In vitro and in vivo methods of propagation towards ex situ conservation

Afr. J. Hort. Sci. (2009) 2:1-12

1
IN VITRO AND IN VIVO METHODS OF PROPAGATION TOWARDS
EX SITU CONSERVATION OF MACADAMIA (Macadamia spp.)
GERMPLASM IN KENYA

Gitonga, L.
1
, Kananga, E.
2
, Ngamau, K.
2
, Muigai, A.W.T.
2
, Gichuki, S.T.
1

and Njogu, N.
1
1
Kenya Agricultural Research Institute, P. O. Box 57811-00200, Nairobi
2
Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-
00200, Nairobi
Corresponding author e-mail: lucygitonga2000@yahoo.com

ABSTRACT
Macadamia is an important nut crop in Kenya grown mainly for export. It is
an attractive cash crop especially among small-scale farmers due to its low
requirement for external inputs. With new superior varieties, farmers replace
old plantations with new varieties. However, old plantations still contain
untapped genetic potential and hence the need for ex situ conservation. Since
it is highly out-crossing, true-to-type clones are propagated through
vegetative means. A study was carried out to evaluate the effectiveness of
cuttage, graftage and direct and indirect regeneration tissue culture
techniques across 39 accessions covering two Macadamia spp. (M.
integrifolia and M. tetraphylla) and the natural (M. integrifolia x M.
tetraphylla) hybrids. Results indicated that cuttings could be used, but took 9
to 15 months before whole plants could be obtained and also the success rate
varied with genotype. All genotypes were amenable to grafting, but with
varying success and the highest (100%) bud break was obtained in
accessions related to M. integrifolia. Direct regeneration from nodal
segments was achieved with up to 85% bud break and shoot multiplication of
up to 12 shoots per explant, while callus induction was possible from
medium to mature cotyledonary tissue in some accessions. Although these
studies are still ongoing, it can be recommended that grafting of seedlings
with the accessions to be conserved and planting them closely in a small field
is an effective and cost-effective method of conserving the material for
several years.

Key words: Conservation, Ex situ, Grafting, In vitro, In vivo, Macadamia

INTRODUCTION
Macadamia is a dark-green, spreading, semi-hard wood tree that belongs to
the family Protaceae of which about 1000 species exist, including the
Banksias, Grevilleas, Stenocarpus, Dryandra, Hakea and Telopea
(McConachie, 1995). Macadamia is rich in unsaturated fatty acids
Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

Afr. J. Hort. Sci. (2009) 2:1-12
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(Macfalane and Harris, 1981) and thus is considered as a healthy food
product as it keeps blood cholesterol levels in check (Onsongo, 2003). It can
be eaten raw, fried, roasted and salted for use as a dessert (Duke, 1983), or as
an ingredient in various confectionery products (Bittenbender and Hirae,
1990; Yokoyama et al., 1990). Oil is also extracted and used in salads and
cooking (Macfalane and Harris, 1981; Duke, 1983) or as a lubricant and in
the manufacture of cosmetics (Bittenbender and Hirae, 1990; Yokoyama et
al., 1990; Onsongo, 2003). The seed cake that remains after oil extraction is
used as a constituent of livestock feed (Woodloof, 1967). Husks are used as
mulch or manure (Bittenbender and Hirae, 1990; Yokoyama et al., 1990),
while broken hard shells are used as fuel in homes and for charcoal-making
(Rosengarten, 1984). Rumsey (1927) also recommends the tree for timber
and ornamental purposes.

Macadamia is indigenous to the subtropical coastal region of Australia
(Storey and Salleeb, 1966). To date, other countries growing macadamia
include Hawaii as the worlds second largest producer after Australia, South
Africa, Kenya, Malawi, Zimbambwe, Guatemala, Brazil, Costa Rica and Fiji
in order of decreasing level of production (Kiuru et al., 2004). Two species;
M. integrifolia and M. tetraphylla are commercially grown and natural
hybrids are also found in macadamia growing areas. Macadamia was first
introduced in Kenya in 1946, but commercial production started in 1968.
Planting materials were mainly distributed in Central, Eastern and Coast
provinces, but the industry did not pick up as expected due to high variability
in yield and quality of nuts. National surveys in 1971 and 1977 revealed high
genetic potential in the existing germplasm in farmers fields that needs to be
conserved for further improvement (Kiuru et al., 2004).

Germplasm conservation can be done in situ or ex situ. In situ conservation
on farm is the continuous cultivation and management by farmers of a
diverse set of populations of a crop in the environment where the crop has
evolved (IPGRI, 2001). However, conservation in farmers fields is
challenged by introduction of newer varieties and several biotic and abiotic
stresses (Vicente et al., 2006). On the other hand, ex situ conservation
(conservation of germplasm outside its natural habitat) is needed to augment
conservation in situ. Techniques of ex situ conservation include gene banks,
seed banks and in vitro methods. Seed banking, the most commonly used
method for ex situ conservation of plant genetic resources accounts for 90%
of the six million accessions conserved globally (IPGRI, 2001). However,
macadamia is highly (>75%) out-crossing (Sedgley et al., 1990) and
therefore seed conservation is of little value. Hence, vegetative propagation
methods are highly recommended for production of true-to-type clones that
In vitro and in vivo methods of propagation towards ex situ conservation
Afr. J. Hort. Sci. (2009) 2:1-12
3
can then be conserved ex situ (IPGRI, 2001). The general objective of the
present study was to develop a method of propagation of specific macadamia
genotypes, while maintaining clonal fidelity of the original genotypes for the
purpose of ex situ conservation. The specific objectives involved evaluating
the response of different macadamia genotypes to graftage, cuttage and tissue
culture techniques for efficient propagation of macadamia.

MATERIALS AND METHODS
Evaluation of Grafting Method on Macadamia Accessions
Scions were obtained from 39 accessions representing M. integrifolia, M.
tetraphyllas and natural M. integrifolia x M. tetraphylla hybrids. The scions
were treated with a fungicide and kept at 4C for 5-10 days before grafting
on 9-15-month-old rootstocks. The cold treatment makes all buds dormant
such that after exposure to room temperature after grafting, bud breaking is
uniform. Single-node scions with leaves trimmed to half were grafted using
top wedge method. The graft union and all cut surfaces were sealed using
candle and bees wax to minimize desiccation. The grafted seedlings were
tended under greenhouse conditions (25C-28C, 85%-100% relative
humidity and 50% light reduction) for three months followed by 50% shade
for one month. Thereafter graft take percentage, number of shoots per
seedling and length of each shoot were recorded. Each treatment consisted of
five seedlings replicated twice.

Evaluation of Cutting Method on Macadamia Accessions
Single-node cuttings from 39 accessions were set in moist sand in
greenhouse tunnels or in pots after cold treatment and covered with
polythene sheet and maintained under greenhouse conditions for 3 months.
Each treatment consisted of five cuttings replicated twice from which
sprouting percentage and rooting frequency were recorded.

Evaluation of Regeneration through Direct Organogenesis
Explants were obtained from shoots of 15-year-old field-grown trees of
KRG-15, MRG-20 (M. integrifolia), and KMB-3 (M. hybrid). Explants were
washed in tap water with a few drops of Tween 80 and rinsed under running
tap water for at least 1 hour, dipped in 70% ethanol for 15 seconds and rinsed
5 times (3-5 minutes each with agitation) using sterile distilled water.
Explants were then sterilized in 10% Jik (3.5% NaOCl equivalent to 0.35%
pure NaOCl) for 10 minutes, rinsed 5 times with sterile distilled water and
air-dried in a laminar-air flow hood before inoculation.

Explant regeneration was initiated on hormone-free Murashige and Skoog
(1962) (MS) medium gelled with 9 g/L Biotec Agar and pH adjusted to
Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

Afr. J. Hort. Sci. (2009) 2:1-12
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5.7. Explants were maintained under growth room conditions (262C, 45-
55% relative humidity, 16 hours photoperiod of about 50 molm
2
s
1
light
intensity provided by cool white fluorescent bulbs) for 4 weeks after which
bud break frequency and number of shoots per explant were recorded. Each
treatment consisted of 25 nodal segments replicated twice and arranged in a
completely randomized design.

Evaluation of Regeneration through Indirect Organogenesis
Regeneration of plantlets from somatic embryos through somatic
embryogenegesis was done using KRG-15 as a test variety. Nuts were
cracked to obtain cotyledons. Zygotic embryos were excised and discarded.
Cotyledons were sectioned into 0.5 cm cubes and initiated on MS medium
gelled with 9 g/L Biotec Agar and pH adjusted to 5.7. They were
maintained under growth room conditions (262C, 45%-55% relative
humidity) for 4 weeks. The age of cotyledons was compared to include
young (nuts one month after anthesis), medium (3 months after anthesis with
the shell starting to harden) and mature (harvested nut with hard brown shell
with or without nut already detached from shell). The effect of five levels of
2,4-dichlorophenoxyacetic acid (2,4-D) concentration (0, 0.5, 1, 2, and 4
mg/l) was evaluated.

Cotyledon cubes incubated on hormone-free medium were transferred to MS
medium supplemented with 2,4-D at 0.5, 1, 2 or 4 mg/l and maintained under
growth room conditions. After callus formation, calli were transferred to half
strength MS medium supplemented with different levels of 6-
benzylaminopurine (BAP) alone or in combination with indole butyric acid
(IBA) or 2,4-D to initiate shooting. Each treatment consisted of 4 cotyledon
sections replicated 5 times and arranged in a completely randomized design.
Greening and callusing frequency was recorded after which callus size was
scored after transfer of calli to different media. All data were subjected to
analysis of variance using SAS (SAS, 2001) and means were separated using
the SNK test at = 0.05.

RESULTS AND DISCUSSION
Effect of Graftage
Successful graft take were obtained in all accessions but with significant
differences in take percentage (Table 1). Graft take ranged from 20% to
90%, while number of shoots ranged from 1 to 4. Number of internodes for
the longest shoot obtained from a scion, as an indication of shoot growth also
ranged from 1 to 4. Length of the longest shoot ranged from 0.5 cm to 22
cm. Graft take was not consistent with Macadamia spp., and it depended on
In vitro and in vivo methods of propagation towards ex situ conservation
Afr. J. Hort. Sci. (2009) 2:1-12
5
season of grafting. However, the general trend was that M. integrifolia had
higher graft take than M. tetraphylla and M. hybrids.

Table 1: Graft take percentage, shoots per scion and internodes of
longest shoot for some Macadamia accessions four months
post-grafting under greenhouse conditions
Accession Morphological
classification
Graft take
(%)
Number of
shoots/scion
Number of
internodes
MRG-25 M. integrifolia 90 2.3a 4.3a
EMB-1 M. integrifolia 90 2.2ab 3.8ab
HAES 333 M. integrifolia 90 2.2ab 4.0a
HAES 508 M. integrifolia 80 2.1ab 2.9ab
EMB-2 M. integrifolia 80 1.9abc 3.4ab
EMB-A M. integrifolia 60 1.5abc 2.8ab
MRG-2 M. hybrid 60 1.0abc 2.1ab
MRU-25 M. hybrid 80 1.6abc 3.7ab
EMB-H M. hybrid 60 0.6bc 2.2ab
KMB-4 M. hybrid 40 0.8abc 1.0b
EMB-T4 M. tetraphylla 90 1.5abc 2.5ab
EMB-T3 M. tetraphylla 80 2.2ab 2.2ab
EMB-T2 M. tetraphylla 50 0.6bc 2.1ab
Means followed by the same letter within a column are not significantly
different at P=0.05, according to the SNK test.


Scions of most of the seedlings grafted on accessions related to M.
tetraphylla KRG-T1, KRG-T2 and KRG-T3 started by producing
inflorescences from which leaflets were later produced ending up in shoots
with shorter internodes than normal (Figure 1). This phenomenon which may
be associated with ecological adaptation was also observed in mature trees of
the same accessions.

Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

Afr. J. Hort. Sci. (2009) 2:1-12
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Figure 1: Macadamia tetraphylla cultivar KRG-T1 showing: a)grafted
seedling with normal shoot, b) grafted seedling with shoot that
was initially an inflorescence showing short internodes and an
inflorescence starting to produce leaves, c) twig of a mature tree
that was initially an inflorescence that later produced leaves.

Species differences in graft take success has been reported by Ryan and
Ryan (1956). Leigh (1973) also reported in his studies on macadamia
propagation in New South Wales, that M. integrifolia grafted satisfactorily
when leaves were trimmed to a short petiole, while in the case of M.
tetraphylla, scion wood that had defoliated naturally was preferred. This may
explain why M. tetraphylla had relatively low percent takes since all scions
were treated similarly. Accumulation of adequate starch reserves in both the
rootstock and the scion is essential for successful graft take. Seasonal change
in carbohydrate build up in macadamia has been reported (Kadman, 1982).
This could explain the differences in percent graft take since different
accessions have different phonological cycles accumulating optimum
carbohydrate levels at different times of the year (Ondabu et al., 1996).

Effect of Cuttage
Most accessions had at least 70% sprouting ability with M. integrifolia
accessions KRG-15 and MRG-20 starting to shoot in the third week with up
to 85% bud break frequency. However 80% of the cuttings shed off the
sprouts after three weeks in the tunnel and started drying. Some cuttings
produced callus at the base of the cuttings six months after planting and only
produced roots after 9 to 15 months. In an earlier study (Gitonga et al.,
1997), massive fibrous root system adequate for transplanting was obtained
after 20 to 27 months and use of rooting hormones was not significantly
a b c
In vitro and in vivo methods of propagation towards ex situ conservation
Afr. J. Hort. Sci. (2009) 2:1-12
7
beneficial, indicating difficulty in rooting of macadamia. Similar to grafting,
some of the cuttings from accessions related to M. tetraphylla EMB-T1,
EMB-T2, EMB-T4, KRG-T1, KRG-T2 and KRG-T3 developed
inflorescences besides shoots (Figure 2).


Figure 2: Sprouting of cuttings from M. tetraphylla cultivar EMB-T1
showing a cutting producing three inflorescences and another
cutting producing three shoots.

Effect of Regeneration through Direct Organogenesis
MRG-20 and KRG-15 maintained well in culture with significant differences
in bud break frequency of 80% and 60%, respectively. Bud break started in
the third week. Shoots per explant ranged from 1 to 3 although some
explants produced up to 12 shoots. KMB-3, EMB-2, MRG-25 and KMB-3
did not maintain well in culture and most of the explants died after the third
week. Efforts to root the in vitro shoots were fruitless and most of the
explants produced excessive calli at the bases (Figure 3).


Figure 3: Direct regeneration of M. integrifolia cultivar KRG-15
showing: a) shoot regeneration from nodal segments after 6
weeks on hormone-free MS medium, and b) excessive callusing
after transfer of shoot masses to MS medium supplemented
with 2 mg/L BAP and I mg/L IBA.
a b
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Afr. J. Hort. Sci. (2009) 2:1-12
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The results of this study indicate genotypic differences in response to in vitro
culture. Debergh (1988) reported genotype to be an important factor to be
considered in tissue culture of plant species. Since explants were initiated on
hormone-free medium in the present study, these differences were associated
with variations in endogenous hormones involved in bud break. Shooting
frequency was low and maintenance of sub-cultured shoots was a problem.
Mulwa and Bhalla (2000) reported successful regeneration and rooting of
shoots from nodal segments of M. tetraphylla, while in the present study M.
integrifolia and M. hybrids were used. This suggests that the tissue culture
protocols need to be species and cultivar-specific.

Rooting of in vitro shoots was not achieved in the present study. Excessive
callogenesis and difficult rooting has previously been reported in woody
plants such as chestnut (Chevre et al., 1983; Piagnani and Eccher, 1988),
Populus x Euamerica trees (Agrawal and Gupta, 1999), neem
(Vantakeswarlu, 2000), and miracle berry (Ongunsola and Iroli, 2007). The
loss of rooting ability is associated with the physiological juvenility of
explant used for tissue culture (Debergh, 1988; Pareek and Mathur, 1999).
Rejuvenation of plant tissues may be achieved through serial grafting
(Mneney and Mantell, 2001).

Effect of Regeneration through Indirect Organogenesis
Greening of explants started from the fourth day on the medium and mature
cotyledons, while young cotyledons shriveled and remained dormant. Low
concentration of 2,4-D of 0.5 mg/l favoured production of green
embryogenic calli in both medium and mature cotyledons. Results are shown
in Figure 4a and Table 2.

Table 2: Effect of age of cotyledon and 2,4-D on greening and callus
formation from cotyledons of M. integrifolia cultivar KRG-15
Age of cotyledon 2,4-D concentration Greening Callusing
Medium 0.5 2.6a 0.6b
Medium 1 2ab 0.7b
Medium 2 1.3c 1.3a
Medium 4 1.3c 0.7b
Mature 0.5 1.3c 0.3bc
Mature 1 0.0d 0.0c
Mature 2 0.7cd 0.0c
Mature 4 0.3d 0.0c
Means followed by the same letter within a column are not significantly
different at P=0.05, according to the SNK test

In vitro and in vivo methods of propagation towards ex situ conservation
Afr. J. Hort. Sci. (2009) 2:1-12
9
When explants were transferred from hormone-free MS medium to MS
medium supplemented with different levels of 2,4-D no significant change
was observed in terms of greening and callus formation, except for the
explants derived from medium cotyledons, which produced rhizoids instead
of shoots (Figure 4b) after transfer to medium supplemented with 2 mg/l 2,4-
D. After transfer of calli to medium supplemented with different levels of
BAP with or without IBA or 2,4-D, results indicated that low levels of BAP
maintained callusing, greening and increase in callus size (Table 3). Some
green shoot primordia were produced in some cotyledons (Figure 4c) but no
shoot regeneration was achieved within the test period. Higher levels of BAP
in combination with 2,4-D produced only white shoots. Regeneration of
shoots from somatic embryos of M. tetraphylla has also been reported by
Mulwa and Bhalla (2006) and this provides promise that regeneration from
M. integrifolia and Macadamia hybrids which are economically important in
Kenya is possible and should be pursued further.


Figure 4: Indirect regeneration from cotyledonary explants of M.
integrifolia cultivar KRG-15 showing: a) embryogenic calli, b)
root regeneration, and c) green shoot primordium.

Table 3: Effect of BAP concentration on callus formation on cotyledons
of M. integrfolia cultivar KRG-15 after 4 weeks in culture
under growth room conditions
Hormone Greening (%) Callus
formation (%)
Callus size (%)
0.5 mg/l BAP 0.35 0.11a 0.65 0.11a 1.55 0.29a
1.0 mg/l BAP 0.30 0.11ab 0.44 0.11abc 0.80 0.29d
1.0 mg/l BAP +
0.5 mg/l IBA
0.11 0.07abc 0.55 0.11ab 1.20 0.27ab
2 mg/l BAP 0.10 0.07c 0.55 0.11ab 1.00 0.24c
Means followed by the same letter within a column are not significantly
different at P=0.05, according to the SNK test

a b c
Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

Afr. J. Hort. Sci. (2009) 2:1-12
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CONCLUSIONS AND RECOMMENDATIONS
All accessions responded to grafting although with varied success depending
on season. Thus, the time of the year for each accession that coincides with
optimum carbohydrate accumulation needs to be established to maximize
graft take. Graftage can be immediately employed for conservation of
macadamia genetic resources. However, due to the challenges associated
with field ex situ conservation in terms of capital and material inputs, grafted
seedlings should be planted closely together in a small piece of land to
conserve the material for several years at minimal cost.

Cuttage is a simple method of multiplying macadamia since cuttings only
require setting in moist sand under high humidity, although they take long to
root and further manipulations should be done to reduce this long period.

Macadamia integrifolia and Macadamia hybrids are amenable to in vitro
shoot regeneration, but the low success and rooting rates are still major
challenges. Further investigations should be conducted to improve shoot and
root regeneration in all the species. An efficient tissue culture protocol could
be used for sustained production of clonal material for ex situ conservation as
well as further breeding through genetic engineering.

Results of this study indicate possibility of shoot regeneration from callus
and therefore these investigations should be advanced further as this method
can be used to produce clonal material as well as generate new varieties
through somaclonal variation.

The three propagation methods, namely grafting, cutting and tissue culture
can be used in combination as seedlings with successful graft take can be
encouraged to elongate and provide more scion wood for further
multiplication as well as provide material for cuttings. Cuttings can be
sprouted to provide explant material for micropropagation.

ACKNOWLEDGMENTS
The authors acknowledge the Kenya Agricultural Productivity Project
through KARI for providing funds for this work, and the Jomo Kenyatta
University of Agriculture and Technology for registration of the first author
for post-graduate studies. Logistical support from the Director, KARI-
Nairobi, and the Center Director, KARI-Thika is appreciated. The generosity
of macadamia growers in providing cuttings and scions, and Peter Nguso for
setting the cuttings and grafting all the accessions is acknowledged.


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Afr. J. Hort. Sci. (2009) 2:1-12
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