Establishment of in vitro culture, plant regeneration, and genetic transformation of Camelina sativa

ISSN 0095�4527, Cytology and Genetics, 2013, Vol. 47, No. 3, pp. 138–144. © Allerton Press, Inc., 2013.Original Russian Text © A.I. Yemets, Yu.N. Boychuk, E.N. Shysha, D.B. Rakhmetov, Ya.B. Blume, 2013, published in Tsitologiya i Genetika, 2013, Vol. 47, No. 3, pp. 14–20.

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INTRODUCTION

The interest in camelina (Camelina sativa (L.)Crantz)—an underutilized member of the familyBrassicaceae—has been recently renewed due to theunique composition of fat in its seeds. The results ofarchaeological studies have shown that this plant hasbeen cultivated in Europe since the time of the BronzeAge [1], where it was probably used as an importantoilseed crop. It was abundant in Europe as a fieldweed, and until the middle of the 20th century cam�elina had been used in agriculture only on a smallscale, including in the Balkans and Ukraine. Nowcamelina is considered as one of the species towardswhich interest is being renewed as a potential alterna�tive to the existing oilseed crops. The oil content of itsseeds is about 38–43%, and the vast majority of fattyacids (>90%) are unsaturated acids, including a signif�icant amount of C20 : 0 eicosadienoic acid, which isrelatively rare in vegetable oils and linolenic (36.2–39.4%), oleic acid (12.8–14.7%), and linoleic (16.3–17.2%) and eicosenoic (14–15.5%) acids [2]. There�fore, camelina is a very promising crop for biodieselproduction [3].

Camelina has several other attractive features as apotential oilseed crop. In comparison with other cru�ciferous, the protein content of the residual mass of itsseeds contains very little glycosinolates [1, 4]. Thecontent of erucic acid is also very low (only 3%) [4];therefore, the plant has important potential for humanand animal nutrition, as well as for industry. For nutri�tional purposes, it can be used as food for animals(birds, cattle, and fish), as a fertilizer, or as a protein

fraction. The oil is used for biodiesel production; inthe food, cosmetics, and pharmaceutical industries; oras a lubricant.

During the years of trials conducted in NorthAmerica, it was established that the C. sativa produc�tivity is comparable with the yield of other oilseeds,including canola [5]. Camelina is a self�pollinatedcrop and largely unaffected by growing conditions. Incomparison with other members of Cruciferae, cam�elina has higher cold� and drought resistance, is notsusceptible to diseases, and is more resistant to thecrucifer, flea, beetle than any other species of Brassica;it is also resistant to the fungi Leptosphaeria maculansand Alternaria brassicae, causing the phoma and alter�naria blight of Cruciferae, respectively [6]. Camelinahas a short vegetative season and can grow in all typesof soil, except clayey soil, and the expenses for its cul�tivation are half the expenses required for growingcanola [7].

Due to these qualities, camelina has a considerablepotential for cultivation in Ukraine. The yield of thiscrop is about 30 dt/ha [8, 9]. Favorable conditions forC. sativa exist in practically all regions of Ukraine.Moreover, the development of biodiesel productionhas a great potential in areas contaminated with radi�onuclides produced by the disaster at the ChernobylNuclear Power Station due to the ability of Brassicacrops to purify soil from radionuclides without accu�mulating them in seeds.

Although earlier there was traditional small�scaleagricultural production of camelina in Ukraine, nowits commercial production is very limited, as well as

Establishment of In Vitro Culture, Plant Regeneration, and Genetic Transformation of Camelina sativa

A. I. Yemetsa, Yu. N. Boychuka, E. N. Shyshaa, D. B. Rakhmetovb, and Ya. B. Blumea

a Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, Kyivb Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, Kyiv

e�mail: [email protected] June 10, 2012

Abstract—The results of establishing an in vitro culture, plantlet regeneration, and rooting of Camelina sativacultivar Peremozhets and cultivar Mirazh are presented. The effective concentrations of sterilizing agents andthe duration of plant material treatment were estimated. The phytohormone ratio, the sucrose concentrationin the nutrient medium that induced the effective formation of C. sativa shoots, and the NAA concentrationfor plantlet rooting have been established. A method of Agrobacterium�mediated transformation of Camelinaby using binary vector pGH217 carrying the reporter β�glucoronidase (gus) gene driven under the 35S CaMVpromoter and nos�terminator, as well as the selective marker hpt gene conferring hygromycin�resistance intransgenic plant, was elaborated.

DOI: 10.3103/S0095452713030031

CYTOLOGY AND GENETICS Vol. 47 No. 3 2013

ESTABLISHMENT OF IN VITRO CULTURE 139

breeding programs for developing new high�yield vari�eties of crops. Nevertheless, the further improvementof the quality of camelina germ plasma to improveyields, increase the size of seeds and oil content, andimprove nutrient profiles is a very important issue. Asignificant contribution could be introduced bygenetic engineering, which requires the developmentof methods for handling this species culture in vitro.

In earlier studies, camelina protoplasts were used asa partner for fusion during somatic hybridization withother Brassica species, where the goal of studies wasthe improvement of these species [10–13]. However,until recently, only a few studies dedicated to theestablishment of an in vitro culture of camelina andplant regeneration have been published. A solution tothis problem was the subject of only one study [14].Recently, a method of embryogenesis from C. sativamicrospores [15] has been described and several tech�niques of camelina transformation have been devel�oped [16–18].

It should be noted that selection of culture condi�tions in vitro and an evaluation of the regenerativeability were performed for only a few genotypes ofcamelina which were cultivated in Northern Europe[14]. Therefore, the goal of this study was the selectionof optimal conditions for the establishment of in vitrocultures and plant regeneration from different types ofcamelina explants from Ukrainian varieties and thesubsequent development of an effective Agrobacte�rium�mediated transformation of the species.

MATERIALS AND METHODS

To establish an in vitro culture, seeds of C. sativacultivar Peremozhets and cultivar Mirazh produced atthe Department of New Cultivars, Gryshko NationalBotanical Garden, National Academy of Sciences ofUkraine, were used. The seeds were sterilized with70% ethanol for 1 min and then treated with sodiumhypochlorite at concentrations of 1 and 1.5% for 5–10 min [16]. After sterilization, the seeds were washedthree times with sterile distilled water for 10 min andplaced in Petri dishes on a half strength MS mediumwithout hormones [19] but containing MS vitamins,2% sucrose, and 0.8% agar (pH 5.7–5.8).

Cotyledons petioles and hypocotyl segments of 5�,7�, 9�, and 14�day�old camelina seedlings were used asexplants to evaluate the effectiveness of shoot regener�ation. Induction of shoots from explants was per�

formed using several variants of nutrient media, basedon the MS medium. The composition of the mediaonly differed by the content of phytohormones andsucrose. In particular, we examined the effect of sev�eral concentrations of benzylaminopurine (BAP) andseveral alternatives of BAP and naphthaleneaceticacid (NAA). The regenerative ability of C. sativa wastested using 12 media (table). Explants were incubatedat 22–24°C for a 16�h photoperiod.

Every three weeks, the material was transferred tofresh nutrient media. Induced shoots were separatedfrom the explants and transferred for further growthand rooting on MS medium or one of its variants con�taining 0.1, 0.5, or 1 mg/L NAA.

Agrobacterium tumefaciens strain LBA4404, con�taining a plasmid pGH217 with reporter gene β�glu�curonidase (gus) under the control of the 35S pro�moter of cauliflower mosaic virus (CaMV) and nos�terminator, and selective marker gene hpt, which pro�vides resistance to hygromycin in transformants, wasused in the genetic transformation experiments. Theplasmid was kindly provided by Dr. V. Radchuk (Insti�tute of Plant Genetics and Crop Plant Research,Gatersleben, Germany) (Fig. 1).

An overnight Agrobacterium culture was grown in20 mL of a liquid LB medium, containing 100 mg/L of

Table. Composition of nutrient media for the in vitro plant�let regeneration of Camelina sativa cultivar Peremozhetsand cultivar Mirazh

Types of nutritient

media

MS macrosalts, microsalts,

and vitamins

Sucrose, g/L

BAP NAA

mg/L

1 + 10 1 –

2 + 20 1 –

3 + 10 2 –

4 + 20 2 –

5 + 10 3 –

6 + 20 3 –

7 + 10 4 –

8 + 20 4 –

9 + 10 1 0.1

10 + 20 1 0.1

11 + 10 2 0.1

12 + 20 2 0.1

noshptP35SGUSP35S

RB LB

nos

Fig. 1. pGH217 construct: LB and RB, left and right T�DNA borders; P35S, 35S CaMV promoter; GUS, β�glucuronidase gene;nos, nopaline synthase terminator; and hpt, hygromycin resistance gene.

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kanamycin and 100 mg/L of rifampicin at 28°C and withconstant stirring on an orbital shaker. Then, the agrobac�teria were purified by centrifugation (4000 rev/min) for5 min. The supernatant was removed, the pellet beforeinoculation was diluted with liquid MS medium to anoptical density in the range from OD600 = 0.01 toOD600 = 0.5.

Petioles and hypocotyls of 5� or 7�day�old seed�lings were used as explants for transformation. Thecocultivation time of explants with Agrobacterium wasfrom 15 min to 1 h. After cocultivation, the explantswere blotted with sterile filter paper to remove anyexcess of Agrobacterium and transferred to an agarmedium for further cocultivation. After 2–3 days, thetransformed explants were placed for 7 days on MSmedia supplemed with phytohormones and 400 mg/Lof cefotaxime for the elimination of Agrobacterium andthen on similar media containing 5 mg/L of hygromy�cin for the selection of transgenic lines. Preliminaryexperiments to study the effect of different concentra�tions (1–10 mg/L) of this selective agent on the viabil�ity of explants were performed to choose an optimalselective hygromycin concentration.

For an histochemical assay of the expression of thegus (uid A) gene (β�glucuronidase gene) in plant tis�sues of C. sativa after Agrobacterium�mediated trans�formation, 5�bromo�4�chloro�3�indolyl glucuronide(X�Gluc) was used. A blue precipitate is formed as aresult of the reaction with this substrate in the places ofenzyme localization in transgenic cells [20].

RESULTS AND DISCUSSION

The optimal conditions for the establishment of anin vitro culture, in particular, the effective concentra�tions of the sterilizing agent and the treatment time ofC. sativa seeds, were established at the beginning of our

experiments. During studying the sterilization effec�tiveness of C. sativa cultivar Peremozhets and cultivarMirazh seeds, we established that, regardless of thetreatment time, with the use of 1% sodium hypochlo�rite contamination of the material was observed on thefifth day. Contamination was detected when seedswere treated with 1.5% hypochlorite for 3 min for cul�tivar Peremozhets and for 5 min for cultivar Mirazh.

An analysis of the effect of the sterilization condi�tions on seed germination revealed that the best optionfor cultivar Peremozhets is 5–7 min treatment with1.5% sodium hypochlorite. In this case, 100% germi�nation of in vitro seeds was observed after 5 min oftreatment and 97% germination was detected after 6–7 min of treatment. Thus, seeds of cultivar Mirazhmust be sterilized by 1.5% sodium hypochlorite for 6–7 min, since under these conditions contaminationwas not observed and the percentage of seed germina�tion was 100% (Fig. 2).

In studies of the regeneration of camelina shoots asthe best types of explants, for the initiation of organo�genesis in vitro, many species of Cruciferae family cot�yledons and hypocotyl explants were used [21, 22](Figs. 3b, 3c). In our experiment, 5�, 7�, 9�, and14�day�old seedlings of C. sativa are grown on themedia described in Materials and Methods, were usedto produce explants.

At present it is known that in several species of theCruciferae family, shoot formation occurs in the pres�ence of cytokinins, which initiates cell division anddifferentiation of shoots, and in the presence of differ�ent combinations of auxin and cytokinins [21–24].The most efficient regeneration of Camelina shootswas determined on media that contain cytokinins incombination with auxins [14].

Thus, to study the regeneration of shoots in culti�vars Peremozhets and Mirazh, we tested a series of

05 6 7 10 15 20

20

40

60

80

100 Peremozhets

Mirazh

Fig. 2. Efficiency of seed germination after treatment by 1.5% sodium hypochlorite solution: vertically, the number of germinatedseeds, %; horizontally, sterilization time, min.



BAP concentrations and combinations of BAP withNAA (table). It should be noted that the presence ofsucrose in the nutrient medium has some effect on theefficiency of callus formation and subsequent shootregeneration. As a result of our study, 12 types ofmedia, which were different by not only the composi�tion of growth regulators but also by the content ofsucrose, were tested. The plant material was trans�ferred every three weeks onto a fresh medium. Afterthree to four weeks of cultivation, the formation ofgreen or yellow–green callus with distinct points ofinitiation of future shoots on all types of media wasobserved on the petiole and hypocotyl segments of 5�,7�, and 9�day�old camelina seedlings. The formationof the callus structure occurred more slowly onexplants of 14�day�old seedlings, and formation ofbuds was not observed on any medium. It means thatyounger cells and tissues must be used for the regener�ation of camelina shoots in vitro. It should be notedthat the efficiency of shoot regeneration on mediasupplemented with BAP and NAA was higher than onmedia containing only BAP. This trend can be foundfor all types of explants derived from 5�, 7�, and 9�day�old seedlings. The highest numbers of shoots wereinduced from petiole and hypocotyl explants of 5� and7�day�old seedlings (Figs. 4, 5).

It was also found that when the sucrose content ina medium was 10 g/L callus formation occurred lessintensely than at 20 g/L; however, this did not signifi�cantly affect the formation of shoots. Thus, in our

experiments, the influence of several concentrations ofphytohormones BAP, the ratio of several concentrationvariants of phytohormones BAP and NAA, and theeffect of two concentrations of sucrose (10 and 20 g/L)on the formation of shoots from petiole and hypocotylsegments of 5�, 7�, 9�, and 14�day�old seedlings wasstudied. Figure 3d shows shoots formed on cotyledonpetiole sections, and Fig. 3e shows shoots formed onthe segment of the hypocotyl.

The regeneration of cruciferous plants in vitro isinfluenced by cytokinins and their combinations withauxins; the rooting of regeneratesd plants is obtainedby the addition of auxin to the medium [25]. Based onthis, the next step of our study was examining theeffect of auxin, in particular, a range of NAA concen�trations, on the process of rhizogenesis of shoots. Ashoot formed and separated from the explant is shownin Fig. 3f; it should be noted that before the studyregenerated shoots (Figs. 3d–3f) were planted on MSand 1/2 MS media; however, the formation of rootsdid not occur. When NAA (0.1, 0.5, and 1 mg/mL)was added to the MS medium, initiation of roots wasonly observed at this phytohormone concentration of1 mg/mL.

Thus, we demonstrated that 5� or 7�day�old seed�lings are best to be used for the induction of shoots ofC. sativa, since the efficiency of regeneration on theirexplants is higher than that of 9�day�old seedlings. Asfor the type of explants, both petiole and hypocotylsegments can be used for the regeneration of shoots.

(a) (b) (c)

(d) (e) (f)

Fig. 3. Shoot regeneration in vitro from C. sativa plant cultivar Peremozhets: (a) 9�day�old camelina seedlings, (b) cotyledonexplants, (c) hypocotyl explants, (d) formed shoots on the cut of cotyledon petiole, (e) plantlet formation on the hypocotylexplant, and (f) shoot separated from the explant. Scale: (a–c) 16, (d) 4, (e) 11, and (f) 7 mm.

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The efficiency of plant regeneration on petiole andhypocotyl explants of cultivar Peremozhets wasslightly higher than that of cultivar Mirazh on severalmedium types (Figs. 4, 5). However, for the regenera�tion of shoots and for rooting, MS media with BAP(4 mg/mL) and NAA (0.1 mg/mL) or with 1 mg/mLof NAA, were selected as the most effective ones.

In addition, we developed an Agrobacterum�medi�ated transformation method of C. sativa. For this pur�pose, a vector construct—pGH217—containing thereporter gene β�glucuronidase (gus) under the controlof the CaMV 35S promoter and the nos�terminatorand selective marker gene hpt, providing resistance tohygromycin in the selected transgenic plants. This vec�tor with the gus gene allows for the quick (within 1 day)visualization of the expression of the transferred for�eign gene in plant cells by the presence of a blue colorin the tissues of transgenic lines [20].

Since petiole and hypocotyls of 5� or 7�day�oldseedlings of C. sativa were used as explants for trans�formation, the study of the effect of hygromycin ontheir survival dedicated to the determination of thehygromycin effective concentration for the selectionof transformants was performed. Hygromycin was

tested at concentrations ranging from 1 to 10 mg/L bythe addition of hygromycin to the shoot regenerationmedium, and after 30 days the survival and regenera�tion rate of tissue explants was estimated. We estab�lished that the most effective hygromycin concentra�tion for selection is 5 mg/L, which is consistent withthe data described previously in [16].

After cocultivation with A. tumefaciens, theexplants were first transferred to MS media containingphytohormones and 400 mg/L cefotaxime for theelimination of bacteria, and then they were againtransferred to the same media with the addition of5 mg/L hygromycin for the selection of transgeniclines (Figs. 6a–6c). The first test of the transformedexplants for GUS�expression was performed threedays after they were transferred to the selectivemedium. The results of the GUS�test are shown inFigs. 6d and 6e. On the selective media, 2–3 weeksafter transformation, callus formation was observed onthe edges of some petiole sections and hypocotyl seg�ments which gave rise to shoots (Fig. 6b). After 2–3 months, on the selective medium, first transgenicC. sativa plants were selected. By histochemical anal�ysis of callus and leaves of the regenerated shoots,

01 2 3 4 5 6 7 8 9 10 11 12

1

2

3

0

1

2

4

5

3

6

7

0

1

2

4

5

3

6

7 (a)

(b)

(c)

Peremozhets

Mirazh

Fig. 4. Efficiency of shoot regeneration on stem sections ofcotyledons of camelina seedlings on various types of media:(a) 5�, (b) 7�, and (c) 9�day�old seedlings; vertically, thenumber of shoots, pcs; horizontally, medium type (table).

01 2 3 4 5 6 7 8 9 10 11 12

1

2

3

0

1

2

4

5

3

0

1

2

4

5

3

(a)

(b)

(c)

Peremozhets

Mirazh

Fig. 5. Efficiency of shoot regeneration from hypocotylexplants of camelina seedlings on various types of media:(a) 5�, (b) 7�, and (c) 9�day�old seedlings; vertically, thenumber of shoots, pcs; horizontally, medium type (table).



expression of the transferred gus gene was detected,confirming the transgenic nature of the obtained lines.

Thus, the developed method of establishing an invitro culture and plantlet regeneration of C. sativa, aswell as Agrobacterium�mediated transformation, canserve as a basis for further biotechnological improve�ment of this species, leading to the improvement of thequality of biodiesel produced from it.

ACKNOWLEDGMENTS

This work was financially supported by the project“Establishment of in Vitro Culture and GeneticTransformation of Camelina in Order to Improve ItsCharacteristics for the Production of Biodiesel” of theResearch Program of the National Academy of Sci�ences of Ukraine “Biomass as Fuel Raw Material”(Biofuels).

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(a) (b) (c)

(d) (e) (f) (g)

Fig. 6. Selection and analysis of transgenic C. sativa: (a) selection of plants on a medium with 5 mg/L hygromycin; (b) formationof callus with regeneration�like structures under the conditions of selective pressure; and (c) a transgenic plant selected on a selec�tive medium. The results of GUS�expression in cells of hypocotyl explants (d) and cotyledons (e) 3 days after transformation, inthe callus formed after 3 weeks on the selective medium (f), and the leaves of regenerated C. sativa plants (g). Scale: (a) 12.8, (b)8, (c) 5, (d) 4, (e) 2.5, (f) 2.3, and (g) 2.1 mm.

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Translated by V. Mittova

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Establishment of in vitro culture, plant regeneration, and genetic transformation of Camelina sativa