Embryogenic calli of Cinnamomum
kanehirae Hayata, are yellowish-white, compact, and granular, which were
induced from young leaves, and were maintained on half-strength
macroandmicro-elements of MS media containing 1 mg L-1 6-Benzyl aminopurine
(BA) plus 0.5 mg L-11-naphthalene acetic acid (NAA). Embryogenic calli were
cultured at 5℃ for 14 d on woody plantmedium (WPM)
with no plant growth regulators, and then somatic embryos appeared on the
surfaceof these calli after they were transferred to 25℃
for another 6 wk. Of somatic embryos, 31.4%germinated that were cultured on WPM
supplemented with 0.2 mg L-1 GA3 and 150 ml L-1 coconutmilk in 2 mo and then
transferred to medium without plant growth regulators for another 10 mowith
2-mo intervals of subculturing. Regenerating plantlets of 5 cm in height were acclimated
andtransplanted to a greenhouse with an 83% survival rate. Subsequently, these
plantlets were transferredto the Botanic Garden of the National Museum of
Natural Science for 3 yr, after which thediameter at breast height was 6 cm , and the height was 4 m .
INTRODUCTIONCinnamomum kanehirae
Hayata wasonce a dominant broadleaf tree species, endemicat elevations of
200~ 2000 m in
themountains of Taiwan
(Liao 1996). It was animportant source of essential oils, terpineol,perfume,
medicines, and cabinet wood.However, this species has become very rareand
endangered by overcutting (Chang et al.2002). At present, the conventional
methodsof propagation by cutting, seeds, and layeringare very slow and do not
guarantee homogeneity.The small seed yield from wild treesis associated with
difficulties of pollinationand damage consumption by birds. Althoughvegetative
propagation was established fromcuttings, the percentage of root formation
wasinsufficient. The percentages of root formationof cuttings of this species
collected from14-yr-old trees were only 20~26%, eventhough they were treated
with 2000~4000ppm indolebutyric acid (IBA) (Kao andHuang 1997).Plant tissue
culture represents an idealand reliable way to proliferate this rare species.In
spite of micropropagation of Cinnamomumspecies having previously
beenpublished (Govinden-Soulange et al. 2007),information on the tissue culture
of C. kanehiraeis scanty. Chang et al. (2002) demonstratedthe
embryo culture of this species andrevealed that tissue culture plants had
bettertree forms, with more-erect stems and morefork-stems than rooted cuttings
of clones.Tsai (2004) investigated callus induction fromspring buds and stem
segments on MS basalmedium supplemented with 3 mg L-1 6-Benzylaminopurine (BA)
and 4 mg L-1 2,4-D in thedark for 3 wk. The calli were maintained
andproliferated but no plantlets regenerated duringthe culture process.This
study proposes a somatic embryogenesissystem by callus induction,
subculture,and regeneration. This system is promisingfor use in investigating
embryo inductionand development of C. kanehirae for propagationMATERIALS
AND METHODSCallus induction and maintenanceSeeds of C. kanehirae collected
from amother tree in the Botanic Garden (NationalMuseum of Natural Science, Taichung , Taiwan
)were packaged in moist sphagnum mossat 5℃ for
3 mo of cold stratification. Seedswhich germinated in a mixture of
peatmoss,perlite, and vermiculite (1:1:1 v/v/v) andplants with heights of 5~ 15 cm were used asthe explants. The
hypocotyls, petioles, andleaf veins were surface-sterilized with 2%NaOCl
supplemented by 1 drop of Tween 20for 15 min and then were rinsed with
sterilizedwater 3 times. These explants were slicedinto lengths of 0.5 cm , cut cross-wise to makemany wounds,
and placed horizontally onthe surface of callus-induction (CI) medium.The CI
medium contained half-strength saltsof MS (Murashige and Skoog 1962) mediumwith
0.5 mg L-1 nicotinic acid, 0.5 mg L-1 pyridoxineHCl, 0.1 mg L-1 thiamine HCl,
100mg L-1 myo-inositol, 170 mg L-1 NaH2PO4, 20g L-1 sucrose, 0.5 mg L-1 NAA (1-naphthaleneacetic
acid), and 2.8 g L-1
gelrite. All mediawere adjusted to a pH of 5.2 prior to autoclaving(15 min at 121℃
; 1 kg cm-2). BA (0, 2.5,and 5
mg L-1) was added to the CI mediumto investigate the effects of callus
induction.Cultures were maintained in the dark at 25±1℃
for 2 mo. Subsequently, 0.5 g
of calli wassubcultured with serial combinations of BAand NAA (0, 1.0, 2.5, and
5.0 mg L-1 BA and0, 0.5, and 1.0 mg L-1 NAA), and the cultureswere stored in
the dark. The increase in freshweight and morphogenesis of calli were
recordedafter 1 mo of culturing.Induction of somatic embryosThe
leaf-derived calli cultured on CIbasal medium supplemented with 1.0 mgL-1 BA
were transferred to the same liquidmedium with the gelrite removed.
Liquidcultures were maintained on a rotary shakerat 125 rpm with a light
intensity of 100 luxfor a 16-h photoperiod. After 3 wk of culture,fractions
sieved through a 40-mesh filter (celldissociation sieve-tissue grinder kit,
SigmaCD-1, St. Louis, MO, USA) were transferredto the same liquid medium for
another 3wk. Masses that could not pass through the40-mesh were transferred to
semisolid woodyplant medium (WPM) (Lloyd et al. 1981) andincubated at 5℃
in darkness for 14 d. Then,the cultures were moved to artificial
lightconditions with a 16-h photoperiod of 2000lux with a light source of
daylight fluorescenttubes (FL-30D/29, 40 W, China Electric,Taipei, Taiwan) at
25±1℃ by subculturingat 2-mo
intervals in WPM. The total numberof somatic embryos was recorded during
theperiod.Germination of somatic embryosIn total, 375 somatic embryos in
differentstages of development were cultured onWPM with or without 0.2 mg L-1
GA3 and150 ml L-1 coconut milk for 2 mo. Thosesomatic embryos which did not
germinatewere transferred to new WPM without GA3or coconut milk every 2 mo. The
definitionof germination was the radicle protrudingand plumule growth. When
small plantletsreached 0.5~ 1.0 cm
in height, they were separatedwith forceps and transferred to WPMwithout plant
growth regulators. The cultureswere exposed to 4000 lux of artificial lightwith
a light/dark cycle of 16/8 h at 25±1℃.The
regenerants were cultured on mediumuntil they were moved to a greenhouse.Transplantation
of plantletsWell-developed in vitro plantlets at 5 cmof height were moved
to a greenhouse for acclimationfor 7 d, then the roots were carefullywashed.
The plantlets were then transferred toa dibble tube containing a peatmoss,
perlite,and vermiculite (1:1:1 v/v/v) mixture for 1wk in a growth chamber.
Healthy plantletswere moved into a tunnel, and the percentagesurvival rate of
the regenerants was recorded.Histological and microscopic studiesRepresentative
samples were removedfrom cultures and put in a formalin, propionicacid, and 70%
ethanol (FPA; 1:1:18) solutionfor at least 4 h at room temperature.
Fixedsamples were dehydrated in a serial ethanolsolution and then infiltrated
with paraffin.Serial tissue sections were obtained using arotary microtome
(Reichert Histostat 820, AOReichert Scientific, Buffalo, NY, USA) andthe
sections were placed on slides and thenoven-dried at approximately 52℃
, arrangedon slide racks, and treated with a schedule ofde-waxing, staining,
and mounting. The preparedslides were observed with a microscope(AXIOSKOP 2, Zeiss , Germany ).
RESULTS Callus
induction and subcultureThe percentage of
calli induced was100% with all 3 kinds of explants (Table1). BA (1~5 mg L-1)
was effective in inducinginducingcalli, but the morphology of the callidepended
on the BA concentration and theexplants. Petioles and hypotocyls of explantscultured
on BA at a lower concentrationformedyellowish-white
compact calli, whilebrownish and soft calli formed with 5 mg L-1BA. Leaf veins
explants producedyellowishwhite,compact and pink, soft calli in all
BAcontainingmedia. The same results of callusinduction were observed in leaves
derivedfrom mature trees (data not shown). Somegranules arose from the surface
of leaf veinson medium containing 1~2.5 mg L-1 BA. Adventitiousroots formed in
primary cultures,and there was noroot formation after 1 or 2generations of
subculture.Leaf-derived calli were maintained byserial subcultures on
half-strength MS mediumsupplemented with BA, NAA, or the2 combined. The fresh
weights of calli weresignificantly increased in medium with 1 mgL-1 BA and 1 mg
L-1 NAA during 1 mo of culture(Table 2). However, most embryogeniccalli were
observed on medium supplementedwith 1 mg L-1 BA and 0.5 mg L-1 NAA.
Severalkinds of calli, including yellowish-brownsoft calli, yellowish-white
soft or compactcalli, and pink soft calli, were observed. Embryogeniccalli were
yellowish-white, compact,and granular and were stably maintainedon medium
containing 1 mg L-1 BA plus 0.5mg L-1 NAA.
Induction
and germination of somaticembryosCell
aggregates (Fig. 1) were sieved outon a 40-mesh filter, cultured on WPM
semisolidmedium, transferred to a refrigerator at 5℃ for
2 wk, and then cultured at 25℃ under2000 lux of
artificial light for another 6 wk.The cell aggregates were then subcultured
onnew semi-solid WPM for 8 wk, and embryogeniccalli formed (Fig. 2).
Pre-embryos withsuspensors which a group of cell developedfrom the fertilized
ovum (Fig. 3) were derivedfrom them and white globular somatic embryosappeared
after 6 wk of culturing (Fig. 4).Histological and morphological
observationsindicated that the developing somatic embryosexhibited no
detectable vascular connectionswith the mother explant and contained avascular
system (Fig. 5), which is one of themost important characteristics of somatic embryodevelopment.Somatic
embryos began to germinateearlier on WPM compared with those culturedon the WPM
supplemented with 0.2mg L-1 GA3 and 150 ml L-1 coconut milk, andslowly turned
brown. The germination percentagewas very low during the first 2 mo.However,
these somatic embryos germinatedstage by stage after they were transferred
toWPM without plant growth regulators. Thepercentage of germination increased
to 11.7%(16/137) and 31.4% (43/137) at 6 and 12 mo,respectively. Compared to 2
mo of GA3 treatment,there was no obvious difference in thesomatic embryo germination
rate in the firstperiod, but it continually rose for another10 mo on WPM with
subculturing at 2-mointervals, and the total germination numberof somatic
embryos with GA3 process wasgreater than those cultured on WPM withoutGA3
treatment (Table 3). There were someabnormal somatic embryos, such as
multicotyledonsor single cotyledons, and theyslowly turned brown and did not
germinate.Normal somatic embryos had a round shape,were white and had a solid
structure; theygerminated with 2 green cotyledons and aradicle and then the
leaves expanded (Figs. 6,7). Plantlets derived from somatic embryosgrew well on
the new WPM, and some secondarysomatic embryos spontaneously developedon the
surface of calli formed on thebase of the plantlets (Fig. 8). These secondarysomatic
embryos could also develop intocomplete plantlets after they were transferredto
WPM. Five-centimeter plantlets were acclimatedin the growth chamber, and
afterbeing transferred to the tunnel, whole plantswere established (Fig. 9).
The survival ratewas 83% (33/40), and the tallest plant was 4 m in height and 6
cm of diameter at breastheight (DBH) after 3 yr in the field
(Fig. 10).
DISCUSSIONIn a review of studies of trees of the Lauraceae,primary
explants for somatic embryogenesisfocused on zygotic embryos of Sassafrasramdaoemse(Hay.)
Rehd (Chen and Wang 1985); C. camphora (L.) Presl (Cheng and Ma
1990, Du and Bao 2005); Persea americana
(Mooney and van 1987, Pliego- Alfrao and Murashige 1988, Wijaksono and Litz
1999a , b); Laurus nobilis L.
(Canhoto et al. 1999); and Ocotea odorifera Me (Catarina et al. 2001)
and somatic embryos of O. catharinensis Mez. (Catarina et al.
2003). In this work, the indirect somatic embryogenesis of C. kanehirae
through the calli derived from young leaf explants might be a good source
of micropropagation for this species. Germination of somatic embryos of the
Lauraceae is difficult, and percentages are very low, at approximately 0% for L.
nobilis (Canhoto et al. 1999), 0~5% for P. Americana (Wijaksono
and Litz 1999b), and very low for C. camphora (Cheng and Ma 1990)
and P. americana
(Mooney and van 1987). Recently, somatic embryogenesis and plant
regeneration from protoplast of Cinnamomum were reported, and the
efficiency could reach 17.5%, which is quite high compared to other plants. In
this work, somatic embryos treated with GA3 for 2 mo germinatedafter they were
moved to WPM with no plantgrowth regulators. After 12 mo of culturing,the
highest percentage was 31.4% and wassignificantly higher than untreated ones.
Theresults were also superior to other previousstudies, but the germination of
somatic embryoswas not synchronized. Although thegermination of somatic embryos
was not regular,we obtained radical root seedlings with ahigh transplantation
rate (83%) in the greenhouse,and the growth capability was strong.Secondary
somatic embryos of the Lauraceaewere differentiated on the followingmedia: MS
basal medium, MS plus 1.0 mgL-1 BA and 0.1 mg L-1 NAA, MS containingmany
combinations of GA3 and BA (or kinetin),and medium plus 2,4-D. The formationof
secondary somatic embryos can proceedby proliferation of calli in former
somaticembryos, and directly on the cotyledon surfaceof previously formed
somatic embryos(Canhoto et al. 1999). Somatic embryos ofavocado that developed
directly from plantedzygotic embryos grew larger, and formed afew secondary
somatic embryos from the baseof each one (Witjaksono and Litz 1999a ). Inthe experiments performed herein,
secondarysomatic embryos developed from calli thathad formed on the base of
somatic embryoderivedplantlets. These secondary embryoswere spontaneously
produced on the WPMbasal medium with no plant growth regulators,and could
regenerate into new plantlets.Secondary embryogenesis represents an
excellentsystem and has the potential for largescalepropagation, especially on
medium free of plant growth regulators.In conclusion, embryogenic calli derived
from young leaves of C. kanehirae were induced on BA-supplemented
medium. These calli were cultured on plant growth regulatorfreeWPM and then
stored at 5℃ for 14 d.Somatic embryos formed on
the surface ofthe calli and could then be regenerated intohealthy plantlets on
the same medium. Coldtreatment and GA3 supplementation hadtreatment and GA3
supplementation hadpositive effects. Secondary somatic embryosformed into calli
from the base of the regenerants.xxxxxxxxA somatic embryogenesis system forC.
kanehirae was successfully established toproduce this rare species in an
efficient and economic manner.
ACKNOWLEDGEMENTSThe authors would like to thank theCouncil of
Agriculture, Taipei , Taiwan , for financiallysupporting
this research under contractno. 94 AS- 5.1.4
-FD-Z2. Mr. Chen Jien-Nan is appreciated for his assistance with
thehistological work.
1) The basal
medium contained half-strength Murashige and Skoog (1962) salts, supplemented
with(mg L-1): myo-inositol (100), nicotinic acid (0.5), thiamine-HCl (0.1),
pyridoxine HCl (0.5), glycine(2.0), NaH2PO4 (170), sucrose (20,000), and
gerlite (2800), the pH was 5.2, and cultures were keptin the dark at 25±1℃.
2)
Approximately 0.5 g of
callus proliferation was measured as the final fresh weight divided by
theinitial fresh weight. Means of 4 replicates with the same letters do not
significantly differ at p < 0.05.
3) YC, soft
yellowish calli; BC, soft brown calli; R, root; YWS, soft yellowish and white
calli; EC,embryogenic calli, yellow-white, compact, and granular calli; PC,
soft pink calli.
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