11.

2 Caribbean Pine (Pinus caribaea Morelet)

G.P. BERLYN, S.J. KOHLS, and A.O. ANORU0 1

1 Introduction
Caribbean pine, (Pinus caribaea Morelet) is the most widely planted conifer in the
tropics (Nikles 1979; Greaves 1980, 1981). The worldwide planting program
encompasses four continents and is approaching 90,000 ha per year, making
Caribbean pine the dominant plantation conifer in the world. The reason for its
immense popularity is: (1) its rapid rate of growth and development; (2) hardiness
with respect to insects and disease; (3) tracheid qualities; and (4) its ability to be
highly productive in diverse environments (e.g., Fig. 1). The selection of Caribbean
pine for tropical reforestation and plantations was primarily predicated on its use for
pulp, but it has also proved suitable in varying degree for products such as lumber,
fence posts, and fuelwood. Thus, it qualifies as a multipurpose rapid-growing tree
species (Goodwin-Bailey and Palmer 1987). However, a major problem is that seed
production outside its natural range is often very low and the cost of importing seed
is prohibitive for many less developed countries (Slee 1967; Gallegos 1983; Okoro
and Okali 1987; Zobel et al. Stahl 1987; Anoruo 1988).
Prior to 1952 the name Pinus caribaea Morelet was used to describe both Pinus
caribaea and another species now designated as Pinus elliottii Engelm, including its
variety P. elliottiivar densa L. & D., (Little and Dorman 1952, 1954). Caribbean pine
is currently divided into three distinct varieties that differ in nuclear DNA content,
habitat, seed size and needle morphology (Table 1). Based on the work ofBarett and
Golfari (1962) and Uickoff (1964), the name Pinus caribaea var. hondurensis is used
for the Central American variety, Pinus caribaea var. bahamensis for the Bahama
and Caicos Island variety, and Pinus caribaea var. caribaea for the Cuban variety
(Table 2). Of these three, variety hondurensis is the most widely planted conifer in
the tropics (Whitmore and Liegell980; Hussain 1987; Anoruo 1988). This is due to
its much greater physiological plasticity (niche dimensions), which permits it to
flourish under the widest range of edaphic, climatic, and physiographic conditions.
Most of the world's planting stock is from two upland seed sources, viz., Pop tun in
Guatemala and Mountain Pine Ridge in Honduras.
Pinus caribaea var. hondurensis is a medium-sized to large tree, 24 to 44 m in
height with a long clear bole and a somewhat rounded crown with dense yellow
green foliage composed of needles 20 to 28 cm in length (rarely shorter than 15 cm
or longer than 30 cm). The leaf buds are encased in silvery white ciliate bud scales

1Yale University, School of Forestry and Environmental Studies, Greeley Memorial Laboratory, 370
Prospect St. New Haven, CT 06511, USA

Biotechnology in Agriculture and Forestry, Vol. 16

Trees III (ed. by Y.P.S. Bajaj)
© Springer-Verlag Berlin Heidelberg 1991
Caribbean Pine (Pinus caribaea Morelet) 255

Fig. 1. Young Caribbean pine plantation, Pindar's River, Jamaica. (About 10 years old)

Table 1. DNA diversity in dormant embryos of Pinus caribaea. (Berlyn et al. 1987)

Variety 2C-DNA DNA No. of needles Cotyledon

Pg" diversity per fascicle" diversity
H H'
caribaea 11.527 0.2987 3 (98.4%) 0.4574 (4- 7)
hondurensis 21.147 0.4191 3 (60.4-98.5%) 0.5964 (5-9)
bahamensis 25.226 0.5574 2 (45.9%) and 0.6027 (4- 9)
3 (54.1 %)
Mean 19.300 0.4251 0.5522

• From mitotic nuclei in germinating root tips.

bFrom LUckoff(1964).
cBased on 153 individuals, numbers in parentheses are the range of cotyledon numbers.

Table 2, Natural distribution of Pinus caribaea. (Anoruo 1988)

Variety Lal. Long. All.

'N 'W m

caribaea 21 ' 30'- 22 ' 30' 82 ' 20'- 84 ' 15' 0- 280
bahamensis 21 ' 40'- 27' 71 ' 40'_79 ' 0-12
hondurensis 12' 10'_18' 83 ' 30'-89 ' 0-1000
Total 12 ' 10'-27 ' 71' 40'-89 ' 0- 1000
256 G.P. Berlyn et at.

that are orange brown to reddish in color. Secondary needles are predominantly
three per fascicle (60.4 to 98.5%, depending on ecotype, Liickoff 1964) with the rest
having mostly two needles per fascicle, but four and five needles per fascicle have
been reported (Liickoff 1964). The bark is resinous, rough, scaly and thick, and is of
grayish color. The inner bark is reddish brown. The wood has a brownish cast and
is relatively light in weight. Pollen is shed in winter and fertilization occurs the
following winter with female cone maturation occurring in the following summer.
The female cones are typically 7.5 cm to 12 cm long, armed, conical in shape and
reddish brown in color with apophyses and umbo raised. The seeds have an
articulated wing that is more loosely attached than that of variety bahamensis and are
distinctly larger than those of variety caribaea (averaging 6.4 mm long as compared
to 5.8 mm). The number of cotyledons varies from 5 to 9, with 7 being the most
common number (34%). This is in contrast to var. caribaea, where the modal value
is 5 and over 57% of the seeds measured by Berlyn et al. (1987) had that number.
By 1973 Caribbean pine had already been planted in over 40 countries (Lamb
1973). Today the plantations range from 55 S in Argentina to 33 N in India and from
sea level to over 2400 meters in Kenya (Table 3). The inability of Pinus caribaea to
produce significant seed crops in many exotic locations is ascribed to geographic
discrepancy from its natural range (Anoruo 1988). Thus, tissue culture regenera tion,
despite its problems (see Berlyn et al. 1986, 1987), is an attractive alternative to
perpetual dependence on relatively high priced imported seed.

2 Vegetative Propagation

Chalmers (1962) had limited success in propagating Caribbean pine by air-layering.

He noted that individuals produced by air-layering from mature trees tended to
flower immediately rather than first growing to tree size. This observation may be
useful in inducing seed crops in seed orchard situations. Later, Slee et al. (1970) and
Lowery (1980) reported that Caribbean pine had good potential for propagation by
air-layering. Lowery (1980) claimed that improved success could be obtained in
air-layering by treating upright branches with indole butyric acid (lBA). However,
he did not evaluate the genetic stability of the resultant plants, and IBA induces
instability in Caribbean pine when applied in tissue culture rooting procedures
(Berlyn et al. 1987). The roots produced in the presence ofIBA tend to be weak and
subject to high mortality. Furthermore, the genetic instability found in the roots was
still present after 6 months in soil.
Slee (1967), working in Queensland, Australia, found that Caribbean pine
could be propagated by grafting. He reported field grafting to be superior to nursery
grafting and that overall bottle grafting produced the best results. In Nigeria, Okoro
(1980) successfully rooted stem cuttings, but growth of the cuttings was initially
slower than that of seedlings. She also propagated the species by grafting but
observed high mortality of grafted individuals and considerable scion-stock
incompatibility.
Caribbean Pine (Pinus caribaea More1et) 257

Table 3. Exotic distribution of Pinus caribaea". (Anoruo 1988)

Country Lat. Long. All.

Argentina 22" _ 55"S 57" - 73"W

Australia 10" - 28"30'S 137"30'- 153"E ±762
Brazil 4"N - 33"S 35" - 74"W ±820
Colombia 12"N _ 40"S 67"31' - 79"W
Congo 3"30'N- 5"S II" - 19"E 150-700
Dahomy and Togo 6" - 12"N I" _ 4"E
Fiji 12" _ 17"S 177" - 180"E
French Guyana 2" - 5"40'N 51 "30' - 54"40'W
Gambia 13"N 14" _ 17"W
Ghana 5" _ IO"N I"E _ 5"W ±490
Guyana I" _ 9"N 56" - 61"W
Hawaii Islands 19" - 27"N 155" - 158"W 1128
India 8" - 33"N 67" - 97"31'E ±914
Ivory Coast 5" _ IO"N 3" _ 8"W
Jamaica 17"40' - 18"30'N 76" - 79"W >820
Kenya 4"30'N- 5"S 34" - 42"E 1150-2438
Madagascar 12" - 25"S 43" - 50"E ±900
Malaysia 1"30' - 6"50'N 100" - 1l9"E 0-120"
Malawi 9"30' _ ]7"S 33" - 36"E
Martinique, Grenada,
St. Lucia and Domonica 12" - 15"30'N 61 " - 62"W
Mozambique 10" - 27"S 30" - 41"W
Nigeria 4" - 14"N 3" - 14"30'E ±1220
Philippines 5"30' _ 18"N 117" - 127"E ±300
Puerto Rico 18" - 18"30'N 65"30' - 67"W
Sierra Leone 7" _ IO"N 10" _ l3"W
Solomon Islands 5" - 11"S 154" - l63"E
South Africa 22° - 35"S 16" - 33"E
Sri Lanka 6" - 9"50'N 79"40' - 81 "50'E 0-1500
Surinam 2" _ 6"N 54" - 58"W
Tanzania I" - 12"S 30° - 400E
Trinidad and Tobago 10" - 11"N 61"W ±600
Uganda 4"N - lOS 29" - 35"E 1070-1830
Venezuela I" - 12"N 60" - 73"W 250-1800
Zaire 5"N _ 13"S 12° - 31"E 900-1200
Zambia 8" - 18"S 22" - 33"E
Zimbabwe 15"30' - 22"31'S 25" - 33"E 900-1830

"Available information up to 1973 (mostly from Lamb 1973).

bWest Malaysia.

3 Tissue Culture Studies

3.1 Micropropagation

Very few papers have been published on tissue culture ofthis species (see Webb and
Santiago 1983; Berlyn et al. 1987); however, as mentioned previously, there is a great
deal of current interest in the topic because of the lack of adequate seed production
in many of the plantations outside the natural range. Using cytokinin as the only
258 G.P. Berlyn et al.

growth phytohormone, Webb and Santiago (1983) successfully produced buds in

culture medium of Schenk: and Hildebrandt (1972). Berlyn et al. (1987) developed
a micropropagation system that produces whole plants that were eventually
transferred to soil. The initial bud induction medium was a half-strength mineral
salt solution devised by Campbell and Durzan (1975) using benzyl amino purine
(BAP) as the only exogenous hormone (CD medium). Later it was determined that
a number of cytokinins were effective in multiple bud induction for this species and
a number of different mineral salt solutions can be used for the micropropagation
of this species. In addition to the minerals, each liter of bud induction medium
contained 0.4 mg thiamine-HCl, 100 mg of myo-inositol, 20 g sucrose, and 8-10 g
Difco Bacto-agar. The optimum pH was 6.0 for the working medium.
Initially the mature embryo from dormant seed was used as the explant source
for bud induction and micropropagation. Procedures have now been developed for
induction of buds from secondary needles, which permits better phenotypic
selection for micropropagation. Seeds are first washed for 1-2 h in vigorously
running tap water before removing the seed coats. The seed coat of Caribbean pine
is thin and easily removed. The decoated seed is surface sterilized with agitation in
10% Clorox (0.5% NaOCI) for 10-15 min and allowed to imbibe in sterile water for
several hours until the female gametophyte is sufficiently hydrated to permit easy
extraction ofthe mature embryo. With this procedure contamination can be limited
to less than 10%. Sixty to 100% ofthe embryos explanted will produce multiple buds,
depending on: (1) seed source and freshness, and (2) environmental conditions,
especially light. The latter factor is especially important; the highest frequency of
regeneration was obtained with low constant light (ca. 75 pE/m2/s) at 25-27"C.
After multiple adventitious bud formation and growth, individual buds are
excised from the mass of multiple buds and explanted (with minimal callus) to bud
growth medium, which can be the original CD medium with a reduction in the BAP
concentration or the rooting medium consisting of half-strength GD medium
mineral salts with no hormones (Gresshof and Doy 1972). All other additives are as
specified in G D except that sucrose is 2%. For root induction, each liter also contains
18 mg of indole butyric acid (lBA). About 60% of the buds will root and form
plantlets (Fig. 2). This is the step that induces the genetic instability, and inves-
tigations are in progress to eliminate this treatment. Vigorous buds will initiate roots
without any auxin, but the process takes several months, and methods need to be
developed for decreasing the time and increasing the percentage of buds that will
root. Temperature, light, and pH for rooting were the same as for bud induction. In
some cases new adventitious buds were regenerated on the base of the adventitious
buds explanted to the rooting medium. These are referred to as r (second order)
regenerated buds.
Some of the regenerated plants were transferred to soil (Fig. 3). Others were
kept in sterile culture for 13 months to be used as a source of axenic spur shoot and
needle explants for bud induction by the same techniques used for bud induction on
embryo explants. For transfer to soil, the rooted buds are washed in running tap
water until all traces of the agar medium are removed and then planted in pots
containing a previously sterilized mixture of peatmoss, potting soil, vermiculite, and
perlite (2:2: I : I). Initially the soil mixture was supplemented with a quarter-strength
modified Knop's nutrient solution (Berlyn 1962; Berlyn and Miksche 1965).
Caribbean Pine (Pinus caribaea Morelet) 259

Fig.2. Micropropagated Caribbean pine plantlet with

developing lateral root system in culture

Fig. 3. Micropropagated
plant of Caribbean pine
after 10 months in soil

For several days after transfer the plants are kept in the shade with a glass beaker
over the tops to keep the humidity high around the foliar surfaces. Each day the
beaker is removed for longer periods and after I week it is no longer required.
However, the roots of these plants exhibited a large variation in nuclear DNA
content due to the auxin used in root induction (Berlyn et al. 1987). After several
months all of the roots of the regenerated plants died, and this total mortality was
repeated in several successive experiments. The mortality has been alleviated (Fig.
3) through the use of ROOTS, a soil/plant enhancer solution (Soilizer Corporation,
25 Science Park, New Haven, Connecticut 06511, USA), that promotes root growth,
nutrient and water uptake, and chlorophyll synthesis, and increases stress resistance
260 G.P. Berlyn et a\.

(Berlyn, Anoruo, and Russo unpubl.). The working solution consists of very low
concentrations of humic acids, extract of marine algae, and Metab (a metabolizer).
Metab is by far the most active ingredient for increasing stress resistance, growth,
development, and chlorophyll content in Pinus caribaea, P. taeda, and P. banksiana
(Berlyn and Beck 1980; Berlyn, Anoruo, and Russo unpubl.). This use of Soilizer
along with a new nutrient solution (Anoruo and Berlyn unpubl.) has greatly reduced
the mortality ofthe culture-regenerated plants, but still they tend to produce mostly
secondary needles and their heigh t growth is considera bIYless than that ofseedlings.
This could be due to the continued genetic instability of the roots, out of phase
development, or both. If the problem is developmental phase, it may be solved by
simple reentrainment through regulation of light, temperature, and water stress
(pseudodormancy) although the species is sylleptic (repeated flushing of buds
without an intervening period of dormancy) or continuously growing (e.g., fox-
tailing) in many environments. Genetic instability of roots must be eliminated at its
source - the induction of the root meristems. This must be done without auxin or,
if necessary, in the presence of auxin but under conditions that prevent instability.
We have examined nDNA (nuclear DNA quantity in picograms) in roots from
tissue culture-regenerated seedlings of Caribbean pine containing genetic instabi-
lity in the cells of the root meristem (due to the auxin used to induce rooting) just
before transfer to soil and after they had grown for 6 months in soil and found that
the nuclear DNA of the root meristem cells was still greatly elevated over the 4C
amount (Fig. 4). It is extremely doubtful if such plants are field-competitive, and the
goal of any tree improvement or regeneration system must be field competitiveness.

3.2 Micropropagation from Secondary Needles

Multiple regeneration from field-grown superior phenotypes is an important po-

tential for tissue culture but there are few reports of successful techniques for tree
species (see Gupta and Durzan 1985). We have developed a successful and

40
45.33%

30

20

10

0 Fig.4. Distribution of nuclear

31 .5 42 52 . 5 63 73 .5 84 94 .5 105 DNA amount in root tips of Carib-
3C 4C SC 6C 7C 8C 9C 10C bean pine plants after 6 months in
DNA, pg soil
Caribbean Pine (Pinus caribaea Morelet) 261

repeatable technique for secondary needles of Caribbean pine. Multiple buds were
initiated from secondary needle explants from 8-month-old plants that had been
regenerated in tissue culture. The most successful treatment was to transfer 2-5-cm
explants from either lateral or terminal branches to half-strength Murashige and
Skoog (1962) medium su pplemented with 5 mgl L BAP, 3% sucrose and 3% sorbitol
(osmoticum). Within 3 weeks after explantation hard green calli formed at the basal
(cut) end of the explants and about 30% of the calli formed buds (Fig. 5). The
explants were grown in a growth chamber with 18.6-h days (photoperiod) at 26 °C
and a light intensity of 75 ,&/m2 Is.

3.3 Somatic Embryogenesis from Secondary Needles

Somatic embryogenesis offers several advantages over micropropagation, viz.,

development of intact plants with better vascular connections between root and
shoot, greater genetic stability because the auxin-induced rooting of buds is ob-
viated, and greater potential for mass production oftrue-to-type plants. At present
most of the work on conifer somatic embryogenesis has concentrated on using
immature and mature embryos (see below) as the explant source (Berlyn 1962;
Durzan 1982; Hakman and Fowke 1987; von Arnold and Woodward 1988).
However, it is especially desirable to use tissue from field-proven trees, but un-
fortunately only a few coniferous species have yielded complete plants from mature
tissue through somatic embryogenesis that are capable of being transferred to soil.
Among conifers at the. time of this writing only Picea abies (Hakman et al. 1985),
Pinus lambertiana (Gupta and Durzan 1986), Picea glauca (Lu and Thorpe 1986),
Larix decidua (Nagmani and Bonga 1985), and Pseudotsuga menziesii (Durzan and
Gupta 1987) have reportedly yielded complete plants through somatic
embryogenesis.
In our studies mucilaginous embryogenic callus was obtained (Figs. 6, 7) from
needle explants of 4-month-old axenically grown shoots growing on half-strength

Fig.S. Buds and elongated shoots initiat-

ed from explants of secondary needles of
Caribbean pine
262 G.P. Berlyn et al.

Fig. 6. Mucilaginous embryogenic calli from secondary needle explants of Caribbean pine showing
early-stage somatic embryos developing from the calli

Fig. 7. Later-stage embryos

developing from mucilaginous callus
of Caribbean pine with extensive sus-
pensor formation

GD (Gresshoffand Doy 1972) medium. Lateral and terminal needle tissue (2-5 cm
long) were transferred to R35D medium (MS salts, 1.25 gil thiamine, 50 mgll
asparagine) with or without mannitol (0.1 or 0.2 M) as an osmoticum and supple-
mented with 1.5 mg/12,4-D and 3.5% sucrose. The R35D medium was developed by
Molecular Genetics, Incorporated (MGI) of Minneapolis, MN, USA. Within I
month after explantation to a dark growth chamber, two distinct types of callus are
visible: a white, hard compact type from the aerial portion ofthe needle explant and
a mucilaginous type arising from the lower surface of the tissue that is in direct
contact with the medium. After 1 month in the dark at 25°C, the calli were exposed
to an environmental regime with a photoperiod of 14.8 h at 25°C with a light
intensity of ca. 150 pElm2 Is. Upon transfer to the light, the compact calli became
green (chlorophyll production) whereas the soft mucilaginous calli remained
Caribbean Pine (Pinus caribaea Morelet) 263

whitish-translucent in color. Paraboloid shaped embryos (Fig. 7) develop from the

mucilaginous calli. The green calli grew well but showed no evidence of
differentiation. Successive transfers of the embryos from the mucilaginous calli
failed to produce cotyledon stage or mature embryos. The development ofthe early
stage embryos was greatly facilitated by the presence of the osmoticum (mannitol).
Further work is required to develop techniques to promote the completion of
embryogenesis and subsequently germination of the mature somatic embryos.

3.4 Somatic Embryogenesis from Embryos

The use of mature embryos of conifers is an advantage over immature embryos

because the immature embryos are available for only a very short period of time
after fertilization in the spring. Generally the efficacy of somatic embryogenesis
from immature embryo explants, under present techniques, falls after induction of
cotyledons (pers. commun. M. Becwar, Institute of Paper Chemistry, IPC, Ap-
pleton, WI USA). Interestingly, Berlyn (1962) could not culture immature embryos
of Pinus lambertiana into plants unless the embryos had differentiated cotyledons
before explantation. This suggests that the initiation or' cotyledons marks a
significant phase in pine embryo development, possibly marking competence for
germination (see Anoruo 1988).
Berlyn (1962) found that Pinus lambertiana and especially Pinus cembra have
a relatively large percentage of "mature" seeds that contain multiple embryos in
various stages of development and hence have a much larger window of availability
of immature embryo explants. It would be a considerable advantage to be able to
obtain immature embryos from mature seed to use in somatic embryogenesis
because most coniferous seeds, especially those ofthe genus Pinus store well and can
be successfully used in micropropagation even after many years in storage. How-
ever, the technique still has the disadvantage that the seeds provide no information
about the phenotype they are capable of producing in the field. Thus it would be even
more advantageous to be able to obtain somatic embryos from tissues (e.g., buds,
leaves, cambial explants) offield-proven mature trees.
In our experiments with Pinus caribaea, axenic cultures from mature embryos
were obtained by the sterilization and extraction procedure described in Section 3.2
above. The media used were those of Norstog and Rhamstine (1967, medium NR
59) and von Arnold and Eriksson (1981, LP medium). Two modified MS formula-
tions of Gupta and Ourzan(1985, 1986), viz. OCR and BM, were also tested. Table
4 lists the phytohormone concentrations and the stages at which they were
administered.
All calli were initiated in the dark at 26°C. After 1 month the calli were trans-
ferred to continuous light at 26°C with a light intensity of 75 pE/m2/s. All calli
were transferred to fresh media every 2 weeks. In the light, as was the case with the
needle explants, two different types of calli formed: compact green and a translucent
white. Only the translucent white calli gave rise to somatic embryos. It appeared that
the embryogenic calli originated from the radical end of the mature embryo
explants. The frequency and media of origin of the explants forming embryogenic
calli is given below. As with the embryogenic calli derived from mature needles, no
264 G.P. Berlyn et a!.

Table 4. Media and sequence ofphytohormones used to induce somatic

embryogenesis in Pinus caribaea var. hondurensis

mgll
Basal medium 2,4-D" BApb KNc

DCR (initiation) 3.0

DCR (1st subculture) O.oJ
LP (initiation) 2.21 1.12
LP (1st subculture) 1.12
59 (initiation) 1.10 1.07
59 (1st subculture) 1.07
Bm-2 (initiation) 11.05 4.5 4.3
Bm-3 (1st subculture) 1.10 0.45 0.43
Bm-4 (2nd subculture) 0.22 0.45 0.43

a2,4-D, 2,4-dichlorophenoxyacetic acid.

bBAP, benzylamino purine.
cKN, kinetin.

soil-ready plantlets have as yet been obtained. The greatest proportion of explants
forming embryogenic calli were in order: (1) LP quarter-strength, 7%; (2) DCR
half-strength, 6%; (3) DCR full strength, 6%; and (4) NR 59 full strength, 1.5%.

3.5 Protoplast Isolation and Regeneration

Protoplasts of Caribbean pine have been isolated and grown for 10 days. Cell
division occurred after 3 da ys. Mucilaginous calli were incubated on a shaker (40-50
rpm) in the dark in a reaction mixture of 1% Worthington Cellulase, 0.1% Pec-
tolyase, 0.4 M Mannitol, 80 mM CaClz, 20 mM MES, and 2 mg/l Bovine serum
albumin (BSA) at pH 5.6 for 4-5 h. The tissue was then washed twice in 10 ml of a
solution containing 0.4 M Mannitol, 80 mM CaClz, 70 mM MES, 0.1 % BSA at pH
5.6. Prior to the last wash the tissue was passed through a 100 pm cell screen. After
washing the cells were cultured on 2 X strength Dachin Agar on a modified
Murashige and Skoog Medium (1962) (0.4 M mannitol with MS salts plus 20 mlll
coconut H 2 0 and 250 mIll glucose). The protoplasts were diluted 1: 1 with the liquid
medium before being layered onto Petri dishes containing a feeder layer of maize
cells (Black Mexican sweet corn).
Similar results using embryo and needle explants as a source of material for
protoplast culture of Pinus caribaea have been obtained using the procedures of
Patel et al. (1984) developed for Pinus coulteri (Berlyn unpubl.) and it is likely that
the protoplast procedures developed for other coniferous species will also work well
in Caribbean pine (e.g., David and David 1979; Kirby and Cheng 1979; David et al.
1982).
Caribbean Pine (Pinus caribaea Morelet) 265

4 Summary and Conclusions

Caribbean pine is the most widely planted conifer in the tropics, including Africa,
Latin America, Asia, and the Caribbean. It has a great deal of genetic variation and
can grow in a wide variety of environments. It is easily regenerated from somatic
tissues, including secondary needles, by micropropagation. It regenerates large
numbers of multiple buds under constant low light and moderate temperatures with
only cytokinins as the hormonal component. To date, rapid rooting requires large
inputs of auxin which causes genetic instability. Vigorous buds will regenerate roots
without auxins and remain genetically stable, but the process takes several months.
Plants with genetically unstable roots have been kept alive in soil for several years
through the use of an organic biostimulant; however, the growth rate ofthese plants
is low and their morphology is altered. The roots remain polymorphic in nDNA
content as opposed to sexually reproduced plants (germinated from seed).
Techniques utilizing exogenous osmotica to increase the osmotic pressure ofthe
culture media have successfully induced somatic embryos from explants from both
mature embryos and fully elongated secondary needles; however, these embryos
have not as yet been induced to produce competent full-term embryos capable of
germination. Protoplasts of Caribbean pine have been isolated and grown for short
periods of time but have not as yet been amenable to either micropropagation or
somatic embryogenesis.
Biotechnology of Caribbean pine has great promise for the future because the
species grows extremely well in many areas outside its natural range but does not
produce sufficient seed in many of these exotic plantations. This creates a demand
for asexually regenerated plants, and conventional vegetative propagation has not
been very promising. Thus tissue culture systems have great appeal and should be
economically feasible. Furthermore, the buds induced in culture are genetically
stable and protoplast techniques should be attainable in the near future. If a root
regeneration program can be developed that does not induce genetic instability, the
system could go into production immediately. Once such a system is already in
place, it can more easily incorporate additional sophistication such as genetic
engineering procedures. Thus the manipulation for specific traits such as disease
resistance and stem form, mod ula tion for different environments, and sustaina bility
of production over entire ecosystems should be practical within realistic time frame.

5 Recommended Protocols

It is unfortunately rather premature to suggest the best protocols because there are only a few papers in
the literature on the tissue culture of this species and the protocols could be greatly improved if more work
was done. However, the recommended protocols work and are a starting point, but in many cases the
yields are quite low (e.g., somatic embryogenesis), and sample sizes must be quite large in order to achieve
relatively few regenerates.

I. Micropropagation
A) Mature embryo explants
a) Wash seeds in rapidly running tap water for l-2h and surface sterilize as in 3-1 above.
266 G .P. Berlyn et al.

b) Explant whole embryos to Petri dishes of CD (Campbell and Durzan 1975) medium at pH 6
containing 3.0 mg/I ofbenzylamino purine (BAP), and 2% sucrose.
c) Allow 1-3 months for buds to develop under low continuous light at 25-26 °C and transfer to
half-strength Gresshoff and Doy (1972) medium (GD 112) containing 1-3 mg/I BAP. Use the
lowest concentration of cytokinin that will permit the buds to continue to grow out.
d) Transfer vigorous buds to G Dl!2 with IS mg!! of IAA. As soon as bud primordia are formed
transfer the micropropagants to GDl!2 without auxin.
e) Transfer vigorous micropropagants to pots containing soil treated to field capacity with 1%
ROOTS, keeping the plants covered with a clear cover and only gradually exposing them to
the Greenhouse atmosphere. Repeat ROOTS monthly until plants are ready for outplanting.
B) Secondary needle explants
a) Surface sterilize as in IAa above. Chop the needles into 2-5-cm pieces and explant to
half-strength Murashige and Skoog (1962) medium (MSI!2) or CDI!2). Supplement the
mineral salts with 5 mg/I BAP, 3% sucrose and 3% sorbitol (osmoticum) and place cultures in
IS.6-h days at 26°C at low light intensity, ca 75 pElm2 Is.
b) Once well-developed buds are formed they can be rooted and transferred to soil as described
in Ac-e above.

2. Somatic embryogenesis
A) Mature embryo explants
a) The explants are obtained by the procedures in lAa above. They are cultured in half-strength
DCR medium (Gupta and Durzan 1985). After I month they can be moved to full-strength
DCR. The calli are initiated in the dark at 26°C and after I month they are illuminated with
low light (ca. 75 pE/m2 /s).Allcalliaretransferred to fresh media every2 weeks. Embryos form
on the white translucent calli. No procedure is currently available to induce completion of
embryogeny and successful germination of the somatic embryos.
B) Secondary needle explants
a) Needles are either obtained from the field and surface sterilized as in IAa above or taken from
axenically grown seedlings or micropropagants.
b) Needle sections 2-5-cm long are explanted to R35D medium (MS salts supplemented with
1.25 gil thiamine, 50 mg/lasparagine, 0.1-0.2 M mannitol, 1.5 mg/12,4-D,and 3.5% sucrose)
and placed in the dark at 25°C. After I month the cultures are presented with and maintained
on a 14.8-h photoperiod and a light intensity of ca. 150 pElm2/s. Embryos form in the
mucilaginous calli, but again no procedures have been developed to induce the completion of
embryo competence for germination.

3. Protoplast isolation and culture

A) Mucilaginous callus explants
Obtain mucilaginous callus as in 2Bb above and incubate on a shaker (40-50 rpm) in the dark in
a reaction mixture of! % Worthington cellulase, 0.1 % Pectolyase, 0.4 M mannitol, 80 mM CaC~,
20 mM MES, and 2 mg/l bovine serum albumin (BSA) at pH 5.6 for 4-5 h. The tissue is then
washed twice in 10 ml of a solution containing 0.4 M mannitol, 80 mM CaC~, 70 mM MES, 0.1 %
BSA at pH 5.6. Prior to the last wash the tissue is passed through a 100 p.m cell screen. After
washing, the ce lis are cultured on2x strength DachinAgar on a modified MS (0.4 M mannitol with
MS salts plus 20 mil I coconut H" 0 and 250 mill glucose). The protoplasts are diluted I: I with the
liquid medium before being layered onto Petri dishes containing a feeder layer of maize cells
(Black Mexican sweet corn).

Acknowledgments. The authors thank Cheryl Larson for assistance with the protoplast techniques. This
work was partially supported by the Program in Forest Biotechnology and the Tropical Resources
Institute ofthe Yale UniversitySchoolofForestryand EnvironmentalStudies, New Haven, Connecticut,
USA.
Caribbean Pine (Pinus caribaea Morelet) 267

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