Dynamic changes in polyamines, endogenous hormones and oxidase activities during rooting of double haploid muskmelon in vitro plantlets

Abstract: 【Objective】Muskmelon is one of the most important fruits in the world, which plays an important role in the adjustment of agricultural planting structure in China. But the narrow genetic basis, the lack of excellent germplasm resources, the long cycle and low efficiency of traditional breeding technology lead to the slow renewal of excellent varieties, which restricts the sustainable development of muskmelon industry. As a breeding method that can quickly and effectively obtain homozygous germplasm, the double monomer culture technology has become a research hotspot for muskmelon breeding workers at home and abroad. Rooting induction culture is an important part of the double haploid tissue culture system, which is directly related to the survival rate and seedling quality of tissue culture plantlets. There are many internal and external factors affecting the rooting process, including en-dogenous hormones, polyamines, related oxidases and exogenous substances such as ethylene and salicylic acid. The study aimed at investigating the changes of endogenous hormones, polyamine contents and related oxidase activities in the rooting culture process of melon double haploid shoots, in order to clarify the physiological mechanism of the response to adventitious root induction, differentiation and elongation, and to provide reference for regulating the rapid rooting of melon double haploid tissue culture plantlets.【Methods】The unfertilized ovary of thick- skinned muskmelon hybrids Jinxiucuiyu was cultured on MS+0.03 mg·L^-1 TDZ+0.2 mg·L^-1 KT+0.01 mg·L^-1IAA+15% coconut juice medium to obtain haploid buds, and then doubled with 700 mg·L-1 colchicines, after identification by flow cytometry, the double haploid buds with the same growth were selected and transfered onto MS+0.1 mg·L^-1IAA+ 30 g·L^-1sucrose+6 g·L^-1 agar medium (pH=5.86) for rooting culture, all of were incubated at (25± 2) ℃, under a 16 h light/8 h dark cycle and 3000-4000 lx light intensity, the contents of endogenous hormones, polyamines and related oxidase activities in the basal stems were measured by liquid chromatography and catechol, colorimetry method at 0, 7, 14, 21 28 d respectively.【Results】During the course of rooting induction, the contents of indoleacetic acid (IAA) and abscisic acid (ABA) decreased firstly and then increased. The content of gibberellin (GA3) rose in the first seven days and decreased between the 7th day and the 28th day. The variation in zeatin nucleoside (ZR) content followed a pattern of“decrease-increase- decrease”. The content of IAA/ABA and peroxidase (POD) activities increased gradually. The IAA/ZR ratio and spermine (Spm), spermidine (Spd), putrescine(Put) contents and indoleacetic acid oxidase (IAAO), polyphenol oxidase(PPO) activities showed a trend of“increase- decrease-increase”, the three polyamines reached the first peak on the 14th day and reached the maximum on the 28th day, the ratio of IAA/ZR reached the first peak on the 14th day and reached the maximum on the 28th day, the activities of IAAO and PPO increased rapidly from 0 to 7 days, reaching the maximum on the 7th and 14th days respectively, and the change range of them was relatively small from 7 to 28 days, and IAA content decreased rapidly from 0 to 7 d, decreased slowly in 7-14 d, increased slowly in 14-21 d, increased rapidly in 21-28 d, ABA content decreased rapidly in 0-14 d, increased slowly in 14-28 d. The correlation analysis showed that there were significant positive correlations between Put and IAA/ABA value, Spm, and between GA3 contents and IAAO activity (p＜0.01), and significant positive correlations between Spm content and IAA/ABA value, Spd, and between Put and Spd contents, POD activities, and between IAAO and PPO activities (p＜0.05), and significant negative correlation between Spd and ABA contents (p＜0.01). It was proved that IAA/ABA ratio and Put content would play an important role in promoting the differentiation and growth of adventitious roots of muskmelon double haploid tissue culture plantlets.【Conclusion】The increase of IAA, ABA contents, IAA/ABA and IAA/ZR values and the decrease of GA3 and ZR contents in a certain range were conducive to adventitious root induction, differentiation and growth, the increase of Spm, Spd Put contents in late tissue culture plantlets was conducive to the formation of adventitious roots. In addition, the changes of IAAO, PPO, POD enzyme activities were closely related to the occurrence and elongation of adventitious roots.