Tingting HU,Shuang LI,Tianshu LU,Fangke GAO,Rongzhe WU

Department of Horticulture and Landscape Architecture,Agricultural College of Yanbian University,Yanji 133002,China

Strawberry(Fragaria ananassaDuch.)is a perennial herb of Fragaria in Rosaceae,with the aliases of Yangmei,Diguo and Shiduopili,which is one of the seven famous fruits in the world.The fruit of strawberry can be eaten in fresh form or can be processed into pulp,juice,fruit wine and can[1].The strawberry fruit are bright in color,taste sour and sweet,contain high organic acid and vitamin contents,and are rich in nutrition and physiologically active components[2-6].Strawberry has the advantages of high adaptability,simple culture and high fruit nutritive value,and thus is suitable for culture in most areas of China.Strawberry produced in the conventional way(seed propagation,stolon propagation,stock plant division propagation)carried various viruses accumulated in vivo due to continuous croppingand long-term vegetative propagation,resulting in decreased fruit quality followed by decreased economic value and commodity value[7].Meanwhile,the rapid propagation system of strawberry established by tissue culture method not only is free of time and location re striction with high propagation coefficient,high survival rate and vigorous growth,but also prevents viral diseases,saving time and labor[8-10].A strawberry variety Bechika introduced from Japan has the advantages of early fruiting,rapid maturation,high yield,good quality,strong disease resistance and long shelf life,and has the disadvantages of short height,deteriorated quality,more deformed fruit and low yield due to infection of virus after being grown for several years,as well as its plants were very expensive.This study performed preliminary test on rooting environment for tissue culture,and domesticated and transplanted tissue culture plantlets so as to provide technique support for large-scale production of virus-free plantlets of strawberry Bechika and rapid breeding of good seedlings.

Materials and Methods

Materials

The tested material was new strawberry stolons collected from Liangshui Town in Tumen City of Jilin Province.The stolons were flushed with flowing water,soaked in 75% ethanol,and flushed with sterile water 4-5 times,and sterilization was performed with 0.1% mercuric chloride for 6-8 min under continuous stirring,which was followed by flushing with sterile water 5-6 times.Stem tips(0.2-0.4 mm)were taken off on ultraclean workbench and cultured on culture medium(MS+BA 2.0 mg/L+NAA 0.1 mg/L+sucrose 30 g/L+agar 8 g/L,pH 5.80)to obtain virus-free tissue culture plantlets.

Methods

Effect of MS concentration on rooting of strawberry BechikaUnder sterile condition,sprouts with different sizes were cultured on 1/4MS,1/2MS,3/4MS and MS culture media which were added with 30 g/L sucrose and 7 g/L agar,respectively,and regulated to pH of 5.8.Three sprouts with different sizes(with a height of about 1 cm,and three leaves)were cultured in each bottle under the condition such as a culture temperature of(25±2)℃,a relative humidity of 70%,an illumination intensity of 1 600 lx and illumination time of 16 h/d.Each treatment had three replications.The rooting of strawberry plantlets was investigated after 25 d.

Effects of auxin kind and concentration on rooting of strawberry BechikaUnder sterile condition,sprouts with different sizes were cultured on the culture medium(1/2MS+sucrose 30 g/L+agar 7.0 g/L,pH 5.8).The concentrations of IBA and IAA were 0,0.1,0.3,0.5 and 0.7 mg/L,respectively.Other culture conditions were the same as"Effect of MS concentration on rooting of strawberry Bechika".

Effect of activated carbon on rooting of strawberry BechikaUnder sterile condition,sprouts with different sizes were cultured on the culture medium (1/2MS+IBA 0.3 mg/L+sucrose 30 g/L+agar 7.0 g/L,pH 5.8),which was added with activated carbon at 0,0.5,1.0,1.5 and 2.0 g/L,respectively.Other culture conditions were the same as"Effect of MS concentration on rooting of strawberry Bechika".

Effect of transplanting substratum on domestication ofstrawberry BechikaThe rooted tissue culture plantlets(with a plant height of about 5 cm)were uncovered,and kept at environmenthumidity.After5 d,the plantlets were transplanted into nutritive disks (L(length)×W (width)×H(height)=(56×32×5)cm3,54 apertures,diameter Φ 6 cm),the substrata watered thoroughly one day before transplanting were sand (A),humus(B),sand+humus=1:1 (C),Perlite+vermiculite=2:1 (D),perlite+vermiculite=1:1 (E)and perlite+vermiculite(1:2 (F).The nutritive disks were placed at locations against the sun,provided with sheds and kept at a relative humidity above 85%.The shed was covered by transparent plastic which was covered by a sunshade net on sunny days while gradually reducing coverage time until the sunshade net was all removed.After 5 d,the transparent plastic was removed,the growth of strawberry was observed,and the survival rate and growth status were investigated after 20 d.

Results and Analysis

Effect of MS concentration on rooting of strawberry Bechika

Different concentrations of MS culture media had different effects on rooting of strawberry plantlets.With MS concentration increasing,the fresh weight of aboveground part of plant increased gradually,while the fresh weight of root presented a trend of increasing at first and decreasing then(Table 1).When the culture medium was 1/4MS,the plants were short and grew weak,and the produced roots were sparse and thin,but the rooting rate was up to 99.4%.In the case of 1/2MS,the length of root reached 2.8 cm and formed a proper proportion with plant height,meanwhile,the roots had a fresh weight up to 89.0 mg,and were greenish white mostly and thicker and stronger than those in the other-Effect of MS concentration on rooting ofstrawberry Bechika.concentration media,with a number as many as 12.0,and therefore,the plants grew vigorously with a rooting rate up to 100%.From 3/4MS,with MS concentration increasing,the roots became short and sparse gradually with the decreasing rooting rate.When the MS culture medium was used,the produced roots were relative fewer and shorter,and the root rate and fresh weight decreased greatly as well(Fig.1).

Effects of auxin kind and concentration on rooting of strawberry Bechika

With IBA concentration increasing,the aboveground part and roots of plant showed a growth trend of increasing at first and decreasing then.When the IBA concentration was 0.3 mg/L,the fresh weight and dry weight of plant root reached their maximums,and the produced roots were more,thicker and stronger,with a length of 4.5 cm (Table 2).When the IBA concentration continued to increase,the growth vigor of roots decreased accordingly.When IAA was added,the aboveground part and root showed a trend of increasing at first and decreasing then with IAA concentration increasing.When the IAA concentration was 0.1 mg/L,the growth vigor of aboveground part and roots of plantachieved the level of significance compared with the control.By comparing IBA with IAA,the root number at the IBA concentration of 0.3 mg/L was more and thicker than at the IAA concentration of 0.1 mg/L,and overall roots produced under IAA were thin(Fig.2),so the IBA concentration of 0.3 mg/L was better.

Table 1 Effect of MS concentration on rooting of Strawberry"Bechika"

Table 2 Effects of IBA and IAA concentration on rooting of strawberry Bechika

Effect of activated carbon on rooting of strawberry Bechika

The fresh weight of aboveground part of plant gradually reduced with increase in concentration of activated carbon,while the fresh weight and dry weight of root as well as root number gradually decreased with the concentration increasing from 0.5 g/L,and when the concentration reached 1.0 g/L,root length was the longest(Table 3).Without activated carbon,the plant roots were in dark color,and the plants were high but grew weak.When the concentration was 0.5 g/L,the fresh weight and dry weight of root were significantly different from those of the control,and the plants were upright and healthy,and grew vigorous with the root being the most and greenish white(Fig.3).With increase in concentration of activated carbon,the roots became thin gradually accompanied by gradually-reduced rootnumber;and furthermore,at the activated carbon concentration of 2.0 g/L,the root length was only 2.2 cm,and the rooting rate was 92.1%.

Effect of transplanting substratum on strawberry domestication

The survival rate was lower in the three combinations using humus and sand than in other three combinations using perlite and vermiculite.Among the three combinations of humus and sand,the survival rate was the lowestat 68.5% in B (humus),followed by that with C(sand:humus=1:1)as substratum,and the highest survival rate was in A (sand).In the case of the treatments combining perlite and vermiculite,F(perlite:vermiculite=1:2)had the lowest survival rate of 90.7%,and E (perlite:vermiculite=1:1)was of the highest survival rate of 98.1% (Fig.4).The strawberry plantlets growing in the substrata containing humus were stronger than in the substrata using perlite and vermiculite and had leaves which were large and deep green in color,while the substrata using perlite and vermiculite were more suitable for the domestication of tissue culture plantlets of strawberry Bechika from the point of survival rate (Fig.5).The formation of strawberry stolons did not need large space for roots,and the larger the root space,the harder the stolon formation,so there was no need for transplanting domesticated plantlets.The plantlets domesticated for 30 d were irrigated with a nutrient solution every 3 d and observed,and it was found that the strawberry plantlets with perlite and vermiculite as substratum became stronger,with the leaves becoming largeraccompanied by deepening color and the stems becoming thicker,i.e.,the plantlets were the same as those in humus(Fig.6).After 90 d,the domesticated plantlets with 8 leaves began to produce stolon and bud to bloom (in March of 2013)(Fig.7).The production of stolon began from the humus substratum,while it was later in the combinations of perlite and vermiculite than in humus by about 15 d.The strawberry plantlets flowered in a large amount and fruited after 120 d.

Table 3 Effects of activated carbon concentration on rooting of Strawberry Bechika

Discussion

Rooting of tissue culture plantlets has been crucial to tissue culture experiments and has been a major focus in this field.The study performed screening the concentration of culture medium MS,the results showed that 1/2MS was suitable for rooting of strawberry Bechika.With increasing MS concentration,the fresh weight and dry weight of aboveground part of plant increased,while the roots presented a trend of increasing at first and decreasing then,and when the culture medium was 1/2MS,the fresh weight of root as well as root number reached the level of significance.This might be due to the fact that nutrition increased with the increasing concentration,while roots did not need too much nutrition in that too much or too little nutrition could inhibit rooting.

Auxins IBA and IAA had a promoting effect on rooting of strawberry,and LIANGet al.[1]found that it was IAA that influenced the growth of main roots and it was IBA that influenced the growth of lateral roots,which also illustrated this point;the number of overall roots was greater with the treatment of IBA than with the treatment of IAA;and though the roots with the treatment of IAA were longer than with the treatment of IBA,they were thinner than those treated by IBA.Consequently,the treatment of IBA was superior to that of IAA.

Activated carbon was widely used in tissue culture,as it could absorb harmful substances from culture media and plant growth,providing dark environment for plants and facilitating rooting;and it could prevent browning during primary culture[10].This study showed that the roots in the treatments added with activated carbon had a color better than those of the treatment without activated carbon,i.e.,the roots in the treatment added with activated carbon showed greenish white with high activity,indicating that activated carbon could adsorb the substances inhibiting root growth.Transplanting domestication was the last step for tissue culture,in which the optimal transplanting substratum was perlite:vermiculite=1:1 among the six substrata,achieving a survival rate close to 100%.Although the plants growing on the combination medium with perlite and vermiculite were little,and weaker than on humus,perlite:vermiculite=1:1 was chosen from the point of survival rate,which accorded with the research result of strawberry domestication by SUNet al.[11].

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