Method for regeneration of coniferous plants by somatic embryogenesis

This invention relates to a method for regeneration of coniferous plants. In particular, this invention relates to the development of a multi-step method that is able to complete the entire somatic embryogenesis regenerative process, from explant collection to planting, for plants of the genus Pinus and Pinus interspecies hybrid plants. This method is well suited for producing clonal planting stock useful for reforestation.

FIELD OF INVENTION 
This invention relates to a method for regeneration of coniferous plants. 
In particular, this invention relates to the development of a multi-step 
method that is able to complete the entire somatic embryogenesis 
regenerative process, from explant collection to field planting, for 
plants of the genus Pinus and Pinus interspecies hybrid plants. This novel 
method is well suited for producing clonal planting stock useful for 
reforestation. 
BACKGROUND OF THE INVENTION 
Reforestation, the controlled regeneration of forests, has become an 
integral part of forest management in order to secure a renewable and 
sustainable source of raw material for production of paper and other 
wood-related products. Forest trees can be regenerated by either sexual or 
asexual propagation. Sexual reproduction of seedlings for reforestation 
has traditionally been the most important means of propagation, especially 
with coniferous species. 
Tree improvement programs with economically important conifers (e.g., 
Pinus, Picea, and Pseudotsuga species) have applied genetic principles of 
selection and breeding to achieve genetic gain. Based on the results of 
progeny tests, superior maternal trees are selected and used in "seed 
orchards" for mass production of genetically improved seed. The genetic 
gain in such an open-pollinated sexual propagation strategy is, however, 
limited by the breeder's inability to control the paternal parent. Further 
gains can be achieved by control-pollination of the maternal tree with 
pollen from individual trees whose progeny have also demonstrated superior 
growth characteristics. Yet sexual propagation results in a "family" of 
seeds comprised of many different genetic combinations (known as 
siblings), even though both parents of each sibling seed are the same. As 
not all genotype combinations are favorable, the potential genetic gain is 
reduced due to this genetic variation among sibling seeds. 
In addition to these genetic limitations, large-scale production of control 
pollinated seeds is expensive. These economic and biological limitations 
on large-scale seed production have caused considerable interest to 
develop in the industry for applying asexual methods to propagate 
economically important conifers. 
The use of asexual propagation permits one to apply what is known as a very 
high selection intensity (that is, propagate only progeny showing a very 
high genetic gain potential). These highly desirable progeny have unique 
genetic combinations that result in superior growth and performance 
characteristics. Thus, with asexual propagation it is possible to multiply 
genetically select individuals while avoiding a concomitant reduction of 
genetic gain due to within family variation. 
Asexual propagation of trees can be accomplished by methods of grafting, 
vegetative propagation, and micropropagation. Grafting, widely used to 
propagate select individuals in limited quantities for seed orchard 
establishment, is not applicable to large-scale production for 
reforestation. Vegetative propagation by rooting of cuttings and 
micropropagation by somatic embryogenesis currently hold the most 
potential for reforestation of coniferous trees. Although vegetative 
propagation by rooted cuttings can be achieved in many coniferous species, 
large-scale production via this method is extremely costly due to 
difficulties in automating and mechanizing the process. This propagation 
method is further limited by the fact that the rooting potential of stock 
plants decrease with time, making it difficult to serially propagate from 
select genotypes over extended periods of time. 
Micropropagation by somatic embryogenesis refers to methods whereby embryos 
are produced in vitro from small pieces of plant tissue or individual 
cells. The embryos are referred to as somatic because they are derived 
from the somatic (vegetative) tissue, rather than from the sexual process. 
Both vegetative propagation and micropropagation have the potential to 
capture all genetic gain of highly desirable genotypes. However, unlike 
conventional vegetative propagation methods, somatic embryogenesis is 
amenable to automation and mechanization, making it highly desirable for 
large-scale production of planting stock for reforestation. In addition, 
somatic embryogenic cultures can easily be preserved in liquid nitrogen. 
Having a long-term cryogenic preservation system offers immense advantages 
over other vegetative propagation systems which attempt to maintain the 
juenility of stock plants. 
The current invention specifically relates to the development of an 
improved cell and tissue culture system for micropropagation of conifers 
by somatic embryogenesis. It was not until 1985 that somatic embryogenesis 
was discovered in conifers (Hakman et al. 1985) and the first viable 
plantlets were regenerated from conifer somatic embryos (Hakman and von 
Arnold 1985). Since 1985 conifer tissue culture workers throughout the 
world have pursued the development of somatic embryogenesis systems 
capable of regenerating plants. The goal of much of this work is to 
develop conifer somatic embryogenesis as an efficient micropropagation 
system for producing clonal planting stock enmasse. In addition, the 
embryogenic micropropagation system interfaces very well with genetic 
engineering techniques for production of transgenic clonal planting stock 
of conifers. 
The two most economically important conifer genera are Picea (spruce) and 
Pinus (pine). There are about 30 species of Picea, largely restricted to 
cooler regions of the northern hemisphere, of which seven species are 
native to North America. Pinus is the largest and most important genus of 
conifers, having approximately 95 species scattered over the northern 
hemisphere. Of these 95 species, 36 are native to North America. (Preston 
1989). 
Those working in conifer somatic embryogenesis have found that there is a 
striking difference between Picea conifers and Pinus conifers as to the 
ease with which somatic embryogenesis can be induced and plants 
regenerated (Tautorus et al. 1991). Indeed, if one evaluates the success 
of somatic embryogenesis in conifers among species of these two important 
genera, it is clear that much more success has been achieved with Picea 
than with Pinus. It is also striking how consistent the success on 
developing somatic embryogenic systems has been among several Picea 
species, whereas the recalcitrance of Pinus has been equally consistent 
across several species. 
Progress in somatic embryogenesis can in part be evaluated by the level of 
success in three important steps of the process: (1) initiation of 
embryogenic cultures, (2) production of fully developed somatic embryos, 
and (3) establishment of somatic embryo plants under field conditions. 
Among Picea species embryogenic culture initiation frequencies are 
relatively high; as high as 95% from immature zygotic embryos, and as high 
as 55% from mature zygotic embryos harvested from fully developed, dry 
seeds (Tautorus et al. 1991). There are numerous reports of production of 
fully developed somatic embryos among Picea species, and several reports 
of establishment and growth of Picea somatic embryo plants in soil. 
Researchers at the British Columbia Research Corporation have reported on 
establishment of interior spruce (a mixture of Picea glauca and Picea 
englemannii) somatic embryo plants under nursery conditions. For example, 
Webster et al. (1990) reported over 80% survival and establishment in 
nursery conditions of interior spruce somatic embryo plants for most of 71 
genotypes tested. Grossnickle et al. (1992) reported the establishment of 
40% of 2000 interior spruce somatic embryo plants in nursery conditions. 
The somatic embryo plants were derived from 15 different genotypes. 
Researchers at the Weyerhaeuser Corporation have reported similar success 
with Norway spruce (Picea abies); over 3000 somatic embryo plants from 17 
genotypes have been established in the field (Gupta et al. 1992). Similar 
success was also reported with Douglas-fir (Pseudotsuga menziesii); over 
2000 somatic embryo plants from 6 genotypes of have been established in 
soil in greenhouse conditions. Thus, conifer somatic embryogenesis workers 
utilizing Picea species (and commercially important Douglas-fir) have been 
successful in developing culture initiation and regeneration systems that 
enable relatively routine production of plants capable of transfer to 
field conditions. The rapid successes in Picea somatic embryogenesis had 
lead to considerable optimism among researchers that commercial 
utilization of conifer somatic embryogenesis for production of clonal 
planting stock of Pinus conifers would be readily achievable. 
However, the progress achieved with somatic embryogenesis in Pinus species 
to date has been much less encouraging than that achieved with Picea 
species. First and foremost in difficulty is the recalcitrance of Pinus 
species for initiation of embryogenic cultures. For example, initiation 
frequencies of about 1 to 5% are routinely cited by those working with 
Pinus species (Gupta and Durzan 1987, Becwar et al. 1988, Jain and Newton 
1989, Becwar et al. 1990). The single report claiming a 54% initiation 
rate from immature zygotic embryos of Pinus strobus (Finer et al. 1989) 
has yet to be repeated or duplicated by others working with this species 
(Michler et al. 1991). Secondly, it is extremely difficult to obtain 
reliable development of Pinus somatic embryos to the fully developed 
(cotyledonary) stage. In addition, subsequent production of plantlets has 
been extremely limited in Pinus species. Tautorus et al. (1992) cited only 
3 of 7 reports which indicated plantlets were obtained via somatic 
embryogenesis in Pinus species. (In contrast, 30 of 43 reports with Picea 
species reported obtaining plantlets via somatic embryogenesis.) Unlike 
the reports with Picea species where several systems have shown potential 
for plantlet production on relatively large scales, the reports of 
plantlet production from Pinus species have yielded few plants. To our 
knowledge there is only one report of successful establishment of Pinus 
somatic embryos in soil (Gupta and Durzan 1987). The authors of this 
report have had limited success in obtaining Pinus taeda somatic embryo 
plants . . . , indeed, only one culture genotype was taken to the plantlet 
stage and only one plant was transferred to soil (see Pullman and Gupta 
1991). To date the only published report of higher numbers of germination 
of Pinus somatic embryos is for Pinus caribaea, where 34 of 69 (49%) 
germinated (Laine and David 1990). However, the authors did not report 
establishment of these plants in field conditions. Thus, for Pinus species 
all three integral parts of the somatic embryogenesis process have not 
progressed to the stages currently achieved with Picea. 
Having a low initiation frequency can severely limit the potential 
applications of somatic embryogenesis in Pinus species for large scale 
production of genetically improved conifers for he following reason. 
Skilled artisans in the field of conifer tissue culture recognize that the 
use of embryogenic cultures derived from juvenile explants (e.g., zygotic 
embryos derived from seed) necessitate that the resulting regenerated 
plants be field tested prior to large scale production. Only selected 
genotypes which show the potential for producing significant genetic gain 
in such a field test will subsequently be propagated by somatic 
embryogenesis. Therefore it will be necessary to screen numerous genotypes 
from desirable parents, initiate embryogenic cultures, cryopreserve each 
genetically different culture, regenerate plants from each genetically 
different culture, field test plants from each genotype, and choose select 
genotypes for large scale production via somatic embryogenesis. Low 
culture initiation frequencies pose severe limitations for implementing 
this strategy. Indeed, an unbeknownst selection process may occur when low 
initiation frequencies exclude a majority of the genotypes. In the case of 
Pinus species where initiation frequencies are very low (e.g., 1 to 5%) 
one could be selecting for embryogenic potential, but selecting against 
improved growth potential (which may be in the 95 to 99% of the genotypes 
eliminated as non-embryogenic). The potential problem of eliminating 
desirable genotypes is exacerbated by the exceedingly low initiation 
frequencies among Pinus species. By contrast, with Picea species where 
initiation frequencies are much higher (approaching 100% from immature 
zygotic embryos of some Picea species) it is much less likely that one 
will eliminate by selection those genotypes which have superior growth 
potential. 
One component of an efficient somatic embryogenesis regeneration system is 
the culture medium. Semi-solid culture media are routinely used during the 
culture initiation, the culture maintenance, and the embryo development 
phases. The culture medium is generally composed of six groups of 
ingredients: inorganic nutrients, vitamins, organic supplements, a carbon 
source, phytohormone(s), and a gelling agent for semisolid media. The two 
gelling agents usually employed for conifer somatic embryogenesis are agar 
and gellan gum, with agar being most commonly used. 
Gelling agent concentration and type are known to influence growth 
responses of certain non-coniferous plant tissue cultures, but the effects 
of gelling agent concentration are varied and complex among different 
plant species and plant tissue types. For example, in a study working with 
rose (Rosa hybrida) tissue cultures Ghashghaie et al. (1991) found that 
increasing the availability of water by lowering a medium's agar 
concentration increased shoot elongation, yet did not improve shoot 
multiplication. Etienee et al. (1991) showed that culturing rubber tree 
(Hevea brasiliensis) explants on cellulose blocks in liquid medium 
increased embryogenic tissue initiation in comparison to culturing on the 
same medium gelled with a standard level of 2 grams of GELRITE.RTM. 
(gellan gum manufactured by Merck, Inc.) per liter of medium (grams/liter 
or g/l). They suggested the increased initiation was due to increased 
water availability of the liquid medium relative to the gelled medium. 
But, they did not determine if culturing explants on medium gelled with 
low levels of GELRITE (e.g, 1 g/l) similarly increased initiation. In 
another study utilizing sugarbeet (Beta vulgaris) leaf discs, Owens and 
Wozniak (1991) obtained more somatic embryos and shoots from leaf discs 
cultured on low levels of gelling agent. However, their results were 
obtained from sugarbeet explants cultured on a filter-paper overlay. The 
study did not directly evaluate how varying gelling agent concentration 
effected somatic embryo production from sugarbeet explants cultured 
directly on the culture medium surface. 
Those working in the field of conifer somatic embryogenesis have mainly 
emphasized medium components other than the gelling agent in attempts to 
improve culture initiation or development of somatic embryos (Tautorus et 
al. 1991). Only four reports have examined the effect of gelling agents on 
conifer somatic embryogenesis (von Arnold 1987, Klimaszewska 1989, Harry 
and Thorpe 1991, and Tremblay and Tremblay 1991). In her study von Arnold 
(1987) compared agar to GELRITE and found no difference between the two 
gelling agents for initiation of embryogenic tissue from mature zygotic 
embryos of Picea abies. The study did not test media gelled with levels of 
agar and GELRITE below 7 and 2 g/l, respectively. Klimaszewska (1989) 
compared the effect of agar versus GELRITE on proliferation and growth of 
Larix embryogenic cultures. Cultures initiated on medium gelled with 7 g/l 
of agar proliferated and grew best when transferred to medium gelled with 
4 g/l of GELRITE. Although her study did not examine the effects of low 
levels of gelling agents on culture initiation, she noted that it was 
difficult to maintain high quality cultures on a medium containing a low 
level of GELRITE (1 g/l). Harry and Thorpe (1991) tested the effect of 
agar and GELRITE concentration on initiation of Picea rubens embryogenic 
tissue, but did not test levels below 6 and 2 g/l, respectively. Tremblay 
and Tremblay (1991) examined the effect of gelling agents on the 
development of Picea abies and Picea rubens somatic embryos. They found 
that GELRITE was superior to agar, in that 3 to 5 times more somatic 
embryos developed on medium gelled with GELRITE than with agar. But, 
similar to the above three studies, concentrations of agar and GELRITE 
below 7 and 2 g/l, respectively, were not tested. 
Researchers in conifer somatic embryogenesis have commonly employed the 
same levels of gelling agents typically used in other plant cell and 
tissue culture research. These traditional gelling agent levels are 6.0 to 
9.0 grams of agar per liter of medium, 2.0 to 4.0 g/l of gellan gum (or 
GELRITE), 6.0 to 10.0 g/l of agarose (a purified form of agar), and 3.5 to 
5.0 g/l of AGARGEL.RTM. (an agar/gellan gum mixture manufactured by Sigma 
Chemical Co.). Although Hakman et al. (1985) employed an agar level of 5 
g/l in a study to induce somatic embryogenic cultures of Picea abies, no 
suggestion was made by the authors of any significance or advantage to 
using this level. Indeed, in subsequent studies these authors exclusively 
used higher levels of GELRITE (3 to 4 g/l) (Hakman and von Arnold 1985, 
von Arnold and Hakman 1988). To our knowledge, no one heretofore has 
explored the efficacy of using low levels of gelling agents for somatic 
embryogenesis among conifers. 
The implementation of somatic embryogenesis in Pinus species for production 
of clonal planting stock is also severely limited by the lack of a 
reproducible multi-step regeneration system. Very few laboratories working 
with Pinus have effectively produced embryogenic cultures or even produced 
cotyledonary stage somatic embryos. Even fewer workers have regenerated 
Pinus plants by somatic embryogenesis (Tautorus et al. 1991). In the cases 
where plants have been regenerated from Pinus embryogenic cultures, both 
the number of responsive culture genotypes and the number of plants 
obtained have been very low. 
The present invention is a multi-step somatic embryo regeneration method 
that is applicable to Pinus species and has demonstrated potential to 
regenerate plants from a diverse range of culture genotypes. The invention 
method also improves the embryogenic culture initiation frequency. This in 
itself is highly significant because it ensures that more embryogenic 
cultures survive to the culture maintenance phase, thereby allowing more 
genotypes to be subsequently available for field testing and production of 
clonal planting stock. 
In U.S. Pat. No. 4,957,866, Gupta et al. teach a process for reproducing 
coniferous plants (i.e. Pinus taeda) via somatic embryogenesis. Direct 
comparisons were performed between the patented process and the method 
taught in the present invention (see Examples 5 and 7 below). The results 
contained in Example 5 clearly showed that the current invention method 
provides a significant improvement in culture initiation when compared to 
the Gupta et al. process. (As noted above, it is vitally important to 
improve the culture initiation method practiced with Pinus in order to 
assure that more embryogenic culture genotypes are initiated and available 
for use in subsequent steps of the regeneration method.) In Example 7 the 
process of increasing the predevelopment medium osmotic potential 
disclosed in the Gupta et al. patent was compared to the method taught in 
the current invention. There the results achieved across several culture 
genotypes were at least equivalent, and in most cases far better, using 
the method of the current invention. 
In U.S. Pat. No. 5,034,326, Pullman and Gupta teach a process for 
reproducing coniferous plants (i.e. Pinus taeda) via somatic embryogenesis 
which involves using activated carbon and high levels of abscisic acid in 
the embryo development medium. In Example 6 the use of high levels of 
abscisic acid and activated carbon in embryo development medium as 
disclosed by the Pullman and Gupta patent was compared to the method 
taught in the current invention. This comparison study found the method 
taught in the current invention to be very effective while, in contrast, 
the patented process was found to be ineffective. 
In U.S. Pat. No. 5,036,007, Gupta and Pullman teach a process for 
reproducing coniferous plants via somatic embryogenesis which involves 
using abscisic acid and osmotic potential variation of the culture medium. 
In addition to utilizing high levels of abscisic acid in combination with 
activated carbon, they also teach using a subsequent embryo development 
medium having very high osmolality levels (preferably in the range of 
about 450 mM/kg). The current invention differs significantly from both of 
the above patented processes (U.S. Pat. Nos. 5,034,326 and 5,036,007). 
First, in the current invention activated carbon is not used in 
combination with abscisic acid. Second, the current invention does not 
require the embryo development medium to have the high osmolality levels 
as taught by Gupta and Pullman (1991). 
Therefore, an object of the present invention is to provide a method for 
mass producing clones of Pinus conifers by the process of somatic 
embryogenesis. 
Another object of the present invention is an improved embryogenic culture 
initiation method for Pinus conifers. 
A further object of the present invention is to provide a multistage 
regeneration protocol which can be utilized effectively on Pinus conifers 
to produce large quantities of plants for field planting. 
Another object of the present invention is to provide a progression of 
steps which, in combination, enable one to complete the somatic embryo 
regeneration method on a number of diverse genotypes of Pinus taeda and 
other Pinus species. 
In addition, it is the object of the present invention to provide a 
progression of steps which, in combination, enable one to complete the 
somatic embryo regeneration method on a number of diverse genotypes of 
Pinus interspecies hybrids (e.g., Pinus taeda.times.Pinus rigida). 
SUMMARY OF THE INVENTION 
These objectives are achieved by a multi-step method for the regeneration 
of Pinus conifer plants by somatic embryogenesis. Although other somatic 
embryogenesis regeneration protocols for conifers have been published, 
none of these methods have proven totally effective with the Pinus species 
in that none enabled the practitioner to reliably proceed from the 
beginning step of explant collection to completion of the regeneration 
process resulting in establishment of plants in field conditions. Our 
invention provides such a multi-step method for Pinus conifer plants. 
There are several advantages inherent with the use of this novel method. 
For example, the method is well suited for large-scale production of 
clonal planting stock of Pinus conifer plants. In addition, the method 
interfaces very well with genetic engineering techniques for mass 
production of clones of genetically modified and improved Pinus trees. 
The method also results in an improved embryogenic culture initiation 
frequency which allows more vigorous cultures to be obtained (which can be 
successfully carried through subsequent stages of the regeneration 
process). Furthermore, the method makes it feasible to include more 
genotypes in subsequent clonal field tests and thereby increase the 
likelihood of being able to select highly productive genotypes. Also, more 
culture genotypes can be quickly proliferated via this method for rapid 
production of clonal planting stock from selected parents. 
The present invention also provides a reliable multi-step regeneration 
method for the recalcitrant Pinus species. It is the combined application 
of the progression of steps in this novel multi-step method that has 
enabled the first successful field planting of many different genotypes of 
Pinus somatic embryos. 
BRIEF DESCRIPTION OF THE DRAWINGS

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The present invention, a multi-step method for the regeneration of plants 
of the genus Pinus and Pinus interspecies hybrid plants by somatic 
embryogenesis, generally comprises the following sequential steps: 
1. placing a suitable explant on culture initiation medium containing a 
sufficient amount of nutrients and plant growth hormones, and a level of 
gelling agent selected from the group consisting of 2.5 to 4.5 g/l of 
agar, 0.5 to 1.5 g/l of gellan gum, 3.0 to 5.0 g/l of agarose, and 1.5 to 
3.0 g/l of AGARGEL, for 2 to 14 weeks under suitable environmental 
conditions to grow a culture containing embryogenic tissue; 
2. transferring the embryogenic tissue culture to culture maintenance 
medium having a sufficient amount of nutrients and plant growth hormones, 
and a level of gelling agent selected from the group consisting of 6.0 to 
9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of 
agarose, and 3.5 to 5.0 g/l of AGARGEL, for a sufficient time under 
suitable environmental conditions to develop a mass of embryogenic tissue 
having a minimum weight of 100 mg; 
3. transferring at least 100 mg of the mass of embryogenic tissue to embryo 
development medium containing a sufficient amount of nutrients, 5 to 33 
mg/l of abscisic acid, a level of gelling agent selected from the group 
consisting of 6.0 to 12.0 g/l of agar, 1.75 to 4.00 g/l of gellan gum, 6.0 
to 8.0 g/l of agarose, and 3.5 to 6.0 g/l of AGARGEL, and 20.0 to 150.0 
g/l of a sugar selected from the group consisting of glucose, maltose, 
sucrose, and combinations thereof for a sufficient time under suitable 
environmental conditions to develop stage 3 somatic embryos; 
4. separating the stage 3 somatic embryos from the development medium and 
partially drying the embryos by exposing the embryos to an atmosphere 
having a high relative humidity (about 99%) for sufficient time to permit 
the embryos to lose about 25% to 75% of their pre-dried weight; 
5. transferring the partially dried somatic embryos to germination medium 
containing a sufficient amount of nutrients, a level of gelling agent 
selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 
g/l of gellan gum, 6.0 to 8.0 g/l of agarose, and 3.5 to 5.0 g/l of 
AGARGEL, and 20.0 to 40.0 g/l of a sugar selected from the group 
consisting of glucose, maltose, sucrose, and combinations thereof for a 
sufficient time under suitable environmental conditions to germinate the 
partially dried embryos; 
6. converting the germinated embryos into acclimatized somatic embryo 
plants; and 
7. field planting the acclimatized somatic embryo plants. 
This method is generally applicable to somatic tissue obtained from the 
Pinus species including, but not limited to, the following: Pinus taeda 
(loblolly pine), P. elliottii (slash pine), P. palustris (longleaf pine), 
P. serotina (pond pine), P. echinata (shortleaf pine), P. clausa (sand 
pine), P. glabra (spruce pine), P. rigida (pitch pine), P. echinata 
(shortleaf pine), P. nigra (Austrian pine), P. resinosa (red pine), P. 
sylvestris (Scotch pine), P. pungens (Table Mountain pine), P. banksiana 
(jack pine), P. virginiana (Virginia pine), P. radiata (Monterey pine), P. 
banksiana (jack pine), P. contorta (shore pine), P. contorta latifolia 
(lodgepole pine), P. ponderosa (ponderosa pine), P. ponderosa arizonica 
(Arizona pine), P. torreyana (Torrey pine), P. coulteri (Coulter pine), P. 
sabiniana (digger pine), P. muricata (bishop pine), P. attenuata (knobcone 
pine), P. leiophylla (Chihuahua pine), P. washoensis (Washoe pine), P. 
jeffreyi (Jeffrey pine), and P. engelmannii (Apache pine); and soft pines 
including Pinus strobus (eastern white pine), P. monticola (western white 
pine), and P. lambertiana (sugar pine), P. albicaulis (whitebark pine), P. 
flexilis (limber pine), P. strobiformis (southwestern white pine), P. 
aristata (bristlecone pine), P. balfouriana (foxtail pine), P. monophylla 
(singleleaf pine), P. edulis (pinyon), P. cembroides (Mexican pinyon), P. 
quadrifolia (Parry pinyon) and P. caribaea (caribbean pine). In addition, 
the current invention is specifically applicable to interspecies hybrids 
of the above mentioned pines including Pinus rigida.times.P. taeda, P. 
serotina.times.P. taeda, and reciprocal crosses. 
Any somatic tissue explant capable of being employed for somatic 
embryogenesis is suitable for use in present method. However, it is 
preferred that the explant be either an immature whole megagametophyte 
containing zygotic embryos or an isolated immature dominant zygotic 
embryo. 
The method of the present invention is not limited to any single culture 
nutrient medium formulation. For example, the basal culture media 
formulations used in Examples 1-10 are listed in Table I below; while 
other specific media formulations employed in Examples 1-10 are listed in 
Table II below. 
TABLE I 
______________________________________ 
Formulations Of Basal Culture Media 
DCR.sup.a 
MSG.sup.b 
COMPONENT CONCENTRATION, mg/l 
______________________________________ 
INORGANIC SALTS 
NH.sub.4 NO.sub.3 400.00 -- 
KNO.sub.3 340.00 100.00 
Ca(NO.sub.3).sub.2.4H.sub.2 O 
556.00 -- 
MgSO.sub.4.7H.sub.2 O 
370.00 370.00 
KH.sub.2 PO.sub.4 170.00 170.00 
CaCl.sub.2.2H.sub.2 O 
85.00 440.00 
KCl -- 745.00 
KI 0.83 0.83 
H.sub.3 BO.sub.3 6.20 6.20 
MnSO.sub.4,H.sub.2 O 22.30 16.90 
ZnSO.sub.4.7H.sub.2 O 
8.60 8.60 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
0.25 0.25 
CuSO.sub.4.5H.sub.2 O 
0.25 0.03 
CoCl.sub.2.6H.sub.2 O 
0.03 0.03 
NiCl.sub.2.6H.sub.2 O 
0.03 -- 
FeSO.sub.4.7H.sub.2 O 
27.80 27.80 
Na.sub.2 EDTA 37.30 37.30 
VITAMINS, AMINO ACID 
Nicotinic acid 0.50 0.50 
Pyridoxine.HCl 0.50 0.10 
Thiamine.HCl 1.00 0.10 
Glycine 2.00 -- 
______________________________________ 
.sup.a According to Gupta and Durzan (1985) 
.sup.b According to Becwar et al. (1990) 
TABLE II 
______________________________________ 
Composition Of Media Commonly Used In The Examples Below 
Initiation/ 
Pre- 
main- devel- Ger- 
tenance opment Development 
mination 
medium medium medium medium 
COMPONENT DCR.sub.1 MSG.sub.1 
KSG.sub.2 
KSG.sub.3 
______________________________________ 
Basal medium.sup.a 
DCR MSG MSG MSG 
CONCENTRATION, g/l 
Inositol 0.50 0.10 0.10 0.10 
Casein 0.50 -- -- -- 
hydrolysate 
L-glutamine 
0.25 1.45 1.45 1.45 
Sucrose 30.00 30.00 -- 30.00 
Maltese -- -- 60.00 -- 
Agar -- 8.00 -- 8.00 
GELRITE Int: 1.00 -- 2.00 -- 
Mnt: 2.00 
Activated -- 5.00 -- 5.00 
carbon 
Auxin.sup.b 
3.00 -- -- -- 
Cytokinin.sup.c 
0.50 -- -- -- 
ABA.sup.d -- -- 11.00-22.00 
-- 
mM/kg 
Medium 145-155 145-165 250-260 145-165 
osmolality 
______________________________________ 
a) Refer to Table I for composition of basal medium. 
b) 2,4dichlorophenoxyacetic acid (2,4D). 
c) N.sup.6benzylaminopurine [or N.sup.6benzyladenine (BA)]. 
d) Abscisic acid 
Gelling agents which are suitable for use in the present method include, 
but are not limited to, the following: agar, gellan gum, agarose (a 
purified form of agar), and mixtures thereof (e.g. AGARGEL.RTM. an 
agar/gellan gum mixture purchased from Sigma Chemical Co.). 
As noted above, heretofore no one has explored the efficacy of using low 
levels of gelling agents for somatic embryogenesis among the more 
recalcitrant Pinus species. The results of our experiments showed that 
gelling agent concentration has a profound effect on both the extrusion 
and the initial proliferation of embryogenic tissue. 
Two parameters characterize the availability of water in a gelled medium: 
(1) gel matric potential--the tenacity with which water is held by the 
solid phase of the gel, and (2) gel expressability--the ease with which 
water is expressed in response to mechanical deformation of the gel. At 
lower concentrations of gelling agents more water is available to plant 
tissue cultures because the water is held less tenaciously by the gel and 
the water is expressed more easily by contact of the explant to the medium 
(gel) surface. 
The osmolality of DCR.sub.1 medium containing different types and levels of 
gelling agents were measured by inserting paper discs into gelled medium 
for 30 seconds, then placing the discs in a vapor pressure osmometer 
(Model 5500 manufactured by Wescor, Inc.). Control measurements of medium 
containing no gelling agents (liquid medium) were taken by loading 10 
.mu.l of liquid on paper discs. The results are listed in Table III below. 
TABLE III 
______________________________________ 
Osmolality Of DCR.sub.1 Medium Containing 
Different Types And Levels of Gelling Agents. 
Medium 
Osmolality 
Gelling Agent (mmol/kg) 
Conc. mean .+-. st. 
Type (g/l) error.sup.a 
______________________________________ 
GELRITE 1 124 .+-. 5 
2 127 .+-. 2 
4 130 .+-. 6 
Agar 4 135 .+-. 4 
8 157 .+-. 2 
12 165 .+-. 3 
Agarose 4 138 .+-. 3 
6 136 .+-. 1 
8 137 .+-. 1 
None.sup.b 0 121 .+-. 4 
______________________________________ 
.sup.a Mean of three measurements. 
.sup.b Liquid DCR.sub.1 medium. 
Measurements of medium osmotic potential (osmolality) showed very little 
change with changing concentrations of GELRITE and agarose. Osmolality 
levels of media gelled with GELRITE, regardless of the GELRITE 
concentration, were similar to liquid medium containing no gelling agent. 
Medium gelled with agarose had slightly higher osmolality levels than 
medium gelled with GELRITE, but osmolality did not change appreciably with 
increasing agarose. Increasing the agar concentration resulted in somewhat 
increased osmolality levels, unlike the other two gelling agents. However, 
based on these measurements it is not likely that differential response of 
explants cultured on different levels of these gelling agents can be 
attributed to osmotic effects. 
These and other results suggest that the positive effect of lower gelling 
agent concentration is due to increased water availability, rather than a 
specific effect of the type of gelling agent. However, one can not rule 
out the possibility that the positive effect of the lower levels of 
gelling agents is due to decreased exposure to inhibitory substances 
(impurities) in the gelling agents. Furthermore, ion availability also 
appears to be dependent on gelling agent concentration and may, therefore, 
be a factor contributing to the positive effect of lower gelling agent 
concentration. 
Thus, a key feature of our multi-step method is the use of low levels of 
gelling agents during culture initiation (see Step 2 of Example 1). 
Specifically, our method is practiced by utilizing culture initiation 
medium containing a level of gelling agent including 2.5 to 4.5 g/l of 
agar, 0.5 to 1.5 g/l of gellan gum. 3.0 to 5.0 g/l of agarose, or 1.5 to 
3.0 g/l of AGARGEL. The preferred gelling agent levels are 3.0 to 4.0 g/l 
of agar, 0.75 to 1.25 g/l of gellan gum, 3.5 to 4.5 g/l of agarose, or 
1.75 to 2.50 g/l of AGARGEL. The common practice in the field of plant 
tissue culture is to use higher levels of gelling agents than we have 
found beneficial for Pinus culture initiation. Indeed, heretofore no one 
has shown or even suggested that using lower than normal levels of gelling 
agents is highly advantageous in initiating conifer embryogenic tissue 
cultures. 
Culture initiation lasts for a period of from 2 to 14 weeks, with the 
preferred period being 3 to 10 weeks. After this period of time the 
embryogenic tissue is transferred for further proliferation and 
maintenance to culture maintenance medium containing a higher level of 
gelling agent. Levels of gelling agents which are suitable for use in this 
method in the culture maintenance medium (as well as the germination 
medium and, if employed, the predevelopment medium) include the following: 
6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of 
agarose, and 3.5 to 5.0 g/l of AGARGEL. The embryogenic tissue is 
maintained on this medium until a mass of embryogenic tissue having a 
minimum weight of about 100 mg has developed (a period of about 1 to 14 
weeks). 
While one may practice the present method without utilizing auxin (e.g., 
2,4-dichlorophenoxy acetic acid) or cytokinin (e.g., N.sup.6 
-benzyladenine) in either the culture initiation medium or the culture 
maintenance medium, it is preferred to incorporate each of them into both 
media. Suitable levels for the present method include about 0.1 to 5.0 
mg/l for auxin and about 0.1 to 1.0 mg/l for cytokinin. 
The embryogenic tissue can be maintained by subculturing at regular 
intervals (usually every 2 to 3 weeks) to new maintenance medium. 
Alternatively, embryogenic tissue can be placed in liquid culture medium 
and grown as a liquid embryogenic suspension (as shown in Example 4 
below). Embryogenic tissue cultures maintained either on semi-solid 
maintenance medium or in liquid suspension can be cyropreserved via 
standard techniques for future use (as shown in Example 8 below). 
After the mass (or masses) of embryogenic tissue has proliferated 
sufficiently such that the culture can be maintained, a tissue mass of at 
least 100 mg (preferably at least 200 mg) is transferred to embryo 
development medium for a period of time sufficient to develop stage 3 
embryos (usually a period of about 3-18 weeks). It should be noted that 
the present method may be practiced by utilizing more than one mass of 
embryogenic tissue. Of course, for large scale production numerous masses 
would be utilized. 
The development medium suitable for use in the present method contains a 
sufficient amount of nutrients, about 20.0 to 150.0 g/l of sugar selected 
from the group consisting of glucose, maltose, sucrose, and combinations 
thereof, and abscisic acid (ABA) in an amount ranging from 5 to 33 mg/l. 
The preferred amount of sugar for use in the development medium ranges 
from about 20 to 70.0 g/l, while the preferred sugar is maltose. The 
preferred osmolality range is from about 120 to 330 mM/kg. The preferred 
range of ABA is about 11 to 27 mg/l. Levels of gelling agents which are 
suitable for use in this method in the embryo development medium include 
the following: 6.0 to 12.0 g/l of agar, 1.75 to 4.00 g/l of gellan gum, 
6.0 to 8.0 g/l of agarose, and 3.5 to 6.0 g/l of AGARGEL. 
While the tissue mass may be cultured on the embryo development medium 
under lighted conditions, it is preferred to culture the tissue mass in a 
dark environment. 
In certain cases it may be preferable to transfer the mass of embryogenic 
tissue from culture maintenance medium to embryo predevelopment medium for 
a period of 1 to 21 days prior to transferring the mass to embryo 
development medium. Embryo predevelopment medium suitable for use in the 
present method has an osmolality level in the range of 120 to 180 mM/kg 
and contains a sufficient amount of nutrients, from 1.0 to 10.0 g/l of 
activated carbon, and from 20.0 to 35.0 g/l of sugar selected from the 
group consisting of glucose, maltose, sucrose, and combinations thereof. 
Levels of gelling agents which are suitable for use in this method in the 
embryo predevelopment medium include the following: 6.0 to 9.0 g/l of 
agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, and 3.5 
to 5.0 g/l of AGARGEL. 
After the stage 3 somatic embryos have developed they are partially dried 
or dehydrated via exposure to an atmosphere having a high relative 
humidity (e.g., greater than 90% up to 99%) for sufficient time to permit 
the embryos to lose about 25% to 75% of their pre-dried weight (usually a 
period of about 2 to 5 weeks). The amount of moisture to be removed an 
embryo depends upon several factors, including the genotype of the embryo, 
the culture medium used, and the storage products contained in the embryo. 
It is well within the ability of a skilled artisan to determine the 
optimum moisture loss necessary to prepare each embryo for germination. 
The partially dried somatic embryos are subsequently transferred to 
germination medium until germination occurs (usually about 1 to 8 weeks). 
These germinated embryos are converted into acclimatized somatic embryo 
plants via the manipulation of environmental factors prior to field 
planting. 
In the present method it is further preferred to cover each of the above 
noted mediums (culture initiation :medium, culture maintenance medium, 
embryo predevelopment medium, embryo development medium, and germination 
medium) with a sterile permeable membrane. The respective embryogenic 
cultures are subsequently placed upon the membrane instead of being placed 
directly upon the medium. The permeablity of the membrane allows the 
free-flow of materials between the culture and the medium. This 
modification greatly facilitates subsequent transfer of embryogenic 
cultures by avoiding direct contact with and disturbance of the cultures 
during transfer (see Example 8 below). 
A number of terms are known to have differing meanings when used in the 
literature. The following definitions are believed to be the ones most 
generally used in the field of botany and are consistent with the usage of 
the terms in the present specification. 
A "cell line" is a culture that arises from an individual explant. 
"Corrosion cavity" is the cavity within the megagametophyte tissue of 
conifers formed by the growth and enlargement of the zygotic embryos. 
"Conversion" refers to the acclimatization process that in vitro derived 
germinating somatic embryos undergo in order to survive under ex vitro 
(nonaxenic) conditions, and subsequent continued growth under ex vitro 
conditions. 
"Cyropreservation" refers to the common process of storing cultures at 
ultra-low temperatures for future use. 
A "dominant zygotic embryo" is one zygotic embryo among the multiple 
embryos formed in conifer seeds due to simple and cleavage polyembryony 
that outgrows the other zygotic embryos and matures in the seed. 
An "embryogenic culture" is a plant cell or tissue culture capable of 
forming somatic embryos and regenerating plants via somatic embryogenesis. 
"Embryogenic tissue" in conifers, is a mass of tissue and cells comprised 
of very early stage somatic embryos and suspensor-like cells embedded in a 
mucilaginous matrix. The level of differentiation may vary significantly 
among embryogenic conifer cultures. In some cases, rather than containing 
well formed somatic embryos, the embryogenic tissue may contain small, 
dense clusters of cells capable of forming somatic embryos. 
"Epicotyl" is the first newly formed shoot to develop and grow after the 
seed leaves (cotyledons). 
An "explant" is the organ, tissue, or cells derived from a plant and 
cultured in vitro for the purpose of starting a plant cell or tissue 
culture. 
"Extrusion" is the process by which zygotic embryos and/or embryogenic 
tissue derived from zygotic embryos emerges or extrudes from the corrosion 
cavity of the megagametophyte of conifer seeds via the opening in the 
micropylar end, when placed in culture. 
"Field planting" is the establishment of laboratory, greenhouse, nursery, 
or similarly grown planting stock under field conditions. 
"Genotype" is the genetic constitution of an organism; the sum total of the 
genetic information contained in the chromosomes of an organism. 
"Germination" is the emergence of the radicle or root from the embryo. 
"Initiation" is the initial cellular proliferation or morphogenic 
development that eventually results in the establishment of a culture from 
an explant. 
"Megagametophyte" is haploid nutritive tissue of the conifer seed, of 
maternal origin, within which the conifer zygotic embryos develop. 
"Micropyle" is the small opening in the end of the conifer seed where the 
pollen tube enters the ovule during fertilization, and where embryogenic 
tissue extrudes from the megagametophyte during culture initiation. 
"Nutrients" are the inorganics (e.g., nitrogen), vitamins, organic 
supplements, and carbon sources necessary for the nourishment of the 
culture. 
A "plantlet" is a small germinating plant derived from a somatic embryo. 
"Regeneration", in plant tissue culture, is a morphogenic response to a 
stimuli that results in the production of organs, embryos, or whole 
plants. 
"Stage 1 embryos" are small embryos consisting of an embryonic region of 
small, densely cytoplasmic cells subtended by a suspensor comprised of 
long and highly vacuolated cells. 
"Stage 2 embryos" are embryos with a prominent (bullet shaped) embryonic 
region that is more opaque and with a more smooth and glossy surface than 
stage 1 embryos. 
"Stage 3 embryos" are embryos with an elongated embryonic region with small 
cotyledons visible. 
"Somatic embryogenesis" is the process of initiation and development of 
embryos in vitro from somatic cells and tissues. 
A "somatic embryo" is an embryo formed in vitro from vegetative (somatic) 
cells by mitotic division of cells. Early stage somatic embryos are 
morphologically similar to immature zygotic embryos; a region of small 
embryonal cells subtended by elongated suspensor cells. The embryonal 
cells develop into the mature somatic embryo. 
A "suspensor cell" is an extension of the base of the embryo that 
physically pushes the embryo into the megagametophyte in conifer seeds and 
is comprised of elongated and highly vacuolated cells. 
A "zygotic embryo" is an embryo(s) which is derived from the sexual fusion 
of gametic cells. 
The following examples are provided to further illustrate the present 
invention and are not to be construed as limiting the invention in any 
manner. 
EXAMPLE 1 
The following multi-step method, when used in combination sequentially, has 
proven effective for regeneration of loblolly pine (Pinus taeda L.) by 
somatic embryogenesis. The procedure is as follows: 
Step 1: Explant Collection And Preparation For Culture 
Immature seed cones were collected from several different loblolly pine 
(Pinus taeda L.) sources located in Westvaco's South Carolina coastal 
breeding orchards near Charleston, South Carolina. The seed cones were 
collected when the dominant zygotic embryo was at the precotyledonary 
stage of development. Using the classification system of Hakman and von 
Arnold (1988), the dominant zygotic embryo at this stage is referred to as 
being at stage 2; that is, an embryo with a prominent embryonic region 
with a smooth and glossy surface, subtended by elongated suspensor cells 
which are highly vacuolated. Embryos which have progressed further in 
their development (to stage 3) will have cotyledon primordia, and will not 
be at an optimum stage of development for culture initiation. Although 
zygotic embryos at an earlier stage of development (stage 1) were also 
used effectively to initiate embryogenic cultures, Stage 2 embryos were 
optimum (and therefore preferred). The stage of zygotic embryo development 
was checked-by extracting megagametophytes from seeds, longitudinally 
dissecting megagametophytes, and removing zygotic embryos for examination. 
This extraction and examination of the zygotic embryos was done under a 
dissection microscope. Loblolly pine cones collected from breeding 
orchards in the Charleston, South Carolina area reach the desired 
precotyledonary stage of development (stage 2) in mid to late July. Based 
on the finding that fertilization in loblolly pine occurred in mid June, 
the optimum stage corresponded to about 4 to 6 weeks post-fertilization. 
Seed cones were harvested from selected trees, placed in plastic bags and 
stored at 4.degree. C. until used for culture initiation. If the cones 
were stored for more than two weeks at 4.degree. C., they were aired and 
dried out weekly (placed at 23.degree. C., ambient laboratory conditions 
for 2-3 hours) to prevent growth of fungi on the surface of the cones and 
concomitant deterioration of seed quality. 
For culture initiation intact seeds removed from seed cones were surface 
sterilized by treatment in a 10 to 20% commercial bleach solution 
(equivalent of a 0.525% to 1.050% sodium hypochlorite solution) for 15 
minutes followed by three sterile water rinses (each of five minutes 
duration). Seeds were continuously stirred during the sterilization and 
rinsing process. 
Step 2: Culture Initiation 
Steps 2-4 were performed in a laminar-flow hood, routinely used to perform 
aseptic plant tissue culture techniques. Megagametophytes containing 
developing zygotic embryos were used as the explant for culture 
initiation. The seed coats of individual seeds were cracked open with the 
use of a sterile hemostat. The intact megagametophyte (which contains the 
developing zygotic embryos) was removed from the opened seed coat with 
forceps. Tissues attached to the megagametophyte, such as the 
megagametophyte membrane and the nucellus were removed from the 
megagametophyte and discarded. The megagametophte was placed on culture 
medium (longitudinal axis of megagametophyte parallel to the surface of 
culture medium) with forceps. The micropyle end of the megagametophyte was 
placed in contact with (but not submerged in) the culture medium. 
Basal salt mixtures which have proven effective for culture initiation 
include the DCR basal salts formulation listed in Table I. (The complete 
formulations of the DCR medium used in the Examples are listed in Table 
II.). The pH of the medium was adjusted to 5.8 with KOH and HCl prior to 
autoclaving at 110 kPa (16 psi) and 121.degree. C. for 20 minutes. Aqueous 
stock solutions of L-glutamine were filter sterilized and added to warm. 
(about 60.degree. C.) medium prior to pouring the medium into culture 
plates. Approximately 20 ml of medium was poured into 100.times.15 mm 
sterile plastic petri plates. 
After megagametophyte explants were placed in culture, the perimeter of the 
plate was sealed with two wraps of AFILM.RTM. (manufactured by American 
Can Co.). The plates were incubated in the dark at a constant temperature 
of 23.degree. C. After about 7 to 21 days, embryogenic tissue extruded 
from the micropyle of the megagametophyte explants. After 28 days in 
culture embryogenic tissue was removed from responsive megagametophyte 
explants and moved to a new position on the same culture plate, or the 
embryogenic tissue was transferred to a new culture plate containing the 
same culture medium as used for initiation. Each individual culture 
derived from an individual megagametophyte explant was kept separate and 
assigned a cell line identification code. 
Over 2000 explants were tested in nine different experiments to illustrate 
the effect of different levels of gelling agents on initiation of pine 
embryogenic tissue. Two culture media were employed in these experiments: 
DCR.sub.1 (Table II) and SH.sub.1 (SH inorganic salts; Schenk and 
Hilderbrandt (1972) with other components as in DCR.sub.1). The pooled 
results from all nine experiments show that more embryogenic cultures were 
initiated on the low level of gelling agents than on normal or high levels 
(see Table IV below). The initiation results are given as both the 
frequency of embryogenic tissue extrusion from the explant and also 
embryogenic tissue proliferation. The frequency of extrusion provides a 
measure of how many explants showed potential for culture initiation, 
whereas the proliferation measures the number which produced vigorously 
growing embryogenic tissue. 
TABLE IV 
______________________________________ 
Summary of Overall Results Of 9 Experiments Conducted To 
Test Effect On Initiation of Pine Embryogenic Tissue (ET) 
Using Different Gelling Agent Levels. 
Gelling Frequency (%) explants 
Agent Level Extrude ET Proliferate ET 
______________________________________ 
Low 305/672 (45) 
112/672 (17) 
Normal 258/672 (38) 
82/672 (12) 
High 236/672 (35) 
72/672 (11) 
______________________________________ 
The levels of gelling agents tested are classified as low, normal, and high 
as follows: The low level of gelling agents (1 g/l of GELRITE, 4 g/l of 
agar, 2 g/l of AGARGEL, and 4 g/l of low gelling temperature agarose) are 
lower than levels traditionally taught for plant tissue culture usage. The 
normal levels (2 g/l of GELRITE, 8 g/l of agar, 4 g/l of AGARGEL, and 6 
g/l of LGT agarose) are levels commonly taught for plant tissue culture 
usage. The high levels (4 g/l of GELRITE, 12 g/l agar, 6 g/l of AGARGEL, 
and 8 g/l of LGT agarose) are generally considered to be higher than 
typically used in plant tissue culture. 
A more detailed summary of each of the nine experiments referenced in Table 
IV is provided by Table V below. 
TABLE V 
______________________________________ 
Summary Of 9 Experiments With Lower Levels Of Gelling 
Than Commonly Used To Initiate Pine Embryogenic Tissue (ET). 
Number.sup.a (%) explants 
Cul- Extrude Proliferate 
Expt Seed ture Gelling ET ET at 10 
No. source medium agent.sup.b 
at 4 weeks 
weeks 
______________________________________ 
1 A45 .times. 
DCR.sub.1 
GELRITE 39 (81%) 
5 (10%) 
A26 Agar 35 (73%) 
12 (25%) 
2 A10 DCR.sub.1 
GELRITE 31 (65%) 
2 (4%) 
Agar 26 (54%) 
4 (8%) 
3 B19.sup.c 
DCR.sub.1 
GELRITE 22 (46%) 
13 (27%) 
Agar 14 (29%) 
7 (15%) 
4 A38 .times. 
DCR.sub. GELRITE 7 (15%) 4 (8%) 
A45 Agar 9 (19%) 4 (8%) 
5 A10 .times. 
DCR.sub.1 
GELRITE 17 (35%) 
4 (8%) 
A45 SH.sub.1 AGARGEL.sup.d 
11 (23%) 
4 (8%) 
6 A38 .times. 
SH.sub.1 GELRITE 12 (25%) 
4 (8%) 
A45 
7 A19 SH.sub.1 GELRITE 37 (77%) 
27 (56%) 
8 A10 .times. 
SH.sub.1 GELRITE 10 (21%) 
1 (2%) 
A45 
9 A45 .times. 
SH.sub.1 LGT.sup.e 
35 (73%) 
21 (44%) 
A33/38 Agarose 
DCR.sub.1 totals: 200 (46%) 55 (13%) 
SH.sub.1 totals: 105 (44%) 57 (24%) 
Overall totals: 305 (41%) 112 (17%) 
______________________________________ 
.sup.a The number of responsive explants per gelling agent are listed. 
Fortyeight explants were cultured on each gelling agent in each 
experiment. 
.sup.b Media gelled with agar contained TC agar (#198202, manufactured by 
Carolina Biol. Supply Co.). 
.sup.c B19 was an interspecies hybrid (Pinus rigida .times. Pinus taeda) 
seed source. 
.sup.d AGARGEL (#A3301, purchased from Sigma Chem. Co.). 
.sup.e LGT (low gelling temperature) agarose (#A6560, purchased from Sigm 
Chem. Co.). 
We purposely conducted these experiments with explants derived from 
genetically different seed sources (including both responsive seed sources 
and recalcitrant seed sources) in order to determine the effects of low 
levels of gelling agents on a broad range of genetic material. The results 
in Table V suggest that there was an interactive effect between the seed 
source and the type of gelling agent used. For example, in experiments 1 
and 2, with seed sources A45.times.A26 and A10, agar resulted in about 
double the proliferation frequency than GELRITE. Whereas, in experiment 3, 
with seed source B19, the opposite was found, and explants on GELRITE 
proliferated more embryogenic tissue than on agar. Thus, one particular 
gelling agent type may not be optimum for use across all seed sources. 
In addition, we purposely conducted several experiments listed in Table V 
(numbers 5-9) on SH.sub.1 medium to verify that the positive effect of 
using low gelling agents was not restricted to the DCR.sub.1 culture 
medium. As indicated previously, proliferation frequencies of 1 to 5% have 
typically been reported for Pinus species conifers. It is clear, 
therefore, from the results shown in Table V that high extrusion and 
proliferation were obtained on both DCR and SH.sub.1 medium with several 
seed sources by using lower than conventional levels of gelling agents. 
The data presented above on the effect of gelling agent levels on extrusion 
and proliferation of embryogenic tissue measured the frequencies of 
culture initiations. Additionally, in eight of the nine experiments we 
also measured culture weight in relation to gelling agent level. The 
results showed that not only were more cultures initiated on media 
containing low gelling agent levels, but that the cultures thus initiated 
were more vigorous and weighed more than cultures initiated on media 
containing normal or high levels of gelling agent. 
FIGS. 1 and 2 in the drawing further illustrate these findings. The 
responsive cultures are ranked from left to right according to culture 
weight. 
Thus, not only did more explants produce vigorous embryogenic tissue, but 
the weight of embryogenic tissue produced was highest using a low level of 
gelling agent. For example, on the low level of GELRITE a total of 13 
cultures proliferated a cumulative total of 1102 milligrams (mgs) of 
embryogenic tissue from seed source B19, whereas on the conventional level 
of GELRITE 6 cultures proliferated a cumulative total of 124 mgs of 
embryogenic tissue (FIG. 1). It is important to obtain rapid growth early 
in the culture establishment process in order to quickly multiply the 
embryogenic tissue for subsequent use. Our results suggest that using 
lower levels of gelling agents than is commonly used in conifer tissue 
culture improves the frequency of rapidly proliferating pine embryogenic 
cultures. 
This is a significant finding for initiation of embryogenic tissue in Pinus 
species, because unlike Picea species, most workers find Pinus initiation 
to be extremely difficult. Any improvement in the initiation process which 
results in more embryogenic cultures being established translates into 
more embryogenic cultures being available for use in the regeneration 
process. Having a higher initiation frequency is critical since it 
increases the probablility of being able to identify superior culture 
genotypes for use in large scale production of clonal planting stock. In 
the past the limited number of embryogenic cultures available for 
regeneration has been a major limitation for implementation of somatic 
embryogenesis in Pinus species. Thus, all three parameters measured--(1) 
extrusion frequency, (2) proliferation frequency, and (3) growth as 
measured by the total weight of embryogenic tissue--were improved by using 
low levels of gelling agents. 
Step 3: Culture Maintenance 
Cultures were maintained on semi-solid medium, i.e., DCR.sub.1 (Table II, 
the same medium as described for culture initiation) by subculturing 
masses of embryogenic tissue every 14 to 21 days to fresh medium. Culture 
maintenance conditions were the same as for culture initiation, except 
that the gelling agent levels contained in the culture maintenance media 
were increased. (It should be noted that the cultures could also be 
maintained as liquid suspension cultures on the same medium devoid of the 
gelling agent.) 
Step 4: Embryo Development 
At the end of a two to three week period on DCR medium, masses of 
embryogenic tissue (about 200 mg each) were either transferred to a 
MSG.sub.1 predevelopment medium or to a MSG.sub.2 development medium (see 
Table II above). The MSG.sub.1 medium contained activated carbon. If the 
embryogenic tissue was placed on a MSG predevelopment medium, after about 
one week it was transferred to a MSG.sub.2 development medium. As noted in 
Table II, the MSG.sub.2 medium contained maltose, a carbon source (Uddin 
1993), and ABA, but did not contain activated carbon. 
All cultures were incubated at 23.degree. C. in the dark. It is preferred 
that the cultures be incubated in the dark rather than light conditions, 
especially during the MSG.sub.2 phase of embryo development out every 21 
days the embryogenic tissue was transferred to fresh embryo development 
MSG.sub.2 medium. After two passages on the MSG.sub.2 medium, cotyledonary 
somatic embryos (stage 3) were visible on the surface of the embryogenic 
tissue. Typically, multiple harvests of cotyledonary somatic embryos were 
made at the end of the second and third passage, and sometimes after the 
fourth passages on MSG.sub.2 medium. Subsequently the embryogenic tissue 
became necrotic and produced very few, if any, cotyledonary somatic 
embryos on MSG.sub.2 medium and the embryogenic tissue was discarded. (It 
should be noted that the original culture from which the embryogenic 
tissue had been derived was concurrently maintained as a stock culture on 
DCR medium as described in step 2.) 
The effect of the ABA level contained in the development medium on 
production of harvestable stage 3 somatic embryos (SEs) of Pinus taeda 
from an individual embryogenic culture genotype initiated from seed source 
A4 was evaluated. Three pieces of embryogenic tissue of about 200 mg each 
were tested on each ABA level, and the results are listed in Table VI 
below. 
TABLE VI 
______________________________________ 
Effect Of Abscisic Acid Levels On Somatic Embryos 
ABA level Number of stage 
(mg/l) 3 SEs harvested 
______________________________________ 
0 9 
11 133 
22 157 
33 114 
______________________________________ 
The results show that very few harvestable stage 3 somatic embryos were 
produced when no ABA was employed in the embryo development medium. 
The effect of abscisic acid concentration in the embryo development medium 
was further evaluated utilizing cultures from different seed sources and 
following the method taught in Steps 1-4 above. The results are shown in 
Table VII below. 
TABLE VII 
______________________________________ 
Effect Of Abscisic Acid Levels On Somatic 
Embryos From Different Seed Sources 
Culture Seed ABA concentration (mg/l) 
code source 5 11 16 21 27 
______________________________________ 
1 A6 6 37 54 98 7 
2 A44 0 10 10 21 72 
3 A26 0 4 39 23 22 
______________________________________ 
As noted above, in the present method it is preferred to incorporate ABA 
into the embryo development media in an amount ranging from 5 to 33 mg/l. 
The more preferred range of ABA is about 11 to 27 mg/l. 
Step 5: Embryo Maturation Drying 
Pine somatic embryos were prepared for germination by a maturation drying 
treatment which reduced their water content by an average of about 50%. 
This technique, referred to as "partial drying" (Kermode et al. 1989) was 
first used to improve germination of immature caster bean seeds (Kermode 
and Bewley 1985). The authors hypothesized that partial drying terminated 
the embryo development process and initiated metabolic processes necessary 
to prepare the embryo for germination and subsequent growth. Roberts 
(1993) used a similiar treatment to improve germination of Picea somatic 
embryos. 
Stage 3 somatic embryos were transferred with forceps to the bottom surface 
of six empty wells of a 12-well plastic plate. The remaining six wells had 
previously been half-filled with sterile water. Typically, not more than 
20 somatic embryos were placed in each empty well. The perimeter of the 
plate was sealed with two wraps of AFILM and incubated for 
approximately 21 days in the dark at 23.degree. C. Our measurements showed 
that the Pinus somatic embryos lost between 35 to 64% of their original 
fresh weight during the partial drying treatment. 
Step 6: Germination 
Partially dried somatic embryos were placed horizontally on the surface of 
MSG]medium. The medium was in 100.times.15 mm sterile plastic petri 
plates. Typically, about 16 to 25 somatic embryos were placed in each 
plate. The perimeter of plates were wrapped twice with AFILM. Plates 
with embryos were incubated in the dark at 23.degree. C. until the embryos 
elongated to approximately 1 to 2 cm (usually about 10 to 14 days). At 
this time the germination process had begun, with the emergence of the 
radicle (root) on some somatic embryos. Plates with the germinating 
somatic embryos were then transferred to a 16-hour fluorescent light and 
8-hour dark photoperiod at 25.degree. C. 
A total of 6585 somatic embryos from 123 different culture genotypes were 
tested for germination via the above 30 procedure. Of these, 2657 (40%) 
germinated from 101 (82%) different genotypes. Forty-six genotypes (38%) 
of somatic embryos had germination frequencies of at least 50%. Six 
genotypes had germination levels above 75%. 
Step 7: Conversion 
The term "conversion" includes the acclimatization process that in vitro 
derived germinating somatic embryos undergo in order to survive under ex 
vitro (nonaxenic) conditions, and subsequent continued growth under ex 
vitro conditions. 
When the length of the roots reached about 2 to 3 cm the germinating 
plantlets were aseptically removed from the plates and placed on moistened 
filter paper in a 100.times.15 mm petri plate. Although plantlets may 
later be placed in sterilized potting mix, they were no longer maintained 
in an axenic environment from this time on. Plants were then transplanted 
into either: (1) sterilized GRACE FORESTRY MIX (a soil mixture 
manufactured by T. R. Grace & Co.) in MAGENTA BOXES (containers 
manufactured by Magenta Corp.); or (2) TECHNICULTURE PEAT PLUGS (peat 
plugs manufactured by Techniculture Inc.). The boxes containing plantlets 
were sealed with AFILM and placed in a growth chamber with a 16-hour 
fluorescent and incandescent light and an 8-hour dark photoperiod at 
23.degree. C. The plantlets in peat plugs were enclosed in a plastic 
container used for growing seedlings and sealed with clear plastic cover 
to maintain a high relative humidity. The container was placed in a growth 
chamber under the same conditions as the boxes. Plantlets were fertilized 
weekly with a nutrient solution containing 50 ppm inorganic nitrogen and 
watered with reverse osmosis treated water as needed in order to keep 
potting mix or peat plugs from drying out. 
When the plantlets formed epicotyls (newly formed shoots approximately 2 to 
4 cm), they were transferred to leach tubes (RAY LEACH "CONE-TAINERS".RTM. 
#SSCUV manufactured by Stuewe & Sons, Inc.). Plantlets in boxes were 
transplanted into leach tubes containing a potting mix (2:1:2 
peat:perlite:vermiculite, containing 602 g/m.sup.3 OSMOCOTE.RTM. 
fertilizer (18-6-12), 340 g/m.sup.3 dolomitic lime and 78 g/m.sup. 
MICRO-MAX.RTM. micronutrient mixture (manufactured by Sierra Chem. Co.). 
Plantlets in peat plugs were inserted directly (peat plug with intact 
plantlet) into potting mix contained in leach tubes. The leach tubes were 
placed in a greenhouse mist chamber. The environmental conditions in the 
mist chamber are as follows: 
(1) Mist was applied for 30 seconds every 30 minutes from 6:00 a.m. to 6:30 
p.m., and for 30 seconds every 60 minutes from 6:30 p.m. to 6:00 a.m.; 
(2) Temperature was maintained at 26 to 31.degree. C. during the day and at 
18 to 20.degree. C. at night; and 
(3) Ambient light was admitted through black polypropylene shade cloth (51% 
shade) covering the greenhouse. Supplemental light from high pressure 
sodium bulbs was provided to produce a total photoperiod of about 16 
hours. 
When the plantlets had grown to approximately 8 to 16 cm in height, trays 
containing the resulting somatic embryo plants in leach tubes were removed 
from the mist chamber and placed on an open bench in the greenhouse for at 
least two weeks for acclimatization. Subsequently, somatic embryo plants 
in leach tube trays were moved to a shadehouse (framed structure covered 
with black polypropylene shade cloth) for approximately two weeks, and 
then to ambient outdoor conditions for an additional two weeks. Somatic 
embryo plants in leach tubes were watered with reverse osmosis treated 
water as required both during the greenhouse, shadehouse, and outdoors 
acclimatization period. 
Following the above procedure, a total of 1567 germinated somatic embryos 
from 91 different culture genotypes were tested for conversion to 
vigorously growing somatic embryo plants. Of these germinants a total of 
328 were converted to vigorously growing somatic embryo plants; a 21% 
conversion frequency. 
Step 8: Field Planting 
Acclimatized somatic embryo plants were carefully removed from the leach 
tubes so that the potting mix remained attached to roots and transplanted 
to a prepared field site. The field plantings were done on two consecutive 
years (1991 and 1992). In the 1991 field planting, 51 somatic embryo 
plants from six different genotypes were planted in the field. The number 
of plants per genotype ranged from 1 to 22. In the 1992 field planting, 
292 somatic embryo derived plants from 61 genotypes were planted in the 
field. The number of plants per genotype ranged from 1 to 28. To date, 335 
of the 343 somatic embryo plants (98%) have survived and appear 
phenotypically normal relative to standard Pinus taeda seedlings planted 
at the same times. 
SUMMARY OF RESULTS 
The present method results in significant improvements both in the number 
genotypes responsive to somatic embryogenesis and in the number of plants 
regenerated from the cultures. While others working in the field of 
somatic embryogenesis have attempted to provide .protocols for Pinus 
species, the present method (here demonstrated employing Pinus taeda) has 
proven to be extremely effective on a broad range of diverse genetic 
material, thereby resulting in the production of large numbers of somatic 
embryos from numerous genotypes. This had not been possible with any Pinus 
species prior to this invention. One should note the importance of the 
sequential application of steps 1 through 7, which enabled successful 
completion of the entire regeneration process and establishment of Pinus 
taeda somatic embryo plants in field plantings. 
EXAMPLE 2 
Pinus rigida is native to eastern North America (New Brunswick) to 
southeastern U.S. (Georgia) and is classified as an Eastern hard pine 
(Peterson 1989). Pinus taeda, a southern yellow pine with native 
distribution which extends as far north as New Jersey, is more productive 
in the southeastern U.S. and gulf states. Interspecies hybrids between 
Pinus rigida and Pinus taeda are of commercial interest because the hybrid 
retains desirable characteristics of each species; namely, the increased 
cold hardiness of Pinus rigida and some of the superior growth potential 
of Pinus taeda. Breeding efforts have resulted in desirable parental 
selections of Pinus rigida and Pinus taeda which yield F.sub.1 hybrid seed 
for production of planting stock for reforestation in regions that extend 
north of where Pinus taeda is productive. Currently production of 
interspecies F.sub.1 hybrid seed is achieved through supplemental mass 
pollination of Pinus rigida with Pinus taeda pollen. But, it is frequently 
difficult to produce large quantities of F.sub.1 hybrid seed due to embryo 
abortion resulting in poor seed production. Production of F.sub.1 hybrid 
clonal planting stock by somatic embryogenesis, therefore, offers a 
potential alternative for efficient large scale production from selected 
superior genotypes of interspecies Pinus hybrids. 
To date, the only report of somatic embryogenesis and plant regeneration 
from interspecies conifer hybrids was with Larix.times.Eurolepis 
(Klimaszewska 1989). There has been no progress, to our knowledge, on 
development of a somatic embryogenesis protocol completing the entire 
plant regeneration process that has proven effective for Pinus 
interspecies hybrids. The present invention solves this problem and 
provides a somatic embryo regeneration system for the interspecies hybrid 
of Pinus rigida.times.Pinus taeda that has demonstrated success with 
established field plantings of F.sub.1 hybrid clonal planting stock. 
We have found the method described in the Example 1 to be effective on 
initiating embryogenic cultures and regenerating F.sub.1 hybrid somatic 
embryo plants of Pinus rigida.times.Pinus taeda. To illustrate this we 
followed Steps 1-3 of the method taught in Example 1 in order to evaluate 
ten genetically diverse Pinus rigida.times.Pinus taeda seed sources 
(labeled B1-B10) for the proliferation of embryogenic tissue from immature 
seeds. The results are listed in Table VIII below. 
TABLE VIII 
______________________________________ 
Proliferation of Embryogenic Tissue from Ten 
Pinus rislida .times. Pinus taeda Seed Sources 
Seed No. seeds 
Percent 
source cultured proliferation 
______________________________________ 
B1.sup.b 55 7 
B2.sup.a 29 14 
B3.sup.a 83 11 
B4.sup.a 95 9 
B5.sup.a 96 13 
B6.sup.a 25 8 
B7.sup.a 40 5 
B8.sup.a 48 6 
B9.sup.c 145 19 
B10.sup.c 145 6 
______________________________________ 
.sup.a Pinus rigida maternal tree supplementally mass pollinated with 
Pinus taeda pollen. 
.sup.b Pinus rigida .times. Pinus taeda F.sub.1 hybrid maternal tree 
supplementally mass pollinated with Pinus taeda pollen. 
.sup.c Pinus rigids maternal tree control pollinated with Pinus taeda 
pollen. 
As previous reports of Pinus species have typically obtained proliferation 
rates of only 1 to 5%, it is clear from the data shown in Table VIII that 
excellent proliferation results were achieved using the present method. 
Somatic embryos were developed from 24 F.sub.1 hybrid culture genotypes 
from 12 genetically different Pinus rigida.times.Pinus taeda parental 
combinations via the procedure taught in Steps 1-4 of Example 1. 
Twenty-one of the 24 F.sub.1 hybrid culture genotypes (88%) were 
responsive and produced harvestable cotyledonary somatic embryos. 
Twenty-five percent of the responsive genotypes produced more than 100 
harvestable somatic embryos per genotype. A total of 1706 cotyledonary 
somatic embryos were harvested from 10 of the 12 seed sources tested. 
Several evaluations conducted with F.sub.1 hybrid embryogenic culture 
genotypes have demonstrated the potential for producing large numbers of 
Pinus rigida.times.Pinus taeda F.sub.1 hybrid somatic embryos via the 
current invention method. Yields of harvestable stage 3 somatic embryos as 
high as 400 to 500 per gram of embryogenic tissue have been obtained. In 
one evaluation utilizing a F.sub.1 hybrid culture genotype 4126 
harvestable stage 3 somatic embryos were obtained from a total of 23 grams 
of embryogenic tissue. This is an average yield per gram of embryogenic 
tissue of 180 harvestable stage 3 somatic embryos. 
Germination of F.sub.1 hybrid somatic embryos of Pinus rigida.times.Pinus 
taeda obtained by following Steps 1-5 of Example 1 were achieved using 
Step 6 of Example 1. A total of 3705 somatic embryos from 23 different 
F.sub.1 hybrid culture genotypes were tested, and 1116 (30%) germinated. 
Germinanting plantlets were obtained from 18 (78%) of the 23 genotypes 
tested. Germination frequencies were as high as 85% for ,one culture 
genotype (139 of 164 somatic embryos). 
Conversion of germinated F.sub.1 hybrid somatic embryos of Pinus 
rigida.times.Pinus taeda obtained utilizing Steps 1-6 of Example 1 were 
achieved by employing Step 7 of Example 1. A total of 399 germinated 
F.sub.1 hybrid somatic embryos from 17 different culture genotypes were 
tested for conversion to vigorously growing plants. Of the 399 germinated 
embryos, a total of 173 were converted to vigorously growing plants; a 43% 
conversion frequency. 
Field plantings of F.sub.1 hybrid somatic embryo plants of Pinus 
rigida.times.Pinus taeda using Steps 1-7 of the Example 1 were achieved by 
employing Step 8 of Example 1. The field plantings were done on two 
consecutive years; 1991 and 1992. In 1991, 57 somatic embryo plants from 
two F.sub.1 hybrid genotypes were field planted. In the 1992 field 
planting, 171 somatic embryo plants from 14 F hybrid genotypes were sown. 
The number of plants per genotype ranged from 1 to 129. To date, 202 of 
the 228 F.sub.1 hybrid somatic embryo Pinus rigida.times.Pinus taeda 
plants (89%) have survived and appear phenotypically normal relative to 
conventional Pinus rigida.times.Pinus taeda F.sub.1 hybrid seedlings. 
EXAMPLE 3 
The method taught in Example 1 was utilized in order to initiate 
embryogenic cultures and regenerate somatic embryo plants of Pinus 
serotina and Pinus serotina.times.Pinus taeda. P. serotina is a species 
native to the southeastern U.S., closely related to Pinus taeda, and of 
potential commercial value for reforestation on poorly drained field 
sites. Explants were derived from immature cones collected from two seed 
sources and tested for culture initiation. Proliferating embryogenic 
tissue was obtained from five different genotypes derived from two seed 
sources. Cotyledonary stage somatic embryos were obtained from one culture 
genotype of seed source C1. Four of 12 somatic embryos germinated. Two of 
the four germinated somatic embryos were established as vigorous plants 
under greenhouse conditions. The somatic embryo plants were similar in 
size and phenotypic appearance to the other Pinus somatic embryo plants 
produced in the first and second examples with P. taeda and P. 
rigida.times.P. taeda. 
EXAMPLE 4 
The following evaluation compared the present method with the process 
taught by Gupta and Pullman in U.S. Pat. No. 4,957,866--particularly the 
method of initiating the embryogenic culture as taught in Step 2 of 
Example 1 of the present method. Immature megagametophyte explants were 
tested from an open-pollinated seed source (A10) and a control-pollinated 
seed source (A45.times.A26). Previous experiments had consistently shown 
that explants derived from these two seed sources provide a range of 
capacity for somatic embryogenesis typically found in loblolly pine; from 
a recalcitrant seed source (A10) to a more responsive seed source 
(A45.times.A26). The culture initiation media compared were 
(1) DCR.sub.1 (see Table II above), and 
(2) BM.sub.1 medium cited in Table 2 of U.S. Pat. No. 4,957,866. In brief, 
this is a modified 1/2P6 basal salts (Teasdale et al. 1986) with 2,4-D 
(11.1 mg/l), kinetin (4.3 mg/l) and BA (4.5 mg/l). After four weeks in 
culture the extruding embryogenic tissue from responsive explants was 
transferred to a maintenance medium as follows: (1) cultures on DCR were 
transferred to new plates of the same medium, (2) cultures on BM.sub.1 
were transferred to BM.sub.2 medium cited in Table 2 of U.S. Pat. No. 
4,957,866. 
The results showed that both embryogenic tissue extrusion and proliferation 
frequency were improved by using the method of the current invention 
relative to the process taught in U.S. Pat. No. 4,957,866 (see Table X 
below). Very importantly, the present method resulted in the proliferation 
frequency of embryogenic tissue approximately doubled for both the 
responsive and the recalcitrant seed sources. 
TABLE IX 
______________________________________ 
Comparison of Embryogenic Tissue (ET) Initiation Frequencies of 
the Method Taught in the Current Invention (Step 1, Example 1) 
and the Process Taught in U.S. Pat. No. 4,957,866. 
Culture Frequency (%) explants 
Initiation 
Extrude ET at 4 weeks 
Proliferate ET at 7 weeks 
Medium A10 A45 .times. A26 
A10 A45 .times. A26 
______________________________________ 
Present Method 
25/48 33/48 7/48 24/48 
(52%) (69%) (15%) (50%) 
U.S. Pat. No. 
20/48 26/48 4/48 10/48 
4,957,866 (42%) (54%) (8%) (21%) 
______________________________________ 
The most striking improvement was on the growth potential of the newly 
initiated embryogenic tissue. Not only did the method of the current 
invention result in more explants of both seed sources producing vigorous 
proliferation of embryogenic tissue, but the culture weight was improved 
by the method of the current invention relative to the process taught in 
U.S. Pat. No. 4,957,866. 
This improvement is graphically illustrated in FIGS. 3 and 4 in the 
drawings, which compares the total weight of each culture genotype 
produced via the different methods as measured after 10 weeks. Employment 
of the present method resulted in a total of cultures from seed source 
A45.times.A26 proliferating a cumulative total of 4.11 grams of 
embryogenic tissue. In contrast, use of the patented process resulted in 
only 10 cultures proliferating a cumulative total of 1.85 grams of 
embryogenic tissue (FIG. 3). 
EXAMPLE 5 
The following evaluation compared the present method with the process 
taught by Pullman and Gupta in U.S. Pat. No. 5,034,326--particularly the 
method of producing stage 3 somatic embryos from embryogenic cultures of 
Pinus taeda as taught in Step 4 of Example 1 of the present method. The 
embryogenic cultures used in this example were derived from the 
experiments described in Example 4. Two somatic embryo development methods 
were evaluated: First, according to the process taught in U.S. Pat. No. 
5,034,326, the embryogenic cultures were initiated on BM.sub.1 medium (see 
Example 4), maintained on BM.sub.2 medium, subcultured onto BM.sub.3 Late 
Proembryo Development Medium, and finally tested for production of stage 3 
somatic embryos on BM.sub.4 Embryo Development medium. After approximately 
3 months on BM.sub.2 medium only 3 of the 10 embryogenic cultures 
initiated from seed source A45.times.A26 on BM.sub.1 employing the 
patented process survived (see FIG. 3, Example 4). The 3 surviving 
cultures were subcultured (every 3 weeks) to BM.sub.3 Late Proembryo 
Development Medium for 9 weeks total time, and then transferred to 
BM.sub.4 Embryo Development medium. In comparison, 12 of the 24 cultures 
initiated from seed source A45.times.A26 on DCR.sub.1 (see FIG. 3, Example 
4) survived after the same 3 month time in culture, were transferred to 
MSG predevelopment medium, and then to MSG.sub.2 embryo development medium 
in accordance Step 4 of Example 1 of the present method. The results are 
summarized in Table XI below. 
TABLE X 
______________________________________ 
Comparison of Stage 3 Somatic Embryo Production of 
the Present Method (Step 4, Example 1) and the 
Process Taught in U.S. Pat. No. 5,034,326. 
Embryo Total No. Total No. Total No 
Development 
of Cultures 
of Pieces.sup.a of ET 
of Stage 3 
Method Tested Tested SEs harvested 
______________________________________ 
Present 12 59 238 
Method 
U.S. Pat. 3 12 0 
No. 5,034,326 
______________________________________ 
It is clear from this evaluation that the method taught in the present 
invention was effective in producing large numbers of Pinus taeda stage 3 
somatic embryos from numerous culture genotypes, whereas the process 
taught by U.S. Pat. No. 5,034,326 was ineffective. Only two of the 13 
cultures tested using the present method did not produce any stage 3 
somatic embryos, whereas all 3 cultures tested on the patented process 
produced zero harvestable stage 3 somatic embryos. While it is possible 
that stage 3 somatic embryos would be produced by the process taught by 
U.S. Pat. No. 5,034,326 if more cultures were screened, it is also evident 
from this evaluation that it would be extremely difficult to maintain 
cultures according to the process taught in U.S. Pat. No. 5,034,326, as 
only 3 of the original 10 cultures survived. It is also possible that the 
process taught in U.S. Pat. No. 5,034,326 might be more effective when 
employed with coniferous species other than Pinus (e.g., Pseudotsuga and 
Picea species). 
EXAMPLE 6 
The following evaluation compared the present method with the process 
taught by Gupta and Pullman in U.S. Pat. No. 4,957,866--particularly the 
method of obtaining somatic embryo development as taught in Step 4 of 
Example 1 of the present method. Embryogenic cultures of Pinus taeda and 
Pinus rigida.times.Pinus taeda used in this experiment were initiated and 
maintained according to method taught in Steps 1-3 of Example 1. 
Three development protocols were tested, with Treatments A and B practicing 
the present method and Treatment C practicing the patented process. In 
Treatment A masses of embryogenic tissue were transferred from culture 
maintenance medium to MSG.sub.1 embryo predevelopment medium for 7 days 
and then transferred to MSG.sub.2 embryo development medium. In Treatment 
B masses of embryogenic tissue, which had been growing on DCR.sub.1 
maintenance medium, were transferred to the same DCR maintenance medium 
(containing 0.5 g/l inositol) for three 21 day subcultures, and then 
transferred to MSG.sub.2 embryo development medium. In Treatment C masses 
of embryogenic tissue were transferred from culture maintenance medium to 
an embryo predevelopment medium (DCR.sub.1 containing 10.0 g/l inositol) 
for three 21 day subcultures, and then transferred to MSG.sub.2 embryo 
development medium. 
In terms of osmolality differences the three embryo development protocols 
tested (Treatments A, B and C) differed as follows: Both Treatments A and 
B, according to the present method, utilized maintenance and 
predevelopment medium with (low) osmolality levels in the range of 145 to 
165 mM/kg, and embryo development medium with (high) osmolality levels in 
the range of 250 to 260 mM/kg. Treatment C similarly utilized maintenance 
medium with osmolality levels in the (low) range of 145 to 155 mM/kg and 
embryo development medium with (high) osmolality levels in the range of 
250 to 260 mM/kg. But Treatment C, in accordance with the patented 
process, differed from Treatments A and B by having a predevelopment 
medium with a (high) osmolality level of 230 mM/kg provided by the high 
level of inositol added to the medium. Thus, Treatment C tested the 
efficacy of using a predevelopment medium with significantly higher 
osmolality levels, in comparison to a either a predevelopment medium with 
low osmolality (Treatment A), or simply maintaining the cultures on a 
maintenance medium with low osmolality (Treatment B) for an equivalent 
period of time. 
The results contained in Table XII below show that the present method 
(Treatments A or B) resulted in higher overall production of both stage 2 
and stage 3 somatic embryos than the process taught in the patent of 
adding a high osmoticum predevelopment step (Treatment C). Only one 
culture genotype (#2) developed slightly more stage 3 somatic embryos by 
following the patented process (Treatment C). It should be noted that the 
only treatment that was effective in inducing production of stage 3 
somatic embryos from culture genotypes maintained as liquid suspension 
cultures was Treatment A; which practiced the method taught in Example 1 
of the present invention. 
TABLE XI 
______________________________________ 
Comparison of Precotyledonary (stage 2) and 
Cotyledonary (stage 3) Somatic Embryo 
Production on Three Different Development Protocols. 
Culture Total no. (stage 2) and stage 3 
geno- somatic embryos produced on 
type Culture development protocol: 
no..sup.a 
Origin.sup.b 
Trt. A Trt. B Trt. C 
______________________________________ 
1 ET (274) 178 (346) 418 
(157) 134 
2 ET (187) 29 (273) 113 
(194) 118 
3 ET (163) 89 (69) 73 
(52) 44 
4 ES (166) 36 (57) 0 (43) 0 
Totals: (790) 332 (745) 604 
(446) 296 
______________________________________ 
.sup.a Cell line 1 was Pinus rigida .times. Pinus taeda. 
.sup.b Cell lines 2-4 were Pinus taeda. 
ET (embryogenic tissue) cultures maintained on semisolid media according 
to Step 3 of Example 1 prior to testing. 
ES (embryogenic suspension) cultures maintained as liquid suspensions 
prior to testing. 
The results in Table XI show that the method of the current invention is at 
least as effective, and for most culture genotypes tested far more 
effective, for producing stage 3 somatic embryos of Pinus taeda and Pinus 
rigida.times.Pinus taeda than the process taught by Gupta and Pullman 
(1990). 
EXAMPLE 7 
The current invention enables one to regenerate large numbers of Pinus 
taeda and Pinus rigida.times.Pinus taeda somatic embryos from embryogenic 
cultures which have been cryopreserved in liquid nitrogen. 
Cryopreservation is an essential component in developing an overall 
strategy for clonal propagation of Pinus species using somatic 
embryogenesis, Until now there has not been an efficient regeneration 
system available for Pinus species to use in conjunction with cryostorage 
procedures, 
Embryogenic cultures were cryopreserved in liquid nitrogen in order to: 
(1) maintain a bank of cultures for retrieval and use after field tests 
have identified superior genotypes; and 
(2) insure against loss of culture genotypes due to contamination, loss of 
vigor associated with culture aging, or other deleterious changes that may 
occur during long-term culture maintenance. 
The following method was very successfully used for cryopreservation of 
both Pinus taeda and Pinus rigida.times.Pinus taeda embryogenic cultures. 
Following the method taught in Steps 1-3 of Example 1 pieces of 
embryogenic tissue (7 to 14 days since their last subculture on the 
culture maintenance medium) were dispersed in liquid DCR.sub.1 medium 
which contained 0.4 molar sorbitol (Klimazewska et al. 1992). Liquid 
embryogenic suspension cultures, produced via the method of Example 4, 
were also used as a source of tissue for cryopreservation. The amount of 
embryogenic tissue from either gelled or liquid medium used was sufficient 
to result in a 30% suspension (e.g., 3 ml volume of embryogenic tissue 
added to 7 ml of liquid medium). Erlenmeyer flasks containing the 
suspension were incubated for 24 hours in the dark on a gyratory shaker 
(100 rpm), and then placed on ice. Five aliquots of the cryoprotectant 
dimethylsulfoxide (DMSO) were added to the suspension to bring final 
concentration of DMSO to 10%. One milliliter aliquots of the cell 
suspension containing DMSO were then transferred to freezing vials (2 ml 
NALGENE Cryovials, Nalge Co.), placed in programmable freezer (Model 9000, 
Gordinier Electronics) and cooled to -35.degree. C. at 0.33.degree. C. per 
minute. The freezing vials were then immersed in liquid nitrogen inside a 
cryobiological storage vessel (Model #CY50945, Thermolyne) for long-term 
storage. 
For retrieval of frozen cultures, individual vials were removed from the 
cryobiological storage vessel and placed in 38.degree. C. water to rapidly 
thaw the frozen cell suspension. The thawed cell suspension was 
aseptically poured from the cryovial onto a sterile NITEX nylon membrane 
(#3-35/16XX, Tetko, Inc.) which had been placed on top of two sterile 
filter papers (Whatman no. 2, Whatman Internation Ltd.) to absorb excess 
liquid from the cell suspension. The nylon membrane containing embryogenic 
tissue was then transferred to maintenance medium, e.g., DCR.sub.1 and 
incubated at 23.degree. C. for 24 hours to allow DMSO to diffuse into the 
medium. The nylon membrane containing embryogenic tissue was removed from 
the medium and transferred to a new plate of maintenance medium. 
Thereafter, the membrane containing embryogenic tissue was transferred to 
a new plate of maintenance medium every 21 days. When sufficient 
proliferation of the embryogenic tissue occurred, individual pieces (about 
200 mg each) were transferred directly to maintenance medium for further 
multiplication or directly to embryo predevelopment medium according to a 
modification of Step 4 of Example 1. The modification was as follows: The 
masses of embryogenic tissue were transferred .onto a sterile NITEX nylon 
membrane (No. 3-35/16XX, TETKO, Inc.) which had been placed on the surface 
of the predevelopment medium. This modification greatly facilitated 
subsequent transfer of the embryogenic tissue masses to embryo development 
medium, by avoiding direct contact with and disturbance of the masses 
during transfer. Instead, the nylon membrane containing the masses was 
easily transferred as a unit to embryo development medium, and also later 
easily transferred to new embryo development medium as described in Step 4 
of Example 1. 
Table XII summarizes somatic embryo yields from embryogenic cultures 
initiated according to Step 2 of Example 1 and cryopreserved as described 
above in comparison to yields from the same culture genotypes which had 
not been cryopreserved. Three of the four cultures tested produced more 
stage 3 somatic embryos after cryopreservation than before. These data 
show that the current invention, when used in combination with the above 
described cryopreservation method, enables one to effectively produce 
large numbers of both Pinus taeda and Pinus rigida.times.Pinus taeda 
somatic embryos from cryopreserved embryogenic cultures. 
TABLE XII 
______________________________________ 
Pine Somatic Embryos (SEs) Harvested from Cryopreserved 
(frozen to -196 .degree. C.) and Unfrozen Embryogenic Tissue (ET). 
No. SEs 
Total No. SEs 
harvested 
Culture 
Parent Cryo- harvested per 
genotype 
Tree.sup.a 
preserved.sup.b 
(No. pieces.sup.c ET) 
piece ET 
______________________________________ 
1 B19 yes 518 (4) 130 
no 347 (3) 116 
2 B19 yes 384 (4) 96 
no 474 (6) 80 
3 A45 yes 194 (3) 65 
no 180 (3) 60 
4 A10 .times. A45 
yes 362 (6) 60 
no 261 (3) 87 
______________________________________ 
.sup.a B19 = Pinus rigida maternal tree supplementally mass pollinated 
with Pinus taeda pollen, A45 = openpollinated P. taeda, and A10 .times. 
A45 = P. taeda maternal tree (A10) .times. P. taeda pollen (A45). 
.sup.b Cryopreserved cultures were in liquid N.sub.2 from 7 to 19 weeks. 
.sup.c Each piece of ET approximately 200 mg fresh weight. 
EXAMPLE 8 
The following experiment was done to test modifications in the form of 
nitrogen and type of gelling agent used in the germination medium, 
MSG.sub.3. The data summarized in Table XIII below used Pinus taeda 
embryogenic cultures initiated and maintained according to Steps 1-3 of 
Example 1 and somatic embryos developed and matured according to Steps 4 
and 5 of Example 1. The somatic embryos were then germinated on either 
MSG.sub.3 medium (according to Step 6 of Example 1) or MSN medium (which 
was equivalent to MSG.sub.3 medium except the L-glutamine nitrogen was 
replaced with an equivalent molar concentration of inorganic nitrogen as 
ammonium nitrate, and GELRITE was replaced with agar). All other 
components of the two media were equivalent and as listed in Table II for 
MSG.sub.3. 
TABLE XIII 
______________________________________ 
Effect of Medium Modifications on 
Pinus taeda Somatic Embryo Germination 
Germina- Germina- 
tion Gelling Culture tion 
Medium Nitrogen.sup.a 
agent genotype.sup.b 
(%).sup.c 
______________________________________ 
KSG.sub.3 
L- agar 1 66 
glutamine 8 g/l 2 14 
1.45 g/l 
MSN NH.sub.4 NO.sub.3 
GELRITE 1 38 
0.8 g/l 2 g/l 2 16 
______________________________________ 
.sup.a In addition, each medium contained 0.1 g/l KNO.sub.3. 
.sup.b Somatic embryos from culture genotypes 1 and 2 derived from parent 
trees A38 .times. A45 and A45 .times. A33/38, respectively. 
.sup.c Fifty somatic embryos of each culture genotype were tested for 
germination on each medium. 
The results showed that germination of embryos from culture genotype 1 was 
highest on MSG.sub.3 with L-glutamine and agar. Whereas, germination of 
embryos from culture genotype 2 was similar on either MSG medium with 
L-glutamine and agar, or on MSN medium with ammonium nitrate (NH.sub.4 
NO.sub.3) and GELRITE. The use of the MSN germination medium, with an 
inorganic form of nitrogen, is advantageous since the inorganic form of 
nitrogen is not heat labile and, therefore, does not require separate 
filter-sterilization as the L-glutamine does in MSG.sub.3 medium. 
Therefore, an additional experiment (see Table XIV below) was done to 
study germination of Pinus taeda somatic embryos from a wide range of 
culture genotypes on MSN medium with ammonium nitrate and GELRITE. 
EXAMPLE 9 
The following experiment was done to verify that large numbers of Pinus 
taeda somatic embryo plants derived from a wide range of culture genotypes 
could be established as planting stock using: (1) the modified germination 
medium used in Example 9, MSN, which contained inorganic nitrogen and 
GELRITE; and (2) the conversion procedure used in Step 7 of Example 1. The 
data summarized in Table XIV below used Pinus taeda embryogenic cultures 
initiated and maintained according to Steps 1-3 of Example 1 and somatic 
embryos developed and matured according to Steps 4 and 5 of Example 1. 
TABLE XIV 
______________________________________ 
Germination and Conversion of Somatic Embryos (SEs) 
Derived from Three Control Crosses of Pinus taeda 
Parent Culture No. Germination 
Conversion 
Tree Genotype SEs Tested 
No. (%) No. (%).sup.a 
______________________________________ 
A45 .times. A10 
1 144 104 (72) 59 (57) 
2 129 38 (29) 19 (50) 
3 85 35 (41) 30 (86) 
4 110 74 (67) 50 (68) 
5 60 22 (37) 17 (77) 
6 61 16 (26) 6 (38) 
A38 .times. A45 
7 73 41 (56) 24 (59) 
8 98 23 (23) 19 (83) 
9 130 21 (16) 8 (38) 
10 63 5 (8) 0 (0) 
11 162 146 (90) 52 (36) 
12 74 37 (50) 27 (73) 
A10 .times. A45 
13 263 72 (27) 45 (63) 
14 75 15 (20) 4 (27) 
15 132 54 (41) 47 (87) 
16 140 76 (5%) 46 (61) 
17 88 59 (67) 43 (73) 
Totals 1887 838 (4%) 496 (59) 
______________________________________ 
.sup.a Conversion calculated as the percentage of vigorous germinated 
somatic embryos which survived and continued to grow ex vitro. 
The results in Table XIV showed that an overall germination frequency of 
44% was obtained from 17 culture genotypes derived from three control 
pollinated trees. Thus, the MSN medium with inorganic nitrogen and 
GELRITE, was generally effective for germinating somatic embryos from 
numerous culture genotypes of Pinus taeda. Somatic embryos from only two 
of the 17 culture genotypes (numbers 9 and 10) of parent tree 
A38.times.A45 had germination levels below 20%. In addition, the results 
in Table XIV showed that an overall conversion frequency of 59% was 
obtained with the methods of the current invention. Somatic embryos from 
only one culture genotype (number 10) did not produce vigorous planting 
stock. The results clearly demonstrated the potential of using the MSN 
germination medium and the conversion method in Step 7 of Example 1 to 
efficiently produce large numbers of Pinus taeda somatic embryo plants for 
field planting. The results presented in Table XIV are unprecedented for 
production of planting stock via somatic embryogenesis of the recalcitrant 
Pinus species conifers. It demonstrates the utility of the methods taught 
in this invention for solving the problem of providing a reproducible 
method for large scale production of Pinus taeda via somatic 
embryogenesis. 
Many modifications and variations of the present invention will be apparent 
to one of ordinary skill in the art in light of the above teachings. It is 
therefore understood that the scope of the invention is not to be limited 
by the foregoing description, but rather is to be defined by the claims 
appended hereto. 
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