Panpan MA，Zongming XlE，Quansheng Ll，Yousheng TlAN，Youzhong Ll

Biotechnology Research Institute，Xinjiang Academy of Agricultural and Reclamation Sciences/Xinjiang Production&Construction Group Key Laboratory of Crop Germplasm Enhancement and Gene Resources Utilization，Shihezi 832000，China

Effect of Different Hormone Combinations on Somatic Embryogenesis in Cotton Cultivar Xinluzao 33（Gossypium hirsutum L.）

Panpan MA，Zongming XlE*，Quansheng Ll，Yousheng TlAN，Youzhong Ll

Biotechnology Research Institute，Xinjiang Academy of Agricultural and Reclamation Sciences/Xinjiang Production&Construction Group Key Laboratory of Crop Germplasm Enhancement and Gene Resources Utilization，Shihezi 832000，China

［Objective］This study was conducted to evaluate the efficiency of somatic embryogenesis and plant regeneration for an upland cotton cultivar Xinluzao33 under the induction of different hormone combinations and thus to determine the optimal hormone combination.［Method］Calli of Xinluzao33（Gossypium hirsutum L.）were in-duced from seedling hypocotyl tissue by a range of DK and BK combinations.Em-bryogenic calli and embryos were induced on callus-inducing medium （CIM）without any hormones.Callus appearance and quality were compared to determine which medium was the optimal for callus induction.Embryogenesis ratio was calculated to determine which medium was the best for somatic embryogenesis and plant regen-eration.［Result］Callus induction rate was 100%in all the 12 hormone combinations. The calli were yellow or kelly，and their texture was loose or soft under low con-centrations of DK combinations，green or white，variably compact under high con-centrations of DK combinations.The calli induced by BK combinations were kelly or green，covering creamy white substance.The best medium for callus induction was DK6 （0.05 mg/L 2，4-D and 0.10 mg/L KT）.Embryogenic calli were successfully in-duced from all the combinations.The efficiency of embryogenic callus induction，embryogenesis，and plantlet regeneration were significantly different among the 12 combinations.The result showed that the embryogenesis ratio was the highest in BK3 combination （0.50 mg/L IBA and 0.50 mg/L KT），72.86%of embryogenic calli differentiated into somatic embryos after being cultured on CIM for 80 d，and 80.93%of the somatic embryos finally regenerated into plants on SEM（somatic em-bryo induction medium）.［Conclusion］These results indicate that hormone combina-tion BK3 （0.50 mg/LIBA and 0.50 mg/L KT）was the best medium for somatic em-bryogenesis and plant regeneration from Xinluzao33.

Gossypium hirsutum L.；Xinluzao33；Hormone combinations；Somatic embryogenesis

S omaticembryogenesisisa critical step to ensuring the success of cotton variety im-provement via mutant screening and gene transfer.Somatic embryogenesis and plant regeneration systems for various elite Chinese cotton varieties have been established［1］.Some of the varieties （lines）such as CCRI24 and YZ1 have been used as model materi-als cotton tissue culture and genetic transformation studies［2-4］.In recent years，somatic embryos and regener- ated plants have been obtained from a series of Gossypium hirsutum L.culti-vars in Xinjiang［5-10］.However，hardly a variety （line）was used as the ac-ceptor material for germplasm creation and gene transformation through so-matic embryos.Itis importantto screen elite cultivars of high efficient somatic embryogenesis in Xinjiang，which will provide excellent receptor for genetic transformation of exoge-nous genes.Xinluzao 33 is one of the upland cotton（Gossypium hirsutum L.） cultivars grown in Xinjiang during re-cent years.It is an early-maturity vari-ety with high yield，resistance to Fusarium and Verticillium wilt，and also suitable for mechanical harvest-ing［11］.It was found to possess a rela-tively strong ability in somatic embryo-genesis and plant regeneration，with an efficiency of somatic embryogene-sis up to 67.2%under the induction of 0.05 mg/L 2，4-D （2，4-dichlorophe-noxyacetic acid）and 0.10 mg/L KT（kinetin）in our previous work［10］.

The efficiency of somatic em-bryogenesis and plant regeneration in cotton is affected by many factors. Among them，concentration of exoge-nous hormones is one of the most im-portant factors，and the influences of different hormones or plant growth regulators are also different［12］.2，4-D and KT are always used in combina-tion for callus induction in cotton tissue culture.The hormone combination of KT and IBA（indolebutyric acid）is also conducive to somatic embryogenesis and plant regeneration in cotton［13］. An in-depth study of hormones would enable the development of culture methods to better enhance the somat-ic embryogenesis response of Xin-luzao 33.

Twelve hormone combinations of DK（2，4-D and KT）and BK（IBA and KT）were designed for callus induction of Xinluzao33 in this study，with an at-tempt to evaluate the efficiency of so-matic embryogenesis and plant re-generation under the induction by a range of hormone combinations and to screen the optimal hormone combina-tion for Xinluzao33.The system for improving Xinluzao33 somatic em-bryogenesis and plant regeneration described here will facilitate the appli-cation of tissue culture and genetic en-gineering for cotton genetic improve-ment in Xinjiang.

Materials and Methods

Plant materials

The seeds of Xinluzao33 were provided by the laboratory of Biotech-nology Research Institute，Xinjiang Academy of Agricultural and Recla-mation Sciences.

Media and culture conditions

MSB medium was used as basic medium of somaticembryogenesis and plant regeneration in cotton，sup-plemented with various hormones and amino acids，etc.［14-15］.All the media were supplemented with 3%sucrose，and solidified with 0.20%-0.25%phy-tagel（Sigma）.The pH of all media was adjusted to 5.8-6.0 with 1 mol/L NaOH before autoclaving at 121℃ for 15 min.All the cultures were performed at（28±2）℃ at a photoperiod of 14 h light with a light intensity of approxi-mately 100 μmol/（m2·s）provided by cool white fluorescent lampsin a growth chamber.

Seed sterilization，germination and callus induction

Mature seeds were sterilized with 75%ethanol for 30 s，then soaked in 10%H2O2for 90 min，washed four times with sterile distilled water and kept in sterile ddH2O overnight for ger-mination.The surface sterilized seeds were cultured on half-strength MS medium with0.20% phytageland maintained at（28±2）℃in dark for 3 d，then under cool white fluorescent light for 3 d.Hypocotyl sections（about 5-7 mm）were inoculated on callus in-ducing medium （CIM），supplement with various combinations of 2，4-D，IBA and KT at different concentrations（Table 1）.After about 30 d，the initial cultures were separated from explants and transferred to fresh CIM medium（without hormone）for subcultures.

Somatic embryogenesis and plant regeneration

After four weeks of culture，light yellow，light green and loose calli were chosen and transferred onto ECM（embryogenic callus differentiation medium，which was MSB supple-mented with 3.80 g/L KNO3）for em-bryogenic callus induction.And em-bryogenic callus emerged in about two months.Grayand yellowish-brown embryogeniccultureswerechosen and transferred onto SEM ［somatic embryo induction medium，which was MSB medium supplemented with 1.00 g/L glutamine，0.50 g/L asparagine，0.25%（w/v）phytagel］for the induction of normally developed somatic em-bryos and subculture every 25 d.After about 20 d，normal somatic embryo-derived seedlings began to appear with high frequency.Regenerated plants with 3-5 leaves and well devel-oped roots were transferred into greenhouse（25±2℃，60%relative hu-midity）.

Statistical analysis

Theexperimentwasarranged following a randomized complete block design with 30 replicates and repeated three times respectively.Data were analyzed using the SPSS program Version 16.0.A probability level of 5%（=0.05）was chosen for all statistical inferences.The analysis of variance and the Least Significant Difference（LSD）test was used to detect differ-ences among treatments.

Table 1 Hormone combinations in callus induction media（CIM）

Results and Analysis

Callus induction and proliferation

A wide range of callus initiation and proliferation responses were ob-served among the hypocotyl explants of all 12 hormone combinations（Table 2）.The two ends of hypocotyls started to swell after they were cultured on DK combination induction media for 5 d and produced a mass of calli within 25 d.As in BK combinations，hypocotyls started to swell as they were induced for 8 d and produced a mass of calli within 35 d.The frequency of callus induction was 100%in all the 12 com-binations.However，the color and tex-ture of the calli were diverse（Table 2）. Light yellow，kelly，green and gray calli were observed from different callus induction media.Some degree of browning was found in all cases.And the texture of the calli ranged from very hard and compact to watery and friable.Calli were yellow or kelly and loose under lower concentrations of 2，4-D and KT （Fig.1A）.When the con-centration of KT was increased to 0.20 mg/L，the calli were green and hard in DK combinations.And the calli were kelly or green，brown and creamy white in all BK combinations（Fig.1B）. The mass of callus proliferation was calculated and analyzed.And the re-sults showed that the indices of callus proliferation in DK combinations were greaterthan in BK combinations（Table 2）.DK6 （MSB supplemented with 0.10 mg/L KT and 0.05 mg/L 2，4-D）was found to be the best for callus induction based on callus color and texture.

Somatic embryogenesis

The yellow，light yellow，or green and loose calli were selected and transferred onto ECM medium for em-bryogenic callus induction.Successiveculture of the induced callus could produce pre-embryogenic calli in one month.Embryogenic calli were first observed as small sectors of a pale grey callus turning from yellowish to brown，friable callus （Fig.1 C and D）. After two months of subculture，em-bryogenic calli emerged in all the hor-mone combinations except DK5（Table 3）.Light yellow，yellow or kelly and loose calli were found to be the main path of embryonic callus and so-matic embryogenesis（Fig.1E）.Most of brown granular calli turned brown and died，and only a few of such calli pro-duced somatic embryos.It always took long time for somatic embryos to ma-ture.A small part of the gray loose or mashed embryonic callus differentiat-ed，but basically deformed and mostly covered the entire medium after they were inoculated for 2 weeks.

Table 2 Effect of different hormone combinations on callus induction of hypocotyls

Hypocotyl segments responded differently to different levels of KT and IBA in callus production and embryo-genesis.The time and efficiency of embryogenic callus differentiation were obviously different among the 12 combinations （Table 3）.After one or two times of subculture on ECM medi-a，embryogenic calli were differentiat-ed in all combinations except DK5. Low concentrations of KT（0.10 mg/L）combined with 2，4-D （0.05 mg/L）in-duced the highest embryogenesis fre-quency （69.17% ）.Embryogenesis took more than 120 d under the induc-tion by high concentrations of KT（0.15 mg/L）in DK combinations，even with low efficiency.In BK combinations，embryogenic callus induction and so-matic embryogenesis efficiencies were increased with the increase of KT concentration when IBA concentration was 0.50 mg/L.However，the differ-ences in embryogenic callus induction and somatic embryogenesis efficiency were not significant when IBA con-centration was 1.00 mg/L （Table 3）. Our best result was obtained with low-er concentrations of IBA （0.50 mg/L）and KT （0.50 mg/L）for producing much higher frequencies of embryo-genic callus and embryos（57.79%and 72.86%，respectively）of Xinluzao33.

Conversion of embryos into plants

Embryogenic calli and embryos at various developmental stages were transferred onto SEM medium for em-bryo induction and plant regeneration（Fig.1F-G）.Somatic embryos induced by the combinations except DK5 could successfully convert into seedlings af-ter 3-4 weeks of culture.And the re-generation efficiency was significantly different among the 12 combinations（Table 3）.A high plant regeneration rate of 80.93%was obtained under BK3 induction.Besides the normal plants，somaclonalvariations were detected during the regeneration pro-cess，such as plantlets with abnormal leaves，cluster axillary shoots and main axillary shoots.

Table 3 Effect of different hormone combinations on differentiation of embryonic callus

The MS medium was used to in-duce rooting for the regenerated plants with 2-3 leaves in this study.Most of the regenerated plants were able to develop root system well in 15 d（Fig.1 H and I）.The regenerated plants with at least 3-5 leaves were cultured with tap water for about 5 d （Fig.1J），and then transferred into pots containing 1∶3（v/v）mixture of sterile soil and ver-miculite in the growth chamber under the same conditions described above（Fig.1 K and L）.The survival rate was above 70%in all the seasons.

Conclusion and Discussion

Plant regeneration through so-matic embryogenesis may be neces-sary for cotton variety improvement through genetic transformation.The regeneration systems for a range of elite Chinese cotton varieties have been established，however，few of them were applied in cotton breeding. Thatwasbecause embryogenesis could not be initiated in many geno-types with the protocols available.It is important to establish a protocol for cotton improvement by genetic trans-formation in Xinjiang.Therefore，we developed the system for plant regen-eration from Xinluzao33，which is con-sidered to be an elite upland cultivar in Xinjiang，China.

The frequency of in vitro callus in-duction and plant regeneration is influ-enced by several conditions including composition of culture medium，ex-plants source and hormones.Hor-mone regimes have been found to be the most important external factors af-fecting somatic embryogenesis［16-18］.In the present research，12 different con-centrations of 2，4-D，KT and IBA combinations were used for callus in-duction from cultivar Xinluzao33.Cal-lus grew quickly and overly proliferated on 2，4-D containing medium，and more slowly on IBA and KT mediums. KT （0.10 mg/L）is much better for cal-lus induction when it is used in combi-nation with low concentration of 2，4-D（0.05 mg/L）.The study showed that DK6（0.05 mg/L 2，4-D and 0.10 mg/L）was the best among the 12 combina-tions for callus induction.The efficien-cy of somatic embryogenesis and re-generation was up to 69.17%and 67.14%，respectively，which was con- sistent with our previous findings［10］. However，the callus proliferation rate was much lower under BK combina-tions induction，and the calli grew rapidly after being transferred onto ECM media.72.86%embryogenic calli differentiated into somatic embryos af-ter 80 d ofculture on ECM，and 80.93%of the somatic embryos con-verted into plants on SEM under the induction with BK3 combinations at low concentrations.Previous studies showed that embryogenesis calli were produced much slowly，and somatic embryos were easy to turn brown and die during the multiplication process［7］. Our studies revealed that BK3 was the best combination for embryogenic cal-lus differentiation and somatic embryo germination.

As the results showed above，so-matic embryogenesis was successful-ly induced by DK and BK combina-tions.High regeneration efficiency was detected especially under the induc-tion by DK6 and BK3 combinations at low concentrations.The differences of callus induction and somatic embryo-genesis between DK and BK combi-nations have been studied.Histologi-cal research showed that 2，4-D is the most effective hormone for tissues to dedifferentiate，but advantageous to re-differentiate during somatic em-bryogenesis.And it will be beneficial to reduce concentration or remove 2，4-D for embryogenic callus induction，when initial callus was induced［19］.It was presumed the vascular bundle cells and marrow cells did not de-dif-ferentiate and the cortex cells pro-duced embryogenic callus on the IBA and KT containing medium［19］.Molecu-lar studies showed that 2，4-D related to nuclear DNA methylation levels. DNA methylation level elevated and gene activity decreased under high concentrations of 2，4-D.Somatic em-bryogenesis-related genes are acti-vated when 2，4-D is removed［20-21］. Auxin signaling pathway and transcrip-tion regulation involved in dedifferenti-ation and re-differentiation during so-matic embryogenesis in cotton were also studied［22］.A number of genes regulating auxin transport and signal-ing pathways have been isolated and identified［23-24］.

In this research，somatic embryo-genesis and regenerated plants of Xinluzao33 were successfully obtained by using IBA and KT.The plants were transferred to soil and grew to maturi-ty.With the protocol described here，we have obtained hundreds of regen-erated plantlets from Xinluzao33，which is important for the application of tissue culture to transgenic cotton breeding.

Acknowledgements

Much assistance from Xinjiang Production&Construction Group Key Laboratory of Crop Germplasm En-hancement and Gene Resources Uti-lization is acknowledged.

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Responsible editor：Qingqing YlN

Responsible proofreader：Xiaoyan WU

Supported by the National Transgenic Major Project of China（2009ZX08009-090B）；the BiologicalGermplasm ProjectofXinjiang Production & Construction Crops（2012BD046）；the TechnologyPlanofXinjiangAcademyofAgriculturaland Reclamation Sciences（81YYD201506）.

*Corresponding author.E-mail：xiezmchy@163.com

Received：August 20，2015 Accepted：September 25，2015