You are on page 1of 12

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

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

2

(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 world’s 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 farmersfields 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…

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

4

5.7. Explants were maintained under growth room conditions (26±2C, 45- 55% relative humidity, 16 hours photoperiod of about 50 µmolm 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 (26±2C, 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 α = 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…

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

Graft take

Number

of

Number

of

classification

(%)

shoots/scion

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

6

a b c
a
b
c

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

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

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).

In vitro and in vivo methods of propagation towards ex situ conservation… 7 beneficial, indicating difficulty

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).

a
a
b
b

Figure 3: Direct regeneration of M. integrifolia cultivar KRG-15 showing: a) shoot regeneration from nodal segments after 6 weeks on hormone-free ½ MS medium, and b) excessive callusing after transfer of shoot masses to ½ MS medium supplemented with 2 mg/L BAP and I mg/L IBA.

Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

8

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…

9

When explants were transferred from hormone-free ½ MS medium to ½ 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.

a b c Figure 4: Indirect regeneration from cotyledonary explants of M. integrifolia cultivar KRG-15 showing:
a
b
c
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

Callus size (%)

 

formation (%)

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

+

0.11 ±0.07abc

0.55± 0.11ab

1.20 ±0.27ab

0.5

mg/l IBA

 

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

Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

10

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.

In vitro and in vivo methods of propagation towards ex situ conservation… 11

REFERENCES

Agrawal, V. and S.C. Gupta. 1999. Rapid micropropagation of Populus x Euramericana trees by callus culture, p. 262-270. In: Pareek, L.K. and P.L. Swarnkar (eds.). Trends in Plant Tissue Culture and Biotechnology. Agro Botanical Publishers. India. Bittenbender, H.C. and H.H. Hirae. 1990. Common problems of macadamia nut. In: Hawaii Research Extension Series 112. College of Tropical Agriculture and Human Resources, HITAHR, University of Hawaii. Chevre, A.M., S. Gill, A. Mouras and G. Salesses. 1983. In vitro vegetative multiplication of chestnut. Journal of Horticultural Science 58:23-29.

Deburgh, P.C. 1988. Micropropagation of woody species: State of the art on in vitro aspects. Acta Horticulturae 227:287-295. Gitonga, L.N., W. Nyakundi and E. Takayama. 1997. Effects of varietal differences on ability to root macadamia cuttings. In: Proceedings of the First National Horticultural Seminar on Sustainable Horticultural Production in the Tropics held on 31 st January 1997 at Jomo Kenyatta University of Agriculture and Technology, Juja. International Plant Genetic Resources Institute. 2001. Ex situ conservation technologies: Thematic research and methodology development:

Regional Report for Sub-Saharan Africa 1999-2000, Rome, Italy. Kadman, A. 1982. Experiments with propagation of macadamia by air- layering. Reprint from CMS Yearbook 1982. Kiuru, P., A.N. Nyaga and L. Wasilwa. 2004. A Review of macadamia research in Kenya. Proceedings of the Macadamia Stakeholders meeting held on 15 th June 2004 at KARI Headquarters, Nairobi. Leigh, D.S. 1973. Notes on macadamia propagation in New South Wales. Reprint from California Macadamia Society Yearbook, 1973. McConachie, I. 1995. The Macadamia story. Macadamia Consultants Pty Limited, Brisbane, Queensland, Australia. Macfalane, N. and R.V. Harris. 1981. Macadamia nuts as an edible oil source. Pryde, E.H., L.H. Prncen and X.D. MukNerjee (eds.). American Oil Chemistry Society. Mneney, E.E. and S.H. Mantell. 2001. In vitro micrografting of cashew. Plant Cell and Organ Culture 66: 49-58. Mulwa, R.M.S. and P.L. Bhalla. 2000. In vitro shoot multiplication of Macadamia tetraphylla L. Johnson. J. Hort. Sci. 75(1): 1-5. Mulwa, R.M.S. and P.L Bhalla. 2006. In vitro plant regeneration from immature cotyledon explants of macadamia (Macadamia tetraphylla L. Johnson). Plant Cell Rep. 25:1281-1286. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Planta. 15:473-497.

Gitonga, Kananga, Ngamau, Muigai, Gichuki and Njogu

12

Ogunsola, K.E. and C.O. Ilori. 2007. In vitro propagation of miracle berry (Synsepalum dulcificum Daniel) through embryo and nodal cultures. African Journal of Biotechnology 7:244-248. Onsongo, M. 2003. Kenya Trees Annual. USDA Global Agricultural Information Network. Ondabu, N., A.N. Nyaga and K. Tominaga. 1996. Macadamia clonal selection in Kenya. Fifth Biennial KARI Scientific Conference, Nairobi, Kenya. Pareek, K. and S. Mathur. 1999. Regeneration of plantlets through tissue culture methods in dicotyledonous trees: A brief review, p. 311-334. In: Pareek, L.K. and P.L. Swarnkar (eds.). Trends in Plant Tissue Culture and Biotechnology. Agro Botanical Publishers (India). Piagnani, C. and T. Eccher. 1988. Factors affecting the proliferation and rooting of chestnut in vitro. Acta Horticulturae 227:384-386. Rumsey, H.J. 1927. Australian nuts and nut growing in Australia. Part 1. The Australian nut. Sidney. Rosengarten, F. 1984. The book of edible nuts. Walker and Company. NY. Ryan, G.F. and E.F. Ryan. 1956. Macadamia propagation. Reprint from California Macadamia Society Yearbook 1956. Sedgley, M., F.D.H, Bell, D. Bell, C.W. Winks, S.J. Pattison and T.W. Hancook. 1990. Self- and cross-compatibility of macadamia cultivars. Journal of Horticultural Science 65:205-218. Statistical Analysis Software. 2001. SAS Institute Inc. Cary, NC, USA. Storey, W.B. and W.F. Salleeb. 1966. Genetics of four vegetative characters in an interspecific macadamia hybrid. California Macadamia Society Yearbook, 12:77-88. Ventakeshwarlu, B. 2002. Micropropagation of elite neem and teak plants and their field evaluation under farmers’ conditions. Proceedings of ‘Review Workshop on Micropropagation Projects under Andhra Pradesh Netherlands Biotechnology Programme’. Hyderabad, India. Vicente, M. C., F.A. Guzman, J. Engels and V.R. Rao. 2006. Genetic characterization and its use in decision-making for the conservation of crop germplasm. In: The Role of Biotechnology in Exploring and Protecting Agricultural Genetic Resources. FAO, Viale delle Terme di Caracalla, 00100 Rome, Italy. Woodroof, J. 1967. Tree nuts, production, processing, products. 1:313-337. Yokoyama, K., K. Wanitprapha, S. Nakamoto and H.C. Bittenbender. 1990. Macadamia nut economic fact sheet. 9. Department of Agriculture and Resources Economics, CTAHR, University of Hawaii.