The Avocado: Botany, Production and Uses. 2nd Edition

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©CAB International 2013. The Avocado: Botany, Production and Uses, 2nd Edition
268 (eds B. Schaffer et al.)
Biotechnological approaches could be
useful for developing avocado rootstocks
because: (i) rootstock selections could be
micropropagated at theoretically low cost;
(ii) identification of somaclonal variants with
increased tolerance of PRR (van den Bulk,
1991) would be possible, especially since
resistance to P. cinnamomi at the cellular level
appears to be similar to that at the whole plant
level (Phillips et al., 1991); and (iii) disease
resistance could be enhanced by transforma-
tion with various classes of genes, e.g.
defensins, genes for pathogenesis-related pro-
teins, ribonucleases, etc. In addition, antisense
transformation for silencing gibberellins (GAs)
biosynthesis genes in order to reduce tree size,
recovery of cultivars showing more cold hardi-
ness or with fruit showing extended shelf life
and on-the-tree storage, are feasible. Finally,
embryo rescue by in vitro culture could be a
very useful tool to recover genotypes from
selected crosses in conventional breeding
programmes.
Molecular biology can be important in tax-
onomic and systematic studies (Chanderbali
et al., 2009). In addition, many genes involved in
the regulation of specific horticultural traits, e.g.
fruit ripening (Smith et al., 1988; Botella, 2002),
insect resistance (Barton et al., 1987; Nickson,
2005), resistance to fungal diseases (Broglie
Avocado breeding by conventional methods is
slow due to heterozygosity and the long juvenile
period of this species (Pliego-Alfaro and Bergh,
1992); however, variability of desirable horticul-
tural traits is almost limitless. Breeding pro-
grammes for scion selections have focused on
improved yield and fruit quality; whereas, for
rootstock selections, the focus has been enhanced
resistance to biotic and abiotic stresses and/or
reduced size. The use of seedlings as avocado
rootstocks has been linked to lack of uniformity
in orchards with respect to production, vigour,
and/or tolerance of soil-borne pathogens or
adverse soil conditions (Gustafson and Kadman,
1970). Phytophthora root rot (PRR), caused by
Phytophthora cinnamomi Rands, is a very seri-
ous problem in avocado orchards, and much
effort has focused on developing rootstocks that
are tolerant of this pathogen (Bergh et al., 1976;
Kellam and Coffey, 1985; Köhne, 1992; Kremer-
Köhne and Mukhumo, 2003; Kremer-Köhne
et al. 2011). Attempts have also been made to
identify material tolerant of saline and calcare-
ous soil conditions (Kadman and Ben Ya’acov,
1980; Kadman, 1985). Rootstock selections
can be vegetatively propagated by the ‘nurse’
seed/etiolation method (Frolich, 1951; Frolich
and Platt, 1972; Brokaw, 1988; see Ernst et al.,
Chapter 9, this volume), a relatively expensive
and time-consuming procedure.
10 Biotechnology
F. Pliego-Alfaro,1 A. Barceló-Muñoz,2 R. LóPez-Gómez,3 E. Ibarra-Laclette,4
L. Herrera-Estrella,4 E. Palomo-Ríos,1 J. A. Mercado1 and R. E. Litz5
1Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad
de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC),
Spain; 2IFAPA, Centro de Churriana, Spain; 3Laboratorio de Fisiología Molecular de
Plantas, Universidad Michoacana de San Nicolás de Hidalgo, Mexico; 4Langebio
Cinvestav, Carretera León, Mexico; 5Tropical Research & Education Center,
University of Florida, USA
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Biotechnology 269
et al., 1991; Stuiver, 2006) and dwarfing (Fagoaga
et al., 2007), have been cloned. Transformation
with these genes allows specific changes to be
made in the genome while maintaining the
genetic integrity of the clone (Schuerman and
Dandekar, 1993). Understanding the mecha-
nisms involved in gene regulation and expres-
sion is necessary to obtain transgenic plants of
horticultural value (Jorgensen, 1993). The number
of fruit and nut crops regenerated by cell cultures
has increased considerably in the last decade
(Litz, 2005). With avocado, different genes have
been introduced into embryogenic cells, gener-
ally derived from immature embryos, and some
regenerants have been obtained; however, more
efforts are needed to improve the conversion rate
of somatic embryos.
In Vitro Morphogenesis
Avocado plants have been recovered in vitro
via shoot culture and somatic embryogene-
sis. The former approach has great utility as
an alternative method for vegetatively propa-
gating proprietary material, particularly new
rootstock selections. Somatic embryogenesis,
involving regeneration from single cells, is
critical for genetic manipulation using
genetic transformation and in vitro mutation
induction.
Shoot culture
Multiplication of plants by stimulation of axil-
lary bud proliferation is the most reliable
micropropagation method for maintaining the
genetic integrity of elite selections (George,
1993). Cultures are initiated from explants
with intact meristem and newly formed shoots
are used as propagules. With woody species
such as avocado, morphogenic competence is
generally greater in physiologically juvenile
than in adult material (Favre and Juncker,
1987; Pliego-Alfaro and Murashige, 1987;
Arrillaga et al., 1991). Micropropagation of
many woody plant species has been addressed
using juvenile material in order to establish
guidelines for multiplication of explants of
adult origin.
Juvenile material
Experiments involving shoot tips or nodal stem
segments of avocado seedlings and related
species have demonstrated the importance of
salt formulation of the basal medium for sur-
vival and shoot proliferation. Murashige and
Skoog (1962) (MS) formulation causes scaly
leaves (González-Rosas and Salazar-García,
1984; González-Rosas et al., 1985; Witjaksono,
1991), leaf tip burn (Witjaksono, 1991),
shoot apical necrosis (Pliego-Alfaro, 1981;
Witjaksono, 1991; Nhut et al., 2008) and leaf
abscission (Pliego-Alfaro, 1981; Witjaksono,
1991), all of which are similar to ammonium
toxicity symptoms in avocado (Lovatt, 1988).
Satisfactory shoot proliferation and survival
can be obtained when the plant growth medium
contains salt concentrations £ MS. Nel et al.
(1983) and Campos and Pais (1996) used MS
formulation with half strength macroelements
to obtain shoot elongation and limited prolif-
eration from nodal explants of Persea indica.
Half-strength MS salts have also been used to
promote axillary bud growth (Vega-Solórzano,
1989; Biasi et al., 1994). Diluted MS nutrient
solution has also been recommended for avo-
cado by Barceló-Muñoz et al. (1990), and
Cooper (1987) used Woody Plant Medium
(WPM) (Lloyd and McCown, 1980) basal
medium. A 100% survival rate has been
obtained on MS medium containing 20 mM
KNO3 without NH4NO3 (Witjaksono, 1991;
Witjaksono et al., 1999a); whereas, concentra-
tions of KNO3 > 20 mM resulted in less growth.
Optimum growth was achieved on modified
MS medium containing 20 mM KNO3 and 10
mM NH4NO3 (Witjaksono et al., 1999a). Nhut
et al. (2008) found that 2% peptone added to
basal MS medium induced shoot formation
and elongation.
Benzyladenine (BA) in the range 4.44–
8.88 mM is the most suitable cytokinin for
proliferation of juvenile shoots of P. indica
(Nel et al., 1983) and P. americana (Nel et al.,
1983; Cooper, 1987; Barceló-Muñoz et al.,
1990; Witjaksono et al., 1999a). BA is superior
to thidiazuron since the latter only stimulates
bud proliferation without shoot elongation from
explants (Mohamed-Yasseen, 1993; Barringer
et al., 1996). Since the presence of BA during
successive subcultures can cause miniaturization
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270 F. Pliego-Alfaro et al.
of shoots, an elongation phase, e.g. subculture
in lower BA dosages in liquid media (Barceló-
Muñoz et al., 1990) or in combination with
auxin (Cooper, 1987) has been used prior to
rooting. According to Barceló-Muñoz et al.
(1990), propagules should not be maintained
for more than 2 weeks in liquid medium to
avoid hyperhydricity. Leaves of hyperhydric
shoots are thicker than those on normal shoots,
showing accumulation of carbohydrates and a
decrease in peroxidase activity (Barceló-
Muñoz, 1995). Hyperhydric shoots are incapa-
ble of survival ex vitro.
Kane et al. (1989) multiplied seedling-
derived P. palustris shoots in liquid MS medium
and plantlets were acclimatized. With avo-
cado, de la Viña et al. (2001) studied the effects
of medium texture, sucrose concentration, and
solid vs. double-phase in combination with
irradiance (35–85 mmol m−2 s−1), on shoot qual-
ity during proliferation. Culture in double-
phase medium caused hyperhydricity of
microcuttings, deformed stomates and poor
development of epicuticular waxes. Increasing
the level of irradiance decreased the content of
leaf chlorophylls and carotenoids but did not
affect hyperhydricity. De la Viña et al. (1999)
reported that avocado plantlets growing in the
presence of high CO2 (100 Pa), high irradiance
(85 mmol m−2 s−1) and 14.61 mM sucrose had
increased ribulose bisphosphate carboxylase
(Rubisco) activity. Photosynthetic activity and
growth rate were enhanced in contrast with
plantlets in the presence of 87.64 mM sucrose;
however, their survival (70%) during acclimati-
zation was not improved compared with plants
grown on high sucrose. Witjaksono et al.
(1999a) observed that proliferating avocado
shoot cultures and plantlets were photosyn-
thetically active on medium supplemented
with 30 gl−1 sucrose and 4.44 mM BA with a 16
h photoperiod (120–150 mmol m−2 s−1). The net
CO2 assimilation rates of shoots and plantlets
in an ambient CO2 environment were 17 ± 2
and 31 ± 7 mmol CO2 m
−2 s
−1, respectively.
When measured at ambient atmospheric CO2
concentration, the net CO2 assimilation rates
for shoots and plantlets were higher for plants
grown in ambient CO2 than those grown in a
CO2-enriched environment, indicating that
reduced photosynthetic efficiency of shoots and
plantlets grown in an enriched atmospheric
CO2 environment. The net CO2 assimilation
rates of in vitro grown plantlets were compara-
ble to those of seedlings ex vitro.
In vitro shoots of avocado could be rooted
mostly in medium containing auxin, e.g. 36.9–
49 mM indole-3-butyric acid (IBA) with 50%
rooting (González-Rosas and Salazar-García,
1984); 4.9–9.8 mM IBA with rooting frequency
of 30% (Barringer et al., 1996); 9.8 mM IBA
with 65% rooting of P. indica shoots in liquid
medium with a filter paper bridge (Nel et al.,
1983); 9.8 mM IBA with 13.9 mM kinetin to root
shoots of P schiedeana (González-Rosas et al.,
1985); or 16 mM naphthalene acetic acid (NAA)
alone (Cooper, 1987) or in presence of 2% pep-
tone (Nhut et al., 2008) for a rooting frequency
of 100%.
Pliego-Alfaro (1988) found that two-step
rooting resulted in 100% rooting frequency of
shoot tips from in vitro seedlings. This proce-
dure includes an induction step in which shoots
are cultured on to 111 mM IBA for 3 days, fol-
lowed by a development step in which the
shoots were transferred to one-third MS
medium with 1 mg l−1 activated charcoal but
without auxin. Witjaksono et al. (1999a) also
obtained high rooting frequency using this pro-
cedure with seedling-derived shoots, although
Biasi et al. (1994) obtained only 45% rooting
from seedling-derived shoots. Variants of the
induction step, including a pulse with 4.92 mM
IBA (Cooper, 1987; Barceló-Muñoz et al.,
1990) can enhance rooting. A quick dip for
1–2 seconds in 4.9–24.6 mM IBA following the
development step has resulted in 100% rooting
of P. indica shoots (Campos and Pais, 1996). In
some cases, the auxin exposure should be car-
ried out in the dark (de la Viña et al., 1996).
Activated charcoal can have a positive effect
on rooting when used in the auxin-free phase
(Pliego-Alfaro, 1988; Barceló-Muñoz et al.,
1990). Successful rooting ex vitro has also been
reported by Cooper (1987); a quick immersion
in a solution of 16 mM NAA was necessary
prior to transplanting the shoots into a fine
pumice-peat substrate. A quick dip for 5 min in
245 mM IBA followed by transfer of shoots into
plug trays with Vegro Klay Mix A resulted in
100% rooting of P. palustris (Kane et al., 1989).
In juvenile shoots, Premkumar et al. (2002,
2003) studied the effect of sucrose in the culture
medium on different physiological parameters
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Biotechnology 271
of rooting and acclimatization. Changes in
sucrose levels did not affect chlorophyll or caro-
tenoid content; however, increasing sucrose
concentration induced a decrease in Rubisco
during rooting which was even more pro-
nounced during the acclimatization phase, sug-
gesting a possible role of this protein as a source
of reduced nitrogen during this phase
(Premkumar et al., 2002). Decreased sucrose
level observed in the leaves was linked to
increased starch content, indicating an improve-
ment in the use of sucrose as well as in starch
biosynthesis during acclimatization (Premkumar
et al., 2003).
Generally, survival of juvenile avocado
material during acclimatization has been accept-
able (about 80%) (Cooper, 1987). However,
inoculation with mycorrhizal fungi, Glomus fas-
ciculatum and G. deserticola, can improve root
and shoot growth, enhance the shoot/root ratio
and increase the content of N, P and K, enabling
plants to tolerate the stress caused by transplant-
ing (Azcón-Aguilar et al., 1992; Vidal et al.,
1992; de la Viña et al., 1996).
Adult material
Early attempts to micropropagate adult phase
shoots were generally unsuccessful. Harty
(1985) used modified MS salts containing
reduced ammonium and elevated concentra-
tion of nitrate (Dixon and Fuller, 1976) and
obtained 90% survival and sixfold multiplica-
tion at 9-week intervals, even though tissue
necrosis was not completely eliminated and
shoots failed to root. Cooper (1987) indicated
that etiolation of mother plants or incubation
of explants for 24 h under continuous light
enhanced bud break; however, proliferation of
shoots did not occur. Zirari and Lionakis (1994)
used material derived from etiolated cuttings
for culture establishment, pointing out that the
use of etiolated explants did not have positive
effects on ‘Topa-Topa’, ‘Fuerte’ or ‘Hass’,
although it improved sprouting of buds from
‘Duke-7’ however, attempts to root micro-
shoots were not successful due to severe shoot
necrosis following transfer to rooting medium.
Zulfiqar et al. (2009) working with ‘ Fuerte’
obtained positive results with modified MS
medium (75% macro and micro salts) and
1 mg l−1 BA during the proliferation phase
(4.8 shoots per explant) or 1 mg l−1 IBA for
rooting (53% rooting). Generally, successful
micropropagation from adult trees requires
selection of explants that have some degree of
tissue juvenility, either as a result of hand-
pruning or partial rejuvenation by grafting adult
tissue on to juvenile seedlings.
Pliego-Alfaro et al. (1987) established cul-
tures of the ‘IV-8’, a highly productive ‘Topa-
Topa’ seedling rootstock and the ‘GA-13’
rootstock, a selection with tolerance of saline
and limestone soil conditions (Kadman and
Ben Ya’acov, 1980), using explants from
actively growing shoots. The ‘IV-8’ rootstock
tree was forced to sprout after heavy pruning,
while adult ‘GA-13’ scions were grafted onto
seedling rootstocks and forced to sprout in the
greenhouse. With both selections, reduced MS
macroelement concentrations and 1.3–4.44
mM BA enhanced bud sprouting and shoot
growth.
Schall (1987) used relatively high BA dos-
ages (22 mM) to stimulate proliferation of adult
‘Fuerte’ explants derived from 2- to 4-year-old
grafted trees; however, after several subcul-
tures, significant oxidation and browning
caused death of the propagules. Although
apical necrosis is a problem during shoot
proliferation, adult shoots of ‘GA-13’ could be
maintained for >1 year on medium with
4.44 mM BA with weekly subcultures and, with
‘IV-8’ rootstock, the double-phase medium
(Pliego-Alfaro et al., 1987) overcame apical
necrosis; however, after several subcultures,
severe hyperhydricity occurred. The combined
use of liquid medium on a rotary shaker
(2 weeks) and double phase medium (6 weeks)
decreased to 20% the percentage of hyperhy-
dricity in proliferating ‘IV-8’ shoots. Replacement
of MS salts with B5 medium (Gamborg et al.,
1968) resulted in more vigorous and elongated
shoots with no leaf necrosis, which could be
continuously maintained in active proliferation
(Fig. 10.1) with a threefold multiplication rate
(Barceló-Muñoz et al., 1999).
Although rooting capacity of adult avo-
cado shoots is very low and does not increase
with subculturing (Pliego-Alfaro et al., 1987),
restoration of rooting competence can be
achieved by grafting adult buds onto in vitro-
germinated seedlings (Pliego-Alfaro and
Murashige, 1987; Barceló-Muñoz, 1995) or
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272 F. Pliego-Alfaro et al.
after successives severe prunings of mother
plants (Barceló-Muñoz et al., 1999). With ‘GA-
13’, the percentage of rooting (about 90%) was
similar to that of juvenile material after 16 suc-
cessive in vitro grafts. The restored rooting
capacity appeared to be a stable trait, since it
was constant for nine subcultures (Barceló-
Muñoz, 1995). Working with ‘IV-8’ rootstock,
Barceló-Muñoz et al. (1999) obtained > 90%
rooting after successive pruning over several
years at ground level.
Rooting capacity is affected by medium
texture (> 70% in liquid medium versus 30% in
solid medium), as well as by the salt formula-
tion of the medium in which the shoots were
proliferated prior to rooting treatment. Shoots
that proliferated in half strength MS rooted sig-
nificantly less (74%) than those in B5 (Gamborg
et al., 1968) medium (90%). Acclimatization of
rooted adult shoots occurred in a peat-moss
substrate with 70% survival after 8 weeks
(Fig. 10.2) (Barceló-Muñoz et al., 1999).
Great variability in shoot proliferation and
rooting responses involving either juvenile or
adult materials could be due to genotype. This
would explain the responses obtained with dif-
ferent cultivars. Therefore, a successful proto-
col developed for one genotype is not
necessarily applicable to others, but could be
used as a guide.
There is also an alternative procedure to
propagate adult avocado material when a lim-
ited number of copies for each genotype is
required. Barceló-Muñoz et al. (2001) micro-
propagated 7 seedling rootstocks showing par-
tial tolerance to R. necatrix while
Cortés-Rodríguez et al. (2011) micropropa-
gated adult Mexican race trees. The procedure
included an establishment phase in B5 medium
supplemented with 0.3 mg/l BA (Barceló-
Muñoz et al., 2001) or in MS medium supple-
mented with 0.5 mg/l BA and 0.1 mg/l IBA
(Cortés-Rodríguez et al., 2011). After 6–8
weeks, axillary buds gave rise to shoots which
were excised, rooted in vitro and acclimatized.
Nodal sections with basal buds were recul-
tured in fresh medium for continuous shoot
production. To enhance rooting capacity,
Barceló-Muñoz et al. (2001) recommended the
use of successive pruning of recalcitrant geno-
types. Using this approach and following the
protocol of Barceló-Muñoz et al. (1999), in
vitro rooting rates in the range 40–100% could
be obtained (Table 10.1).
Embryo culture
The avocado fruitlet has a high abscission rate.
Therefore, very few fruit from controlled crosses
remain on the tree which is a serious obstacle
in avocado breeding programmes. In vitro cul-
ture of immature embryos allows plant recov-
ery from interesting crosses which would
otherwise be lost (Sharma et al., 1996). The first
attempt of avocado embryo rescue was
described by Skene and Barlass (1983), who
cultured immature embryos from abscised avo-
cado fruit in order to recover genotypes
Fig. 10.1. Adult avocado shoots of the ‘IV-8’
rootsctock at the proliferation stage in double-
phase medium.
Fig. 10.2. Micropropagated adult avocado plants
of the ‘IV-8’ rootstock at the acclimatization phase.
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Biotechnology 273
obtained after hybridization. They used liquid
medium with diluted MS macroelements and
2.2 mM BA (M1); embryos needed to be at least
6 weeks old to survive, and about 30–50% of
shoots formed roots. Therefore, to assure sur-
vival, shoots had to be grafted onto greenhouse-
grown seedlings.
Perán-Quesada et al. (2005) studied the
anatomical differentiation and storage product
accumulation during development of avocado
zygotic embryos, and results were correlated
with in vitro germination capacity. Histo-
differentiation was completed 100 days after
pollination (DAP) in 16–18 mm-long embryos,
while maturation started 125 DAP with
embryos 24–26 mm in length, and was linked
to accumulation of starch granules and protein
bodies. Interestingly, in previous investiga-
tions, Sánchez-Romero et al. (2002) had shown
that storage proteins began to accumulate in
smaller embryos (7–8 mm in length), although
most protein was found at the end of the
growth period. Moreover, they also indicated
that storage proteins represent 83% of total
proteins in avocado embryos. When examin-
ing the germination capacity in vitro, in rela-
tion to the developmental stage, Perán-Quesada
et al. (2005) found that embryos 64–95 DAP
show a > 20% rooting capacity which
increased to > 60% between 125–204 DAP.
Germination rates > 90% were observed with
305 DAP embryos and were linked to low
moisture content and complete physiological
maturity. Pliego-Alfaro (1988) also obtained
nearly 100% plant recovery from excised
embryo axes with a piece of cotyledon from
mature avocado seeds on MS basal medium.
Investigating the different germination require-
ments of the embryos at different developmen-
tal stages, Sánchez-Romero et al. (2007) found
that very immature embryos (5 mm long) ger-
minated better in liquid M1 medium while
15-mm long embryos responded better on
semi-solid M1 medium, although cotyledon
removal was necessary. These authors indi-
cated that cotyledons may be involved in
avocado embryo dormancy. In vitro germina-
tion of 16–22-mm long embryos can be
improved following a partial desiccation proc-
ess (Sánchez-Romero et al., 2003; Márquez-
Martín et al., 2007).
To increase germination capacity of very
immature embryos, Márquez-Martín et al.
(2009) developed a system whereby < 10 mm-
long avocado embryos underwent an in vitro
maturation phase. This included culture on B5
medium (Gamborg et al., 1968) supplemented
with Jensen’s amino acids (Jensen, 1977), 88
mM sucrose and 6 g l−1 agar, prior to germina-
tion on M1 medium. They obtained a 65% ger-
mination rate and healthy and vigorous plants.
Histological studies revealed that at the end of
the in vitro maturation period, histodifferentia-
tion had been achieved and starch granules and
proteins were abundant. This technique could
be used as a reliable method for plant recovery
in controlled crosses in avocado breeding pro-
grammes (Pliego-Alfaro and Litz, 2007).
Callus cultures
In early studies, avocado callus was established
from different explants although there was no
evidence of plant regeneration. Culture of cot-
yledons, etiolated stem sections, peduncles
and leaf petioles on Nitsch medium (Nitsch,
1951; Schroeder, 1977), stem sections on MS
medium with 1.3 mM BA and 5.7 mM indole-3-
acetic acid (IAA) (Blickle et al., 1986) or 10 mM
BA and NAA (Phillips et al., 1991), as well as
Table 10.1. In vitro rooting percentages of avocado
seedling rootstocks after successive prunings at
ground level. Shoots were rooted according to the
protocol of Barceló-Muñoz et al. (1999).
Genotype
Number of successive prunings at
ground level
12 3
BG70 100 %
rooting
BG194 98 %
rooting
BG81 72 %
rooting
BG71 No
response
90 %
rooting
BG181 No
response
100 %
rooting
BG183 No
response
No
response
100 %
rooting
BG189 No
response
40 %
rooting
48 %
rooting
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274 F. Pliego-Alfaro et al.
stem and leaf sections on Anderson medium
(Anderson, 1975) with 1 mM 2,4 dichlorophe-
noxy acetic acid (2,4-D) (Young, 1983) resulted
in active callus proliferation (Schroeder, 1977;
Young, 1983) in complete darkness (Schroeder,
1971) or under low light (Young, 1983).
Blumenfeld and Gazit (1971) compared the in
vitro responses of cotyledon and mesocarp-
derived calluses, and reported that cotyledon
callus synthesized its own cytokinin and could
be grown on a medium without this hormone.
Growth requirements for callus prolifera-
tion from seedling-derived stem explants have
been evaluated extensively for avocado and
other Persea species, i.e. P. indica, P. borbonia
and P. nubigena (Aaouine, 1986). Generally
satisfactory callus growth for all species was
obtained on medium consisting of half con-
centration of MS major salts except for MgSO4;
MS minor salts, vitamins with 1.4 mM 2,4-D
and 4.9 mM 2-isopentenyladenine (2iP)
(Aaouine, 1986). Cell suspensions could be
established in 0.5 mg l−1 picloram from callus
derived from ‘Hass’ avocado peel initiated on
MS medium supplemented with 0.1 mg l−1
picloram (Prusky et al., 1996).
Adventitious root development was
observed after several subcultures of fruit
pericarp-derived callus on Nitsch medium sup-
plemented with 59 mM IAA (Schroeder et al.,
1962), or at the base of etiolated stem sections
which had formed callus on Nitsch or MS
medium with 29.4–147 mM 2iP (Schroeder,
1980). No histological studies were conducted
to determine if the roots were derived from the
callus or from tissues of the original explant.
Adventitious shoots have been induced in
the presence of 4.4 mM BA and 2.5 mM IBA.
Explants were etiolated internodal sections
from in vitro germinated ‘Lula’ avocado seed-
lings. The regeneration rate was about 6% with
1–2 regenerated shoots per explant (R. Perán-
Quesada, IFAPA-Centro de Churriana, Málaga,
Spain, 2001, personal communication).
Somatic embryogenesis
Embryogenic avocado cultures have been
induced in the presence of picloram (Pliego-
Alfaro, 1981; Mooney and van Staden, 1987;
Pliego-Alfaro and Murashige, 1988; Raviv et al.,
1998; Witjaksono and Litz, 1999a) from zygotic
embryo explants of various sizes and develop-
mental stages, ranging from globular (0.1 mm)
to heart and cotyledonary (1.2 mm) (Mooney
and van Staden, 1987; Pliego-Alfaro and
Murashige, 1988; Witjaksono and Litz, 1999a).
Raviv et al. (1998) induced embryogenic cul-
tures from cotyledon segments excised from
7–10 mm embryos. Witjaksono and Litz
(1999a) demonstrated that an induction basal
medium consisting of B5 (Gamborg et al., 1968)
major salts with MS minor salts and organic
addenda was superior to MS medium. Using
small fruitlets (£ 0.3 cm) and explanting the
bisected ovules that contain zygotic embryos at
pre-globular to globular stages, induction fre-
quencies of about 25% can be achieved
(Witjaksono, University of Florida, Tropical
Research and Education Center, Homestead,
Florida, 1997, personal communication)
Embryogenic avocado cultures consist of
proembryonic masses (PEMs) and early stages
of hyperhydric somatic embryos, and first
appear 8–25 days after explanting on induction
medium. Although early reports described the
newly initiated cultures as being embryogenic
callus, it is clear in retrospect that they are typi-
cal embryogenic cultures that consist of pro-
embryonic cells and PEMs. This is confirmed
by the high magnification photographs of all
published reports and the histological prepara-
tions of Mooney and van Staden (1987), and
later confirmed by Witjaksono and Litz
(1999a,b) and Witjaksono et al. (1999b).
Embryogenic cultures have been maintained
on induction medium based upon MS basal for-
mulation (MSP) (Pliego-Alfaro and Murashige,
1988) or on Dixon and Fuller medium supple-
mented with 3 mg l−1 each of isopentenyladeno-
sine and benzothiazole-2-oxyacetic acid
(Mooney and van Staden, 1987). The medium
and long-term maintenance of embryogenic avo-
cado cultures on semi-solid medium and in sus-
pension was described in detail by Witjaksono
and Litz (1999a). They observed that proliferation
of PEMs is optimum on semi-solid MSP induction
medium formulation. Maintenance on semi-solid
medium requires subculture at 3–5 week inter-
vals, and only the smallest PEMs are inoculated
onto fresh medium.
Embryogenic culture maintenance is opti-
mal in liquid MS medium that has been modified
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Biotechnology 275
to contain 12 mg l−1 NH4NO3 and 30.3 mg l−1
KNO3 (MS3:1P), which results in the highest
culture fresh weight increase compared with
other medium formulations (Witjaksono and
Litz, 1999b); MS3:1P is supplemented with
30–50 g l−1 sucrose, 100 mg l−1 myo inositol,
4 mg l−1 thiamine HCl and 0.41 mM picloram
(Witjaksono and Litz, 1999a, b). Embryogenic
suspension cultures are initiated by inoculating
0.5–1.0 g PEMs into 40 ml or 80 ml liquid
MS3:1P medium in 125 ml or 250 ml
Erlenmeyer flasks, respectively, maintained on
a rotary shaker at about 100–125 rpm and sub-
cultured at 2-week intervals. Newly initiated
SE-type suspension cultures (see below) must
be sieved and only < 0.8 mm PEMs are used as
inoculum for subculturing.
Witjaksono and Litz (1999a) recognized
two types of embryogenic avocado cultures: (i)
genotypes that proliferate as PEMs in the pres-
ence of auxin (PEM-type); and (ii) genotypes in
which heart and later stages of somatic embryos
can develop in the presence of auxin (SE-type).
The latter response (SE-type) appears to be
more common. Witjaksono and Litz (1999a)
also demonstrated that the time required for
loss of embryogenic potential is genotype-
dependent, varying from 3 months for ‘Yon’ to
> 2 years for ‘Esther’. Loss of embryogenic
potential is associated with increasing disor-
ganization of PEMs. Maintenance cultures on
semi-solid medium are incubated in darkness
at 25°C, whereas suspension cultures are incu-
bated in semi-darkness.
Early reports indicated that somatic
embryos develop following transfer of embryo-
genic cultures on to semi-solid medium with-
out picloram (Mooney and van Staden, 1987;
Pliego-Alfaro and Murashige, 1988). The
absence of picloram, however, is not a prereq-
uisite for somatic embryo development, as
somatic embryos of the SE-type cultures can
develop in the presence of picloram (Witjaksono
and Litz, 1999a). Raviv et al. (1998) also
observed that cotyledonary-stage somatic
embryos can develop on semi-solid prolifera-
tion medium supplemented with 9.04 mM
2,4-D and 2.22 mM BA. These somatic embryos
are generally hyperhydrous, and cannot
develop to maturity. Somatic embryo develop-
ment in avocado is genotype-dependent;
SE-type cultures produce somatic embryos
readily, whereas PEM-type cultures are less
efficient in this respect (Witjaksono and Litz,
1999a,b; 2002; Márquez-Martín et al. 2012).
Witjaksono and Litz (1999b) demonstrated that
various factors could affect development of
good quality somatic embryos, and recom-
mended the use of the MS formulation, while
better results have been reported by Perán-
Quesada et al. (2004) following the use of B5
major salts. According to Perán-Quesada et al.
(2004), MS formulation favours formation of
somatic embryos at early development stages,
while maturity is enhanced by B5 major salts.
Increasing the gelling agent and sucrose con-
centrations affects both size and number of
somatic embryos. The optimum response for
recovery of high quality somatic embryos
occurs on medium supplemented with 6–7 g
l−1 gellan gum and 90 g l−1 sucrose; however,
this concentration of sucrose also suppresses
somatic embryo development. Better results
have been obtained when using 10 g l−1 agar
(Sygma A-1296) in comparison with 6–7 g l−1
gellan gum (Márquez-Martín et al., 2011). High
gellan gum concentrations together with high
sucrose content in the maturation medium are
effective for reducing the occurrence of hyper-
hydricity. Perán-Quesada et al. (2004) also
observed a positive effect of sugar on embryo
maturation, but Márquez-Martín (2007) indi-
cated that combined effects of high agar con-
centration and high sugar decreased embryo
quality; embryos turned dark beige and their
surface became irregular. Supplementing mat-
uration medium with 20% (v/v) filter-sterilized
coconut water has also been shown to improve
the quality of recovered somatic embryos
(Witjaksono and Litz, 2002). Somatic embryos
mature in darkness at 25°C. The effect of absci-
sic acid (ABA) has also been studied in avo-
cado somatic embryo maturation but its role is
unclear; ABA seems to increase the number of
globular translucent embryos (Perán-Quesada
et al., 2004)
Well developed, opaque and mature
somatic embryos (³ 0.5 cm diameter) (Fig. 10.3)
are transferred individually to semi-solid ger-
mination medium, which is similar to mainte-
nance medium, but without picloram, and
supplemented with 4.44 mM BA and 2.89 mM
gibberellic acid (GA3) (Witjaksono and Litz,
1999b). After 4–6 months on germination
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276 F. Pliego-Alfaro et al.
medium, only a few somatic embryos develop
shoots or roots only or are bipolar. Shoot devel-
opment from somatic embryos is generally
£5% (Mooney and van Staden, 1987; Pliego-
Alfaro and Murashige, 1988; Witjaksono and
Litz, 1999b). Other reports, including Raviv
et al. (1998), Witjaksono et al. (2009) and
Avenido et al. (2009), indicated that 11, 20 and
26.9%, respectively, of somatic embryos were
able to form shoots. In a recent study, Encina
et al. (2010) were able to get up to 35% shoot
sprouting following a two-step regeneration
system which involves a culture period in liq-
uid germination medium, supplemented with
glutamine followed by transfer to solid medium
of the same composition. A similar approach
was followed by Palomo-Ríos (Departamento
Biología Vegetal, University of Málaga, Spain,
2010, unpublished data), who obtained similar
sprouting percentages following culture of
somatic embryos of ‘Duke-7’ for 3 days in the
liquid germination medium of Witjaksono and
Litz (1999b) and subsequent transfer to solid
medium, of the same composition, for three
recultures of 5 weeks each (Fig. 10.4). Very few
somatic embryos (about 1%) developed both a
shoot and a root. In fact, most mature avocado
somatic embryos are not bipolar (Mooney and
van Staden, 1987; Pliego-Alfaro and Murashige,
1988; Witjaksono and Litz, 1999b). Pliego-
Alfaro and Murashige (1988) ascribed this
anomaly of avocado somatic embryo develop-
ment to failure of either the apical or root mer-
istem to become organized.
To compensate for the low recovery of
shoots from somatic embryos, Witjaksono
et al. (1999a) and Perán-Quesada et al. (2004)
demonstrated that shoot tips and nodal sec-
tions from somatic embryo shoots can be
micropropagated (see above). This procedure
can be utilized for producing a large number of
shoots from the relatively few somatic embryos
derived from protoplasts and embryogenic
cultures, respectively (Witjaksono et al., 1998,
1999b). Avocado shoots derived from somatic
embryos can be individually rooted according
to the two-step procedure of Pliego-Alfaro
(1988). Individual shoots (1.5–2-cm long with
1–3 leaf primordia and non-expanded leaves)
are pulsed for 3 days on medium supplemented
with 122.6 mM IBA before subculture on basal
medium in GA7 vessels. Culture conditions
included a 16 h photoperiod (100–120 mmol
m−2 s
−1 provided by cool white fluorescent
bulbs) at 25°C (Witjaksono and Litz, 1999b).
However, Perán-Quesada et al. (2004) recom-
mend the use of a lower IBA concentration
(4.92 mM) during the 3-days pulse to avoid
excessive callus formation at shoot base.
Fig. 10.3. Well-developed, white-opaque, mature
avocado somatic embryos.
Fig. 10.4. Sprouted somatic embryos after culture
in liquid germination medium for 3 days
(Witjaksono and Litz, 1999a) followed by tranfer to
solid medium of the same composition during
three recultures of 5 weeks each.
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Biotechnology 277
Raharjo and Litz (2005) and later Avenido
et al. (2009) demonstrated that somatic embryo
shoots could be micrografted on seedling root-
stocks. The former obtained success rates of
59–100%, depending on the scion genotype,
and reported success rates of 52–76% when
somatic embryo shoots were grafted ex vitro on
seedling rootstocks.
Protoplast isolation, culture and plant
regeneration
Protoplast technology at one time was impor-
tant in many breeding programmes, although it
has been largely eclipsed by other strategies,
i.e. genetic transformation and in vitro muta-
genesis and selection. Protoplasts can be used
for genetic engineering, e.g. somatic hybridiza-
tion and direct DNA transfer, with the advan-
tage that a new genotype may arise from each
manipulated protoplast. However, regenera-
tion from single cells is frequently associated
with a high frequency of somaclonal variants
(Roest and Gilissen, 1993).
Avocado protoplasts have been isolated
from non-morphogenic callus for studying sun-
blotch viroid replication (Blickle et al., 1986)
and from fruit mesocarp tissue to study fruit rip-
ening (Percival et al., 1991). Witjaksono et al.
(1998) demonstrated that protoplasts isolated
from embryogenic cultures underwent somatic
embryo development under appropiate condi-
tions. Protoplasts are isolated by incubating
0.8–1.2 g of 8–14-day-old suspension cultures
in 60 × 15 mm Petri dishes in a mixture of 1.5
ml enzyme solution and 2.5 ml avocado proto-
plast culture medium. The enzyme solution
consisted of filter-sterilized 1% cellulase
Onozuka RS, 1% Macerase R 10, 0.2%
Pectolyase Y-23, 0.7 M mannitol and salts
(Grosser and Gmitter, 1990). The protoplast
culture medium (MS-8P) is a combination of
avocado maintenance medium without piclo-
ram and in which NH4NO3 has been replaced
by 3.1 g l−1 glutamine, and Kao and Michayluk
(1975) organic addenda as modified by Grosser
and Gmitter (1990). Medium osmolarity is
maintained with 0.15 M sucrose and 0–0.55 M
mannitol as needed. Protoplasts are purified
following filtration through 45-mm mesh stain-
less steel screen and further purified by gradient
centrifugation according to Grosser and
Gmitter (1990) and Witjaksono et al. (1998).
Embryogenic protoplasts ranged in size from
20 to 40 mm. Consistently high yields of > 3 ×
106 protoplasts g−1 can be obtained from
embryogenic cultures maintained in liquid
medium for > 8 months (Witjaksono, University
of Florida, Tropical Research and Education
Center, Homestead, Florida, 2000, personal
communication).
Avocado protoplasts can be cultured
either in liquid (Witjaksono et al., 1998) or in
agarose-solidified (Witjaksono et al., 1999b)
medium; however, the former is a more effi-
cient and simpler procedure for recovery of
somatic embryos. Protoplasts develop as
microcalluses and PEMs in plant growth
medium. The recovery of PEMs from proto-
plasts in liquid medium is dependent on
medium osmolarity, source of nitrogen, plating
density and interaction of osmolarity and nitro-
gen source. Only PEMs have been recovered in
medium with 0.4 M osmolarity, while only
microcalluses develop in medium with 0.6 M
osmolarity. With 0.4 M medium, replacement
of NH4NO3 of MS formulation with 3.1 g l−1
glutamine increases the frequency of occur-
rence of PEMs. A plating density of 0.8 × 105
ml−1 also results in more PEMs than a higher
plating density of 1.6 x 105 ml−1. Therefore, the
optimum conditions for recovery of PEMs from
protoplasts are provided by 0.4 M MS−8P with
a protoplast density of 0.8 × 105 ml−1.
Protoplasts are cultured in 2–3 ml liquid
medium distributed in 60 × 15-mm sterile plas-
tic dishes sealed with Nescofilm® and main-
tained in darkness at 25°C. Under these
conditions, 5% of the protoplasts underwent
the first division after 5 days of culture. The
plating efficiency after 12 days was 25% and
PEMs were visible 14 days after plating. The
number and mass of protoplast-derived PEMs
is dependent on subculture age, dilution rate
and their interaction. The recovery of PEMs is
optimum when the cultured protoplasts are
subcultured after 2–3 weeks at diluted density
of 20–40 × in MS−8P medium with 1.5 M
osmolarity for 1 month. Protoplast-derived
PEMs develop as somatic embryos on matura-
tion medium, and mature somatic embryos
have germinated, with a low frequency of
conversion.
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278 F. Pliego-Alfaro et al.
Protoplast isolation, culture and regenera-
tion are genotype- and culture-age dependent
(Witjaksono, University of Florida, Tropical
Research and Education Center, Homestead,
Florida, 2000, personal communication).
Cultures consisting of highly organized PEMs
and somatic embryos yield only a few proto-
plasts that do not divide. Old cultures consist-
ing of disorganized PEMs yield high numbers
of protoplasts that form microcalluses without
somatic embryo development. Cultures that
have been maintained in liquid for at least
6 months are usually good sources for regener-
able protoplasts.
A goal of avocado protoplast technology
has been to create somatic hybrids between
avocado and sexually and graft-incompatible
Persea species that are resistant to Phytophthora
root rot (PRR), e.g. P. borbonia, P. cinnerascens
and P. pachypoda. Witjaksono addressed this
objective by fusing protoplasts derived from
embryogenic cultures with leaf mesophyll
protoplasts from in vitro-grown Persea spp.
plantlets, and with protoplasts isolated from
non- morphogenic callus of the Persea spp.
Somatic hybridization occurred between
avocado and P. cinnerascens, and somatic
embryos were recovered; however, the fre-
quency of recovery was low and the apical
meristem failed to organize on the somatic
hybrid embryos (Witjaksono, University of
Florida, Tropical Research and Education
Center, Homestead, Florida, 2000, personal
communication).
Induced mutations
The exploitation of induced mutations in horti-
cultural crops has had a long and successful
history (Joint FAO/IAEA, 2010), although it has
largely been superceded by genetic transfor-
mation. According to the Joint FAO/IAEA
Mutant Variety and Genetic Stock Database,
currently there are about 3100 mutant varie-
ties. If current animosity to genetically modi-
fied (GM) crops prevails in the European Union
and elsewhere, the directed in vitro selection
for a specific trait of mutated embryogenic cul-
tures can be a very efficient process for avo-
cado improvement.
The selection ‘D9’, which shows some
promise as a rootstock with moderate resist-
ance to PRR, was derived from an irradiated
‘Duke’ seedling. De la Cruz et al. (1993, 1995,
1998) and Sánchez-Colín et al. (1990) described
the irradiation of ‘Hass’ budwood and observed
variability with respect to fruit set, flowering
time, plant height, etc. More recently, Fuentes
et al. (2009) irradiated zygotic embryos of
‘Duke 7’ at a LD50 of about 25 Gy, and micro-
propagated the resulting plants in an effort to
recover plants with tolerance to saline condi-
tions. Screening for salt tolerance occurred at
an LD20 of 157 mM NaCl.
Witjaksono and Litz (2004) irradiated
embryogenic cultures of ‘Fuerte’ and ‘T362’,
and determined an LD50 for both genotypes at
35 Gy. Subsequent studies by Witjaksono
et al. (2009) and Avenido et al. (2009) involved
the irradiation of embryogenic cultures of
‘Semil’ and unknown Indonesian dooryard
trees and regeneration of plants. The subse-
quent work plan calls for selection in vitro for
resistance to the culture filtrate of Phytophthora
cinnamomi and regeneration of putatively
PRR-resistant plants (R. Litz, University of
Florida, Tropical Research and Education
Center, Homestead, Florida, 2011, personal
communication).
Micrografting and nucellar culture
for viroid elimination
Avocado sunblotch viroid (ASBVd), a member
of the Avsunviroidae family, causes sunblotch
disease, a serious production problem of avo-
cado in some areas of its range. There is no
resistance to the disease in the genus, and the
viroid is spread by pollen, root grafting,
mechanical injury, etc. ASBVd replicates in
chloroplasts unlike other pathogenic microor-
ganisms. Viroids in the Pospiviroidae family
replicate in the nucleus and can be eliminated
by meristem culture (Durán-Vila et al., 1988),
micrografting (Navarro et al., 1975) or nucellar
cultures (Bitters et al., 1972). Suárez et al.
(2005; 2006) demonstrated that micrografting
and nucellar culture were ineffective for
eliminating ASBVd from infected avocado
materials.
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Biotechnology 279
Genetic transformation
The genetic transformation of avocado has
been based upon the well-defined protocol for
regeneration from embryogenic suspension
cultures as described above. Growth of embry-
ogenic suspensions of the PEM type can be
suppressed by as much as 50% by 50 mg l−1
kanamycin sulphate, whereas 50% growth
suppression on semi-solid medium requires
100 mg l−1 kanamycin sulphate (Cruz-
Hernández et al., 1998). Complete suppression
of growth of embryogenic cultures occurs on
semi-solid medium containing 200 mg l−1 kan-
amycin sulphate.
Cruz-Hernández et al. (1998) utilized a
two-step selection procedure for recovery of
genetically transformed embryogenic avocado
cultures (PEM type) that were resistant to
kanamycin and that expressed the uidA
(b- glucuronidase) gene. Embryogenic cultures
on semi-solid maintenance medium were
gently abraded with a soft camel hairbrush and
then inoculated with log phase acetosyrin-
gone-activated disarmed Agrobacterium tume-
faciens strain 9749 ASE2, which harboured a
co- integrate vector pMON9749 containing a
selectable kanamycin sulphate-resistant marker
gene (nptII) and the reporter gene uidA, both of
which were driven by the 35S CaMV constitu-
tive promoter. The PEMs were co-cultured with
A. tumefaciens in liquid maintenance medium
for 3 days at 100 rpm. Agrobacterium tumefa-
ciens was eliminated by incubating the cultures
in maintenance medium supplemented with
200 mg l−1 cefotaxime and 50 mg l−1 kanamy-
cin sulphate. An initial selection for antibiotic
resistance in liquid maintenance medium con-
taining 50 mg l−1 kanamycin sulphate for 2–4
months was followed by a second, more inten-
sive selection with 100 mg l−1 kanamycin sul-
phate for 2 months. The cultures were finally
cultured in maintenance medium containing
200 mg l−1 kanamycin sulphate. Somatic
embryo development occurred on maturation
medium without kanamycin sulphate, followed
by subculture onto maturation medium con-
taining 200 mg l−1 kanamycin sulphate.
Transformed somatic embryos were recovered
that stained positively for uidA in the X-GLUC
reaction (Jefferson, 1987), and the integration
of nptII and uidA into the avocado genome was
confirmed by PCR and Southern hybridization.
Transgenic plants were not regenerated in that
study.
Inhibition of ‘Hass’ embryogenic suspen-
sion culture growth by phosphinothricin (the
active ingredient of Basta® or Finale®) herbicide
was reported by Raharjo et al. (2008), who
observed that 3 mg l−1 phosphinothricin inhib-
ited the growth of embryogenic cultures, and
was utilized at this rate for selection of cultures
transformed with the pGPTV-BPDF1.2. The
construct contained the reporter gene uidA, the
bar gene that confers resistance to phosphi-
nothricin and the antifungal pdf1.2 defensin
gene, all of which were under the control of
the 35S CaMV constitutive promoter. The large
fraction of embryogenic ‘Hass’ suspension cul-
tures in their logarithmic growth phase were
collected on sterile filtration fabric (1000 mm
pore size), and about 300 mg was abraded
with a sterile brush on sterile filter paper. The
tissue was then co-cultured with log phase
acetosyringone-activated A. tumefaciens strain
EHA 105 with pGPTV-BPDF1.2 in 50 ml of liq-
uid medium for 3 days. The PEMs were then
transferred to liquid maintenance medium sup-
plemented with 200 mg l−1 cefotaxime and 500
mg l−1 carbenicillin, and after two weeks were
subcultured into fresh liquid maintenance
medium with 200 mg l−1 cefotaxime, 500 mg
l−1carbenicillin and 3 mg l−1 phosphinothricin
for three months. Embryogenic cultures were
selected continuously with phosphinothricin,
and stained periodically for uidA until they
were apparently completely transformed.
Transformed embryogenic cultures were trans-
ferred to somatic embryo development medium
that contained 3 mg l−1 phosphinothricin.
The shoots from somatic embryos trans-
formed with pdf 1.2 were excised and grafted
in vitro on decapitated ‘Peterson’ seedling root-
stocks (Raharjo and Litz, 2005). Transgenic
shoots were excised and grafted on ‘Peterson’
rootstocks in the nursery, and transformed
scion shoots were later air layered and self-
rooted plants were recovered (Raharjo et al.,
2008). ‘Hass’ plants were resistant to foliar
application of the herbicide Finale®.
An alternative protocol for genotypes,
such as ‘Duke-7’, which show poor prolifera-
tion in liquid media, was developed by Palo mo-
Ríos et al. (2007, 2012). These authors inoculated
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280 F. Pliego-Alfaro et al.
isolated globular somatic embryos of this culti-
var with A. tumefaciens AGL1 strain harbour-
ing the plasmid pBINUBIGUSInt. This binary
vector contains nptII and uidA as marker genes.
Agrobacterium-inoculated embryos were cul-
tured in solid selection medium supplemented
with 50 mg l−1 kanamycin and 250 mg l−1
timentine for a month. Subsequently, kanamy-
cin concentration in the selection medium was
increased up to 100 mg l−1 and 12% of inocu-
lated embryos proliferated in this selection
medium. Transgenic somatic embryos (Colour
Plate 50A,B) and plants expressing the GUS
gene (Fig. 10.5) could be recovered from five
independent embryogenic lines. This transfor-
mation protocol has been used to study the
usefulness of fluorescent proteins as visual
markers during the transformation of avocado
somatic embryos. Palomo-Ríos et al. (2011c)
obtained transgenic embryogenic lines expres-
sing the green (GFP) or red fluorescent protein
under the control of the constitutive promoter
CaMV35S. After three months of culture in the
solid selection medium, all kanamycin resist-
ant somatic embryos showed a level of fluo-
rescence significantly higher than control
embryos. Conversion experiments are currently
in progress to analyze the expression of fluo-
rescent proteins in plants.
In vitro conservation
Genetic diversity within the genus Persea, and
in particular, within the species Persea ameri-
cana is very large (Lahav and Lavi, 2002),
although it is being threatened by the progres-
sive decrease in tropical and subtropical for-
ests. Hence, the maintenance and increase of
existing germplasm collections is urgently
needed, although it is a task hampered by high
costs of land, labour and orchard management.
Moreover, avocado manipulation at the somatic
cell level requires continuous supply of embry-
ogenic material to overcome the decrease in
embryogenic capacity over time; hence, there
is also a need for in vitro conservation methods
for selected embryogenic lines.
Low temperature storage
Vidales et al. (2011) were able to preserve axil-
lary buds of Mexican genotypes (‘Criollo’) for
270 days at 5°C in a medium with low ionic
strengh (0.25–0.5× MS salts); however for
Guatemalan × West Indian hybrids, Valladares
(2005) found a temperature of 12.5°C to be
more suitable. Along this line, Vidoy-Mercado
et al. (2008) found a temperature of 17 °C to be
adequate for conservation of juvenil material
of the Guatemalan genotype ‘Anaheim’ as well
as adult material of PC4 genotype (a selection,
of unknown origin, tolerant to the fungus
R. necatrix); moreover, molecular analysis
using 15 SSR markers indicated that material
was genetically stable after a year storage. Raya
(2004) was able to maintain embryogenic
masses in storage for 150 days at 15°C in a
medium with 0.5× MS mineral elements, 30g
l–1 sucrose and 30g l–1 manitol.
Cryopreservation
Cryopreservation, involving storage of materi-
als int liquid nitrogen (at −196°C), is now a
viable option for most types of plants in germ-
plasm collections and for storing culture col-
lections (Reed, 2008). Therefore, it could be a
very useful tool for backing up field collections
as well as for long-term maintenance of embry-
ogenic cultures.
Attempts to cryopreserve avocado buds of
Mexican genotypes were carried out by Vargas
Fig. 10.5. Six-month-old transgenic plant having
incorporated the GUS gene.
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Biotechnology 281
(2008), who found that the presence of brassi-
nolide in the medium enhanced bud sprouting
after 1 h storage at −20°C. However, more
efforts are needed to establish reliable proto-
cols for avocado bud storage at ultra-low tem-
peratures (Vidales et al. 2011).
Efendi and Litz (2003) described the effect
of cryogenic storage on embryogenic avocado
cultures and the successful recovery of somatic
embryos from cryopreserved cultures. Two pro-
cedures were described: (i) stepwise cooling
(−1°C/min from room temperature to −75°C
followed by rapid cooling to −196°C); and (ii)
rapid cooling ((vitrification) from room tem-
perature to −196°C). In both cases, following
removal of vials from liquid nitrogen and rapid
warming at room temperature, cultures were
thoroughly washed with maintenance medium
and plated on semi-solid maintenance medium.
To induce embryo development, cultures were
transferred to embryo maturation medium.
In a more recent study, Guzmán-García
and Sánchez-Romero (2011a) cryopreserved
embryogenic cultures for 5 months using the
drip-vitrification method (Panis et al., 2005).
They reported a negative effect of cryopreser-
vation on proliferation of embryogenic cul-
tures, and opaque white embryos > 4 mm long
were less abundant following cryopreserva-
tion. Cryopreserved embryos showed a 13%
germination rate versus 5% for control embryos.
In another study, Guzmán-García and Sánchez-
Romero (2011b) found that a 7–14 days 0.3M
sucrose pretreatment prior to cryopreservation
accelerated the initation of cell division and
enhanced proliferation after cryostorage.
Molecular biology
Gene isolation
Many avocado genes have been cloned and
sequenced; most of them have been isolated
from pulp of ‘Hass’ fruit, e.g. cellulase (Cass
et al., 1990), cytochrome P-450 (Bozak et al.,
1990), polygalacturonase (Kutsunai et al., 1993)
and ACC oxidase (McGarvey et al., 1992).
Owino et al. (2002) reported the isolation and
cloning of two ACC synthase genes (PA-ACS1,
PA-ACS2) and a putative ethylene response sen-
sor (PA-ERS1) expressed during fruit ripening
and wounding, and also in response to 1-MCP
and propylene treatments. A fragment of the
ethylene receptor was amplified from seedless
fruit of ‘Arad’ in a study of mesocarp discolora-
tion (Hershkovitz et al., 2009a). An evaluation
of the expression of genes related to ethylene
production during chilling stress by quatitative
real-time PCR (qRT-PCR) was performed by
Hershkovitz et al. (2009b). Cell wall metabo-
lism during fruit growth and ripening is impor-
tant for post-harvest fruit handling. Tateishi et al.
(2007) isolated several b-galactosidase genes
and evaluated their expression in relation to
ethylene production.
In relation to the genetic basis of fruit size,
Dahan et al. (2011) isolated two cDNAs,
PaCYCA1 and PaCYCB1, encoding two mitotic
cyclins in young normal fruit tissues of ‘Hass’
avocado. Accumulation of transcripts of these
genes gradually decreased in average-size fruit
while they decreased earlier in small fruit, con-
comitant with an earlier arrest of cell division.
In contrast, m-RNA levels of Pafw2.2-like, a
negative regulator for cell division, showed
higher accumulation in small fruit compared
with average-size fruit at all growth stages
examined (Dahan et al. 2010).
The disease anthracnose, caused by the
fungal pathogen Colletotrichum gloeosporio-
ides Penz., affects avocado fruit in the orchard;
two genes involved with this infection have
been isolated, a D12 fatty acid desaturase (Wang
et al., 2004) and an ubiquitin conjugating
enzyme (Liu et al., 1998).
Avocado is a ‘palaeopolyploid plant’, an
evolutionary ‘outspot’ among flowering
plants, representing a basal linage (the mag-
nolild clade) near the origin of the flowering
plants (Soltis et al. 1999; Chanderbali et al.,
2008; Soltis et al. 2011; see also Chanderbali
et al., Chapter 3, this volume). This is partially
based on isolation and bioinformatic analysis
of avocado SEPALLATA MADS box gene
homologues PEam AGL9.1 and PEam AGL9.2
(Zahn et al., 2005). The chloroplast matK
sequences have also been used for plant taxo-
nomic classification in the Lauraceae family
(Rohwer, 2000).
Avocado fruit are a source of allergens that
can elicit diverse IgE-mediated reactions,
including anaphylaxis in sensitized individu-
als. Sowka et al. (1998) identified the major
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282 F. Pliego-Alfaro et al.
avocado allergen as an endochitinase protein
encoded by the gene Prs a1.
Differential gene expression analysis
Only a small fraction of the genes present in
the genome of an organism is expressed in a
particular cell type or under particular condi-
tions. The regulation of gene expression during
growth and development, and in response to
biotic or abiotic stimuli, can lead to changes in
the level of accumulation of diverse popula-
tions of transcripts. The characterization of
genes expressed during these different circum-
stances provides invaluable information con-
cerning many biological processes of interest.
The initial efforts to characterize differen-
tially expressed genes were based on differen-
tial screenings of cDNA or genomic libraries.
In these screenings, mRNAs were isolated from
plants exposed to the desired experimental
conditions or from tissues of plants at a particu-
lar developmental stage. These types of
approaches mainly detect cDNAs representing
abundant transcripts. Starret and Laties (1993)
analyzed ethylene and wound-induced genes
in the preclimacteric phase of ripening avo-
cado fruit and mesocarp discs of ‘Hass’. These
authors selected nine clones showing various
patterns of expression during wounding and
fruit ripening. Dopico et al. (1993) cloned and
characterized genes with differential expres-
sion during low temperature storage of ‘Hass’
fruit. Some of the sequenced fragments showed
homology with cystein proteinase inhibitor
(oryzacystatin), polygalacturonase from tomato,
thaumatin-like protein and endochitinase.
Genomics
New technologies and strategies for manipulat-
ing the genome have revolutionized the study
of genetics. Genomics focuses on the analysis,
mapping and sequencing of genomes. Large-
scale sequencing efforts in model organisms
are having a profound impact on the under-
standing of many biological phenomena occur-
ring in living organisms. The sequencing of
cDNA clones has been an important part of
large-scale sequencing projects. The goal of
these efforts is to determine the nucleotide
sequence from the highest number of expressed
genes in an organism. In this strategy, a partial
single-pass DNA sequence is generated from
cDNAs randomly picked from libraries. These
clones, namely ESTs for Expressed Sequence
Tags, have been extremely useful in gene dis-
covery, and their number increases regularly in
model organisms. EST clones are very useful in
the comparative analysis of genes; in many
cases the amino acid sequence deduced from
a new DNA sequence gives a prediction of the
possible function of the gene. EST analysis can
also provide information about the genes that
are highly conserved in plants (Olmedo et al.,
2006). In avocado, there are several large-
scale sequencing projects. Nearly 10,000 EST
sequences from early development of the
flower of ‘Hass’ have been collected by the
Floral Genome Project (Albert et al., 2005) and
85,000 more will be produced as part of the
Ancestral Angiosperm Genome Project (AAGP),
funded by the US National Science Foundation
(Chanderbali et al., 2008; see Chanderbali
et al., Chapter 3, this volume). This information
has been useful for designing flower microar-
rays and performing comparative gene exp-
ression analysis with flowers of different
angiosperms. Chanderbali et al. (2009) are
investigating the ancestral programme of avo-
cado flower development using this methodol-
ogy. In addition, custom microarrays targeting
6,068 genes collected from Persea floral buds
by the Floral Genome Project (Albert et al.,
2005) identified 4,797 significantly differen-
tially expressed genes (P < 0.05) among 8 sam-
pled tissues: inflorescence buds, premeiotic
floral buds, outer tepals, inner tepals, stamens
(including staminodes), carpels, initiating fruit
and leaves (Chanderbali et al., 2009).
The Universidad Michoacana de San
Nicolás de Hidalgo (Michoacán, México) and
the Laboratorio Nacional de Genómica y
Biodiversidad (CINVESTAV-IPN-Unidad
Guanajuato, México) have initiated avocado
fruit EST transcriptomic and genomic projects.
In the ESTs project, cDNA libraries from peel,
pulp and seed of ‘Criollo Mexicano’ have been
generated. Approximately 11,755 clones have
been sequenced by the Sanger method and
compared with the avocado flower data base.
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Biotechnology 283
From the analysis of the seed library, 190 prob-
able contigs and 1463 singletons have been
obtained, whereas the analysis of the pulp
library reported 622 contigs and 2958 single-
tons. In total, 5233 possible unigenes have
been identified (R. López-Gómez, Universidad
Michoacana de San Nicolás de Hidalgo,
Morelia, Michoacán, México, 2012, personal
communication).
The analysis of cDNAs from the pulp fruit
library revealed the presence of some very
abundant transcripts (Table 10.2). The most
abundant sequences match with metallothe-
onein genes. This result suggests that metallothe-
onein genes dominate the avocado fruit
transcriptome, as has been reported for other
fruits, e.g. pineapple (Moyle et al., 2005), mango
(Pandit et al., 2010), banana (Clendennen and
May, 1997) and kiwi fruit (Ledger and Gardner,
1994). Metallotheoneins are involved in metal
homeostasis, and have been reported to increase
during biotic and abiotic stress conditions (Mir
et al., 2004); however, until now, there is no
explanation for the function of metallotheoneins
during fruit development and ripening. An ole-
osin gene is the second most abundant transcript
in avocado fruit. Oleosins are the most abun-
dant oil body-associated proteins (Frandsen
et al., 2001). Previous studies of oil bodies from
mesocarp tissues in avocado and olive (Olea
europaea L.) did not show the presence of this
protein (Ross et al., 1993). This apparent contra-
diction could be due to the different avocado
genotypes used in that work, or that this protein
does not form the structure of the avocado oil
bodies. The abundance of this gene in the mes-
ocarp suggests that it participates in the storage
of triacylglycerides in this tissue. A gene encod-
ing an acyl carrier protein is also present at high
levels in the avocado pulp library. The presence
of these genes suggests strong lipid metabolism
in avocado fruit. Pathogenesis related genes,
such as endochitinase and protease inhibitor,
are also abundant in the fruit library. It has
been reported that endochitinase and chitinase
are abundant messengers in banana fruit
(Clendennen and May, 1997), where they are
involved with fruit softening. Other genes
related to metabolism are expressed abundantly
in avocado fruit, such as fructose biphosphate
aldolase, polyubiquitin, photosystem II and
endopeptidase (Table 10.2).
Two putative transcription factors, the
Ethylene Response Factor (ERF) and the Auxin-
repressed protein-like protein, have also been
identified in avocado fruit ESTs. These two
genes have been reported during the ripening
of different fruit, and it has been suggested that
they can act as crossroads between ethylene
and auxin during fruit ripening and develop-
ment (Reddy and Poovaiah, 1990; Wang et al.,
2007; Trainotti et al., 2007; Chaabouni et al.,
Table 10.2. Some of the most abundant genes expressed in criollo avocado fruit (Persea americana var
drymifolia).
EST-unigene Abundance E Value Plant species
Metallotheonein type 1 322 0 Persea americana
Metallotheonein type 2 316 1e−20 Arachis hypogea
Metallotheonein type 3 117 9e−22 Populus balsamifera
Oleosin 135 7e−15 Theobroma cacao
Endochitinase 106 0 P. americana
Fructose biphosfate aldolase 60 0 P. americana
Polyubiquitin 29 9e−124 Picea sitchensis
Photosystem II Protein 26 5e−32 Eucalyptus globulus
Universal stress protein (ERF) 26 6e−45 Populus trichocarpa
Auxin-repressed protein-like protein 23 3e−33 Manihot esculenta
Acyl carrier protein (chloroplast) 23 1e−42 Camellia oleifera
Peptidase, putative 17 3e−30 Ricinus communis
B12D- like protein 17 7e−38 Wolffia arrhiza
Catalase 16 6e−156 Mesembryanthemum
crystallinum
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284 F. Pliego-Alfaro et al.
2009). The B12D-like protein gene is another
messenger founded in mesocarp; this is a pro-
tein of unknown function that has been iso-
lated from senescing sweet potato (Huanga
et al., 2001). Fruit ripening is considered by
some authors to be a specialized form of senes-
cence (Seymour et al., 1993). Antioxidant sys-
tems (López et al., 2010) have an important
role in both senescence and fruit ripening,
including enzymes such as superoxide dis-
mutase, catalase and peroxidase. Catalase is
abundantly expressed in avocado mesocarp.
Recently, platforms for massively parallel
DNA sequencing such as Solexa/Illumina
(Bennett et al., 2005), 454 Life Sciences
(Margulies et al., 2005) and the Applied
Biosystems SOLiDTM platforms (Picardi et al.,
2010) have been developed. These systems have
dramatically changed the way to investigate
the functional complexity of transcriptomes
(Delseny et al., 2010). These technologies are
also powerful for identification of genes, struc-
ture of transcripts, non-coding RNAs and alter-
native splicing. Transcriptomic analysis of Persea
americana var. drymifolia was focused on study
sets of genes involved in several physiological
processes, e.g. leaf and root growth and devel-
opment, flower development, and fruit ripening
(E. Ibarra-Laclette Langebio, CINVESTAV-
Unidad Guanajuato, México, 2012, personal
communication). Additionally, leaf and flower
transcriptomes of Hass and Bacon cultivars have
been generated for SNP identification using sec-
ond generation sequencing (Kuhn et al., 2011).
Djami-Tchatchou and Straker (2011) have
undetaken the first transcriptome analysis of
the interaction between avocado fruit and
Colletotrichum gloeosporioides. Many catego-
ries of genes predicted to function in meta-
bolism, signal transduction, transcriptional
control, defence, stress and transportation
processes have been identified. The overall
goal isto select candidate genes differentially
expressed in avocado fruit as result of C. gloe-
osporioides infection. In a similar study
Mahomed et al. (2011) sequenced ESTs from
Phytophthora cinnamomi infected avocado
root cDNA libraries; approximately 20 putative
defence related genes have been identified
among which a metallothionein, a thaumantin
and the pathogenesis related protein Psmel
were found to be differentially regulated.
The Avocado genome sequencing project
The avocado genome consists of 12 chromo-
somes with a genome size of proximately 980
million base pairs, which appears to be a
medium sized plant genome when compared
to other species, e.g. wheat (15,000 Mb) or lil-
ies (120,000 Mb) (Sharon et al., 1997).
As part of a hierarchical whole sequenc-
ing strategy, a library of bacterial artificial chro-
mosome (BAC) clones has been constructed
and fingerprinted (L. Herrera-Estrella, Langebio
CINVESTAV, Unidad Guanajuato, México,
2012, personal communication). The BAC
library comprised 92,160 clones with an aver-
age insert size of 122 kbp, representing an esti-
mated 12-fold redundant coverage of the
genome. This BAC library was constructed by a
team at the Arizona Genomics Institute. Using
the end sequences and fingerprints from 31,629
clones a physical map was constructed to sup-
port the genomic sequencing effort. This strat-
egy will be complemented by whole genome
shotgun sequencing approaches using both
Roche Genome Sequencer (GS XL–Titanium
and GS XL+ platforms) and SOLiD sequencer
instruments.
To generate de novo avocado genome
draft, conventional, BACs paired-end (PE)
Sanger reads, single-end (SE) and PE 454 reads
from Roche GS XL–Titanium and/or GS XL+
sequencers, as well as PE reads from SOLiD
sequencer, were combined. To date, 55,824
Sanger PE sequences (approximately 0.03-fold
genome sequence coverage) have been gener-
ated. Additionally, fragmented genomic DNA
was used to generate 40 read sets on Roche
GS-Titanium and GS-Plus sequencers, produc-
ing 50,430,834 reads with 340.35 and 543.87
bp average lengths, respectively (19.8 Gpb of
sequence data; approximately 20.2-fold
genome sequence coverage). These data sets
were supplemented with 16,331,539 PE 454
reads (approximately 4.5-fold genome
sequence coverage; sequencing libraries were
constructed with insert sizes of 3, 5 and 8 kbp).
Finally, 50-pb PE reads (125,213,740) for a sin-
gle library of approximately 2000-bp sheared
DNA fragments were obtained using the SOLiD
system (approximately 6.2 Gpb of sequence
data; 6.4-fold genome sequence coverage). In
total, reads collection represent ~30-fold
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Biotechnology 285
redundant coverage of the Persea amricana
var. drymifolia genome (L. Herrera-Estrella,
Langebio CINVESTAV, Unidad Guanajuato,
México, 2012, personal communication). An
estimation of the number of genes represented
in the avocado draft assembly, using a combi-
nation of different annotation pipelines, sug-
gests that the avocado genome has about
50,000 genes.
Since avocado is considered among the
basal angiosperms, the functional and struc-
tural analysis of the avocado genome will help
provide a better understanding of plant evolu-
tion, as well as the molecular processes that
regulate fleshy fruit development and other
important traits. The avocado genome will also
facilitate the development of genomic tools for
the detailed characterization of the existing
genetic diversity of this economically impor-
tant plant species and to establish more effec-
tive breeding strategies to identify genes
important for the development of new avocado
selections with increased productivity, better
quality and nutritional fruit value, pathogen-
resistance and tolerance to different types of
adverse environmental conditions.
Potential for Biotechnology
in Avocado Improvement
In vitro mutagenesis and selection
Disease control
Phillips et al. (1991) established callus from
different tissues of avocado cultivars differing
in their susceptibility to PRR, e.g. ‘Topa-Topa’
(susceptible), ‘Duke-7’ (fairly resistant) and
‘Martin Grande’ (moderately resistant).
According to these authors, in vitro responses
following inoculation with Phytophothora cin-
namomi were quite similar to those observed
in the field. Moreover, although no clear differ-
ences in infection rate could be detected
between the two tolerant cultivars, in both
cases a hypersensitive type of reaction was
observed, e.g. rapid necrosis occurred in cells
surrounding the inoculated area. That report
clearly showed that resistance to PRR operates
at the cellllular level.
Efficient in vitro selection can be obtained
when fungal toxins or pathogen culture filtrates
are used as selective agents and these agents
cause symptoms at the cellular level (van den
Bulk, 1991). The most efficient way to proceed
with such studies would involve induction of
mutations in vitro followed by in vitro selec-
tion, i.e. irradiation of embryogenic avocado
cultures and selection for resistance to cinnam-
omin (Nespoulous et al., 1992) or P. cinnamomi
filtrates (Witjaksono, 2000). This approach has
been successfully used in apple to obtain
increased resistance to Phytophthora cactorum
(Rosati et al., 1990).
Somatic hybridization
In the graft-incompatible (with sub-genus
Persea) Persea, sub-genus Eriodaphne, the spe-
cies P. borbonia, P. cinnerascens and P. pachy-
poda show complete resistance to infection by
Phytophthora cinnamomi. Working with
Brassica napus, Sjödin and Glimelius (1989)
transferred resistance to Phoma lingam by
asymmetric somatic hybridization. The reports
by Witjaksono (1997) and Witjaksono et al.
(1998) on avocado protoplast regeneration
could serve as the basis to fuse resistant geno-
types of subgenus Eriodaphne with cultures
derived from sensitive or partially tolerant gen-
otypes of subgenus Persea.
Genetic transformation
Several fungal avirulence genes have been
cloned (van den Ackerveken et al., 1992;
Joosten et al., 1994; Valent and Chumley, 1994;
Stuiver, 2006). They share common traits and
appear to be involved in signal transduction
cascades where protein-protein interactions
and dephosphorylation events take place.
These observations suggest that plants might
have developed transduction signals, which
are common to a wide range of pathogens. This
knowledge could be used to design synthetic
resistance genes capable of responding to dif-
ferent pathogens (de Wit, 1995).
NPR1 is a key regulatory gene of the sali-
cylic acid-mediated systemic acquired resist-
ance in Arabidopsis (Cao et al., 1997). In
different species, it has been shown that consti-
tutive expression of AtNPR1 in transgenic
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286 F. Pliego-Alfaro et al.
plants induces a higher production of
pathogen-related genes and also resistance to
a wide range of different pathogens (Cao
et al., 1998; Lin et al., 2004; Wally et al., 2009).
Palomo-Ríos et al. (2010, 2011a) obtained
several transgenic avocado embryogenic lines
containing the NPR1 gene from Arabidopsis
thaliana in an attempt to recover plants that are
resistant to the fungus Rosellinia necatrix.
Hydrolytic enzymes, e.g. b-1-3 glucanase
and chitinase, play an important role in consti-
tutive and inducible defence reactions due to
their role in degradation of fungal cell walls
(Broglie et al., 1991). Punja and Raharjo (1996)
enhanced tolerance of several fungal patho-
gens in carrot using a chitinase gene from
tobacco, but they were unsuccessful when a
petunia chitinase gene was used. They con-
cluded that the type of chitinase protein
expressed, the fungal pathogen and the plant
species can greatly affect the final response.
The expression of antifungal proteins in trans-
genic plants, such as the AFP from Aspergillus
giganteus, is an alternative to the use of hydro-
lytic enzymes. AFP is secreted by Aspergillus
giganteus and shows a potent antifungal activ-
ity against filamentous fungi. The expression of
this gene enhanced fungal resistance in trans-
genic rice plants (Coca et al., 2004). In avo-
cado, Palomo-Ríos et al. (2011b) obtained ten
embryogenic lines transformed with the AFP
gene; attempts are now in progress to obtain
regenerants from these lines. Mitter et al. (2011)
are attempting transformation of avocado
embryogenic cells with hairpin RNA constructs
targeting Phytophthora cinnamomi essential
genes, as a new approach to control this impor-
tant avocado disease.
Viroid resistance
Avocado sunblotch viroid (ASBVd) is a serious
problem in some areas, and no resistance to
this disease occurs in avocado populations (see
Dann et al., Chapter 13, this volume). The
expression of double-stranded RNA-specific
ribonucleases in transgenic plants has been an
efficient approach to enhance plant resistance
to viroids, and also to some viruses in potato
(Sano et al., 1997), tobacco (Ishida et al., 2002)
and chrysantemum (Ogawa et al., 2005). In
avocado, Litz et al. (2010) demonstrated that
transformation of ASBVd-infected avocado
with the pac1 ribonuclease gene from
Schizosaccharomyces pombe is effective in
eliminating the viroid.
Insect control
There are currently two approaches for engi-
neering increased insect resistance in plants:
genes from Bacillus thuringiensis coding for a
toxin active against insects, mainly Lepidoptera
(Barton et al., 1987; Fujimoto et al., 1993;
Nickson, 2005), and plant genes involved in
inhibition of insect proteases (Hilder et al.,
1987) or a-amylases (Altabella and Chrispeels,
1990). However, transgenic plants expressing
protease inhibitors are not very effective against
insect pests, likely due to the low potency of
protease inhibitors and to insect adaptation
(Christou et al., 2006). In avocado, there are
four important lepidopteran pests; Amorbia
cuneana (western avocado leaf roller),
Sabulodes aegrotata (omnivorous loopers),
Stenoma catenifer (avocado seed borer) and
Cryptoblades gnidiella (honeydew moth) (see
Peña et al., Chapter 14, this volume). To control
the latter, Wysoki et al. (1975) used prepara-
tions of Bacillus thuringiensis (Bt) as a biologi-
cal insecticide. Transformation of avocado with
Bt genes could also be an useful tool for
enhancing tolerance of lepidopteran pests;
however, the genetic transformation of peren-
nial species with Bt genes has not been evalu-
ated for its potential impact on the development
of insect resistance over time.
Dwarfing rootstocks
Use of high density avocado plantings is an
interesting option for increasing fruit produc-
tion, especially in areas where tree vigour is
low (Winer, 2007), with the added advantage
that harvesting is cheaper from compact trees.
However, tree size reduction is a key compo-
nent of this type of planting; e.g. in avocado,
the use of mechanical pruning and treatments
with compounds inhibiting GA biosynthesis
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Biotechnology 287
are methods used to achieve this goal and are
costly (see Hofman et al., Chapter 15, this
volume).
Biotechnological techniques could be
useful to obtain rootstocks with a dwarfing
growth habit. The characterization of key
enzymes in GA biosynthesis linked to the con-
served function of these enzymes between spe-
cies has enabled the level of bioactive GAs in
crop species to be altered. Consequently, plant
height can be modified (Hedden and Phillips,
2000). Several approaches have been used to
achieve this goal, e.g. Fagoaga et al. (2007)
transformed the citrus hybrid rootstock Carrizo
citrange with antisense constructs of Cc GA
20-oxidase and resulting plants showed a dwarf
phenotype. In addition, they showed a bushy
growth habit suggesting a possible role of GAs
in auxin biosynthesis and/or transport. In pop-
lar however, Busov et al. (2003) used sense
transformation with GA2ox, a key enzyme cat-
alyzing GA1 deactivation and hence reducing
levels of active GA1; Transgenic plants showed
great reduction in size although, in this case,
the dwarf phenotype was reversible by apply-
ing GA to the shoot apex. More recently, Zhu
et al. (2008) significantly reduced the size of
apple plants following transformation with the
Atgai (gibberellic acid insensitive) gene. In the
avocado, only the rootstock Colín V-33
(Sánchez-Colín and Barrientos-Priego, 1987)
has been selected for dwarfing habit (in Mexico)
and therefore, alteration of GA metabolism in
the rootstock could result in smaller tree size
and more intensive plantings.
Cold hardiness
Acclimatization of plants to low temperatures
has been associated with the increased expres-
sion of certain genes (Guy, 1990). The resulting
polypeptides could be acting as cryoprotect-
ants (Lin et al., 1990). In cyanobacteria, Wada
et al. (1990) improved chilling tolerance
through modification of fatty acid saturation.
Kodama et al. (1994) enhanced cold tolerance
of tobacco following transformation with a
fatty acid desaturase gene. Another approach
has been the introduction of antifreeze protein
genes obtained from polar fish (Hightower
et al., 1991). The expression of these proteins
seems to inhibit crystal formation. In avocado,
moderate cold hardiness is a trait of the
Mexican race. West Indian race genotypes are
very cold-sensitive and those of Guatemalan
race show an intermediate response (see
Schaffer et al., Chapter 7, this volume).
In Arabidopsis thaliana, the molecular
basis of cold acclimation involves a common
cis acting regulatory element, the C repeat/
Dehydration responsive element that has the
conserved core sequence CCGAC. This ele-
ment is present in one or more copies in the
promoters of many cold-induced genes, includ-
ing COR 6.6, COR 15a, COR 47 and COR 78
of A. thaliana and BN 115 of Brassica napus
(Baker et al., 1994; Stockinger et al., 1997;
Yamaguchi-Shinozaki and Shinozaki, 1994).
All COR genes from A. thaliana are coordi-
nately upregulated by CBF 1, CBF 2 or CBF 3,
a family of cold- and drought-inducible tran-
scriptional activators that bind to promoters
containing a CRT/DRE element (Jaglo-Ottosen
et al., 1998; Kasuga et al., 1999; Liu et al.,
1998; Thomashow, 2001). Although freezing
tolerance is a complexly inherited trait, manip-
ulation of a single gene (CBF1) has been shown
to improve whole plant freezing tolerance of
Arabidopisis (Jaglo-Ottosen et al., 1998). CBF
genes are key regulators of cold acclimation
and over expression of CBF 1, 2 or 3 can
improve the freezing tolerance of Arabidopsis.
Dhekney et al. (2007) transformed papaya
(Carica papaya L.) with C-repeat binding factor
genes to enhance cold-tolerance in this spe-
cies. Increasing cold tolerance through genetic
engineering of Guatemalan and West Indian
genotypes could extend the growing area for
these types of avocados.
Fruit shelf-life
Fruit of Mexican and Guatemalan cultivars can
be stored on the tree for up to 4 months after
reaching maturity, thereby facilitating handling,
transportation and marketing strategies (Whiley,
1992). Ripening is initiated only after harvest-
ing. ‘Hass’ now accounts for about 90% of
world avocado export market (see Crane et al.,
Chapter 8, this volume) and fruit is available for
most of the year. Avocado fruit can ripen on the
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288 F. Pliego-Alfaro et al.
tree and are often not easily. Therefore tropical
production areas must rely on several cultivars
in order to provide fruit year round. Avocado is
a climacteric fruit, and therefore control of eth-
ylene biosynthesis can extend fruit shelf-life
(Meyer and Terry, 2010; Zhang et al., 2011; see
Hofman et al., Chapter 15, this volume).
ACC synthase, a key enzyme in ethylene
biosynthesis, has been cloned in several spe-
cies (Bapat et al., 2010). Tomato plants that
have been transformed with antisense ACC
synthase show about 99% inhibition of ethyl-
ene synthesis compared with nontransformed
plants (Klee et al., 1991). Exegesis (formerly
Agritope, Beaverton, Oregon, 1997, personal
communication) utilizes S-adenosylmethionine
hydrolase (SAMase) to convert SAM to a non-
toxic by-product that is recycled within the
plant cell, so that SAM cannot be converted to
ACC. Bacterial ACC deaminase causes ethyl-
ene synthesis to be reduced by 90–97% in
tomato (Klee et al., 1991; Klee, 1993).
Efendi (2003) described the transforma-
tion of avocado embryogenic cultures with
S-adenosylmethionine hydrolase (samase) to
block ethylene biosynthesis in Antillean (West
Indian race) avocados with the goal of control-
ling fruit ripening and promoting on-the-tree fruit
storage. These transgenic plants are under obser-
vation in a screened enclosure, and have not
flowered (R.E. Litz, University of Florida, Tropical
Research and Education Center, Homestead,
Florida, 2011, personal communication).
Shortening the juvenile period
Insight into the molecular basis of flowering has
resulted in the potential for shortening the juve-
nile period of woody plants. For example, Peña
et al. (2001) transformed citrus with the AtLFY
gene. Regenerated plants showed precocious
flowering; however, the apple homologue was
ineffective with this species (Kotoda et al.,
2003). Other genes involved in precocious
flowering, such as AP1 have been introduced
into woody plants with controversial results.
The AtAP1 also induced precocious flowering
in citrus (Peña et al., 2001), while its apple
homologue MdAP1 over-expressed in ‘Fuji’
apple and drastically reduced the juvenile
period (Kim et al., 2006), while it had no effect
on ‘Orin’ apple (Kotoda et al., 2003).
The FT protein (Flowering locus T) is one
of the main components of the so-called
‘florigen’ in Arabidopsis (Kobayashi et al.,
1999). The FT protein moves from leaves to
the apex interacting with FD protein and acti-
vates flower identity genes such as AP1 (Abe
et al., 2005). Transformation of Poncirus trifoli-
ata (Endo et al., 2005) and apple (Tränkner
et al., 2010) with CtFT and MdFT genes,
respectively, induced early flowering in these
species. The use of these early flowering geno-
types in conventional breeding programmes
linked to successive back crosses with the gen-
otype of interest could be a very interesting
tool. Using this approach, genotypes of succes-
sive progenies are selected for the trait of inter-
est, e.g. fungal resistance and early flowering,
while in the last cross genotypes showing fun-
gal resistance and late flowering phenotype are
selected. Using this approach Lewis and
Kernodle (2009), were able to introduce patho-
gen resistance into tobacco in a much shorter
period than what is required using conven-
tional approaches. So far, in the avocado the
AtLFY gene has been introduced to promote
early flowering (I. Perea and R.E. Litz, University
of Florida, Tropical Research and Education
Center, Homestead, Florida, 2011, unpub-
lished data).
Conclusions
Biotechnology could become very powerful in
avocado breeding during this decade.
Micropropagation of shoots via axillary branch-
ing is being used in breeding programmes for
fungal resistance to generate a limited number
of copies of selected genotypes during the
selection process, although it cannot yet be
used commercially. Moreover, very significant
improvements have been obtained in embryo
rescue techniques, thereby allowing efficient
recovery of genotypes from selected crosses.
Recent advances in avocado genomics
have allowed collection of nearly 10,000 EST
sequences from early development of the avo-
cado flower. Mexico has initiated the EST avo-
cado fruit and the avocado transcriptome
projects, resulting in the identification of a total
Schaffer_Ch10.indd 288Schaffer_Ch10.indd 288 9/18/2012 7:02:22 AM9/18/2012 7:02:22 AM

Biotechnology 289
of 5233 unigenes. Some of the most abundant
genes expressed in criollo avocado fruit have
been identified; among them are metallothe-
oneins involved in metal homeostasis, two
putative transcription factors, Ethylene
Response Factor and Auxin-repressed protein-
like gene, as well as pathogenic related genes,
i.e. an endochitinase and a protease inhibitor.
Other genes involved in the interactions of
avocado fruit with the fungus Colletotrichum
gloeosporioides as well as the interaction of
avocado root with the pathogen Phytophthora
cinnamomi have also been identified; these
studies will facilitate the design of more effi-
cient strategies to manage these important
diseases.
Establishment of embryogenic lines from
immature zygotic embryos is now routine.
However, efforts are needed to improve embry-
ogenic culture induction in explants of adult
origin such as nucellus. Methods for transform-
ing avocado embryogenic cultures are currently
available and, in some cases, plants have been
recovered from transgenic embryos, although
the methodology to convert transformed
somatic embryos into plants needs to be
improved. Continuing advances in gene clon-
ing linked to improvements of avocado regen-
eration will be key factors for improving this
important crop species using biotechnology.
Use of currently available cryopreserva-
tion techniques could be useful for preserving
selected embryogenic cell lines, and avoiding
the loss of embryogenic competence which
occurs with time. Efforts are needed to apply
these techniques to avocado shoot tips or
embryonic axes, in order to use this material as
backup for avocado germplasm collections
and to facilitate international movement of
important genetic resources, particularly from
the great genetic repositories of the USA, Israel,
Mexico and Australia.
Acknowledgments
Support provided by research projects AGL2008-
05453-C02-01 and AGL 2011-30354-C02-01
(PLAN NACIONAL I+D+I, MEC, Spain), the
California Avocado Commission and the USDA
TSTAR Special Grants programme (USA),
Campo Experimental Uruapan CIRPAC-INIFAP
Michoacan, Consejo Nacional de Ciencia y
Tecnologia (CONACYT-México) research
projects MOD-ORD-03-07 000126262 and
53062 as well as Coordinación de la Investigación
Científica-Universidad Michoacana de San
Nicolás de Hidalgo research project 2.2 (México),
is gratefully acknowledged.
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