and subjected to flow cytometry for cell multiplication identification. Using the immature endosperm of A. arguta as explants, sampling was set when the immature endosperm began to form, and samples were taken every 10 days for a total of four times. The samples were inoculated onto callus induction medium, and the callus induction status was surveyed every 10 days. Subsequently, the endosperm callus was inoculated onto different differentiation media, and the callus differentiation status was regularly monitored to determine the optimal sampling time and the best endosperm callus induction and differentiation media.【Results】Starting from the formation of the endosperm, samples were collected every 10 days for stereomicroscopic observation of seed morphology. As the seeds matured, the seed coat color gradually transitioned from light brown to dark brown, while the endosperm texture evolved from loose and incompletely filled to firm, hard, and fully enclosed. The embryo grew larger with progressively hardened texture, becoming easier to separate from the endosperm. This study effectively minimized embryo interference. After inoculation, the callus induction and growth were observed and recorded every ten days. The endosperm was induced to form callus tissue. With the increase of culture time, the callus became compact and differentiated into plants. Subsequent studies explored synergistic effects of NAA and 6-BA at different concentrations. After 60-day callus induction, researchers compared growth patterns across hormone ratios and sampling intervals. Significant variations in optimal induction media were identified across cultivars during different growth phases. The optimal culture medium was 1/2MS with NAA at 0.05, while 6- BA concentrations varied significantly (0.5, 2.0, 1.0 and 1.0). Under identical cultivation conditions, optimal sampling times varied significantly depending on maturity stages. Material TH1, TH2, TH3 and Longcheng No.2 showed optimal sampling at 60-70 days, 70-80 days, 65 days and 70-80 days post-flowering, respectively. Through observing callus differentiation under different media, we found that medium Ⅰ and Ⅱ strains simultaneously differentiated roots and shoots, with Ⅱ＞Ⅰ in shoot differentiation. The root systems of medium Ⅲ strains showed no bud differentiation, though overall differentiation numbers were relatively low. As cultivation time extended, browning became more pronounced, ultimately yielding unsatisfactory results. Flow cytometry analysis confirmed all differentiated seedlings were octaploid, validating this conclusion. Literature reviews indicate that transferring callus tissue to MS+1.0 mg·L-1 2, 4-D+3.0 mg·L-1 Zt medium not only increased genetic variation but also significantly boosted plant differentiation, providing a basis for subsequent experiments. The results of the diploid detection by flow cytometer showed that the parental material of TH1, TH2, TH3 and their common parent material, and the parental material of Longcheng No.2 were all tetraploid. Using regenerated plants as controls and TH1 embryo tissue culture buds as reference for relative DNA content, the results showed that the regenerated plants were hexaploid, octoploid and octoploid (rather large). Physiological measurements revealed polyploid plants exhibited significantly longer leaves, wider leaf margins, larger stomatal openings, and reduced stomatal density compared to tetraploid plants, demonstrating better environmental adaptability.【Conclusion】Few studies have been conducted on the endosperm culture from A. arguta. This paper investigated the cultivation of endosperm from different reproductive stages of artificially domesticated wild A. arguta varieties. By regularly comparing the induction and differentiation of callus, it aimed to determine the optimal sampling time, endosperm callus induction medium, and differentiation medium. Through flow cytometry for ploidy identification, multiple polyploid plants were differentiated. The study found that the reproductive stage significantly affected the sampling time and tissue culture conditions. The reproductive stage discussed in this paper was incomplete, and future research can improve the endosperm tissue culture system by adding more varieties at various reproductive stages. Additionally, by investigating the traits of the offspring with different ploidy levels, superior varieties can be selected for breeding studies.