东方百合明星无性快繁体系的建立

牛俊乐 杨美纯 王燕 农艳丰 岑湘涛

Abstract：【Objective】Propagation system established with sterile leaves of Oriental lily Love Story as material to provide technical reference for industrialized production of its seed-bulbs. 【Method】Sterile scales and leaves from flower organs culture were used as material and MS was used as basic medium， adding different concentrations of 6-BA and 2，4-D to induce adventitious buds and bulb-lets； using different concentrations of sugar to induce bulb-lets expansion and rooting； perlite， peat and vermiculite were mixed as the transplanted matrix of bulb-lets. 【Result】MS+1.5 mg/L 6-BA was appropriate for inducing adventitious buds with sterile scales and induction rate was 75.0%； the suitable sterile leaves differentiation bulb-lets medium was MS+2.0 mg/L 2，4-D and induction rate was 91.7%； the MS medium added 70.0 g/L sugar was suitable to induce bulb-lets expansion and rooting； perlite∶peat∶vermiculite=1∶1∶1 was the best transplanted matrix of plant-lets with bulb-lets and roots， transplanting survival rate got 100.0% in this matrix.【Conclusion】Oriental lily Love Story could be established asexually rapid propagation system by sterile leaf.

Key words： Oriental lily Love Story； tissue culture； bulblets； rapid propagation

CLC number： S682.29 Document code： A Article：2095-1191（2016）09-1528-05

摘要：【目的】以東方百合品种明星无菌叶为材料建立快繁体系，为其种球产业化生产提供技术支撑。【方法】以花器官培养获得的无菌小鳞片及叶片为材料，以添加不同浓度6-BA、2，4-D的MS培养基诱导不定芽及小鳞茎产生；以不同浓度的蔗糖诱导小鳞茎的膨大及根的产生；以珍珠岩、草炭、蛭石的混合基质作为小鳞茎移栽基质。【结果】用 6-BA诱导无菌小鳞片产生不定芽时以1.5 mg/L为宜，诱导率为75.0%；用2，4-D诱导无菌叶片产生小鳞茎时以2.0 mg/L为宜，诱导率为91.7%；添加70.0 g/L蔗糖的MS培养基适宜小鳞茎膨大及根的产生；带小鳞茎及根的组培苗在珍珠岩∶草炭∶蛭石=1∶1∶1的基质中移栽成活率最高，达100.0%。【结论】东方百合品种明星可利用无菌叶片建立无性快繁体系。

关键词： 东方百合明星；无菌叶；小鳞茎；无性快繁

0 Introduction

【Research significance】Lily， a perennial herb bulbous plant， belongs to Lilium of Liliaceae. Oriental hybrids are one of the four cut-flower lily varieties being larger flower， rich color and fragrance， and have been preferred by majority of consumers. The bulbs of commercial Oriental lily have low reproductive rate， and are easily infected by virus and so on， so the qualities of their seed-bulbs and cut-flowers were declined gradually； while a large of fine quality bulbs could be produced through tissue culture technology in a short time to meet the demand of cut-flower market（Tang et al.， 2003； Zhou et al.， 2010； Liao et al.， 2012）. Therefore， to establish the asexual rapid propagation system of Oriental lily Love Story， could ensure its species cha-

racteristics， and improve its propagation coefficient to shorten breeding period， which would provide technical references for industrial production of its seed-bulbs. 【Research progress】Currently， many studies on tissue culture of different lily varieties were carried out and reported. According to the source of explants， the used explants of lily can be divided into three categories viz.， scale， leaf and floral organ. Scale is used as explants to induce adventitious buds. Fu et al.（2008） screened out fit concentration of NAA to differentiate adventitious buds derived from sterile scales of Oriental lily. Tiber by adopting orthogonal polynomial regression method and mathematical model. Wang et al.（2013） studied the effects of different types and concentrations of hormones on differentiation of adventitious buds derived from sterile scales of Tiber， Rodina and Constanta plantlets. The results showed that， 1.0 and 2.0 mg/L of 6-BA were suited for differentiation of adventitious buds from sterile scales of Tiber and Rodina， while KT and TDZ were not suited for them. For lily variety Constanta， 1.0 mg/L KT was more suitable for differentiation of adventitious buds. Leaf was also used as explants to induce adventitious buds， and 2，4-D in medium was found to be the important regulated factor on somatic embryogenesis of adventitious buds induction derived from scale leaves of Lilium brownie var. Viridulum（Liu et al.，1997）. Zhao et al.（2005） compared the effects of NAA， 6-BA， TDZ， and 2，4-D on leaves to differentiate adventitious buds of five lily varieties， it found that different lily species presented different sensibility to exogenous hormones； TDZ used alone or combining with low concentration of 2，4-D were suited for Yellow Lilium regale and Polyana； 0.05 mg/L NAA was suited for Lilium var. Golden Sun lily and Red lily； 6-BA combing with low concentration of 2，4-D was more suited for Lilium var. Snow Queen lily. Floral organs were used as explants to induce adventitious buds. Liu et al.（2007） used floral organs of four lily varieties as explants for tissue culture， it found that different lily varieties showed different induction rates of adventitious buds from floral organ， and different floral organs of the same variety were also found to have significant differences in induction rate； the adventitious buds induced sequence from easy to hard in order was filament， receptacle， ovary， corolla， stylus and anther. By using floral organs of Lilium var. Manissa to induce adventitious buds， Nong et al.（2014） found that， 3.0 mg/L 6-BA had significant effect on differentiation of adventitious buds， its highest differentiation rate could be reached to 95.5%. 【Research breakthrough point】At present， the commercial seed-bulbs of Oriental lily are commonly cultured by using plantlets. So far， there are no relative researching reports on commercial seed-bulbs of Oriental lily Love Story in domestic， and its fewer clones derived from sterile leaves are reported. 【Solving problem】The present study aimed to establishing the asexually rapid propagation system of Oriental lily Love Story by sterile leaf， which would provide technical references for industrialized production of its seed-bulbs.

1 Materials and methods

1. 1 Experimental materials

The sterile bulblets derived from flower organ culture of Oriental lily Love Story were used as materials.

1. 2 Experimental methods

1. 2. 1 Induction of adventitious buds The sterile scales were taken from the sterile bulblet with tweezers to cut out the top， the intermediate and bottom parts of leave were cut into 0.5 cm2 of pieces， followed by culturing in MS medium supplied with 0-2.0 mg/L 6-BA. The new growing adventitious buds were counted after culturing for 40 days.

1. 2. 2 Bulblets induction of sterile leaves The sterile leaves derived from the above-mentioned induced adventitious buds were cut out the top， then the middle and lower parts were cut into about 1.0 cm to culture in MS medium supplied with 0-3.0 mg/L 2，4-D， the induced bulblets were counted after culturing for 50 days.

1. 2. 3 Expansion culture and rooting induction of sterile bulblets The sterile above-mentioned bulblets induced by sterile leaves were inoculated on MS medium supplied with 30.0-90.0 g/L sugar for bulblets expansion and rooting induction. The multiple of enlargement， rooting rate and average roots per bulblet were counted after culturing for 90 days.

1. 2. 4 Transplanting culture The 50% carbendazim was mixed with different transplanted matrix（perlite， peat， vermiculite， sawdust， garden soil） as the amount of 40 g/m3， followed by covering with plastic for 3 days. The rooting bulblets（>0.3 cm in diameter） were removed from the culture room to domesticate for 7 days in greenhouse. The roots of bulblets were washed out adhering media， the bulblets were divided into 2 groups according to its diameter（0.3-0.5 cm， >0.5 cm） and immersed in 0.1% carbendazim solution for 15 min， followed by transplanting into four different mixed matrixes with equal proportion of perlite， peat， vermiculite， sawdust， or garden soil. The survival rate and plant height of bulblets were counted after culturing for 30 days.

The above-mentioned culture process conditions were as follows： 25-28 ℃ of the culture room temperature， 12-14 hours/day of lighting time companied with 1500-2000 lx of light intensity.

2 Results and analysis

2. 1 Effects of different concentrations of 6-BA on adventitious buds differentiated from sterile scales

During the process of scale culture， the scales began to wrinkle on the surface after inoculating for 8 days， and the scale incision site of two ends appeared little beige bumps after 14 days， followed by greening and developed into adventitious buds after 25 days.

From the results shown in Tab.1， it found that in the range of 0-2.0 mg/L 6-BA， the differentiation rate of adventitious buds showed change trends of increasing firstly and decreasing at latter with the increase of 6-BA dosage in MS medium. By adding with 1.5 mg/L 6-BA， the adventitious buds grew strong and differentiated well， and its differentiation rate reached the highest as 75.0%. The dense adventitious buds with a lot of callus found to be differentiated when added 2.0 mg/L 6-BA； because the induced adventitious buds were too dense and unhealthy， so it was not good for further multiplication. Therefore， MS+6-BA 1.5 mg/L was the suitable culture medium for differentiating adventitious buds of sterile scales.

2. 2 Effect of 2，4-D on bulblets differentiated of sterile leaves

During the culture process of leaf sections， it found that sterile leaves began to enlargement after inoculating for 7 days， subsequently， little yellowish protuberance was formed at the both ends of incision， and the bulblets were formed after 30 days of culture. The callus was not found during the whole culture process.

From the results of Tab.2， it showed that in the range of 0.5-2.0 mg/L 2，4-D， the number and size of bulblets differentiated were increased gradually， while the leaf number presented decreasing trend. When MS medium added with 2.0 mg/L 2，4-D， the differentiation rate of bulblet reached the highest as 91.7%. With the increase of 2，4-D concentration（above 2.0 mg/L）， the number of bulblet was decreased gradually， the diffe-

rentiation rate decreased to 42.9% by supplying 3.0 mg/L 2，4-D in MS medium， and no leaf was found to grow on bulblet.

2. 3 Effect of sugar on expansion and rooting of bulblet

As shown in Tab.3， the results showed that the rooting rate of all bulblets could reach to 100.0% when MS added with 30.0-90.0 g/L sugar. The expansion times of bulblet gradually increased with the increase of sugar concentration， while the average roots of different treatments presented change trends of increasing firstly and decreasing at latter. By adding 90.0 g/L sugar in MS medium， the bulblet grew strong， and its expansion times reached the highest as 3.0， the average roots per bulblet reached 5.4. By supplying 70.0 g/L sugar in MS medium， although the expansion times of bulblet was 2.6， the average roots per bulblet reached the highest as 6.3. Taking into account both of expansion state and rooting effect， MS+70.0 g/L sugar was considered as the suitable culture medium for expansion and rooting of bulblet.

2. 4 Effects of different transplanted matrices on transplanting survival rate

After transplanting for 10 days， the rooting bulblets of different matrices treatments started to grow new leaves. From the results of Tab.4， it found that the survival rate of bulblets for the 1st matrix treatment（perlite， peat and vermiculite） reached to 100.0% when bulblet diameter were 0.3-0.5 and >0.5 cm in size， and their average height found to be the highest as 4.5 cm. Furthermore， the survival rates of bulblets with 0.3-0.5 cm in size were 12.5%-44.3% higher than those of bulblets with >0.5 cm in size for other 3 treatments. The survival rate and average height of bulblets for the 4th treatment（perlite， peat， vermiculite and sawdust） were found to be higher than the 3rd treatment（perlite， peat， vermiculite and garden soil）， because that the ventilation ability and water permeability of added sawdust were better than that of garden soil. Being without supplement of vermiculite， the ventilation ability and water permeability of the 2nd treatment（perlite， sawdust and garden soil） were lower than the other three treatments added with ventilation， which resulted in the lowest survival rate. By comprehensive consideration， the matrix combination of perlite，peat and vermiculite（1∶1∶1） was more suitable for transplanting of Oriental lily love story.

3 Discussion

Lily traditional propagation modes include generative propagation and vegetative propagation. Generative propagation require long period and easily produce mutation， vegetative propagation was characterized by very low propagation coefficient for inducing adventitious buds by using scales investment method（Lan， 2011）. Therefore， during propagation process of lily plantlets， the tissue culture and rapid propagation of lily bulb are of important significance to improve its propagation coefficient， get a lot of virus-free high-quality bulbs and keep its species characteristics.

In recent years， there are many reports on the propagation system established of lily. By using the bulb scale and leaf of three oriental lily varieties as explants， Hu et al.（2007） studied the effects of 6-BA， KT， NAA and 2，4-D in MS medium on organogenesis of oriental lily. The results of adventitious buds and bulblets diffe-

rentiated from scale indicated that， the small bulbs were wrapped tight when formed in culture medium added with 2，4-D， poorer quality bulblets with serious vitrification were found in medium with high concentration of 6-BA. It also found that 6-BA was very important to adventitious buds differentiation， while KT could obviously shorten differentiation time. The results of orthogonal test showed that， KT and 2，4-D had a little effects on the formation of adventitious buds； the result of adventitious buds differentiated leaves indicated that， the initiation culture time， differentiation efficient and types of leaves were found to be different being variety differences. In the present study， 6-BA was used to choose for adventitious buds differentiation of lily variety Love Story scales， it found that 1.5 mg/L 6-BA was suitable for differentiation of scales， adventitious buds and the differentiation rate reached to 75.0%， which was similar as the reported results of Liu and Lin（2002） and Wang et al.（2013）.

By comparing the effects of 2，4-D， IAA and NAA on callus formation of L. brownie var. Viridulum sterile leaves（Liu et al.， 1997）， it found that callus induction rate were higher than the other treatments whether 2，4-D used alone or combining with cytokinin. Cytological observations results showed that， during the process of somatic embryogenesis formation， somatic embryos could be obtained directly without callus inducing stage when supplied 2，4-D in culture medium. In the study of Oriental lily. Tiber leaves to induce callus by using 2，4-D in MS medium， Fu et al.（2008） found that after a period of culture， the surface of explants could induce small absinthe-green embossmen， which some of them could be formed into callus and others were directly formed into bulblets. This similar phenomenon was also observed in the present study by using 2，4-D to induce callus from leaves， and bulblets were found to directly form from small embossmen of explants surface without obvious callus.

During the rooting process of lily plantlets， Wang and Lin（2005） indicated that both of 1/2MS and MS media were advantageous to plantlets rooting； Luo et al.（2000） found that MS medium without any hormones was the best medium for rooting induction of Longiflorum. During the culture process of bulblets expansion of Lilum davidi var. Unicolor（Hoog） Cotton ， MS medium adding with 60.0-90.0 g/L sugar could significantly promote the bulblets formation， and the highest weight reached to 99.30 mg when added 90.0 g/L sugar in medium（Wei et al.， 2014）. As to the rooting culture of Manissa， the rooting rate found to be reached to 100.0% and the average roots was 6.9 when added 60.0 g/L sugar in MS medium. In this trial for inducing bulblets expansion and rooting of Oriental lily Love Story， the similar results were found by supplying 70.0 g/L sugar in MS medium， the expansion times of bulblet was 2.6 and the average roots per bulblet reached the highest as 6.3.

The transplanting matrix is one of the important factors for plantlet survival of lily， different lily plantlets need different suitable transplanting matrices. Luo et al.（2000） tried to use river sand， peat and perlite alone or in combination（1∶1） to study the transplant survival rate of Longiflorum， the results indicated that the best matrix for transplant was river sand and peat with ratio of 1∶1； Li et al.（2014） adopted peat， vermiculite， humus soil and dry chicken manure as matrix with ratio of 1∶1∶1∶1 to transplant lily plantlets， and the survival rate of plantlets with >0.5 cm in diameter found to be 86.7%. The perlite and humous（3∶1） screened was the best mixed matrix for Acapulco， the survival rate found to be reached to 100.0% and the plantlet grew well（Zhao et al.， 2003）. In our experiment， the matrix combination of perlite， peat and vermiculite（1∶1∶1） was more suitable for transplanting of Oriental lily Love Story， the survival rate of bulblets in diameter of 0.3-0.5 and >0.5 cm could be achieved 100.0%.

4 Conclusion

The present results showed that， by using sterile scales of adventitious bud derived from flower organ culture as explants， the established rapid propagation system of Oriental lily Love Story could be used for its industrialized production of seed-bulbs.

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