Effects of CuSO4 and Uniconazole on Mature Embryo Culture in Japonica Rice

Shan He,and Zou Deng-tang

College of Agriculture,Northeast Agricultural University,Harbin 150030,China

Introduction

Tissue culture is the basis for the development of genetic engineering breeding and functional genome,the establishment of a genetic stability,conversion efficiency and easy regeneration of transgenic research receptor system is critical.Rice is one of the world's major food crops.Since Nishi and Yamada had induced callus and regenerated small plantlets from the mature embryos for the first time,rice tissue culture and rapid progress also received a greater achievement (Lin,2010).Meanwhile,there are some problems of rice tissue culture,such as low regeneration rate.There are many factors that influence callus regeneration rate of the rice mature embryo,some of the metal ions and growth regulators play vital roles in improving regeneration.Zhou et al.(2006) pointed that addition of CuSO4in differentiation medium contributed to callus differentiation.As a high efficient plant growth regulator,uniconazole has been widely used for the growth of rice plantlets (Ji,2004).This article studied the effects of different concentrations of CuSO4and uniconazole on the callus induction and plantlet differentiation with Kongyu 131,Longjing 24 and Dongnong 425 as test materials,screened out the optimum concentration of CuSO4and uniconazole for improving rice mature embryo induction and differentiation rates,optimized rice tissue culture system and laid a foundation for further researches on rice transformation.

Materials and Methods

Test materials

Rice mature embryos of Kongyu 131,Longjing 24 and Dongnong 425 which were mainly planted in Heilongjiang Province were used as test materials.Three varieties were kindly provided by Rice Breeding Institute of Northeast Agricultural University,Harbin,China.

Screening CuSO4 concentration

5.0,10.0,15.0,20.0,and 25.0 μmol · L-1concentrations of CuSO4were added in the induction and subculture media,respectively,and 0.0 μmol · L-1concentration as the control medium,then 10 culture dishes per treatment,and 20-25 mature embryos in each culture dish.The callus induction rates were calculated as the formula after 12 days.After 28 days in culture,the growth of callus was investigated with the relative growth rate of callus tissue as index for determining the optimal concentration of CuSO4in induction and subculture media.Embryogenic calluses were transferred to corresponding concentration differentiation media.The numbers of embryogenic callus were 4-5 per Erlenmeyer flask on differentiation media,10 Erlenmeyer flasks per treatment.After 40 days,the green plantlet differentiation rates were calculated as the formula.

Screening uniconazole concentration

0.25,0.50,1.00,and 2.00 mg · L-1concentrations of uniconazole were added in the induction and subculture media,and 0.0 μmol · L-1concentration as the control medium.The callus induction rates were calculated as the formula and average of embryo bud lengths was measured after 12 days.Embryogenic callus was transferred to corresponding concentration differentiation media after 28 days.The numbers of embryogenic callus were 4-5 per Erlenmeyer flask on differentiation media and 10 Erlenmeyer flasks per treatment.After 40 days,the green plantlet differentiation rates were calculated as the formula,the average of the plant height,the average of main root length and the plantlet root activity were measured.

Test method

The mature seed coat was removed from mature plump rice,soaked in 75% ethanol for 1 min,then rinsed one time with sterilized distilled water,and mercuric chloride solution (0.1%) for 10 min for sterilization.It was then rinsed three times with sterilized distilled water.Total 20 seeds were cultured on each callus induction medium and incubated in the dark at (26±2)℃ for 12 days.Embryogenic callus (Ⅱ) was picked out according to the identification standard by Armstrong and Green (1985) and then transferred to subculture media at (26±2)℃.After 28 days (14 days per generation) in culture,embryogenic callus was transferred to differentiation media.The numbers of embryogenic callus were 6-8 per flask on differentiation media.It was then cultured at 28℃ day/20℃ night temperature under 3 000 Lx light intensity for 16 h each day to induce adventitious.After 40 days,observed germination situation.The numbers of green plantlets were counted from various treatments.

The compositions of all the media were as the followings.Induction media: NMB basal medium N6 macroelement+MS microelement+MS ferric salt+B5 organic compound)+2,4-D 2.0 mg · L-1+6-BA 0.5 mg · L-1+CH 300 mg · L-1+Pro 500 mg · L-1+3% sucrose+0.7% agar;subculture media: NMB basal medium+2,4-D 1.0 mg · L-1+6-BA 0.5 mg · L-1+CH 500 mg · L-1+Pro 500 mg · L-1+3% sucrose+0.7% agar;and differentiation media: MS basal medium+NAA 0.5 mg · L-1+6-KT 2.0 mg · L-1+3% sucrose+0.7% agar.

Data analysis

Callus induction rate (%)=number of rice producing callus/number of rice inoculated×100%

Relative growth rate of callus=(weight of after cultivation callus–weight of before cultivation callus)/ weight of before cultivation callus

Green plantlet differentiation rate (%)=number of green plantlets differentiation/number of callus inoculated×100%

Data was analyzed with Microsoft Excel2003 and DPS v7.05.

Results

Effect of different concentrations of CuSO4 on callus induction and differentiation

Table1 showed that the induction rates,relative growth rates and green plantlet differentiation rates of callus were significantly different among three varieties because of different genotypes,all the treatments were higher than those of the controls.At a certain concentration,the callus induction rates,the relative growth rates and the green plantlet differentiation rates of three varieties increased to maximum,then decreased with the increasing addition of CuSO4.When concentration of uniconazole was 15.0 μmol · L-1,callus induction rates of three varieties were the highest,reaching 88.0%,68.0% and 59.2%,compared with other treatments,callus induction rates reached the extremely significant level.Callus relative growth rate of Kongyu 131 reached the highest at 20.0 μmol · L-1CuSO4concentration,and other treatments reached a significant level;callus relative growth rate of Longjing 24 reached the highest at 15.0 μmol · L-1,and other treatments reached a significant level except with 10.0 μmol · L-1;callus relative growth rate of Dongnong 425 reached the highest at 15.0 μmol · L-1,and other treatments reached a very significant level except with 10.0 and 20.0 μmol · L-1.When concentration of CuSO4was 15 μmol · L-1for Kongyu 131 and Dongnong 425 and was 10 μmol · L-1for Longjing 24,green plant regeneration rates were the highest,reaching 58.5%,60.6% and 34.8%,increasing by 26.4%,27.0% and 19.5%,respectively compared with the controls.This result showed that increasing the concentration of CuSO4in culture media made a contribution to improve the rice mature embryo callus induction and the green plant regeneration rates.

Table1 Effects of different Cu2+ concentrations on callus induction and differentiation of rice mature embryos

Effects of different concentrations of uniconazole on callus induction

It could be seen in Table 2 that plant growth retardant uniconazole had significantly inhibited on the growth of bud,and the impacts strengthened with the increasing of the uniconazole concentration.At a certain concentration,callus induction rates of three varieties increased to maximum,then decreased with the increasing addition of uniconazole.When the concentration of uniconazole was 0.50 mg · L for Kongyu 131 and Dongnong 425 and 0.50 mg · L-1for Longjing 24,callus induction rates were the highest,reaching 80.0%,53.6% and 67.0%,increasing by 5.5%,11.6% and 17.5%,respectively compared with the controls.Compared with other treatments,callus induction rates reached the extremely significant level.All the results indicated that the suitable concentration of uniconazole in induction medium could significantly improve the induction rate of rice mature embryo callus.

Table2 Effect of uniconazole on embryonic bud growth and callus induction

Effect of different concentrations of uniconazole on callus differentiation

Table3 illustrated that uniconazole had promotion effects on green plantlet differentiation.The promotion effects increased to maximum then decreased with the increasing addition of uniconazole at a certain concentration.When the concentration of uniconazole was 0.50 mg · L-1for Kongyu 131 and Dongnong 425 and 0.50 mg · L-1for Longjing 24,green plant regeneration rates were the highest,reaching 60.5%,71.8% and 67.0%,increasing by 29.9%,39.1% and 17.3%,respectively compared with the controls.Compared with other treatments,green plant regeneration rates reached the extremely signif icant level.

With the increase of uniconazole concentration,the plant height decreased and the root length increased.Regeneration seedlings of the control groups were the highest,but the root systems were relatively the weakest.Compared with other treatments,plant heights and root lengths reached the extremely signif icant level.

Crop root is active absorption organ and synthesizer organ,and the situation of root growth vigor directly affects the level of aboveground growth,nutritional status and output levels.Fig.1 showed that the root activities of three varieties regenerated seedlings increased to maximum firstly,and reached maximum values at 0.50 mg · L-1.Those were 0.196 mg · g-1· h-1,0.263 mg · g-1· h-1,and 0.404 mg · g-1· h-1,respectively,then decreased with the increasing addition of uniconazole.All the results indicated that the suitable concentration of uniconazole (0.50 mg · L-1) could significantly improve green plant regeneration rate and play an important role in reducing plant height,increasing root length and improving root vigor of seedlings.

Table3 Effect of uniconazole on regeneration rate of subculture embryonic callus

Fig.1 Effect of uniconazole on regenerated plantlet root activity

Discussion

Low callus induction and differentiation rates of production type rice mature embryo are one of the major factors of restricting genetic transformation of rice,and also one of the main factors of limiting technology improvement of rice.Genotype,the explants and medium compositions are the major reasons of the difference of culture force among different varieties.In this experiment,concentrations of the microelement and plant growth regulators in the culture media were adjusted,in order to obtain the appropriate concentration of CuSO4and uniconazole for improving induction rate and differentiation rate of rice mature embryo. Cu2+is a plant growth necessary element,but also a cofactor for many important enzymes in plants,such as ascorbic acid oxidase and polyphenol oxidase.A variety of enzymes containing Cu2+involved in electron transfer,protein and carbohydrate biosynthesis,nitrogen metabolism and other important physiological and biochemical metabolisms in the plant growth process (Purnhauser and Gyulai,1993).Adding 0.01-0.02 mmol · L-1Cu2+in the subculture media could decrease browning rate of japonica rice in cold region and in the differentiation medium could get more regenerate plants (Zhao and Song,2013).The induction rate,differentiation rate and plant regeneration rate reached the highest value with 1 μmol · L-1Cu2+in the induction and differentiation culture medium (Hou et al.,2008).The browning rate of callus reduced and the differentiation rate was improved greatly by adding copper in differentiation culture medium for hsien rice (Huang et al.,2010).Our study pointed out that the callus induction rates of three varieties significantly increased with the appropriate concentration of CuSO4(10-15 μmol· L-1)in the induction medium and subculture medium,compared with the control group increased by 8%,19%,and 17.2%,respectively.The qualities of callus were significantly improved,and relative growth rates of callus showed significant difference compared with the control group.The callus differentiation rates were significantly improved by adding 5.0-10.0 μmol· L-1CuSO4in the differentiation media.

Uniconazole is an efficient plant growth regulator,which affects the kaurene oxygenase activity,and restrains synthesis of endogenous GA3to reduce the level of endogenous GA3and IAA (Wang et al.,1997).Fu et al.(2005) found that adding 1.00 and 0.50 mg · L-1uniconazole to induction and subculture media showed a promotion effect on inducement and subculture growth of embryonic callus from immature embryos in maize,with no harmful influence to differentiation and regeneration of embryonic callus.Addition 0.25 mg · L-1S-3307 to differentiation medium was shown to have significant promotion effect on differentiation rates.It was studied that under salt stress,adding 10 μmol · L-1and 50 μmol · L-1concentrations of uniconazole to medium made growth situation of Lycium ruthenicum callus was superior to CK,but also promote activities of POD,SOD and APX (Wang,2011).The test results showed that 0.25-1.00 mg · L-1uniconazole was the promotion factor of the rice mature embryo callus induction and subculture growth,which increased the callus differentiation rates.But 2 mol · L-1uniconazole had inhibiting effect on the induction and differentiation rate of mature embryo callus,and plant height of regeneration plantlets was the longest,and root length was the shortest.

Through the research,we found that callus induction and differentiation rates of rice mature embryo were similar between CuSO4and uniconazole treatments,namely the promotion effects increased to maximum and then decreased with the increasing addition concentration.At optimal CuSO4and uniconazole concentrations,the induction and differentiation rates could reach the highest.Since the addition of uniconazole could improve a variety of enzyme activity of containing copper ion,which provided theoretical basis for our future exploration of uniconazole and copper ion use.Whether combined treatment of copper ion and uniconazole has the interaction effect on callus induction and differentiation of rice mature embryo,and whether it's cooperative or just simple superposition still need us to go further research.

Conclusions

The experiment showed that the induction and differentiation rates of callus from mature embryos in rice could be improved by suitable concentration of CuSO4in the medium.The callus induction rates of three varieties significantly increased and callus relative growth rates reached the highest at 10.0-15.0 μmol · L-1concentration of CuSO4in the medium.Supplemented with uniconazole concentration to 0.50-1.00 mg · L-1in the media,the induction and green plantlet differentiation rates of three varieties reached the highest.And the root activities of three varieties were the most vigorous when the concentration was 0.50 mg · L-1.

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