U.S. patent number PP14,538 [Application Number 09/482,645] was granted by the patent office on 2004-02-17 for mint plant named `sambhav`.
This patent grant is currently assigned to Council of Scientific and Industrial Research. Invention is credited to Janak Raj Bahl, Mahendra Pandurang Darokar, Sunita Dhawan, Suman Preet Singh Khanuja, Sushil Kumar, Tiruppadiripuliyur Ranganathan Santha Kumar, Nirmal Kumar Patra, Dharmendra Saikia, Sarita Satapathy, Ajit Kumar Shasany, Arun Kumar Tripathy.
United States Patent |
PP14,538 |
Khanuja , et al. |
February 17, 2004 |
Mint plant named `Sambhav`
Abstract
The present invention relates to a novel, insect tolerant, high
yielding essential oil and menthol yielding mint plant named
`Sambhav`, which is a cultivar of Mentha arvensis L.. The mint
plant of the present invention has been developed as a result of
planned experiments which devised a procedure for early selection
of somaclonal variants at the in vitro stage of the variety of
Mentha arvensis.
Inventors: |
Khanuja; Suman Preet Singh
(Lucknow, IN), Shasany; Ajit Kumar (Lucknow,
IN), Dhawan; Sunita (Lucknow, IN), Darokar;
Mahendra Pandurang (Lucknow, IN), Satapathy;
Sarita (Lucknow, IN), Kumar; Tiruppadiripuliyur
Ranganathan Santha (Lucknow, IN), Saikia;
Dharmendra (Lucknow, IN), Patra; Nirmal Kumar
(Lucknow, IN), Bahl; Janak Raj (Lucknow,
IN), Tripathy; Arun Kumar (Lucknow, IN),
Kumar; Sushil (Lucknow, IN) |
Assignee: |
Council of Scientific and
Industrial Research (New Delhi, IN)
|
Family
ID: |
31188790 |
Appl.
No.: |
09/482,645 |
Filed: |
January 18, 2000 |
Current U.S.
Class: |
PLT/259 |
Current CPC
Class: |
A01H
5/02 (20130101) |
Current International
Class: |
A01H 005/00 () |
Field of
Search: |
;PLT/259 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
SP.S. Khanuja, et al., A Rapid procedure for isolating somaclones
of altered genotypes in Mentha arvensis., J. Med. Aroma. Plant Sci.
20 (1998) 359-361. .
A.K. Shansany, et al. High regenrative nature of Mentha arvensis
internodes., Journal of Biosciences 23 (1998) 641-646..
|
Primary Examiner: Campell; Bruce R.
Assistant Examiner: Haas; W C
Attorney, Agent or Firm: Ladas & Parry
Claims
I claim:
1. A new and distinct variety of Mentha arvensis plant, as herein
illustrated and described.
Description
Botanical designation: Mentha arvensis L.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The origin of the mint plant of the present application is a single
somaclonal variant of cultivar `Himalaya` (U.S. Plant Pat. No.
10,935). The present invention relates to a novel, insect tolerant,
high yielding essential oil and menthol yielding mint plant named
`Sambhav`, which is derived from Mentha arvensis L.. The mint plant
of the present invention has been developed as a result of planned
experiments for the development of an insect tolerant mint plant
with high oil and menthol yield which devised a procedure for early
selection of somaclonal variants at the in vitro stage variety of
Mentha arvensis. The plant of the present invention can be
propagated vegetatively by suckers and is genetically stable for
commercial cultivation. The plant type of the present invention is
unique because it has an extra wide canopy and height surpassing
all existing varieties. This allows for better sunlight capture and
foliage production which ultimately produce high oil and menthol
yield.
2. Description of Related Art
Mentha arvensis L. var piperescens Holmes (menthol or Japanese
mint) is an industrial crop that is widely cultivated for its
essential oil from which menthol is purified by crystallization
through freezing. Menthol and other terpenoids present in the
dementholated oil of Mentha arvensis are used in the food,
perfumery and pharmaceutical industries. In the varietal
improvement programme, the genetic alternations leading to the
enhancement in the tolerance against pest and disease and
improvement of other characteristics which improve the yield and
quality of essential oil is most desirable. Insect tolerance is
desired particularly against Spilarctia obliqua in Mentha arvensis
L., one of the most damaging pests of mints in India. Since the
mint plant is of considerable interest to the industrial world,
programs for the isolation of desirable clones with improved
terpene accumulation and suitable agronomic traits are being
pursued in several laboratories. However, due to the inherent
problem of seed setting in mints, conventional breeding programs
are severely hampered. In vitro high efficiency procedures for cell
and callus cultures and shoot regeneration from axillary buds and
leaf explants have been reported in some species of the genus
Mentha, especially the commercially important species M. piperata
and M. spicata. However, in M. arvensis proliferation from limited
explants such as nodes, terminal and axillary buds and distal
segments of leaf petiole have relatively low levels of efficiency.
There have been attempts to develop new varieties or genotypes by
alternative methods like clonal selection, mutation breeding and
somaclonal variation in mint plants (S. P. S. Khanuja, A. K.
Shasany, S. Dhawan, S. Kumar, Rapid procedure for isolating
somaclones of altered genotypes in Mentha arvensis. J Med. Aroma.
Plant Sci. 20 (1998) 359-361.). Applicants have reported high
efficiency protocols for rapid detection and selection procedures
for development of somaclonal variants through molecular approaches
in Mentha arvensis (S. P. S. Khanuja, A. K. Shasany, S. Dhawan, S.
Kumar, Rapid procedure for isolating somaclones of altered
genotypes in Mentha arvensis. J Med. Aroma. Plant Sci. 20 (1998)
359-361). Applicants have also successfully defined the condition
and media to restrict the emergence of somaclonal variation for
stable micropropagation purposes (A. K. Shasany, S. P. S. Khanuja,
S. Dhawan, U. Yadav, S. Sharma, S. Kumar, High regenerative nature
of Mentha arvensis internodes. Journal of Biosciences 23 (1998)
641646.).
SUMMARY OF THE INVENTION
For the present invention, Applicants utilize the already reported
protocol (S. P. S. Khanuja, A. K. Shasany, S. Dhawan, S. Kumar,
Rapid procedure for isolating somaclones of altered genotypes in
Mentha arvensis. J Med. Aroma. Plant Sci. 20 (1998) 359-361.) to
generate capture the somaclonal variations in larger frequencies.
Applicants then screen the somaclones for their tolerance to
Spilarctia obliqua by using a novel method for rapid and dependable
selection of tolerant clone(s) at the tissue culture stage. The
somaclones that were determined to be insect tolerant were then
subjected to screening using instar larvae of lepidopteran insect
pest Spilarctia obliqua.
The selected tolerant pants are then tested for their stability,
essential oil content, menthol content and biomass yield.
Consequently, the plant `Sambhav` was selected for unmatchable
vigor of shoots, higher menthol production and increased insect
tolerance through field evaluation.
Accordingly, the invention provides a novel mint plant of Mentha
arvensis named `Sambhav`, which is developed employing tissue
culture techniques, said plant of the present invention possessing
the following combination of characteristics: a. the plant is
highly tolerant to foliage feeding insect pests, especially
Spilarctia obliqua; b. the plant possesses vigorous and rapid
vegetative growth with high regenerability covering at least a 85
cm canopy area and a height of at least 73.5 cm attained in a
maximum of 100 days; c. the plant has a distinct molecular profile
by random amplified polymorphic DNA (RAPD) using 20 OPJ primers and
20 MAP primers which distinguishes the plant from the other
existing varieties, d. the plant shows tolerance to leaf spots,
rust and powdery mildew as in the parent variety `Himalaya`, e. the
plant has the follows characteristics: light greenish leaves,
whitish flowers with a distinct morphology of a single main stem
with branches coming out of the lower nodes imparting a shape of
up-side down open filled umbrella to the canopy allowing equal
distribution of sunlight, thus prevents yellowing and fall of lower
leaves; f. the plant is able to produce the highest herbage when
compare to the other control plants, and g. the plant yields oil
containing 75 to 80% menthol;
The plant of the present invention is developed through an unique,
rapid in vitro screening method. This method is not limited to
Mentha arvensis.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows field views of the morphological features of the
`Sambhav` plants at 70 days.
FIG. 2 shows field views of the morphological features of the
`Sambhav` plants at 100 days.
FIG. 3 shows the rapid growth of the `Sambhav` plant and its canopy
at 70 days.
FIG. 4 shows the rapid growth of the `Sambhav` plant and its canopy
at 100 days.
FIG. 5 shows instar larvae infected individual clones of other
plants in comparison with clone of `Sambhav`. Instar larvae is
larvae that attacks mint plants.
FIG. 6 shows the Genetic similarity of the new clone `Sambhav` with
other varieties of Mentha arvensis.
BOTANICAL DESCRIPTION OF THE PLANT `SAMBHAV` 1. Genus: Mentha. 2.
Species: arvensis L. 3. Family: Lamiaceae. 4. Common name: Japanese
mint/corn mint/menthol mint. 5. Plant height: 73.5.+-.3.27 cm. 6.
Plant canopy: 85-90 cm. A. Stem: 1. Shape.--Round to quadrangular,
hard, woody. 2. Unit of branches (main, upper, middle, lower)
Number of Fingers.--Right hand: 5. Left hand: 5. 2. Number of
branches.--7 to 24. 3. Main branch.--18 to 37. 4. Upper branch
(30.sup.th node).--3 to 6. 5. Middle branch (20.sup.th node).--3 to
8. 6. Lower branch (10.sup.th node).--4 to 10. 7. Main branch (From
lower to upper nodes in cm).--1.0 to 6.0. B. Leaf: Apex shape
acute.--Base shape Attenuate. Shape.--Lacerate. Surface.--Hairy and
rough. Texture.--Moderately thick and rigid. Margin.--Moderately
deep serration (14 to 50 numbers). Size.--Moderately broad.
Area.--16.1 cm.sup.2 (the average leaf lamina area of 41 leaves of
a single branch). Length.--8.56.+-.0.56 cm. Width.--5.51.+-.0.34
cm. Petiole length.--1.7.+-.0.17 cm. Fragrance.--Methanol smell.
Vein.--Colour green (144A). Colour of surface.--Light green (137B)
for upper surface, Yellow green (139C) or lower surface. Number of
trichomes per leaf.--1280. Trichome ratio (lower leaf/upper
leaf).--3.10. C. Time of flowering: April-May (60 to 70 days after
planting first flowering detected). D. Lasting of blooms: Continue
till harvesting (110 days after planting). E. Flowers: Arranged in
whorls surrounding the stem at the base of lateral leaves. 1.
Size.--4.65 mm long. 2. Flower shape.--Tubular. 3. Inflorescence
size.--Continues to grow as indefinite racemose. 4. Pedicel
length.--1.1 to 2.5 mm. 5. Pedicel colour.--Yellow green (145C). 6.
Calyx diameter.--1 mm, four fused. 7. Calyx colour.--Yellow green
group (145B). 8. Corolla.--Purplish white, four, fused to a bell
shaped corolla tube (76C-D), 3 to 4 mm long. 9. Anthers.--Four,
ocidimetary, come out of the corolla tube. 10. Stigma.--Bifid,
Purple (76A). 11. Colour of ovaries.--Yellow green group (151A).
12. Oil content in the fresh herb (%).--0.77 to 0.8. 13. Oil
quality.--Menthol content (%): 75 to 80%. Congealing point: 2 to
21.degree. C. Herbage (Shoot biomass (Q/100 m.sup.2).--2.10
Bihar hairy caterpillar (Spilarctia obliqua) is a pest of a
polyphagus nature which infects mint crops frequently in Terai and
in the northern Indian plains. The infestation is sometimes so
heavy and unmanageable that it may lead to more than 80% complete
loss of foliage and consequently the oil yield proportionately.
Therefore, it is desirable to explore the possibility of developing
insect tolerant high yielding clones of mint plants. The popular
menthol producing mint plant variety `Himalaya` developed in 1996
by Central Institute of Medicine and Aromatic Plants (hereinafter
referred to as CIMAP) (U.S. Plant Pat. No. (10,935). This was the
beginning of specific improvements in insect tolerance through
generation of somaclonal variation while simultaneously looking for
improved plant type with higher essential oil and menthol
yields.
Encouraged by Applicants' own initial leads in the laboratory for
rapid detection and isolation of somaclonal variants by using the
protocol (S. P. S. Khanuja, A. K. Shasany, S. Dhawan, S. Kumar,
Rapid procedure for isolating somaclones of altered genotypes in
Mentha arvensis. J Med. Aroma. Plant Sci. 20 (1998) 359-361),
Applicants generated 3000 independent somaclones. These clones were
subjected to detection of molecular variation at the tissue culture
stage through RAPD profiling. The DNA was isolated from 40 mg of
leaf tissue and Polymerase chain reactions (PCRs) were carried out
in 25 .mu.l volume in a reaction tube containing 25 ng of DNA, 0.2
unit of Taq DNA polymerase, 100 pl each of dNTPs, 1.5 mM MgC.sub.12
and 5 p mol of decanucleotide primers. The amplifications were
carried out using a thermal cycler (MJ Research, USA). The
amplified products were loaded in 1.2% agarose gel containing 0.5
.mu.g ml.sup.-1 of ethidium bromide and photographed by Polaroid
system. Twelve decamer primers having the Sequence ID NOs 1-12
AAATCGGAGC (SEQ ID NO:1), GTCCTACTCG (SEQ ID NO: 2), GTCCTTAGCG
(SEQ ID NO:3), TGCGCGATCG (SEQ ID NO:4), AACGTACGCG (SEQ ID NO:5),
GCACGCCGGA (SEQ ID NO:6), CACCCTGCGC (SEQ ID NO:7), CTATCGCCGC (SEQ
ID NO:8), CGGGATCCGC (SEQ ID NO:9), GCGAATTCCG (SEQ ID NO: 10),
CCCTGCAGGC (SEQ ID NO: 11), CCAAGCTTGC (SEQ ID NO:12) were used to
analyze all the in vitro regenerated clones. Out of 3000
regenerated clones, 245 showed variations at the DNA level in the
RAPD profiles compared to the control plant "Himalaya".
The individual molecular variants selected through the RAPD
analysis of somaclones were then subjected to screening against the
larvae of lepidopteran insect pest Spilarctia obliqua.
For this purpose, a new strategy was devised by subjecting the in
vitro growing clones to attack by actively feeding 3.sup.rd instar
larvae. The larvae were released right in the culture tubes
containing individual clones on the rooting medium (FIG. 5). Most
of the shoots of the clones were eaten away by these larvae within
2-3 days. However, three clones showed the least feeding by the
larvae. In these tubes, only initial bites could be observed and
nonfeeding was also conspicuous by typical symptoms of stalled
growth in the starved larvae. These larvae were then transferred to
other clone tubes, where they resumed feeding. This led Applicants
to believe that the three clones must have some characteristics not
liked by the feeding larvae. So Applicants again confirmed this by
releasing another set of actively feeding 4.sup.th instar larvae
into the tubes containing these three identified clones. This
process was repeated three times and each time, the larvae showed
non-preference to the clones and stopped feeding.
Applicants then hardened these three "insect-non-preferred" clones
namely, CIMAP/GRB 1-06, `Sambhav` and CIMAP/GRB 5-15 and
transferred them to the glasshouse in pots. Among these three
clones, `Sambhav` showed growth characteristics that were much
higher in height and shoot proliferation. `Sambhav` (This GRB) was
multiplied in vitro from interodal explants through stable
micropropagation protocol developed in the laboratory (A. K.
Shasany, S. P. S. Khanuja, S. Dhawan, U. Yadav, S. Sharma, S.
Kumar, High regenerative nature of Mentha arvensis internodes.
Journal of Biosciences. 23 (1998) 641-646.) for genetic uniformity
into about 1000 plantlets. Randomly 100 regenerated shoots from the
clone were tested for variation in their profiles using the above
described 12 random primers. Complete uniformity was observed among
these clones without any variation from the control mother plant
`Sambhav` but the profiles were clearly distinct from other mint
varieties including `Himalaya`.
Breeding History
The plant `Sambhav` can be propagated vegetatively through suckers
for cultivation. The plant was developed by performing the RAPD
analysis, selecting different somaclones followed by force feeding
by insect to screen for insect tolerance character. The selected
plant was grown in the glass house and the suckers obtained were
field planted for multiplication. The suckers from the
multiplication plots were taken for planting in the main field for
field trials. Replicated field trials were conducted following
normal agromonic practices by planting multiplied suckers in
January 1998 and January 1999 in RBD fashion. Different growth and
yield characteristics were recorded. For field trials, 10
m.times.10 m plots were prepared by adding only FYM 1.5 ton per ha.
The field experiments were carried out in the farm field of
CIMAP.
The seeds and fruits were not checked as the plant is propagated
vegetatively through suckers.
The plants of the present invention was described after 100 days of
planting during the month of May when the maximum temperature
remained between 32 to 42.degree. C. and the minimum temperature
remained between 25 and 32.degree. C.
The overall objective of the present invention was not only to
develop insect tolerant genotypes but to simultaneously also have a
better plant type with high menthol content and herbage yield for
better productivity. So, the plant of present invention was tested
in the field for oil yield, menthol content and herbage production
against the checks. Replicated field trials were conducted
following normal agronomic practices by planting multiplied suckers
in January, 1998 and January, 1999 for 2 consecutive years in RBD
fashion and diffent growth and yield characteristics were recorded
(Table. 1). For field trials, 10 m.times.10 m plots were prepared
by adding only FYM 1.5 ton per ha. Astonishingly, the `Sambhav` was
able to out-compete all existing varieties in its rate of growth.
It was so rapidly growing that it could cover the inter-row spacing
of 80 cm completely within a period of 90-100 days, which was not
the case for the other control varieties taken (FIG. # 2). The
plant canopy covered a space of 85 to 90 cm in 110 days in
comparison to Himalaya(62-70 cm), Shivalik (50-56 cm) (unpatented),
Gomti (70-75) (unpatented); Kosi (65-72) U.S. Plant Pat. No.
12,426, Kalka (40-60 cm) (unpatented) MAS-1(40-50 cm) (unpatented).
This was the most desirable advantage to the plant because it
produced the highest amount of oil and menthol.
Natural infestation of Spilarctia obliqua in the field absent
insecticidal spray was noted in both the 1998 and 1999 crop stands.
The clone `Sambhav` had less than 10% leaf damage compared to 86%
in the case of Gomti, 58% in Himalaya, 50% in Kosi in 1998. A
similar trend was also recorded in 1999 when leaf damage was 70%,
50% and 46% for Gomti, Himalaya and Kosi respectively, compared to
6% in case of GRB `Sambhav`. The plant of the present invention
also produced the highest amount of essential oil per unit area in
comparison to the other control plants in both successive yield
trials. The total menthol yield consequently, was the highest due
to more oil and herbage yield (Table 1). The plant was named
`Sambhav` (means "Possible") because the expression of its genotype
made this a rare but most desirable combination of traits.
Thus, the plant of the present invention `Sambhav` has a very high
tolerance to Spilarctia obliqua attack, high growth rate and
regenerability and produces the highest total herbage, oil and
menthol yield per unit area in comparison to the other control
plants and thereby being a unique and novel genotype which can be
exploited for commercial cultivation superior to other available
mint varieties for menthol production.
TABLE 1 Comparative growth and yield characteristics of plant of
invention "Sambhav" (GRB 2-18) in relation to the existing Japanese
mint varieties Property Himalaya Shivalik MAS-1 Kalka Plant height
60.9 + 6.2 68.25 + 3.4 43.2 + 1.1 51.6 + 2.7 (cm) Canopy (cm) 62-70
50-56 40-50 40-60 Leaf number 40.2 + 4.2 49.6 + 4.8 40.0 + 1.4 38 +
2.0 Branch length 40.4 + 6.8 44.2 + 8.7 36.6 + 9.0 45 + 9.7 Branch
number 29.2 + 4.0 22.4 + 1.7 13.6 + 1.7 27.2 + 1.1 Leaf length 7.1
+ 0.5 7.3 + 0.3 7.5 + 1.0 7.4 + 0.8 Leaf breadth 4.5 + 0.2 5.0 +
0.2 3.8 + 0.5 4.0 + 0.3 Petiole length 1.75 + 0.3 1.40 + 0.1 1.6 +
0.5 1.5 + 0.5 Oil % 0.7 0.5 0.5 0.8 Menthol % 80 77 88 82 Herbage
yield 1.25 0.75 0.50 0.80 (Q per 100 m.sup.2) Oil yield (Kg 0.85
0.35 0.27 0.64 per 100 m.sup.2) Property Gomti Kosi Sambhav Plant
height (cm) 74.2 + 7.3 66.2 + 3.1 73.5 + 3.3 Canopy (cm) 70-75
65-72 85-90 Leaf number 46.0 + 2.8 50.4 + 3.8 44.2 + 2.2 Branch
length 58.4 + 14.6 52.0 + 5.7 50.1 + 8.8 Branch number 28.4 + 2.9
30.4 + 1.7 35.1 + 2.8 Leaf length 7.7 + 0.6 7.40 + 0.7 8.6 + 0.6
Leaf breadth 4.7 + 0.2 4.60 + 0.5 5.50 + 0.3 Petiole length 1.4 +
0.1 1.4 + 0.3 1.70 + 0.2 Oil % 0.5 0.8 0.8 Menthol % 73 76 77
Herbage yield 1.60 1.70 2.10 (Q per 100 m.sup.2) Oil yield (Kg per
0.83 1.42 1.61 100 m.sup.2)
TABLE 2 Comparison of "Sambhav" with other existing varieties of
the same botanical and market class of Mentha arvensis Character
cv.MAS-1 cv.Kalka cv.Shivalik 1. Leaf:stem ratio 1.2 1.0 0.8 2.
Stem colour Upper green GREEN Green (141B) (141C), lower (141C),
lower lower pig- pigmented red pigmented red mented red purple
(71B) purple (71B) purple (71B) Stiffness Hard Hard Woody Thickness
at 5.sup.th 6.8 3.6 10.0 internode (mm) 3. Leaf Colour Green (139C)
Green (139C) Green (138A) Length (cm) 6.46 6.7 5.2 Width (cm) 2.3
2.4 2.8 Area (cm.sup.2) 8.2 8.7 10.1 4. Petiole length 1.1 1.2 0.6
(cm) 5. Flower colour Whitish Whitish Whitish 6. Flower length 3.72
3.48 4.66 (mm) 7. Calyx colour Green (143B) Green (143B) Green
(141B) with red purple with red purple streaks (71B) streaks (71B)
8. Stigma colour White White Purplish (71C) 9. Disease incidence to
Rust Tolerant Resistant Susceptible Alternaria -- Resistant
Susceptible leaf blight Corynespora Susceptible Tolerant
Susceptible leaf spot Powdery Susceptible Susceptible Tolerant
mildew 10. Pest infestation Spilarctia obliqua Susceptible
Susceptible Susceptible Character cv.Gomti cv.Himalaya cv.Sambhav
1. Leaf:stem ratio 1.0 1.5 1.7 2. Stem colour Green (143B) Green
(143C) Green (144A) lower lower lower pigmented purplish (70A)
purplish (59B) purple (77A) Stiffness Woody Hard Woody Thickness at
5.sup.th 7.9 8.0 10.0 internode (mm) 3. Leaf Colour Green (138A)
Green (138B) Green (137B) Length (cm) 6.4 6.2 8.6 Width (cm) 3.1
3.6 5.1 Area (cm.sup.2) 13.3 15.4 16.1 4. Petiole length 0.7 1.5
1.7 (cm) 5. Flower colour Whitish Pinkish white Purplish white
(76C) 6. Flower length 4.74 3.72 4.65 (mm) 7. Calyx colour Green
(141B) Green (143C) Green (145B) 8. Stigma colour Purplish (71C)
White Purple (76A) 9. Disease incidence to Rust Susceptible
Resistant Resistant Alternaria Susceptible Resistant Resistant leaf
blight Corynespora Susceptible Tolerant Tolerant leaf spot Powdery
Tolerant Tolerant Tolerant mildew 10. Pest infestation Spilarctia
obliqua Highly Susceptible Tolerant Susceptible
The plant genotype `Sambhav` of the present invention is a
herbaceous perennial with a single tall upright stem possessing
several lateral branches coming out from the lower nodes laterally
rising in a fashion to give a shape of an open filled umbrella
turned upside down. This special arrangement of branches
facilitates the distribution of the captured sunlight equally to
all the leaves and hence avoids shading, reducing lower leaf fall,
which prevents economic loss to the plant. The chromosome number of
the plant is 2n=96. The colour codes are in accordance with the
"R.H.S. colour chart" published by The Royal Horticultural Society,
80 Vincent Square, London SW1P 2PE, 1995.
Evidence of uniformity and stability
No variants of any kind (morphological or molecular) has been
observed since 1997 indicating the stability and uniformity of the
genotype. Further, the comparative herbage and oil yields of
`Sambhav` were significantly higher in comparison to other
varieties/genotypes in different years and seasons. Due to vigorous
vegetative growth this genotype can be harvested earlier without
reducing the yield of herbage, oil or menthol. The traits of insect
tolerance against S. obliqua is unprecedented and stable.
Statement of distinction
The genotype `Sambhav` possesses a very high level of insect
tolerance against leaf damage by S. obliqua larvae, which is unique
and unprecedented by any known variety. Additionally, it has a
distinct canopy of one straight main stem with many lower branches
arranged like an open filled umbrella turned upside down which is
characteristic to this genotype only. The genotype of the present
invention has the highest biomass and highest oil yield unit area
in comparison to others. The total menthol yield of the new
genotype is higher per unit area in comparison to other genotypes.
Its genetic make up is distinct in terms of the DNA profile.
Randomly Amplified Polymorphic DNA analysis
The RAPD profiles of the plant `Sambhav` establish its distinct
identity as completely different from the parent plant `Himalaya`
as well as the known released varieties. The plant of the present
invention was developed by screening molecular variants among
somaclones already differentiated as distinct, unique and novel at
the DNA level. The plant of the present invention has desirable
morphological and economical traits in a rare unmatchable
combination and is available only by Applicants in CIMAP. No
variation in the RAPD patterns was observed in the analysis of the
micropropagated as well as field raised population in successive
generations indicating the stability of the genotype. The 20 MAP
primers (MAP 01 to MAP 20) with the Sequenc ID NOs 1-20 AAATCGGAGC
(SEQ ID NO:1), GTCCTACTCG (SEQ ID NO:2), GTCCTTAGCG (SEQ ID NO:3),
TGCGCGATCG (SEQ ID NO:4), AACGTACGCG (SEQ ID NO:5), GCACGCCGGA (SEQ
ID NO:6), CACCCTGCGC (SEQ ID NO:7), CTATCGCCGC (SEQ ID NO:8),
CGGGATCCGC (SEQ ID NO:9), GCGAATTCCG (SEQ ID NO:10), CCCTGCAGGC
(SEQ ID NO:11), CCAAGCTTGC (SEQ ID NO:12), GTGCAATGAG (SEQ ID
NO:13), AGGATACGTG (SEQ ID NO:14) AAGATAGCGG (SEQ ID NO:15),
GGATCTGAAC (SEQ ID NO:16), TTGTCTCAGG (SEQ ID NO:17), CATCCCGAAC
(SEQ ID NO:18), GGACTCCACG (SEQ ID NO:19), AGCCTGACGC (SEQ ID
NO:20) and 20 OPJ primers (Operon Technologies Inc, USA) were used
for the analysis and similarity indices were computed to generate a
similarity matrix among existing varieties and the plant `Sambhav`
(Table 3) The OPJ primers (01 to 20) were obtained from Operon
technologies, USA. The MAP primers were used to develop a unique
and distinct RAPD profile of the Plant.
TABLE 3 Similarity indices of different control plants analyzed in
comparison to `Sambhav` Gomti Himalaya Kosi MAS-1 Kalka Shivalik
Sambhav 1.00 0.90 1.00 0.89 0.94 1.00 0.91 0.92 0.92 1.00 0.85 0.88
0.85 0.89 1.00 0.92 0.93 0.91 0.90 0.87 1.00 0.87 0.73 0.78 0.82
0.83 0.84 1.00
TABLE 4 Genetic similarity of the new clone `Sambhav` with other
varieties of Mentha arvensis. ##STR1##
From RAPD analysis, the profiles were studied and similarity
indices were calculated which were put into a matrix. This matrix
was used to produce a graphic phenogram by means of UPGMA
(unweighted pair group method with arithmetic average) cluster
analysis (FIG. 1). As represented in the phenogram the clone of the
invention is quite different from the other varieties. The
diversity of the genotype of the plant of invention `Sambhav` is
13%, 28%, 22%, 18%, 17% and 16% from the varieties Gomti, Himalaya,
Kosi, MAS-1, Kalka and Shivalik. The highest difference in terms of
polymorphic profiles was observed for the genotype of the invention
with the parent plant `Himalaya` from which it was developed as a
somaclone. The plant genotype of the invention `Sambhav` was most
similar in terms of polymorphism with Gomti. In total the
distinctiveness of the clone in RAPD profiles was established as
total polymorphism detected.
Comparison of Sambhav with the check varieties
The new genotype `Sambhav` was so rapidly growing that it could
cover the inter-row spacing of 80 cm completely within a period of
90-100 days, which was not the case of other control varieties
taken (FIG. # 2). The plant canopy covered a space of 85 to 90 cm
in 110 days in comparison to Himalaya (62-70 cm), Shivalik (50-56
cm), Gomti (70-75), Kosi (65-72), Kalka (40-60 cm) and MAS-1_(40-50
cm). The herbage yield of the plant 2.8 Q per 100 m.sup.2 against
1.25, 0.75, 0.50, 0.80, 1.60, 1.70 for Himalaya, Shivalik, MAS-1,
Kalka, Gomti and Kosi. Similarly the oil yield was highest (0.8%)
in case of the genotype. The genotype though has less menthol
percentage, when the total menthol yield per 100 m.sup.2 is
estimated by converting the total oil yield per 100 m.sup.2 against
the menthol percentage (77.times.1.61/100) and it comes to 1.24 kg,
which is much higher than other varieties. The menthol yield values
for other varieties are 0.68, 0.27, 0.24, 0.52, 0.60,1.08 kg per
100 m.sup.2 for Himalaya, Shivalik, MAS-1, Kalka, Gomti and Kosi
respectively. This was the most desirable economical advantage to
the plant with commercial value as it produced highest amount of
herbage when compared to the other control plants and ultimately
the oil and menthol per unit area in comparison to other genotypes
while being the most tolerant to insect pest attack.
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* * * * *