Micropropagation of adult avocado
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Plant Cell, Tissue and Organ Culture 58: 11–17, 1999.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
11
Micropropagation of adult avocado
Araceli Barceló-Muñoz1 , Carlos L. Encina2 , Elvira Simón-Pérez1 & Fernando Pliego-Alfaro3
1 Centro de Investigación y Formación Agraria, Cortijo de la Cruz, s/n, 29140 Churriana,Málaga, Spain; 2 Estación
Experimental La Mayora (CSIC), Algarrobo-Costa, Málaga, Spain; 3 Departamento de Biologı́a Vegetal, Facultad
de Ciencias, Campus de Teatinos, s/n, 29071 Málaga, Spain (∗ Requests for offprints; Fax: 34-952621868)
Received 10 June 1997; accepted in revised form 30 September 1999
Key words: mature explants, morphogenesis, Persea americana Mill., tissue culture
Abstract
A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock.
Cultures were initiated from basal shoots obtained after pruning a tree back to ground level. Buds sprouted in
Murashige and Skoog solid medium with macroelements at half strength and a 1.3 µM benzyladenine supplement.
To induce proliferation, shoots were cultured for 2 weeks in liquid medium in a rollordrum with the Gamborg salt
formulation and 1.3 µM benzyladenine, followed by 6 weeks in double phase conditions (solid medium with a
layer of liquid medium on the top) using the same salt formulation and two different benzyladenine supplements,
2.8 µM in the solid phase and 0.4 µM in the liquid phase. Ninety percent of the shoots rooted after a 3-day culture
in liquid medium in a rollordrum with MS macroelements at 0.3× and 4.9 µM indolebutyric acid, followed by
transfer to solid medium in the absence of auxin but with 1 g l−1 activated charcoal. Survival rate during the
acclimatization in the greenhouse was 70%.
Abbreviations: BA – benzyladenine; IBA – indolebutyric acid; MS – Murashige & Skoog
Introduction vestigation was undertaken to develop a method to
micropropagate adult avocado.
The avocado is a highly heterozygous species and the
use of seedling rootstocks results in high variability Materials and methods
in field behaviour; e.g., resistance to biotic and abi-
otic stress and productivity of individual trees (Ben A 10-year-old tree of an orchard in full production,
Ya’acov, 1987). Currently, selected genotypes can be located at La Mayora Experimental Station (Málaga)
propagated vegetatively using the Frolich technique was chosen. This tree was a combination of the cul-
(Frolich and Platt, 1972), which involves grafting of tivar Hass (scion) and a Mexican seedling (IV-8) as
the material to be rooted on a nurse seedling, followed rootstock, and had been selected due to its high pro-
by etiolation of the lower part of the growing scion. duction and low biennial-bearing index over a 4-year
Roots emerge at the etiolated base of the grafted shoot. period (Pliego-Alfaro et al., 1987).
This method is expensive and its applicability is geno-
type dependant (Fernández-Galván and Galán-Sauco, Initiation of cultures
1987). Thus, development of vegetative propagation
methods is desirable for avocado. In vitro culture has The tree was pruned back to ground level and sprouts
given positive results when juvenile material was used arising from the basal part of the rootstock were
(Cooper, 1987; Barceló-Muñoz et al., 1990); however, used for culture establishment. After removal of the
poor responses were obtained whith adult explants leaves, the 20–25-cm long shoots were disinfected in a
(Cooper, 1987; Pliego-Alfaro et al., 1987). This in- 0.5% sodium hypoclorite solution with a few drops of12
Tween 20 for 10 min. Afterwards, they were washed quality of varying the length of time, 4–8 weeks, under
with water and divided into 1- to 1.5 cm long nodal the double-phase conditions were assessed.
sections with a lateral bud. Depending upon the size Finally, an experiment was carried out to test the
of the bud, two to four leaf covers were removed. Af- effects of different salt formulations; that is, MS with
terwards, these explants were again disinfected for 10 macroelements at half strength or Gamborg salt mix-
min and washed three times with sterile water prior to ture (Gamborg, 1966) on shoot proliferation. Organic
culture in test tubes. addenda in all cases were those of MS medium. The
A modified MS medium (Murashige and Skoog, effects of two cytokinins, zeatin and BA, were as-
1962), with macroelements at half strength (MS 0.5×) sessed. In this experiment, the culture system was 2
and a 1.3 µM BA supplement, solidified with A-1296 weeks in liquid medium in a rollordrum followed by 6
agar (Sigma Chemical) at 6 g l−1 , was used for culture weeks on double-phase medium. In the liquid medium
initiation (Pliego-Alfaro et al., 1987). (rollordrum), the cytokinin concentration was 1.3 µM,
A replica of the IV-8 rootstock was obtained us- while in the double phase the cytokinin concentrations
ing the Frolich technique (Frolich and Platt, 1972) were 2.8 and 0.4 µM in the solid and liquid media,
and planted in the field. After this tree had flowered, respectively.
nodal sections from actively growing shoots of the up-
per part of the tree were established in vitro following
the procedure indicated above; although in this case, Rooting and acclimatization of the shoots
the Gamborg salt formulation (Gamborg, 1966) was
used instead of the modified MS basal mixture. Nodal The standard rooting procedure used was that de-
sections with lateral buds from basal sprouts of the veloped by Barceló-Muñoz et al. (1990) for juvenile
original pruned tree, IV-8, were also established in this avocado; e.g., 1.5–2-cm long shoots were incubated
medium to be used as controls. for 3 days in solid MS medium with macroelements
at one-third and 4.9 µM IBA. Afterwards, they were
transferred to the same medium without auxin but with
Shoot multiplication 1 g l−1 activated charcoal (Sigma Chemical).
To optimize rooting, the effects of culturing the
All experiments were carried out with material from shoots in liquid medium in the rollordrum (5 rpm)
basal shoots of the pruned tree. Using 1.5-cm long during the first (with auxin) or second (without auxin)
shoots which had sprouted from the nodal sections phases of the rooting process were studied. Material
on the initiation medium, a stock proliferating in a for this experiment was obtained from a stock prolif-
double-phase medium was established following the erating with the alternate method using the modified
procedure of Pliego-Alfaro et al. (1987). The double- MS salt formulation and BA as cytokinin (1.3 µM in
phase medium had a lower solid phase and an over liquid medium in the rollordrum, 2.8 and 0.4 µM in
layed liquid phase. The solid phase contained MS me- the solid and liquid media, respectively, of the double
dium with macroelements at half strength and 2.8 µM phase).
BA, while the liquid phase had the same composi- A second experiment was carried out to test the
tion but the BA concentration was 0.4 µM. After one effect of the salt formulation used in the multiplica-
subculture, 1-cm long apical shoots or nodal sections tion medium, MS 0.5× or Gamborg, on the rooting
with lateral buds obtained from this stock, were used capacity of the shoots. Material had been multiplied
to study the effect, on reactivation of bud growth, of using the alternate method, with BA as cytokinin. In
culture in liquid medium in a rollordrum (5 rpm). The this experiment, liquid medium was used during the
effects of different BA concentrations (0–1.3 µM) as first stage of the rooting process (3 days). Over 300
supplements to MS medium with macroelements at shoots were used per treatment.
half strength were evaluated. In the second experi- Finally, the rooting capacity of shoots derived from
ment, the effect of an alternate culture system; e.g., the clonal tree obtained by the Frolich technique (Fro-
culture in liquid medium in a rollordrum (2 weeks) fol- lich and Platt, 1972), in relation to shoots obtained
lowed by culture in double-phase conditions (4 weeks) from the pruned tree, was evaluated. In this experi-
on shoot proliferation was evaluated. Continuous cul- ment, liquid medium was used during the first part (3
turing under double-phase conditions was used as a days with auxin) of the rooting process. The mater-
control. After this experiment, the effects on shoot ial was obtained from nodal sections with lateral buds13
Table 1. Effect of BA in liquid medium, on a rollordrum,
which had sprouted in the culture initiation medium on bud elongation on explants from mature avocado trees.
with Gamborg salt mixture (Gamborg, 1966). Data taken after 2 weeks. Apical shoots were initially 1
In vitro rooted plants were transplanted to trays cm long. Different letters indicate significant differences
with 3×3×5.5-cm cells with De Baat Spec Mixt 20/80 at 99% level
(Coevorden, Holland) rooting sustrate and maintained Apical Shoots Nodal Sections
for 4 weeks under polyethylene tunnels with 100% rel- BA Length Number of leaves Length Number of
ative humidity and 110–120 µmol m−2 s−1 irradiance. (µM) (cm) (cm) leaves
During this period, plants were exposed daily to in- 0 1.3 a 4.9 a 0.3 a 2.3 a
creasing periods (5 min the first day, 5 h the last day) of 0.4 1.5 a 5.6 a 0.3 a 1.9 a
low relative humidity (30–40%), as indicated by Marín 1.3 1.9 b 5.4 a 0.6 b 3.3 a
and Gella (1987). After 4 weeks, plants could be trans-
ferred to open tunnels. Temperature in the greenhouse
fluctuated in the range 15–30◦C.
Table 2. Effect of alternate culture in double-phase medium and
liquid medium with agitation in a rollordrum, in relation to
General conditions of the experiments continuous culture in double-phase medium (Control)
Distilled-deionized water was used to prepare all me- Main shoot No of No axillary Axillary shoot
dia. The pH was adjusted to 5.74 before adding the length, (cm) leaves shoots, (cm) length, (cm)
agar. The solid medium was heated in the autoclave Double-phase 2.6 4.0 2.9 0.3
for 7 min (121◦C, 0.1 MPa) to melt the agar, then it Liquid medium in a
was distributed in aliquots of 25 ml into 25×150-mm rollordrum/double- 3.3a 5.2b 4.7a 0.4a
phase
Bellco test tubes. Finally, it was sterilised for 15 min.
In the double-phase culture, 3 ml of liquid medium a Significantly different from control (double-phase) at 99%.
b Significantly different from control (double-phase) at 95%.
were added on top of the sterile solid medium in the
laminar flow hood. In the experiments carried out in
the rollordrum, only 5 ml of liquid medium were used.
Temperature was maintained at 25±1◦C during Results
day and night. Irradiance level was 45 µmol m−2 s−1 ,
provided by Grolux lamps with a 16-h photoperiod.
Effect of culture on liquid medium, with agitation in a
Data taken and statistical analysis rollordrum
In multiplication and shoot elongation experiments, Previous observations of Pliego-Alfaro et al. (1987)
data were taken on main shoot length, number of had shown that continuous culture in double-phase
leaves, number and length of axillary shoots and conditions induced hyperhydricity and swelling of the
appearance of necrotic or hyperhydric shoots. buds which showed rather slow growth. To overcome
In rooting experiments, number and length of the this problem, the effect of culturing on liquid medium
roots, as well as appearance of necrosis or hyperhydri- in a rollordrum in the presence of different BA concen-
city were evaluated at 2-week intervals over a 6-week trations (0, 0.4 and 1.3 µM) was studied. With the two
period. types of propagules used, best results were obtained
In multiplication experiments, 20–25 shoots were in the presence of BA 1.3 µM, where apical shoots
used per treatment and they were kept for at least 5 virtually doubled their length (Table 1). After 2 weeks,
subcultures. On rooting, unless other specifications are the shoots had elongated and only showed mild hy-
given, 30–50 shoots were used per treatment and the perhydricity symptoms; however, if the material was
experiments were repeated twice. For normal distri- kept longer under these conditions, elongation con-
butions, a randomized block design and the LSD test tinued but severe hyperhydricity symptoms appeared
were used (Statgraphics Program). For binomial dis- (data not shown). Thus, for apical or lateral buds, a
tributions, the χ 2 -test was chosen (Sokal and Rohlf, reactivation period in liquid medium of 2 weeks is
1981). sufficient.14
Table 3. Effect of different combinations of salt formulations, Table 4. Effect of liquid vs solid media during the two stages of
Gamborg and MS 0.5×, and cytokinins, BA and zeatin, on the rooting process in the root formation capacity of adult avocado
shoot proliferation of adult avocado. Different letters indicate shoots. Different letters indicate significant differences at the 99%
significant differences at 99% level level for percentage of rooted cultures, and 95% level for number
of roots per cutting
Mineral Growth Main shoot length No axillary
salts regulators (cm) shoots % rooted cultures No roots/rooted cutting
MS 0.5X BA 2.9 a 3.2 b Solid–liquid 63.6 b 2.8 b
Zeatin 4.3 b 0.1 a Liquid–solid 72.2 b 2.4 ab
Gamborg BA 4.0 b 3.2 b Solid–solid 30.0 a 1.5 a
Zeatin 4.4 b 0.2 a Liquid–liquid 76.9 b 2.6 b
Effect of successive subculturing using an alternate shoots with swollen bases appeared under these condi-
culture system tions. Thus, another experiment was carried out to test
the effect of a salt formulation of lower ionic strength
After observing the positive results that culture on (Gamborg formulation versus the control, MS formu-
liquid medium with agitation had on shoot growth, lation with macroelements at half strength; each salt
an alternate culture system was examined; i.e., cul- mixture was tested in combination with BA or zeatin).
ture on agitated liquid medium followed by culture on Main shoot length was slightly higher in the presence
double-phase medium versus the continuous culture of zeatin; however, dramatic differences were ob-
on double-phase conditions (control). The alternate served in formation of axillary shoots, with BA giving
culture system allowed the production of longer shoots better results than zeatin, independently of the salt for-
with more leaves, and significantly increased the num- mulation used (Table 3). The mineral formulation did
ber of axillary shoots (Table 2). Moreover, the shoots not affect the number of axillary shoots. Axillary shoot
growing continuously in double-phase conditions had length in the presence of BA was in the range of 0.3–
succulent bases which formed callus and severe hype- 1.3 cm. No differences in the number of leaves were
rhydricity symptoms were noticeable. Shoots growing detected among treatments; however, leaves found in
alternately in double-phase and liquid media were not the presence of zeatin were much larger than those
succulent and had a more normal appearance but the formed in the presence of BA. No necrotic leaves or
percentage of hyperhydricity was high (70%). At this shoots with swollen bases could be observed in cul-
time, an experiment was carried out by varying the tures grown with the Gamborg formulation, so this salt
time of culture in double-phase conditions (4, 6 and mixture was chosen as standard.
8 weeks), after the 2-week period of elongation in
liquid medium in a rollordrum. The length of time Rooting and acclimatization
in double-phase conditions did not affect proliferation
capacity; however, at the 6-week treatment only 20% The effect of medium consistency, solid versus liquid,
of the cultures showed mild hyperhydricity symptoms, in the rooting process was studied in the first exper-
while at the 8-week treatment symptoms of apical iment. Four treatments were used: solid–solid (con-
necrosis were noticeable. In view of the above obser- trol), liquid–solid, solid–liquid, liquid–liquid. The
vations, the 6-week treatment was chosen as standard medium consistency had a strong influence in the root-
(data not shown). Using this alternate culture system ing of avocado (Table 4). Higher rooting percentages
(2 weeks in agitated liquid medium followed by 6 were obtained when the first part of the rooting process
weeks in double-phase conditions), shoot cultures can (3 days) took place in liquid medium independently
be continuously maintained in active proliferation. of the medium consistency during the second phase,
liquid or solid; however, if the second phase was li-
Effect of salt formulation and type of cytokinin on quid, virtually 100% of the shoots were hyperhydric,
proliferation rate but when it was solid, shoots were normal, so the
liquid–solid combination was chosen as standard.
Stock cultures could be maintained by the altern- Cuttings started to root after 1 week and the pro-
ate method; however, sometimes necrotic leaves and cess continued over 5 weeks in all treatments. The15
Figure 1. Rooted adult avocado shoots of the IV-8 rootstock. The
Gamborg salt mixture was used in the proliferation phase. Root-
ing was accomplished in two stages; i.e., 3 days in liquid medium
with auxin (4.9 µM IBA) and subsequent transfer to solid medium
without auxin but with 1 g l−1 activated charcoal (5 weeks).
number of roots/rooted cutting fluctuated; i.e., the
lowest number was obtained in the solid–solid media,
while no significant differences were obtained among
the other treatments (Table 4).
In the second experiment, the effect on rooting
of the salt mixture used in the shoot multiplication
phase was evaluated. The average rooting percentage
Figure 2. Five-month-old micropropagated adult avocado plant of
obtained with shoots from the MS 0.5× formulation the IV-8 rootstock.
was 74%, while 90% rooting was obtained when the
shoots were taken from the stock grown on the Gam-
borg salt mixture (Figure 1). These differences were
statistically significant at p ≤ 0.01. culturing under these conditions induced succulence
In the last rooting experiment, the effect of the in shoot bases and the appearance of hyperhydric cul-
physiological state of the mother plant, pruned down tures. Moreover, after 9 to 11 subcultures there was a
to ground level or not pruned, was evaluated. The per- progressive slowing of growth of apical shoots, with
centage of rooting of shoots from the pruned tree was formation of many axillary buds which did not elong-
92% while that from the unpruned tree was only 50%. ate (Pliego-Alfaro et al., 1987). Drew (1988) in Carica
Using the established micropropagation procedure, papaya obtained reactivation of growth after culturing
survival rate during the acclimatization phase was 70% buds in liquid medium in a rollordrum during short
after 8 weeks under in vivo conditions. A 5-month-old time periods. This treatment gave excellent results in
micropropagated plant is shown in Figure 2. avocado since it allowed the production of elongated
shoots with enlarged leaves which could be used for
further multiplication under double-phase conditions.
Discussion Culture in liquid media in the rollordrum may have im-
proved supply of nutrients, plant growth regulators and
The avocado, together with many other woody plants, oxygen to the propagules (George, 1993). Moreover,
shows a decrease in morphogenetic capacity with age in avocado, when using the double-phase method, it
(Cooper, 1987; Pliego-Alfaro and Murashige, 1987) is critical to control the volume of the liquid medium
and, until now, attempts to micropropagate adult ma- added on top of the solid phase and to have an ex-
terial have not been successful. Shoot proliferation tended period (around 6 weeks) to allow shoots to
of adult material in a modified solid MS medium recover from the period growing in liquid medium.
supplemented with BA was not feasible due to the The need of extended time periods to allow propagules
high incidence of apical necrosis (Pliego-Alfaro et al., to recover from continuous exposure to liquid medium
1987). This problem could be overcome by using the has also been indicated by Aitken-Christie and Jones
double-phase culture technique. However, continuous (1987) and Arrillaga et al. (1992).16
In avocado, Pliego-Alfaro et al. (1987) had shown pacity of newly formed basal sprouts from a pruned
that reduction of MS macroelements to half strength tree was higher than that of shoots taken from the up-
was beneficial for shoot development. When this salt per part of a tree which was a clonal replica of the
formulation was used in the alternate multiplication former. The increase in rooting capacity of the mater-
method (liquid medium in a rollordrum followed by ial sprouting after heavy pruning, has been explained
double-phase) some cultures had succulent bases and by Fortanier and Jonkers (1976) as a consequence of
a few necrotic leaves; in contrast, use of the Gam- the increase in physiological vigour which takes place
borg formulation (Gamborg, 1966) induced normal due to a better availability of water and nutrients,
growth. This formulation has 43% the total nitrogen while Franclet (1983) indicates that the sprouts per-
of MS 0.5× and a smaller (0.23 vs. 0.34) NH+ 4 /N ra- form better than adult shoots, because they come from
tio. Our results in the IV-8 avocado rootstock confirm meristems which have remained juvenile. The method
previous observations of other authors (Vieitez et al., described here allows, for the first time, successful
1983; Preece, 1995) about the importance of mineral micropropagation of an adult avocado rootstock, the
elements in the regulation of morphogenesis in vitro. IV-8 selection.
The formation of large leaves in the presence of
zeatin has also been observed in other species (López-
Encina et al., 1994), but its use in avocado is not Acknowledgements
recommended due to its negative effects in axillary
shoot formation. The authors are grateful for the support provided by
The use of liquid medium with agitation had a the Instituto Nacional de Investigaciones Agrarias,
highly significant effect in promoting rooting of the INIA Grant 8186 and the Comisión Interministerial de
IV-8 adult avocado rootstock. This observation had Ciencia y Tecnología, Grant AGR 95-0588-CO2-01.
been previously reported in difficult to root Malus cul-
tivars by Sriskandarajah and Mullins (1981). These
authors indicated that agitation itself enhanced rooting
References
since culture in stationary liquid medium did not give
good results; probably, the better auxin and nutrient Aitken-Christie J & Jones C (1987) Towards automation: Radiata
uptakes could explain these positive effects. However, pine shoot hedges in vitro. Plant Cell Tiss. Org. Cult. 8: 185–196
according to Loach (1988), it is also necessary to take Arrillaga I, Lerma V & Segura J (1992) Micropropagation of ju-
into account the importance of cell growth and syn- venile and adult flowering ash. J. Am. Soc. Hort. Sci. 117:
346–350
thesis of new components during the rooting process. Barceló-Muñoz A, Pliego-Alfaro F & Barea JM (1990) Micropro-
The need to maintain an adequate turgor pressure for pagación de aguacate (Persea americana Mill.) en fase juvenil.
cell growth is well known (Green, 1968). The use Actas Hort. 1: 503–506
of liquid medium enhances water availability and this Ben-Ya’acov A (1987) Avocado rootstock-scion relationships.
South Afric. Avocado Growers’ Assoc. Yrbk. 10: 30–32
could have favourable effects on root formation. The Blazich FA (1988) Mineral nutrition and adventitious rooting. In:
important role of mineral nutrients in rhizogenesis is Davis TD, Haïssig BE & Sankhala N (eds) Adventitious Root
well established (Blazich, 1988). The salt mixture Formation in Cuttings (pp. 61–69). Dioscorides Press, Portland,
used on the multiplication stage also affected the root- OR
Cooper PA (1987) Advances in the micropropagation of avocado
ing capacity of the shoots; best results were obtained (Persea americana Mill.). Acta Hort. 212: 571–575
with the salt formulation of lower ionic strength, Gam- Drew RA (1988) Rapid clonal propagation of papaya in vitro from
borg, which is consistent with previous results of Haun mature field-grown trees. HortScience 23: 609–611
Fernandez Galván D & Galán Sauco V (1987) Adaptabilidad de
and Cornell (1951) who observed a decrease in rooting
distintos patrones de aguacate (Persea americana Mill.) a la
capacity of Pelargonium shoots when increasing the propagación clonal. Actas II Congreso SECH 1: 51–58
level of macroelements of the solution used to fertil- Fortanier EJ & Jonkers H (1976) Juvenility and maturity of plants as
ize the mother plants. Moe and Andersen (1988) also influenced by their ontogenetical and physiological ageing. Acta
Hort. 56: 37–44
found a negative correlation between nitrogen content Franclet A (1983) Rejuvenation: theory and practical experiences
of microcuttings and their rooting capacity. in clonal silviculture. In: Zsuffa L, Rauter RM & Yeatman CW
Severe pruning has been shown to increase (eds). Clonal Forestry: Its Impact on Tree Improvement and
morphogenetic capacity of the newly formed sprouts Our Future Forests (pp. 96–134). 19th Meeting Canadian Tree
Improvement Association, Toronto, ON
in different species (Howard et al., 1989; Selby et al., Frolich EF & Platt RG (1972) Use of etiolation technique in rooting
1990). In the IV-8 avocado rootstock, the rooting ca- avocado cuttings. Ca. Avoc. Soc. Yrbk. 55: 97–10917 Gamborg OL (1966). Aromatic metabolism in plants. II. Enzymes Murashige T & Skoog F (1962) A revised medium for rapid growth of the shikimate pathway in suspension cultures of plant cells. and bioassays with tobacco tissue cultures. Physiol. Plant. 15: Can. J. Biochem. 44: 791–799 473–497 George EF (1993) Plant Propagation by Tissue Culture. Part 1. The Pliego-Alfaro F & Murashige T (1987) Possible rejuvenation of Technology. Exegetics Ltd., Edington adult avocado by graftage onto juvenile rootstocks in vitro. Green PB (1968) Growth physics in Nitella: a method for continu- HortScience 22: 1321–1324 ous in vivo analysis of extensibility based in a micro-manometer Pliego-Alfaro F, López-Encina C & Barceló-Muñoz A (1987) technique for turgor pressure. Plant Physiol. 43: 1169–1184 Propagation of avocado rootstocks by tissue culture. South Afric. Haun JR & Cornell PW (1951) Rooting response of geranium Avocado Growers’ Assoc. Yrbk. 10: 36–39 (Pelargonium hortorum, Bailey var. Ricard) cuttings as influ- Preece JE (1995) Can nutrient salts partially substitute for plant enced by nitrogen, phosphorus and potassium nutrition on the growth regulators? Plant Tiss. Cult. Biotechnol. 1: 26–37 stock plant. Proc. Am. Soc. Hort. Sci. 58: 317–323 Selby C, Lee R & Harvey BMR (1990) Partial rejuvenation of 20- Howard BH, Jones OP & Vasek J (1989) Long-term improvement year-old sitka spruce by lower crown pruning. In: Rodríguez R, in the rooting of plum cuttings following apparent rejuvenation. Sánchez Tamés R & Durzan DJ (eds) Plant Aging: Basic and J. Hort. Sci. 64: 147–156 Applied Approaches (pp. 373–376). Plenum Press, New York Loach K (1988) Water relations and adventitious rooting. In: Davis Sriskandarajah S & Mullins MG (1981) Micropropagation of TD, Haissig BE & Sankhala N (eds) Adventitious Root Form- Granny Smith apple: factors affecting root formation in vitro. J. ation in Cuttings (pp. 102–116) Dioscorides Press, Portland, Hort. Sci. 56: 71–76 OR Sokal RR & Rohlf FJ (1981) Biometry. The Principles and Practice López-Encina CL, Barceló-Muñoz A, Herrero-Castaño A & Pliego- of Statistics in Biological Research. WH Freeman & Company, Alfaro, F (1994) In vitro morphogenesis of juvenile Annona San Francisco, CA cherimola Mill bud explants. J. Hort. Sci. 69: 1053–1059 Vieitez AM, Ballester A, Vieitez ML & Vieitez E (1983) In vitro Marín JA & Gella R (1987) Acclimatization of the micropropag- plantlet regeneration of mature chestnut. J. Hort. Sci. 58: 457– ated cherry rootstock ‘Masto de Montañana’ (Prunus cerasus 463 L.). Acta Hort. 212: 603–606 Moe R & Andersen S (1988). Stock plant environment and sub- sequent adventitious rooting. In: Davis TD, Haissig BE & Sankhala N (eds) Adventitious Root Formation in Cuttings (pp. 214–234) Dioscorides Press, Portland, OR
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