CN108834900B

CN108834900B - Cultivation method for inhibiting oxidative browning and endophyte contamination of explants in tea plant tissue culture

Info

Publication number: CN108834900B
Application number: CN201810855401.3A
Authority: CN
Inventors: 魏书; 卡斯卡伊·阿拉卡萨米; 沙马拉·鲁鲍比·费丁南
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2018-07-31
Filing date: 2018-07-31
Publication date: 2022-03-11
Anticipated expiration: 2038-07-31
Also published as: CN108834900A

Abstract

The invention discloses a culture method for inhibiting oxidative browning of explants and endophyte contamination in the process of tea tree tissue culture, and relates to the inhibition of endogenous catechin biosynthesis in the process of tea tree tissue culture. In this method, 2 μM phenylalanine ammonia lyase inhibitor 2-aminoindole-2-phosphonic acid (AIP) was added to B5 minimal medium to inhibit the biosynthesis of polyphenolic compounds in tea plant explants and significantly reduce the expression of The content of gallocatechin (EGC), and then inhibit the browning of callus; by adding 2mg/L 6-benzylaminopurine (BAP), 1mg/L thidarone (TDZ), 10 mg/L in B5 medium % coconut water, and 300mg/LL-glutamine and 2μM AIP, can significantly increase the callus induction rate and growth rate of tea leaves. In addition, this technology can eliminate the contamination of endophytic bacteria in the explants by adding 150mg/L of timentine and 30mg/L of gentamicin to the B5 medium and culturing tea leaf explants with them for 12-24 days.

Description

Culture method for inhibiting explant oxidation browning and endophyte pollution in tea tree tissue culture

Technical Field

The invention relates to the field of tea tree tissue culture and biotechnology, and the core of the invention is a new technology which applies an endogenous polyphenol synthesis inhibitor and an antibiotic capable of effectively eliminating tea tree endophyte in a tea tree culture medium and inhibits explant browning and endophyte pollution in tea tree tissue culture.

Background

The tea belongs to the genus camellia and originates from China. Cultivars have several varieties, such as assam, chinese, etc. (Coasta et al, 2007). Compared to the Amam type variety, the leaves of the Chinese species are smaller, resistant to drought and low temperatures (Harler 1964), contain very high levels of polyphenols (16-30% dry weight) (Almajano et al 2008; Graham 1992). Biotechnological research on the in vitro genetic improvement of chinese tea varieties has been difficult to succeed not only because of the oxidation and browning of explant polyphenols, but also probably because of the contamination of endophytes present in large quantities in field tea plants. So far, the problems of brown stain, necrosis, endophyte pollution and the like of the explants of the tea trees are difficult to overcome by applying the conventional tissue culture technology, so that the tissue culture of the tea trees is slow in progress, which is a bottleneck of the development of the biotechnology of the tea trees.

In plants, phenolic compounds can form catechol due to the action of polyphenol oxidase (EC 1.1418.1) (PPO), and can be further oxidized to form highly colored o-quinones (Sapers)&Hicks 1989; Janovitz-Klapp et al 1990). The o-quinone compounds combine with other o-quinone compounds and proteins, reducing sugars, etc. to form polymers that precipitate in browned plant tissue (McEvily et al 1992). Silencing of PPO expression has been successfully achieved in non-browning cultures

Apple (Xu 2013). In addition, phenylalanine ammonia lyase (EC 4.3.1.24) (PAL) is the first enzyme in the phenylalanine pathway, converting L-phenylalanine to trans-cinnamic acid (Hahlbrock)&Scheel 1989). Subsequent reactions result in the production of some cinnamates (caftaric acid and dicaffeoyltartaric acid) and quinic acid derivatives (5-caffeoylquinic acid and 3, 5-dicaffeoylquinic acid). Studies have shown that this process leads to browning of lettuce (Tom-s-Barber-n et al 1997). Some chemicals such as calcium chloride and 2, 4-dichlorophenoxyacetic acid (2,4-D) can control browning of plant tissues by inhibiting PAL activity, thereby reducing biosynthesis of phenolic compounds ((Tom a s-Barber a n et al 1997). some reducing agents such as L-cysteine or ascorbic acid can lead to reduced necrosis of plant tissues (Enriquez-Obregon et al 1999; Olhoft and Somers 2001). the use of different adsorbents and antioxidants in the culture medium can reduce browning (necrosis) of tissues, but they may have a significant negative impact on callus induction, Agrobacterium-mediated transformation and VIR gene expression (Rana et al 2016). 2-aminoindole-2-phosphonic acid is an analog of phenylalanine and inhibits certain plant PAL activities and phenolic compound synthesis. However, the effect of AIP on browning of tea plant explants is uncertain, and because of the high polyphenol content in tea, it is necessary to do this.

Contamination with fungi and bacteria and endophytes of explants is often a major obstacle in plant cell or tissue culture. Bacterial endophytes are widely present in medulla, sclerenchyma, xylem lumen, and parenchyma, causing plant culture loss and affecting micropropagation efficiency. In strawberry, the presence of various endophytes, the combined use of the antibiotics tylosin, streptomycin sulphate and gentamicin, was found to be effective in eliminating endophyte contamination (Tanprasert and Reed 1997). Wang et al (2016) recently discovered 24 endophytic bacteria in the tea varieties Zijuan and Yunjin 10, which belong to six species of staphylococcus and the like. Therefore, there is a need for a method for effectively eradicating tea tree endophytes and inhibiting polyphenol synthesis by AIP to reduce browning of explant tissue of tea tree of China species.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a culture method for inhibiting browning of an explant and endophyte pollution in tea tree tissue culture, so as to solve the technical problem of browning of the explant caused by polyphenol oxidation and endophyte pollution in the tea tree tissue culture process.

The invention is realized by the following technical scheme:

the invention provides a culture method for inhibiting explant oxidation browning and endophyte pollution in tea tree tissue culture, which comprises the following steps:

step 1, obtaining an operation object

(1) Washing fresh tea tree seeds with tap water, removing episperms, soaking overnight, separating 'floating seeds' and 'settling seeds' by using a filter, and selecting 'settling seeds' as culture materials;

(2) transferring the seeds selected in the step 1 into a sterile 500 ml conical flask, washing twice with water, and soaking overnight in 4% carbendazim; washing the carbendazim-treated seeds with sterile distilled water for the next day, sterilizing with 70% ethanol for 3 minutes, sterilizing with 0.1% mercuric chloride for 4 minutes, and rinsing with sterile distilled water for three times to obtain sterile seeds;

(3) drying the seeds obtained in the step 2 by absorbing dry water on Whatman No. 1 filter paper, removing coats of the seeds by using a sterile knife and tweezers under a sterile condition, placing the seeds on an MS (Sigma) culture medium with the strength of 1/2, and allowing the seeds to germinate and grow into test-tube plantlets;

(4) cutting seedling, segmenting twig, and propagating in 1/2 strength MS culture medium containing 2 mg/L6-Benzylpurine (BAP) and 0.1mg/L indole-3-butyric acid (IBA) for further use;

step 2, explant culture

The procedure obtained in step 1.4 was cultured for 12-24 days in Gamborg B5(B5) (Sigmaaldrich) medium to which the following compounds were added: 2-3.5. mu.M 2-aminoindole-2-phosphonic Acid (AIP), kinetin (2 mg/L6-Benzylaminopurine (BAP), 1mg/L Thiadazole (TDZ), 10% coconut water and 300mg/L L-glutamine, 150mg/L timentin and 30mg/L gentamicin sterilized with a 0.2 μ M filter membrane.

The invention has the following beneficial effects:

the invention can effectively inhibit the browning of the tea tree explant caused by polyphenol oxidation, eliminate the pollution of endophytes, improve the induction rate and the growth stamp of the explant, and overcome the technical bottleneck of tea tree tissue culture.

Drawings

FIG. 1, callus induction of leaf explants of tea tree of China species after addition of hormone antibiotics and AIP to culture medium B5 (a) sterile shoots grown for 6-8 weeks on 1/2MS culture medium; (b) growing expanded shoots on 1/2MS medium supplemented with 2mg/L benzylpurine and 0.1mg/L indolebutyric acid; (c, d) endophyte contamination status after one week of leaf explant growth on antibiotic-free medium; (e) leaf explants grown on medium containing antibiotics for 12 days without endophyte contamination (f) browning of calli cultured on B5 medium without AIP for 12 weeks (g) non-browned milky white callus cultured on B5 medium with AIP for 12 weeks;

FIG. 2, high performance liquid chromatography for the quantitative analysis of extracts from AIP-treated and untreated mulberry tissues;

EGCG, epigallocatechin gallate; EC, epicatechin; c, catechin; EGC, epigallocatechin; CAF caffeine;

FIG. 3 high performance liquid chromatograms of standard reagent mixtures (a), AIP-treated (b) and untreated (c) callus extracts;

peak 1, epigallocatechin; peak 2, caffeine; peak 3, catechin; peak 4, epicatechin; peak 5, epigallocatechin gallate; peak 6, gallic acid; peak 7, Gallic acid Catechin gallate.

Detailed Description

The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, the reagents and other materials used in this example are commercially available products, and the germplasm resources used in this example are all available from national germplasm resource banks.

Materials and methods

Plant material

The test material is healthy tea tree seeds planted in 8-year old agricultural resistant plants in the Experimental tea garden of agriculture university of Anhui.

Preparation of explants

The seeds can be easily sterilized without significant browning compared to the leaves and stems of tea trees planted in the field. Thus, the experimental seeds were sterilized as test materials using the following surface sterilization protocol. The fresh seeds were washed with tap water, the testa removed and soaked overnight. Using a filter, "floating seeds" and "settled seeds" are separated. The seeds were transferred to a sterile 500 ml Erlenmeyer flask, washed twice with water and soaked overnight in 4% carbendazim. The carbendazim treated seeds were rinsed with sterile distilled water for the next day, sterilized with 70% ethanol for 3 minutes, then sterilized with 0.1% mercuric chloride for 4 minutes, and rinsed with sterile distilled water three times. The surface-sterilized seeds were dried by blotting dry water on Whatman No. 1 filter paper, and the seed coat was removed under aseptic conditions with a sterile knife and tweezers. The seeds were then placed on 1/2 strength MS medium and allowed to germinate and grow into test tube plantlets. Seedlings were cut and shoots were segmented and propagated on 1/2 strength MS medium containing 2 mg/L6-Benzylpurine (BAP) and 0.1mg/L indole-3-butyric acid (IBA) (FIGS. 1a, b).

In vitro callus induction

Cutting young leaves from aseptic seedlings, placing on Gamborg B5(B5) culture medium which is sterilized at high temperature and has pH of 5.8, and adding different concentrations of tylosin (50,100,150mg/L) and gentamicin (10,20,30mg/L) to inhibit bacterial endophyte. The experimental results showed that all leaf explants without antibiotic addition were contaminated with endophytes (fig. 1, c, d)). When cultured on the medium with 150mg/L of timentin and 30mg/L of gentamicin for 12 days, the endophyte contamination rate of the explants is reduced to zero (figure 1e, table 1). In addition, experiments compared the effect of AIP at concentrations of 0.5, 1, 1.5, 2, 2.5 and 3 μm to inhibit browning of callus. Experimental results show that the callus without AIP added on the B5 culture medium is obviously browned (figure 1f), and the callus with 2 mu m AIP added is milky white and does not have obvious browning (figure 1 g). The AIP concentration is lower than 2 mu M, the browning inhibition effect is limited, and is higher than 2 mu M, and the inhibition effect is not obviously enhanced. In addition, the present study further compared the effect of mixed AIP, BAP (1, 1.5, 2, 2.5mg/L) TDZ (0.1, 0.5, 1, 2.5mg/L), 10% coconut water, 300mg/L L-glutamine on the induction and growth of wounded tissues on B5 medium. The results show that 2. mu.M AIP works best with 2.0mg/L BAP +1.0 mg/LTDZ. The hormones, coconut water and antibiotics used in the experiment are all sterilized by a filtration method. The B5 medium was autoclaved. All aseptic operations are finished on a clean bench.

High performance liquid chromatography

The content change of catechin and caffeine in the tea tree callus cultured on the AIP-containing culture medium and the B5-free culture medium respectively is analyzed and compared by using a modern High Performance Liquid Chromatography (HPLC) analysis technology. The extraction of polyphenols and caffeine from explants was carried out according to the method of Ashihara et al (2010). Briefly, about 200mg were ground to a fine powder in liquid nitrogen and extracted with 80% methanol water containing 20mM sodium diethyldithiocarbamate. The supernatant after centrifugation (12000g x 20 minutes) of the mixture was vacuum-dried and subjected to HPLC analysis by machine detection in 2ML of distilled water by the method referred to Kerrio et al (2013). The compound was quantified by establishing a standard curve according to Kochuo et al. All experiments were performed in triplicate and the data analyzed using the SPSS 17.0 statistical software package. The experimental results showed that AIP treatment reduced the catechins of each class analyzed to varying degrees, but only Epigallocatechin (EGC) to a statistically significant level (p <0.05) (fig. 2 and 3).

TABLE 1 application of Timentin and gentamicin to eradicate endophytes from explants of tea trees of the Chinese species

^*No significant difference (p) between the same row data and the same letter>0.05), dunken multiple comparison test. There were three replicates per treatment, 30-90 calli per replicate.

TABLE 2, 6-Benzylaminopurine (BAP) and Thidiazuron Effect on callus induction of leaf explants of Camellia sinensis cultured for 2 weeks on B5 Medium

Bold data are significantly different from other data in the same column (P < 0.05).

The phenomenon of explant tissue browning and even necrosis which often occurs in the process of tea tree tissue culture is a bottleneck of tea tree in-vitro tissue culture and research and application of tea tree biotechnology. Based on the scientific principle that the tea tree explant is high in polyphenol content and easy to oxidize and poison the tea tree explant tissue to cause browning, the invention applies a phenylalanine ammonia lyase inhibitor 2 mu M2-amino indole-2-phosphonic Acid (AIP) in a polyphenol synthesis way, and effectively inhibits the browning of the tea tree explant by inhibiting the synthesis of catechin. The induction and growth of the callus are effectively enhanced by the mixed use of cytokinin, natural coconut milk and L-glutamine. Through experiments of antibiotics with different concentrations, the invention also discovers that the tea tree explants are cultured for 12 days on the B5 culture medium containing 150mg/L of tylosin and 30mg/L of gentamicin, and the pollution of tea tree endophytes can be eliminated. The method has theoretical significance and practical value.

Claims

1. suppress the culture method of explant oxidative browning and endophyte pollution in tea tree tissue culture, it is characterised in that the method comprises the following steps:

Step 1. Get the operation object

(1.1) Wash the fresh tea tree seeds with tap water, remove the outer seed coat, soak them overnight, use a filter, separate the "floating seeds" and "settled seeds", and select "settled seeds" as the culturing material;

(1.2), transfer the seeds selected in (1.1) to a sterile 500 ml conical flask, wash twice with water, soak in 4% carbendazim overnight; rinse the carbendazim-treated seeds with sterile distilled water the next day , sterilized with 70% ethanol for 3 minutes, then sterilized with 0.1% mercuric chloride for 4 minutes, and rinsed three times with sterile distilled water to obtain sterile seeds;

(1.3), dry the seeds obtained in (1.2) with Whatman No. 1 filter paper, remove the seed coat under sterile conditions with a sterile knife and tweezers, and then place the seeds in 1/2 strength MS medium , wait for it to germinate and grow into test-tube seedlings;

(1.4), cut the test tube seedlings obtained in (1.3), and segment the shoots, in a solution containing 2 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L indole-3-butyric acid (IBA). 1/2 strength MS medium for expansion and reproduction, ready to use;

Step 2. Explant culture

Put the operation object obtained in step 1 into the B5 medium for 12-24 days, and the medium is also added with the following compounds: 2-3.5uM 2-aminoindole-2-phosphine sterilized by a 0.2um filter membrane Acid (AIP), 2 mg/L 6-benzylaminopurine (BAP), 1 mg/L Thiidalone (TDZ), 10% Coconut Water and 300 mg/L L-Glutamine, 150 mg/L Typha Mentin and 30mg/L gentamicin.