Field of the invention
The present invention relates to novel culture media compositions for rapid propagation
of Eucalyptus camaldulensis More specifically the invention relates to modified Standard
Hoagland solution that is useful for soil less mini-cutting production technique for E.
Camaldulensis.
Background and prior art
Eucalyptus camaldulensis commonly known as River Red Gum is a tree belonging to the
genus Eucalyptus. It is one of around 800 in the genus. It is a plantation species in many
parts of the world but is native to Australia where it is widespread especially beside
inland water courses. The tree can grow to 45 metres tall; it has smooth bark, ranging in
colour from white and grey to red-brown which is shed in long ribbons (Plantnet (1999-
2008). "Eucalyptus camaldulensis Dehnh". Royal Botanic Gardens & Domain Trust,
Sydney. Australia). The tree has a large, dense crown of leaves. The base of the bolecan
be covered with rough, reddish-brown bark (Mullins, B (1979). Australian Eucalypts.
Sydney: A. H. & A. W. Reed). The juvenile and adult leaves are stalked, with the adult
leaves broad at the base, tapering to the tip. The adult leaf colour is a dull blue-green. The
leaf also contains several to many oil-producing glands in the un-veined areas of the leaf
(Brooker, M. I. H.; Kleinig, D. A. (1990). Field Guide to Eucalypts: Volume 1 South-
eastern Australia. Sydney).
Red gum is so named for its brilliant red wood, which can range from a light pink
through to almost black, depending on the age and weathering. It is a popular timber for
wood turners, particularly if old and well-seasoned.
It is also popular for use as firewood. The wood makes fine charcoal, and is successfully
used in Brazil for iron and steel production. In addition, this plant is used for beekeeping
in Brazil and Australia.
It is one of the most widely planted eucalypts in the world (ca 5,000 km2 planted) (NAS,
1980a). Plantations occur in Argentina, Arizona, Brazil, California, Egypt, Kenya,
Morocco. Nigeria. Pakistan, Senegal, Sierra Leone, Spain, Sri Lanka, Sudan, Tanzania,
Upper Volta, Uruguay, and Zimbabwe. The areas of significance to humans of
Eucalyptus camaldulensis include agricultural, ecological, cultural, and recreational
significance. The speed of growth of the tree makes it a useful plantation timber.
Apiarists also use the tree's flowers for honey production. E. camaldulensisis important
in supporting the ecology of its habitat through providing food, and shelter for breeding.
Culturally, the species is an iconic part of Australia.
Red Gum is renowned globally for its fast growth, high levels of drought tolerance and
adaptability to a diverse climatic conditions and soils, which made this a 'choice' and not
by a 'chance' species among tree growers. Since demand is burgeoning and nursery
production centers/units are unable to supply vigorous seedlings from conventional
coppice-producing chain (time constraint) and are looking for effective surrogate
propagation methods.
For rapid production of the plant clonal propagation have been adapted. (A tissue culture
method for rapid clonal propagation of mature trees of Eucalyptus torelliana and
Eucalyptus Camaldulensis, P. K. Gupta, U. J. Mehta and A. F. Mascarenhas, Plant Cell
Report (1983) 2:296-299.)
Clonal propagation is an extensively used strategy to raise the economic potential of
eucalypt species and hybrids by multiplying desirable genotypes. It is done with
moderate degree of sophistication in most forestry business for strategic improvement in
productivity. The stem cutting (conventional) technique is most common and widely used
method of propagation due to its ease of handling compared to other micro-propagation
methods.
However, this method presents some difficulties in the production process, such as poor
rooting and plants formation in certain clones, which consequently affects their
deployment in the planting programme. The poor rooting of certain clones by using
conventional cuttings is a major constraint to cloning, and has been attributed to the
maturation degree of the plant material leading to the adoption of various techniques for
"rejuvenating" the mature plants material into the ''juvenile" stage.
Mass vegetative propagation has become an important tool for increasing the
competitiveness of the forestry-based industry. This method reaches its highest potential
when it is used to establish clonal forests of hybrids endowed with better wood quality
and higher volumetric growth. However, in several hardwood species, most notably in
Eucalyptus, the popular method of rooting stem-cuttings has limitations, for example,
rapid loss of rooting competence due to ontogenetic aging, intra-clonal variation resulting
from topophysis, and poor quality of the root system, that in combination negatively
affect genetic expression of some clones. Two alternative super-intensive systems, one
micro- and one mini-cutting, for cloning Eucalyptus at a commercial scale have been
applied successfully. These systems have shown great potential for offering technical and
economic advantages not available from conventional stem cuttings. The micro-cutting
system uses the apices obtained from micro-propagated plantlets, while the mini-cutting
system is based on the rooting of auxiliary shoots from rooted stem-cuttings. In both
systems the plants are managed intensively to produce small cuttings. Field clonal hedges
are replaced by indoor hydrophonic mini-hedges, which provide plantlets or rooted
cuttings with a high degree of juvenility. The success of the system is also dependent on
achieving optimal nutrient status in the resulting mini-cuttings. Compared to stem-
cuttings, the micro- or mini-cuttings systems have improved rooting potential, rooting
speed, and root system quality, as well as reduced costs. Additionally, these systems offer
propagules with increased uniformity, and greatly reduced topophysis effects. The
development of these super-intensive cloning systems has set the stage for a new phase of
mass vegetative propagation of Eucalyptus and other hardwood species. ("Current
techniques and prospects for the clonal propagation of hardwoods with emphasis on
Eucalyptus"; DE ASSIS Teotopio Francisco, FETT-NETO Arthur G., COUTO
ALFENAS Acelino; Plantation forest biotechnology for the 21st century 2004
(2004), pp. 303-333). The mini cuttings system has been under commercial operation in
Brazil, Australia and South Africa (Titon, 2001 and Titon eta/., 2006) for species such as
£. grandis, Eglobulus. E. nittens. etc.
Subsequent to production of minicuttings, these young saplings should be nurtured in
proper growing/rooting medium as it plays a key role to achieve higher survival as well
as in development of early vigorous root system. Successful production is largely
dependent on the chemical and physical properties of growing medium. An ideal growing
media used in a commercial nursery should not only be cost effective but also be free
from weeds and diseases. The medium should also be well drained and yet retain
sufficient moisture.
Once novel and proper growing medium are identified, high yielding clones can be mass
multiplied and used for commercial forestry, where continuous and reliable supply of
stock is the need of the hour.
For the purpose of mini cuttings Standard Hoagland solution have been used which is
more directed towards improving growth of annual crops (agriculture/ horticulture) and
its nutrient composition is mostly suited to tissue composition/constituents of crop plants.
Table 1 shows the constituents of a standard Hoagland solution.
As plants require all 16 essential elements to complete its life cycle, its usage depends on
plant types. In tree species (perennials) due to its hardy wood nature, there is an alteration
in tissue constituents, which leads to either higher dose of nutrient requirement or
preferential uptake i.e., need of one or more elements in higher dosage at particular
growth stage. Thus many modifications of the Standard Hoagland solution have been
tried like the once used in Brazil, South Africa that are suitable for Eucalyptus speices
like globulus, grandis or nitens.
Nutrient mix used in commercial nursery elsewhere (Brazil, South Africa etc)
The nutrient mix consist of a mixture of calcium nitrate (555 g m"3), ammonium sulphate
(200 g m"3), phosphoric acid (70 g m"3), potassium chloride (210 g m"3), magnesium
sulphate (150 g m"J), boric acid (3.33 g m ), zinc sulphate (0.15 g m" ), copper sulphate
(0.40 g m"3), manganese sulphate (1.67 g m"3), iron sulphate (5.22 g m"J), EDTA (6.96 g
m"3) and sodium molybdenum (0.05 g m"3).
However these nutrient mediums could support minicutting production for only 2-3
harvest cycles.
Thus there is need to provide proper medium which would provide appropriate nutrients
as well as take care of the rooting and other problems associated with prior art methods.
Objects of the invention
It is an object of the present invention to overcome the drawbacks of the prior art.
It is another object of the present invention to provide plant nutrition management in
mini-cutting based propagation to get vigorous growth of hedge plants.
It is yet another object of the present invention to provide growing media for vigorous
saplings from minicuttings sets.
It is yet another object of the present invention to provide media adapted to provide
minicuttings grown in the same with enhanced survival rate, rapid rooting and growth of
saplings.
It is yet another object of the present invention to provide media adapted to provide
minicuttings grown in the same with improved intrinsic physiological traits such as more
leaf nitrogen content, carbon fixation rates, carboxylation efficiency and higher leaf water
status.
It is yet another object of the present invention to provide growing media adapted to
provide continued production of upto 5-6 harvest cycles of minicuttings from the hedge
plants.
Summary of the invention
A culture media comprising a nutrient solution mix and a growing media intended for ex
vitro propagation,
said nutrient solution mix comprising 10.1 - 10.5% by weight KNO3, 9.0 - 10 % by
weight CaN03. 5.05 - 5.5 % by weight KH2P04 , 6.0 - 6.5 % by weight MgSO4, 0.5 -
0.75% by weight FeSO4 , 0.25 - 0.4% by weight EDTA, 0.35 - 0.4% by weight MnCl2,
0.15 - 0.25% by weight CuSO4, 0.25 - 0.5% by weight Zn SO4, 0.15 - 0.25% by weight
H3BO30.1 - 0.2% by weight. Ammonium molybdate
A culture media comprising a nutrient solution mix and a growing media intended for ex
vitro propagation.
said nutrient solution mix comprising 10.1 - 10.5% by weight KNO3, 9.0 - 10 % by
weight CaN03. 5.05 - 5.5 % by weight KH2P04 , 6.0 - 6.5 % by weight MgSO4, 0.5 -
0.75% by weight FeSO4 , 0.25 - 0.4% by weight EDTA, 0.35 - 0.4% by weight MnCl2.
0.15 - 0.25% by weight CuSO4, 0.25 - 0.5% by weight Zn SO4, 0.15 - 0.25% by weight
H3BO3.0.1 - 0.2% by weight, Ammonium molybdate
and growing media comprising ecosand, vermiculite and compost.
Brief description of the accompanying figures
Fig. la illustrates the efficient rooting behavior without any hormone treatment (6000
ppm IBA was tried).
Fig. lb illustrates the percent rooting showing significant, suggesting native auxin level
would be sufficient to induce rooting.
Fig. 2. illustrates variations in intrinsic physiological traits between mini cutting
seedlings and coppice seedlings.
Fig. 3. illustrates increment in plant height (cm) and TSP (mg/gFW) of mini cutting
plants in relation to coppice plant.
Detailed description of the invention
The present invention provides culture media composition for mini-cutting based
technique of clonal propagation of E. Camaldulensis. The composition is found to be
useful for clonal propagation of large number of plantable seedlings of E. Camaldulensis.
Mini-cutting based propagation is the economy of space. Minicuttings technique provides
more harvest in less time and less space. For instance to meet the demand of 20 million
cuttings per annum through coppice shoots, land area required would be approximately
55 acres, whereas, for mini-cuttings a mere 2 acres is only required.
The soil less mini-cutting production technique for R camaldulensis with the fusion of
the nutrient medium of the present invention offers the following advantages:
• Economic - reduced labour costs, chemical costs, land area requirements
Seedling quality -higher ability to produce roots, better root system, better stem
• Reduced time - plants come out of mist chamber quicker (fewer disease
problems), time from setting to production of plantable seedlings shorter
compared to stem-cuttings
Accordingly the present invention discloses media composition comprising a first media
where the hedge plants are grown and a second media, which is a growing rooting media.
Nutrient Solution mix:
The hedge plants were grown in liquid nutrient medium to harvest mini-cuttings. The
nutrient medium has been formulated to get vigorous growth of hedge plants in order to
increase harvest cycles of mini-cuttings. The nutrient solution mix is a modification of
the Standard Hoagland solution. The standard Hoagland solution (SHS) normally (in
hydroponics) is a blend of total-nutrient mix (contain essential elements) largely
recommended for crop plants (annuals). The present invention discloses a nutrient mix
(NM), which is a modified standard Hoagland solution, and provides excellent result in
inducing more number of roots, new roots, root and shoot growth with better leaf area
and greenness under commercial nursery. Apart from this, plants pumped with nutrient
mix showed improved intrinsic physiological traits such as more leaf nitrogen content,
carbon fixation rates, carboxylation efficiency and maintained higher leaf water status
etc., than standard Hoagland solution pumped plants.
A unique ionic/chemical composition of the nutrient medium for E. camaldulensis has
been formulated which is provided in Table. 2.
Table. 2. Nutrient mix unique for E. Camaldulens/s used for producing minicuttings
Growing medium:
The growing rooting medium, ecosand has an amazing ability to absorb, hold, release and
exchange different nutrients/ions. It is a mineral with infinite, 3-dimentional honey comb
like structure that allows it to loose and gain water reversibly. Being negatively charged
(by nature) that makes it to attract certain cations. The added benefit is it does not wade
out over time but remains in soil to help in improving nutrient and water retention. Acting
as a natural wetting agent, it is an excellent amendment for non-wetting sands and to
assist water distribution through soils. Ecosand is used as a viable growing/rooting
medium to sets of minicuttings before subjecting them to mist chamber conditions.
In mini-cuttings propagation, ecosand has been used as a supplement to vermiculite
(normally used in commercial eucalyptus nursery) for realizing its potential (Table. 3).
Table. 3. Physical and Chemical Properties of ecosand:
The present invention uses a mixture of ecosand with vermiculite and leaf compost as a
rooting medium. This facilitates early rooting and establishment of young seedlings
compared to contemporary medium thereby making seedlings healthy to utilize available
nursery resources efficiently. There was an improvement of 20% in seedling survival
rate over contemporary medium.
Modified NM strengthens the growth and vigour of hedge plants by providing enough
nourishment, which facilitates production of more minicuttings harvest in less time span.
Ecosand helps in improving rooting ability and survival rates by holding water for long
(wetting agent) and releasing required nutrients slowly.
Together, these two makes a viable blend in improving overall vigour of seedlings and
reducing the nursery duration by 2 months, which has enormous potential from the views
of nursery production scale and cost benefits in Eucalyptus industry.
The invention is now described by way of illustrative non-limiting examples with
reference to figures.
Example 1
A nutrient solution mix was prepared containing said nutrient solution mix comprising
1010 g/lOOOL KN03, 900 g/lOOOL CaN03. 505 g/lOOOL KH2P04. 600 g/lOOOL MgSO4.
50 g/lOOOL FeSO4 .25 g/lOOOL EDTA , 35 g/lOOOL MnCl2, 15 g/lOOOL CuSO4 , 25
g/lOOOL ZnSO4, 15g/1000L H3BO3, lOg/lOOOL Ammonium molybdate in water.
Rooting mediums were made by mixing ecosand with vermiculite and leaf compost in the
ratio of 1:3:1. 1:2:1 and 1:1:1
Example 2
Provided below is the schematic diagram of the process of minicutting of B.
Camaldulensis.
Mini cuttings -> Mist Chamber -> Hardening -> Open Nursery -> Total Duration
25days + 25days + lOdays + 60days = 4 Months
60-65 day old plants from open nursery (produced from conventional coppice shoot
method) was initially selected and kept in nutrient mix tank for 25 days (tanks were
housed in a polythene enclosure with air temperature of 33-35°C, RH of 60-65% and
natural sun light with 30% cut-off were maintained). These hedge stumps act as source
of minicuttings. The nutrient mix was fed continuously (cycled and recycled) using
motorized pump (to create aeration). The DO (dissolved oxygen), pH and EC (Electrical
Conductivity) of nutrient mix were maintained regularly (using portable DO, pH/EC
meters) to facilitate proper ion exchange and nutrient uptake by hedge plants. The
nutrient mix was changed (replace completely) once in a fortnight. On 25th day,
minicuttings from these hedge plants were harvested/cut and continued till 30th day as
and when sets were ready for harvest. These sets were positioned immediately in rooting
trays containing Ecosand: Vermiculite: Compost mix and placed in mist chamber
(regulated misting to maintain temperature: 32-35°C and RH: 80-85%) for another 25
days. Then these seedling trays were moved to hardening area (under 50:50 shade area
with regulated sprinklers) for 10 days to create robustness. The hardened seedlings were
moved to open (nursery) area (completely under natural environments) and irrigated
twice daily until they were lifted for field planting.
Example 3
Minicuttings made out from the examples mentioned above showed efficient rooting
behavior without any hormone treatment (6000 ppm IBA was tried) (Fig. la). The
percent rooting was also significant (Fig. lb), suggesting native auxin level would be
sufficient to induce rooting. Since root system developed in minicuttings during initial
organogenesis is as top roots, when exposed to congenial growth condition; grow
vigorously leading to early establishment. The increased intrinsic physiological and
biochemical mechanisms corroborated further the apparent improvement in growth rates.
Example 4
Improvement in intrinsic physiological traits in hedge plants and mini-cuttings over stem
cuttings saplings
Further, in order to examine the reason behind the difference in growth rates between
mini cutting obtained from Nutrient solution mix and ecosand based growing medium
and coppice seedlings, variations in intrinsic physiological traits were determined.
The hedge plants treated with the continuous nutrient solution and the mini-cuttings made
out of these (13-15 days after setting), showed considerable improvement in intrinsic
carbon fixation (photosynthesis) and associated physiological traits along with leaf
Nitrogen status compared to coppice seedlings, thus indicating hedge plants and mini-
cuttings have higher intrinsic physiological efficiencies. When compared, performance
of mini-cuttings appreciably outplayed the coppice seedlings (similar age) (Table. 4) in
overall growth behavior. The mini-cuttings resembled a miniature plant with more
vitality (ready to plant in the field).
Index: C-fixation: Carbon fixation (u.mol/m2/s), SCMR-SPAD Chlorophyll Meter
Reading, a unit less invasive index for leaf nitrogen status, T. rate- Transpiration rate
(mmol/m2/s)
Example 5
Further, in order to examine the reason for difference in growth rates between mini
cutting and coppice seedlings, variations in intrinsic physiological traits were determined
using portable photosynthesis system and chlorophyll meter.
The hedge plants (HPs) treated with continuous NM solution and MCs made out of these,
showed considerable improvement in intrinsic carbon fixation (photosynthesis) and
associated physiological traits along with leaf N status compared to coppice seedlings
(ONP) and young CSs (ONP) respectively (Fig. 2). This indicates that HPs and MCs
developed by this technique have intrinsic superior physiological efficiencies vigour.
To substantiate further, total soluble protein (TSP) content from each plant sets was
extracted and determined. The values suggested that leaves of MCs had higher TSP both
at open nursery as well at field conditions (Fig. 3) compared to their coppice shoot grown
plants.
Minicuttings grown plants posses largely all intrinsic physiological, biochemical
efficiencies that would facilitate them to acclimatize early and perform better under any
given natural field conditions are superior to conventional coppice plants.
Example 6
Comparative field performance of plants grown from minicuttings and coppice shoots
under natural field conditions:
Plants (established) grown on field made from minicuttings were compared to same aged
plants grown from coppice shoots source.
Once the seedlings (whether from minicuttings or coppice shoot source) were planted in
the field, no external supply of nutrient solution to minicuttings was made. The native
nutrients present in the soil are the only source for growth of these two sets of plants.
Since minicuttings had luxurious consumption of nutrients during production stage,
retained the ability of higher intrinsic physiological rates, though barely matched to
efficiencies at nourishment stage.
Increment in plant height and leaf area of saplings derived from mini-cuttings is
considerably high in relation to coppice plants of similar age (composite of all plant sets
planted at different time periods) during similar growth period (3 months after planting).
To examine the reasons for improved performance in mini-cuttings, intrinsic
physiological traits has been determined, which indicated marked differences in carbon
fixation, associated traits coupled with root extraction associated water relation traits. The
extent of enhancement of C-fixation rates and leaf N traits was to a tune of 48 to 33
percent respectively. Similar increment in transpiration rates (represents water
harnessing ability of roots) was around 28%.
Example 7
Improvement in rooting and survival of the sampling due to the Ecosand based growing
media
The samplings were initially grown under NM solution (hydroponics way of minicuttings
production). Once hedge plants were ready to make minicuttings, sets were made and
planted in Ecosand mix medium for rooting for survival. Different combination mix of
Ecosand with vermiculite and leaf compost have been tried to get desired effect on
rooting percentage and saplings survival. Though the experiments, were conducted up to
30 DAP (Day after Planting), as initial survival is crucial part in nursery management, we
presented the data of 20 DAP. Between 20 and 30 DAP, a marginal difference in rooting
and survival percentage was observed.
The result obtained is shown in table 5
Example 8
Improvement in the quality of the sampling due to the Ecosand based growing media
Number of leaves, leaf area and shoot length had also shown marked increase in Ecosand
based mix. Once survival is established, the effect needs to be seen till it goes out as full-
fledged saplings. Hence data up to 75 DAP has been provided.
All the variables recorded were statistically significant at p=0.01% level.
We claim:
1) A culture media comprising a nutrient solution mix intended for ex vitro
propagation.
said nutrient solution mix comprising 10.1 - 10.5% by weight KNO3, 9.0 - 10 %
by weight CaN03, 5.05 - 5.5 % by weight KH2P04 , 6.0 - 6.5 % by weight
MgSO4, 0.5 - 0.75% by weight FeSO4 , 0.25 - 0.4% by weight EDTA, 0.35 -
0.4% by weight MnCl2, 0.15 - 0.25% by weight CuSO4, 0.25 - 0.5% by weight
Zn SO4. 0.15 - 0.25% by weight H3BO3. 0.1 - 0.2% by weight, Ammonium
molybdate
2) A culture media comprising a nutrient solution mix and a growing media intended
for ex vitro propagation,
said nutrient solution mix comprising 10.1 - 10.5% by weight KNO3, 9.0 - 10 %
by weight CaN03. 5.05 - 5.5 % by weight KH2P04 , 6.0 - 6.5 % by weight
MgSO4 , 0.5 - 0.75% by weight FeSO4 , 0.25 - 0.4% by weight EDTA, 0.35 -
0.4% by weight MnCl2. 0.15 - 0.25% by weight CuSO4, 0.25 - 0.5% by weight
Zn SO4, 0.15 - 0.25% by weight H3BO3. 0.1 - 0.2% by weight, Ammonium
molybdate
and growing media comprising ecosand, vermiculite and compost for
strengthening root system, such that the nutrient mix and growing media are
provided sequentially.
3) The culture media as claimed in claim 1 or 2, wherein said KNO3 is preferably
10.1% by weight.
4) The culture media as claimed in claim 1 or 2, wherein said CaNCb is preferably
9.0% by weight.
5) The culture media as claimed in claim 1 or 2, wherein said KH2P04 is preferably
5.05% by weight.
6) The culture media as claimed in claim 1 or 2, wherein said MgSO4 is preferably
6.0% by weight.
7) The culture media as claimed in claim 1 or 2, wherein said FeSO4 is preferably
0.5% by weight.
8) The culture media as claimed in claim 1 or 2, wherein said EDTA is preferably
0.25% by weight.
9) The culture media as claimed in claim 1 or 2, wherein said MnCl2 is preferably
0.35% by weight.
10) The culture media as claimed in claim 1 or 2, wherein said CUSO4 is preferably
0.15% by weight.
11) The culture media as claimed in claim 1 or 2. wherein said ZnSO4 is preferably
0.25% by weight.
12) The culture media as claimed in claim 1 or 2, wherein said H3BO3 is preferably
0.15% by weight.
13) The culture media as claimed in claim 1 or 2, wherein said Ammonium molybdate
is preferably 0.1 % by weight.
14) The culture media as claimed in claim 2, wherein said growing media comprises
said ecosand. said vermiculite and said compost in the ratio of 2:3:1 to 1:1:1
15) The culture media as claimed in any of the preceding claims wherein said ex vitro
propagation is of Eucalyptus camaldulensis

A culture media for ex vitro propagation of plants, comprising nutrient mix comprising
10.1 - 10.5% by weight KNO3, 9.0 - 10 % by weight CaNO3. 5.05 - 5.5 % by weight
KH2PO4, 6.0 - 6.5 % by weight MgSO4, 0.5 - 0.75% by weight FeSO4. 0.25 - 0.4% by
weight EDTA. 0.35 - 0.4% by weight MnCl2. 0.15 - 0.25% by weight CuSO4. 0.25 -
0.5% by weight Zn SO4, 0.15 - 0.25% by weight H3BO3. 0.1 - 0.2% by weight,
Ammonium molybdate. A culture media comprising said nutrient mix where the hedge
plants are grown and growing rooting media. The nutrient mix, provides excellent result
in inducing more number of roots, new roots, root and shoot growth with better leaf area
and greenness under commercial nursery. The growing media is a mixture of ecosand
with vermiculite and leaf compost, which improves rooting ability, and survival rates by
holding water for long and releasing required nutrients slowly.