AsPac J. Mol. Biol. of Molecular Biology and Biotechnology, 2008
Asia Pacific Journal Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato 25
Vol. 16 (2) : 25-33
Efficient regeneration and Agrobacterium tumefaciens mediated
transformation of recalcitrant
sweet potato (Ipomoea batatas L.) cultivars.
Rolando García González1,2*, Danalay Somonte Sánchez2, Zurima Zaldúa Guerra1,
Jesús Mena Campos2, Alina López Quesada2, Rolando Morán Valdivia2,
Ariel D. Arencibia2, Karla Quiroz Bravo3 and Peter DS. Caligari3
Departamento de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Chile.
Avenida San Miguel, No.3605.
Centro de Ingeniería Genética y Biotecnología de Camagüey. A.P. 387, C.P. 70100. Camagüey, Cuba.
Instituto de Biología Vegetal y Biotecnología, Universidad de Talca. 2 Norte 685, Talca, Chile.
Received 20 June 2007 / Accepted 15 May 2008
Abstract. Sweet potato is a major world crop and its behavior under in vitro culture is genotype dependent. We studied several
factors influencing the regeneration and transformation efficiency of two recalcitrant cultivars: Jewel and CEMSA 78354. Growth
regulators, explant preparation and removal of apical dominance were evaluated in order to optimize the regeneration steps. At
the same time, the influence of environmental conditions for the interaction between Agrobacterium tumefaciens and leaves were
evaluated to obtain higher transformation efficiencies.
For Jewel, the best results were obtained when intact leaf explants were cultivated on MS medium supplemented
with 0.5 mgL-1 indol-3-acetic acid for four weeks (Regeneration frequency, RF= 2.02). However, for CEMSA 78354 the best
results were obtained by culturing leaf explants on MS medium supplemented with 1.0 mgL-1 paclobutrazol and 1.0 mgL-1
naftalenacetic acid (RF= 0.98).
Optimal transformation conditions were obtained for both cultivars by co-cultivating leaf explants with Agrobacterium tumefaciens
in liquid MS medium for 24 hours at 28ºC in stationary cultures in the dark. Acetosyringone influence on the transformation
efficiency was found to be dependent on the co-cultivation temperature but it did not increase the transformation efficiencies.
Molecular evidences by PCR, Southern blot and Dot blot demonstrated the effectiveness of the transformation procedure. The
protocol described here is currently in use to produce transgenic sweet potatoes from different cultivars with high efficiency.
Keywords. Ipomoea batatas; organogenesis; Paclobutrazol; Agrobacterium tumefaciens mediated transformation;.
INTRODUCTION isolation and regeneration (Sihachakr, 1987), and plant regen-
eration from roots, petioles, stems and leaves (Gosukonda
Sweet potato (Ipomoea batatas Lam.) is a perennial, herba- et al., 1995). A relatively high success in developing an effi-
ceous, dicotyledonous species of the morning glory family cient system of embryogenic suspension cultures for a wide
Convolvulaceae that is grown as an annual crop in many range of sweet potato genotypes especially for commercial
countries. It is one of the most versatile, and yet under-ex- cultivars was obtained but the methods was time consuming
ploited, crop species in the world, its current annual produc- and quite difficult to establish (Liu et al., 2001). Sweet potato
tion is estimated at 133 million tons, mainly growing in 100 has been transformed using different systems including
developing countries and being placed among the five most microprojectile bombardment (Prakash and Varadarajan,
important food crops in over 50 of these. Despite its major 1992) electroporation of protoplasts (Garcia et al., 1998),
importance sweet potato has not received significant attention Agrobacterium rhizogenes (Otani et al., 1993) and Agrobacterium
from plant biotechnologists (Midmore et al., 2003). tumefaciens (Otani et al., 2001). In general, the results on sweet
In recent years, only a few groups have reported molecular potato genetic transformation have shown low efficiency of
biology manipulation results in terms of increasing its nutri- a high genotype dependent response (Otani et al., 2003; Song
tional quality (López et al., 1996) and to give pest resistance et al., 2004; Shimada et al., 2006; Luo et al., 2006).
(Newel et al., 1995). Other groups have reported results on Sweet potato is considered a highly recalcitrant species in
sweet potato conservation (Blakesley et al., 1996), protoplast *Author for Correspondence.
Abbreviations: PBZ, Paclobutrazol; BAP, 6- benzilaminopurine; IAA, Mailing address: Departamento de Ciencias Agrarias y Forestales, Uni-
Indol-3-acetic acid; NAA, Naftalenacetic acid; Zea, Zeatin; Btt, Bacillus versidad Católica del Maule, Chile. Avenida San Miguel, No.3605. Talca.
thuringiensis tenebrionis. E-mail: email@example.com
26 AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato
terms of regeneration and transformation because the above regeneration media. It was calculated as:
mentioned cultivar responses to in vitro treatments (Aloufa, RF= (RS/TNE).
2002) and this has limited the applications of genetic engi-
neering technologies (Newel et al., 1995; Gosukonda et al., Where: RS, is the total number of individual rooted shoots
1995). It also shows a high genetic diversity (Wang et al., 1998) and TNE, is the total number of explants.
and its major anthocyanins and polyphenolic compounds are Statistic analyses were performed to test the differ-
genotype dependent (Islam et al., 2002) which could explains, ences between treatments using ANOVA and LSD Test
along with other factors, its genotype dependent responses at p<0.05.
to in vitro manipulation.
Here, is reported an efficient methodology for sweet Effect of Plant Growth Regulators (PGRs) on organogenic
potato regeneration and transformation. Two very impor- regeneration. The effect of auxins and cytokinins supplied
tant cultivars, which are both considered as very recalcitrant in the medium both, combined or alone, were evaluated for
to be in vitro manipulated were studied, namely: Jewel and the induction of organogenic regeneration. 48 growth regu-
CEMSA 78354. This research was done in two steps. In the lator combinations were evaluated to ascertain the optimum
first step, different growth regulator relationships and four response. Naftalenacetic acid (NAA) was combined with
procedures for leaves explant manipulation were studied in 6-Benzilaminopurine (BAP) and Zeatin (Zea), while Indol-3-
order to establish an efficient medium for shoot induction. acetic (IAA) was combined with the same cytokinins. PGRs
In the second step factors that affect Agrobacterium tumefaciens concentrations employed in the experiment were:
mediated transformation, such as induction of vir genes, time
Naftalenacetic acid (NAA): 0.25 mgL-1; 0.50 mgL-1; 0.75 mgL-1.
of co-cultivation, temperature and co-culture conditions, Indol-3-acetic (IAA): 0.25 mgL-1; 0.50 mgL-1; 0.75 mgL-1.
were tested. 6-Benzilaminopurine (BAP): 0.25 mgL-1; 0.50 mgL-1; 0.75 mgL-1.
Zeatin (Zea): 0.25 mgL-1; 0.50 mgL-1; 0.75 mgL-1.
Effect of explant preparation on the organogenic response of
MATERIALS AND METHODS sweet potato leaves. Leaf explants from Jewel and CEMSA
78345 cultivars grown during four weeks on propagation
Plant material. Sweet potato plants of Jewel and CEMSA medium were prepared following four different treatments:
78354 cultivars were kindly donated from the collection CL: Leaves with four cut edges but without cutting into the
of the Instituto Nacional de Investigaciones en Viandas Tropicales lamina; ML: Leaves cut on their basal and apex ends but
(INIVIT) from Santo Domingo, Cuba. Plants were propa- without cutting into the lamina; LL: Leaves cut on their lateral
gated in glass tubes containing 7 ml of MS salts and vitamins edges but without cutting into the lamina; WL: Whole leaves
(Murashige and Skoog, 1962) of solid medium supplemented without any cut edges.
with 3% sucrose, 0.5 mgL-1 IAA and 6 gL-1 agar-agar (Difco),
with the pH adjusted to 5.6-5.7 prior to autoclaving. For all Effect of Paclobutrazol (PBZ) on sweet potato regeneration.
the regeneration and transformation experiments, explant The addition of PBZ to the best regeneration media for
donor plants were grown in the propagation medium for Jewel cv. and CEMSA 78354 cv. to increase the regeneration
four weeks. frequencies was evaluated. For Jewel cv., Paclobutrazol (0.75
mgL-1; 1.0 mgL-1; 1.5 mgL-1, 2.0 mgL-1) was combined with
Growth regulator studies and explants manipulation for IAA (0.5 mgL-1). For CEMSA 78354 cv., PBZ at the same
organogenic regeneration. MS basal salt mixture with vita- concentrations was combined with NAA (1.0 mgL-1).
mins, supplemented with sucrose 3% and solidified with
6.0 gL-1 agar-agar (Difco), was used for all the experiments. Agrobacterium tumefaciens mediated transformation experi-
The pH was adjusted to 5.6-5.7 before autoclaving (20 min; ments. In all the transformation experiments, the Agrobacte-
121 ºC; 138 kPa), and all growth regulators were added prior rium tumefaciens C58C1-pGV2260 (Strr; Spcr; Rifr; Ampr) strain
autoclaving except zeatin riboside, which was filter-sterilized was employed. It contains the binary plasmid pDEBtt, a
and added to the cooled medium. All experiments were set derivative from pDE1001, harboring the Bacillus thuringiensis
up in five repetitions for each growth regulator combination. subsp. tenebrionis (Btt) δ-endotoxin gene under the regulation
Each repetition was composed of six leaves. Leaf explants of the 35S promoter from CaMV and the tnos terminator, as
were incubated at 25 ºC and in a 12/12 h photoperiod. Leaf well as the nptII gene as selectable. Agrobacterium tumefaciens
explants were prepared by removing the four ends of the was kept before use on selective solid YEB medium supple-
lamina, and then cultivated with the axial side of the leaf mented with rifampycin (100 mgL-1), spectinomycin (100
expanded and fully in contact with the medium. All the mgL-1) and streptomycin (100 mgL-1). For transformation,
regeneration experiments were evaluated four weeks after Agrobacterium tumefaciens isolated colonies were inoculated into
planting in the tested media. a 5 ml miniculture of YEB liquid selective medium, as above,
Regeneration Frequency (RF) was used to choose the best for 24 hours, at 28 ºC and 100 r.p.m. At the time, 50 μl were
AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato 27
taken from the initial miniculture and were inoculated into 50 ers for amplification were designed considering the 5´ and 3´
ml of selective YEB medium for transformation and grown ends of the δ-endotoxin gene from Bacillus thuringiensis subsp.
for 24 hours, at 28 ºC and 100 r.p.m on a shaker. tenebrionis as: Primer 1: 5´-ATGAATCCGAACAATCGAAG-
3´; Primer 2: 5´-ATTCACTGGAATAAATTC-3´.
Agrobacterium tumefaciens mediated transformation condi- The cry3A gene from Bacillus thuringiensis subsp. tenebrionis
tions. The influence of previously culturing leaf explants (1.9 kb) labeled with P32 was used as a probe for blotting the
in MS liquid medium (0 hour; 12 hours; 24 hours) combined PCR products (Morán et al., 1998).
with different transformation periods (16 hours, 24 hours;
48 hours) in MS liquid medium was evaluated. For the trans- Dot blotting of transgenic plants. RNA was isolated from
formation step, 5 ml (3 x 106 CFUml-1) of the Agrobacterium transgenic sweet potato leaves (1 g of fresh tissues) by a modi-
tumefaciens culture were inoculated into 45 ml of MS liquid fied CTAB protocol (Kim et al., 2000). Dot blot (Sambrook
transformation medium. Ten explants were used per repeti- et al., 1989) analysis to detect transgenic mRNA was done on
tion and each treatment had six repetitions. total RNA at two concentrations (10 μg and 20 μg) isolated
from PCR positive transgenic plants. Labeled cry3A gene was
Effects of environmental factors and acetosyringone on used as a probe to detect transcripts from the recombinant
Agrobacterium tumefaciens mediated transformation ef- Btt gene in transgenic sweet potato plants.
ficiency. The influence of co-culturing leaf explants with
Agrobacterium tumefaciens under dark or light conditions Southern blot analysis. 40 µg of total DNA from transgenic
(110 mEm-2s-1) on the transformation efficiency was evalu- plants were digested with KpnI and EcoRV. KpnI cuts out
ated along with co-cultivating in stationary or slow agitation the 5´ end (+5) of the Bacillus thuringiensis subsp. tenebrionis
(25 r.p.m.). The effect of temperature and vir gene inducers δ-endotoxin gene in the construct while EcoRV cuts inside the
on the transformation efficiency were also analyzed by co- toxin gene (+619) giving two fragments of the gene of 619
cultivating leaf explants with Agrobacterium tumefaciens at 25ºC bp and 1281 bp. The enzymes cuts neither anywhere in the
or 28ºC with different concentrations of acetosyringone rest of the construct or in the left or right T-DNA borders.
(0 mgL-1; 2 mgL-1; 4 mgL-1). For these experiments, 5 ml The Btt δ-endotoxin gene labeled with P32 was used as probe
(3 x 106 CFUml-1) of an Agrobacterium tumefaciens suspension to detect the integration pattern for sweet potato transgenic
were inoculated into 45 ml of MS liquid medium. lines. Hybridization was performed according to previous
Agrobacterium tumefaciens was eliminated from the infected reports (Morán et al., 1998).
tissues by washing three times the leaf explants with distilled
sterile water followed by washing them for 15 minutes in MS
liquid medium supplemented with cefotaxime (Roussel) at
500 mgL-1. Subsequently the explants were planted on their RESULTS AND DISCUSSIONS
respective regeneration medium.
Organogenic regeneration from leaf explants.
Selection of the transgenic shoots and evaluation criteria. All Effect of growth regulators on organogenic regeneration.
the regeneration media were supplemented with 50 mgL-1 of Leaves from Jewel cv. and CEMSA 78354 cv. had the high-
kanamycin, as selectable markers. Two controls were included est frequency of regeneration when they were cultivated on
in the study: un-transformed explants were planted both on basal MS medium supplemented with auxins alone in the
regeneration medium supplemented with kanamycin and on media. As shown in Table 1, leaves from Jewel showed best
kanamycin free regeneration medium. All the transformation results in the treatment supplemented with IAA 0.5 mgL-1
experiments were evaluated six weeks after planting on the (RF= 1.06; LSD, p<0.05). For CEMSA 78354 cv., the best
selective regeneration medium. results were achieved by cultivating leaf explants in basal
To select the best regeneration conditions, the Effective medium supplemented with NAA 1.0 mgL-1 (RF= 0.66;
Transformation Frequency (ETF= KRS/TNE), where KRS LSD, p<0.05).
is the number of kanamycin resistant shoots already individu- CEMSA 78354 cv. gave half of the Regeneration Fre-
alized and regenerated in selectable propagation medium and quency as compared to Jewel cv., confirming that regeneration
TNE is the total number of transformed explants. All the response of sweet potato is strongly genotype dependent
transformation experiments were randomly designed with (Aloufa, 2002). However, relatively, root and callus initiation
6 repetitions per treatment and 10 explants per repetition. was induced for CEMSA 78354 cv. and NAA had a stronger
effect on shoot regeneration instead IAA as is shown in
Polymerase chain reaction and Southern blot of the PCR Table 1. Organogenic formation of shoots and roots was
products. PCR and Southern blot to the PCR products were observed arising from the cut edge of the petiole (2-3 mm)
performed on 69 and 55 kanamycin resistant clones both still remaining on the leaf lamina. Organogenic roots were
from Jewel cv. and CEMSA 78354 cv. respectively, according able to regenerate shoots de novo on the regeneration medium
to the procedure described by (Goyenechea et al., 1991). Prim- after four weeks of culture and this increased the regeneration
28 AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato
Table 1. Summary of the regeneration media that induced the
highest Regeneration Frequencies in sweet two potato cultivars,
Jewel and CEMSA 78354. RF: Regeneration frequency, p<0.05.
Calli formation is expressed in milligrams of fresh weight. Evalu-
ation was done four weeks after planting on each regeneration
Jewel CEMSA 78354
RF Calli (mg) RF Calli (mg)
MS+IAA (0.5 mgL-1) 1.06 ± 0.09 10 ± 0.81 0.27 ± 0.09 25 ± 0
MS+IAA (0.75 mgL-1) 0.66 ± 0.05 13 ± 0.81 0.25 ± 0 25 ± 1.41
MS+IAA (1.0 mgL-1) 0.25 ± 0 25 ± 0.82 0.25 ± 0 44.3 ± 4.18
Figure 1. Effect of PBZ treatments on sweet potato regeneration
MS+NAA (0.5 mgL-1) 0.6 ± 0.08 15 ± 0 0.25 ± 0.04 25 ± 0.87
efficiency. RM (Regeneration medium) for: Jewel cv., MS+IAA (0.5
MS +NAA (0.75 mgL-1) 0.25 ± 0 24.3 ± 1.25 0.25 ± 0.04 23 ± 0
mgL-1); CEMSA 78354 cv., MS+NAA (1.0 mgL-1). Regeneration
MS+NAA (1.0 mgL-1) 0.25 ± 0 24.3 ± 1.25 0.66 ± 0.03 30.3 ± 1.88
medium for each cultivar supplemented with Paclobutrazol at the
MS+BAP (0.5 mgL-1) 0.25 ± 0 26 ± 0.87 0.1 ± 0 45 ± 4.08
following concentrations: RM+1, RM+PBZ (0.75 mgL-1); RM+2,
MS+BAP (0.75 mgL-1) 0.1 ± 0.08 75 ± 7.07 0 94 ± 3.26
RM+PBZ (1.0 mgL-1); RM+3, RM+PBZ (1.5 mgL-1); RM+4,
MS+BAP (1.0 mgL-1) 0.1 ± 0 77 ± 2.16 0 94 ± 0.81
RM+PBZ (2.0 mgL-1). For both cultivars, PBZ-1 to PBZ-4 are
MS+Zea (0.22 mgL-1) 0.4 ± 0 12.3 ± 1.24 0.25 ± 0.20 12 ± 0
MS basal medium supplemented only with PBZ: PBZ-1, MS+PBZ
MS+Zea (0.5 mgL-1) 0.25 ± 0.20 25 ± 0.87 0.25 30.3 ± 1.7
MS+Zea (0.75 mgL-1) 0.33 ± 0.12 25 ± 4.08 0 30 ± 0.82 (0.75 mgL-1); PBZ-2, MS+PBZ (1.0 mgL-1); PBZ-3, MS+PBZ (1.5
mgL-1); PBZ-4, MS+PBZ (2.0 mgL-1). RF: Regeneration frequency.
n=30; p<0.05. Evaluation was done four weeks after planting on
each regeneration medium.
efficiency, giving a higher number of individualized shoots.
Regeneration of sweet potato has been reported previously
using combined treatments of auxins and cytokinins (Gosu- ent-kaurene, a giberellic acid precursor, increases sucrose and
konda et al., 1995; Al-Jubory and Skirvin, 1991). However, starch concentrations in plant tissues (Maki et al., 2005) and
none of the previous work documented plant regeneration reduces shoot tip dominance both by diminishing endog-
by using auxins treatments with no cytokinins. Probably, enous levels of giberellic acid and by interfering with endog-
endogenous concentrations of auxins and cytokinins are enous interactions between auxins and citoquinins (Passian
also strongly genotype dependent in sweet potato and should and Bennett, 2001; Kongbangkerd and Wawrosch, 2003).
be taken into consideration to establish any regeneration
protocol. Effect of explant preparation on the organogenic response of
sweet potato leaves. Leaf explants prepared as the whole
Effect of PBZ on the regeneration efficiency. To reduce lamina without any cuts gave the best regeneration frequen-
shoot dominance PBZ was included in the best regeneration cies (Figure 2), both for Jewel cv. (RF= 1.9; LSD, p<0.05)
medium for both cultivars. PBZ increased the regeneration regenerated on basal MS basal medium supplemented with
efficiency for CEMSA 78354 cv., but this effect did not IAA (0.5 mgL-1) and CEMSA 78354 cv. (RF= 0.85; LSD,
increase the regeneration efficiency for Jewel cv. (Figure 1). p<0.05), regenerated on MS basal medium supplemented
On the other hand, when PBZ was added to the media at a with PBZ (1.0 mgL-1) and NAA (1.0 mgL-1). Management
level of over 1.0 mgL-1 it induced high frequency of callus of intact leaf explants also homogenized shoot production
formation instead of roots or shoots. For CEMSA 78354 cv. since 90% of leaf explants in Jewel cv. and 60% in CEMSA
the best regeneration frequencies were obtained by supple- 78354 cv. produced at least one organogenic shoot.
menting the MS basal medium with 1.0 mgL-1 of NAA and Injured plant tissues produce calli as a defensive response
1.0 mgL-1 of PBZ (LSD, p<0.05) (RM+2 medium). Hyper- under in vitro culture (Tisserat, 1985) and it could be a critical
hidricity of the explants was obtained for PBZ concentrations factor in the reduced shoot differentiation in sweet potato
over 1.5 mgL-1 in the medium. High regeneration frequencies cut leaves. Wound responses can be mediated by endogenous
were obtained by combining thydiazuron and naftalenacetic auxin production by the tissues, this endogenous production
acid with PBZ in beans, giving short plants with reduced could interact with those auxin concentrations supplied in the
leaf area (Veltcheva and Svetleva, 2005). In our work, the medium and could also reduce the organogenic responses
isolated effect of PBZ in the media, with no other growth of the tissues.
regulators (PBZ-1, PBZ-2, PBZ-3, PBZ-4), did not produce
efficient regeneration responses in sweet potato as shown in Agrobacterium tumefaciens mediated transformation
the Figure 1, but it did induce shoot and root regeneration of leaf explants.
as well as callus formation. Agrobacterium tumefaciens mediated transformation
The above described results can be explained because conditions. Explant pre-culture in liquid medium before
PBZ inhibits oxidative steps in the biosynthesis pathway of transformation was shown to be genotype dependent and
AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato 29
to depend on the co-culture time of the leaf explant and
Agrobacterium tumefaciens. For Jewel cv., there were not statis-
tical differences (LSD, p<0.05) between giving a pre-treat-
ment or not for transformation times different to 24 hours
of co-culture. However, co-culture time seemed to be more
important in this cultivar since the highest Transformation
Frequencies were obtained with 24 hours of co-culture in
liquid medium, either with a previous pre-culture of the
explants (ETF=0.8; LSD, p<0.05) or not (ETF=0.83; LSD,
p<0.05), as shown in Figure 3. Figure 2. Effects of leaves preparations on sweet potato regenera-
On the contrary, the best Transformation frequencies tion efficiencies for Jewel cv. and CEMSA 78354 cv. WL: Leaves
for CEMSA 78354 cv. were obtained by co-cultivating the without any cut on its lamina. ML: Leaves cut from half lamina to
explants with Agrobacterium tumefaciens for 24 hours without the tip. LL: Leaves cut on their lateral ends. CL, leaves prepared as
any pretreatments (ETF=0.57; LSD, p<0.05). CEMSA 78354 a square, cut all around their edges. RF: Regeneration frequency.
cv. is probably adversely susceptible to cultivation in liquid n=30; p<0.05. Evaluation was done four weeks after planting on
medium and it could affect the regeneration capability after the regeneration medium.
transformation since the lack of regeneration for the pre-
cultured explant was associated with higher explant death in
those explants (data no shown).
Co-cultivation times longer than 24 hours caused hydrata-
tion and death of leaf tissues following an early yellowing
and browning of the tissues. This explant response is prob-
ably not associated with kanamycin toxicity because, in the
untransformed leaf controls, the toxic effect of antibiotics
is expressed from the seventh day of culture on selective
regeneration medium and starts as brown zones around the
leaf tips which spread progressively to the central zone of the
leaf lamina. Transformation periods less than 24 hours gave
the lowest Regeneration frequencies in Jewel cv. (ETF=0.13,
LSD, p<0.05), probably due to the short time for interactions
between plant tissues and Agrobacterium tumefaciens. Figure 3. Effective transformation efficiency (ETF) for leaves of
two sweet potato cultivars transformed during different preculture
Effects of environmental factors and acetosyringone on Agro- and co-culture conditions. X-axis represents the combination of
bacterium tumefaciens mediated transformation efficiency. co-cultivation period (hours) and preculture treatment in liquid
medium (hours) for leaves. Co-culture time/Preculture time in
Environmental conditions for co-cultivation also influenced
liquid medium. n=90; p<0.05. Evaluation was done six weeks after
transformation efficiencies for Jewel cv. and CEMSA 78354 planting on the regeneration medium supplemented with kanamycin
cv. (Figure 4). Optimal results were obtained when leaf ex- as selectable.
plants from Jewel cv. (ETF= 0.73, LSD, p<0.05) and CEMSA
78354 cv. (ETF= 0.53, LSD, p<0.05) were cultivated in the
dark without agitation. Transformation under dark conditions
has been reported for several species (Liu et al., 2004) and the Effective Transformation Frequency of 0.8 (LSD, p<0.05).
higher transformation efficiencies could be related to higher According to the results shown (Figure 5), transformation
stability of the vir protein inducers which can be accumu- efficiency increased from 22 ºC to 28 ºC for both cultivars
lated and could cause a stronger virulence of Agrobacterium but at 30 ºC it was strongly reduced in line with previous
tumefaciens. Transformation under slow agitation produced work suggesting that T-DNA transfer machinery works more
higher Agrobacterium cell densities and more homogeneous efficiently under temperatures below 28 ºC (Fava Ditt et al.,
suspension in the co-cultivation medium than stationary 2005). T- Pili formation to attach Agrobacterium cells to the
co-cultivation, but Agrobacterium colonies attached to the plant cell for T-DNA transmembrane transport is reduced
leave tissues appeared to be less developed. Thus, colonies at temperatures over 28 ºC (Lai et al., 2000). However, it has
were more strongly attached to the explant in stationary co- also been recognized that a number of virB proteins shows
cultivated leaves. important expression patterns without loosing biological
It was observed that sweet potato transformation be- activity at 28 ºC (Jakubowski and Krishnamoorthy, 2003)
haved better at 28ºC without the addition of acetosyringone, and they could play a key role on oncogenesis.
CEMSA 78354 cv. showed an Effective Transformation On the other hand, the transformation efficiency must
Frequency of 0.66 (LSD, p<0.05) while Jewel cv. reached an be considered based on the complex interactions between
30 AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato
The influence of acetosyringone on the transformation
efficiency was found to be dependent on the temperature
for both cultivars (LSD, p<0.05). Leaf explants transformed
at 28 ºC without acetosyringone produced higher ETF
(Figure 6) than those transformed with 2 mgL-1 or 4 mgL-1
of acetosyringone (LSD, p<0.05). However, no differences
were found for explants transformed with or without ace-
tosyringone at 22 ºC while explants transformed at 25 ºC
gave higher ETF when they were transformed with 4 mgL-1
of acetosyringone (LSD, p<0.05). Probably, acetosyringone
addition is not necessary for sweet potato at high transfor-
mation temperatures, at least under these transformation
Figure 4. Influence of darkness/light and agitation/stationary conditions. Sweet potato leaves produced a large variety and
co-culture on the transformation efficiency for sweet potato Jewel higher amounts of phenolyc compounds than other tissues
and CEMSA 78354 cultivars. Stat: Stationary co-culture; Agit: Co- (Okamoto et al., 2000; Huang et al., 2004) and that basal level
culture under agitation (25 rpm). ETF: Effective transformation of phenolyc compounds could be enough to induce the
frequency. n=90; p<0.05. Evaluation was done six weeks after
T-DNA transfer mechanism.
planting on the regeneration medium supplemented with kanamycin
as selectable. Use of acetosyringone in sweet potato transformation
protocols seems to be dependent on the genotype being
transformed. The optimal concentration of AS determined
by Newell et al. (1995) (39.24 mgL-1 AS), Otani et al. (1998)
(10 mgL-1 AS) were different. For instance, it is always recom-
mended to take into consideration the chosen cultivar and its
own phenolyc composition to succeed on the transformation
steps instead replicating any previous experience.
Molecular evaluation of Kmr sweet potato plants. Kanamycin
selection both on the regeneration and micropropagation
steps was efficient for Jewel cv. and CEMSA 78354 cv.
Untransformed control did not regenerated any shoots for
both cultivars when they were cultivated on regeneration
medium supplemented with kanamycin at 50 mgL-1, nor
Figure 5. Effect of temperature and acetosyringone on the ef-
individual untransformed plants grow or form roots on se-
ficiency of sweet potato leaves transformation by Agrobacterium tu-
mefaciens transformation. Temperature (ºC)/acetosyringone (mgL-1). lectable propagation medium supplemented with kanamycin
ETF: Effective transformation efficiency. n=90, (p<0.05). Evalua- at 25 mgL-1. For PCR detection of the cry3A gene, 69 plants
tion was done six weeks after planting on the regeneration medium from Jewel cv. and 55 plants from CEMSA 78354 cv. gave
supplemented with kanamycin as selectable. 90 transformed leaf explants which were evaluated, giving
68 and 53 PCR positive plants, respectively. Southern blot
hybridization of the cry3A PCR products confirmed the
amplification of the foreign gene from sweet potato DNA
(Figure 6). The untransformed control did not show bands
Agrobacterium tumefaciens and the plant tissue. Firstly, Agrobac- by PCR or Southern blot hybridization.
terium tumefaciens, reach higher cell densities when it is grown Transcript cry3A mRNA was detected in leaves tissues
at 28 ºC (Birch, 1997) and a higher number of bacterial cells for two selected PCR positive plants of Jewel cv. (C-27) and
must influenced on the plant-bacterial interactions. Secondly, CEMSA 78354 cv. (C-1) by Dot blot (Figure 7A). RNA from
phenolyc compounds from wounded tissues as defensive untransformed sweet potato plant Jewel cv. and CEMSA
response could be also higher at temperatures over 25ºC 78354 cv. as negative controls, did not show transcript signal.
(Birch, 1997), being those compounds an important fac- Row B, shows mRNA detection from transgenic lines in Jewel
tor for the T-DNA transfer event. For sweet potato it has cv. (C-27, lanes 4-5) and CEMSA 78354 cv. (C-1, lanes 1-2)
been found higher oxidative responses between 28º-32ºC which are stronger according to RNA amounts placed in
(Ankembauer and Nester, 1990) and the application of reduc- each hole; mRNA concentration was estimated to be around
ing compounds into the media affected the transformation 1 pg/10 μg of total RNA.
efficiencies in leaf explants transformed by Agrobacterium Genomic Southern blot from C-27 gave a single hy-
tumefaciens affecting the acetosyringone induced vir genes bridization for the KpnI digestion at 5 kb, indicating that
expression (Okamoto et al., 2000). there could be a single copy of the cry3A inserted for this
AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato 31
A) A) B)
Cultivar Repetition Infected Total Kmr PCR+ plants ETF+ 1
explants shoots (cry3A) Figure 7. Molecular analysis of transgenic sweet potato plants
1 30 23 22 0.73 obtained during this study. A) mRNA Dot blot hybridization (cry3A
Jewel 2 30 25 25 0.83 transcripts) from transgenic sweet potato clones. Row A: Total
3 30 21 21 0.70 RNA from Bacillus thuringiensis subsps. tenebrionis (Btt) at different
Total 90 69 68 0.75 concentrations (Positive controls, Lanes 1-4). Lane 1: 5 ng; Lane 2:
1 30 17 16 0.53
10 ng; Lane 3: 50 ng; Lane 4: 100 ng. Lane 5: Negative control, 20
CEMSA 2 30 18 18 0.60 µg of total RNA from an untransformed sweet potato plant Jewel
78354 3 30 20 19 0.63 cv. Row B: Total RNA from transgenic sweet potato plants. Lane
Total 90 55 53 0.59 1: 10 µg of total RNA from clone C-1, CEMSA 78354 cv. Lane
2: 20 µg of total RNA from clon C-1, CEMSA 78354 cv. Lane 3)
Figure 6. A) PCR products from kanamycin resistant sweet potato 20 µg of an untransformed sweet potato plant CEMSA 78354 cv.
leaves. C-: Negative control, PCR performed to DNA from untrans- (Negative Control); Lane 4) 10 µg of total RNA from clone C-27,
formed sweet potato plants. C+: Positive control, PCR performed Jewel cv.; Lane 5) 40 µg of total RNA from clone C-27, Jewel cv.
to DNA from Bacillus thuringiensis tenebrionis. Jewel: PCR performed B) Genomic Southern blot hybridization from C-27 Jewel cv.,
to five (5) kanamycin resistant shoots from Jewel cv. C-78354: PCR harboring the cry3A gene (1.9 kb). Lane 1: Molecular weight marker
performed to four (4) kanamycin resistant clones from CEMSA in the original gel. Lane 2: 40 µg of total DNA from C-2., KpnI
78354 cv. B) Efficiency of sweet potato transformation determined digested and hybridized with the probe. Lane 3: 40 µg of total DNA
by PCR from leaves of kanamycin (Kmr) resistant plants. ETF+1: from C-27, EcoRV digested and hybridized with the probe. Lane 4:
Effective transformation frequency but calculated considering PCR Negative control, 40 µg of total DNA KpnI from untransformed
positive plants for the cry3A gene. sweet potato plants digested and hybridized with the probe. Lane
5: Negative control, 40 µg of total DNA from untransformed
sweet potato plants EcoRV digested and hybridized with the probe.
C) DNA construct for the transformation experiments. LB: Left
border in the T-DNA; 35S: 35S promoter from the Cauliflower
transgenic clone (Figure 7B, lane 2). Digestion with EcoRI, mosaic virus (CaMV); Ω: Omega fragment from Tobacco Mosaic
which has an internal restriction site in the cry3A gene, gave Virus (TMV); cry3A: deltaendotoxin gene from Bacillus thuringiensis
two bands, confirming that only a single copy of the gene tenebrionis; tnos: Nopalin synthetase terminator; pnos: Nopalin
was present. No signal was detected for untransformed sweet synthetase promoter; nptII: Neomycin phosphotransferase gene;
potato plants from Jewel cv.. 3’OCS: 3 Octopin synthetase terminator; RB: Right border in the
In conclusion, we have succeeded in our aim to increase T-DNA. SalI, EcoRV, KpnI: Restriction sites for every enzyme
the regeneration and transformation efficiency in sweet po- employed in the Genomic Southern Blot experiments.
tato by synchronizing the regeneration and transformation
steps and also by identifying the specific factors that affects
each cultivar. Addition of PBZ could increase the regenera-
tion efficiency for those cultivars with strong shoot domi-
nance. In general, it is a model that should be applied to any
effort to transform sweet potato since it is documented that ACKNOWLEDGMENTS
the results will depend on the genotype. On the other hand,
we demonstrated that by using an organogenic protocol it The authors would like to thanks to Dr. Gene Nester from
is possible to obtain an efficient transformation procedure, Washington University and Dr. William Velander from Uni-
not only by the number of putative transgenic plants but versity of Nebraska for their suggestions and English cor-
also because the reduction of the time to produce them. The rection. Also, we would like to thanks to Dr. Justo González
protocol generated from this study is now being the used to Olmedo from Centro de Bioplantas and Dr. Rafael Gómez
produce transgenic sweet potato in our research programs Kosky, for their critical review of the original results that
with high efficiency. helped us to write this final version.
32 AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008 Efficient regeneration and transformation of recalcitrant sweet potato
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