Plant husbandry – Process – Manipulating seed or seedling
Reexamination Certificate
2001-03-08
2002-09-17
Jordan, Charles T. (Department: 3643)
Plant husbandry
Process
Manipulating seed or seedling
C047S05810R
Reexamination Certificate
active
06449899
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for inducing improved seed germination in a high altitude, medicinal and endangered plant,
Podophyllum hexandrum
Royle
BACKGROUND OF THE INVENTION
Podophyllum hexandrum
Royle is an important medicinal plant, which grows in the inner ranges of Himalayas at an altitude of 2700-4200 m from Kashmir to Sikkim (Badhawar and Sharma 1963), extending up to south west of China.
The rhizomes of
Podophyllum hexandrum
are rich in podophyllotoxins and have anticancer and antitumor properties (Kamil and Dewick, 1986). The podophyllotoxins are lignans whose semi-synthetic derivatives-etoposide (VP-16-213) and teniposide (VM-26) are approved drugs for the treatment of testicular and lung cancer (Stahelin and Warburg, 1991).
P. hexandrum
has the maximum podophyllotoxins up to about 4% from dried roots as compared to 0.25% of
P. peltatum
(Jackson and Dewick, 1984a).
Ever increasing demand for this drug in modern medicine, coupled with its existing use in traditional system of medicine has resulted in ruthless uprooting of the underground parts of the plant leading to intense collection coupled with the lack of organized cultivation. Consequently,
P. hexandrum
has been declared as an endangered species (Airi et al., 1997, Bhadula et al., 1997), thus, expeditious methods are required for its sustained propagation and organized utilization. Moreover, since Podophyllum generally grows at high altitudes of the Himalayas and inaccessible locales, it is all the more essential to cultivate these plants in large numbers (Badhawar and Sharma, 1963) for their sustainable utilization.
Early attempts in domestication of Podophyllum were through multiplication of rhizomes. However, since rhizomes are also the source of podophyllotoxins, considerable loss is incurred either in terms of propagules (when uprooted) or important harvestable materials of pharmaceutical importance (when conserved) (Sadowska et al., 1997). The other infrequent method of propagation is through seed germination. Seed germination is erratic and is further limited by long periods of seed coat imposed or mechanical and endosperm dormancy which cannot be generally broken by traditional methods (Badhawar and Sharma, 1963). More than the germinability of the seeds, the structural barrier in the seedlings i.e. the hypocotyl (Purohit and Nautiyal, 1986) may also reduce the chances of its survival in nature (Nautiyal et al., 1987).
Thus, it is not surprising that despite the urgent necessity of mass propagation and conservation of Podophyllum, the work done till date has been limited only to academic interests with respect to the small number of plants propagated. The major objective in the propagation of endangered species is conservation of valuable heterogeneity. An improved method of uniform seed germination which also leads to successful field establishment will help in removal of existing problems which hinders successful plant establishment and large scale non-destructive propagation. The germinative propagation will also help in raising large scale population of heterogeneous plants of representative gene pool.
Reference may be made to the work of Troup, (1915) wherein he reported the (i) the presence of a dormancy period of about two to three years in the seeds that were collected immediately after fruit ripening (ii) sporadic germination which commenced after 3 months and continued up to 7 months of sowing (iii) failure to break the seed dormancy after soaking the seeds for 8 days in water (iv) rather sporadic and forced germination under abnormal conditions at an altitude of 660 m above mean sea level. A similar report of Bhadawar and Sharma (1963) wherein they also indicated the (i) the presence of a dormancy period of about 9-10 months in the seeds and poor germination of about 7-45% (ii) different treatments like soaking of seeds in luke warm water for 24 hrs, 60% H
2
SO
4
for five minutes and chipping of seeds with a blade all failed to break seed dormancy and induce germination The drawbacks include (i) the reports does not provide a method for germination of seeds in large numbers (ii) the time taken for complete seed germination was relatively long (iii) seed germination was poor (iv) there is no mention of growth conditions required for seed germination and hence, the method cannot be reproduced (v) a long dormancy period of about 9-10 months which could not be overcome by any of the treatments tested; therefore, the time required for seed germination could not be shortened (vii) the treatments that were employed for breaking seed dormancy were not effective.
Rust and Roth, (1981) studied the seed production and seedling establishment in the other related species—
Podophyllum peltatum
wherein they found that under natural conditions in 14 hectares of Quercus-Liriodendron forest that was cut 90 years ago, the seeds planted below ground showed 24.4±3.2% germination with very high seedling mortality rate (98.7%). The major objective of this paper was to study seed production potential and seedling establishment under natural conditions so as to study the natural processes controlling seed germination and seedling establishment. The drawbacks are (i) poor seed germination (24.4%) and (ii) seedling mortality rate beyond acceptable limits.
Reference may be made to Nautiyal et al., (1987), wherein no germination of fresh seeds at 20° C. as compared to 66% and 88% germination in seeds stored for one month at low temperatures with and without Gibberellic acid treatment respectively. The drawbacks are (i) no germination of fresh seeds (ii) requirement of one month of low temperature storage prior to germination (iii) time required for seed germination is considerably long and (iv) germination even after storage was comparatively low.
Reference may be made to Choudhary et al., (1996) wherein relatively poor germination of 3 months old sterilized seeds on moistened filter papers in Petri dishes at room temperature (10-22° C.) under light and dark conditions with and without scarification. While under dark conditions, scarified seeds germinated (60.0%) after 45-60 days of sowing, very poor (8.89%) germination under light conditions was achieved. In non scarified seeds up to 78.88% and 14.44% germination under light and dark condition was reported after 120 days of seed sowing. The drawbacks are (i) the method described is cumbersome and long (ii) time required for seed germination under all conditions is long, (iii) cumbersome and labour intensive methods like scarification are required (iv) data on seed germination at room temperature makes it a non-reproducible and unsatisfactory because of the fluctuations in physical parameters, (v) maximum germination is relatively poor i. e. 78.88% and (vi) there is no mention of field establishment of seedlings.
Reference may be made to Choudhary et al., (1998) wherein the cultivation method for
P.hexandrum
through seeds is described. Following their previous method for seed germination, seedling development and true leaf emergence (4 months after seed germination) under natural conditions is reported. The drawbacks are (i) the previous protocol which was employed for germination and seedling establishment was very long and cumbersome, (ii) rate of seed germination under recommended cultural practices is not reported, (ii) the actual percent or the number of seedlings that were transferred has not been reported, (iii) seedling mortality rate which is very crucial for its survival has not been mentioned and, (iv) details about the actual percent or number of true leaf emergence was not given.
Reference may be made to Singh et al., (1999) wherein the seed germination behaviour among different populations of
P. hexandrum.
Under controlled conditions, up to 95% seed germination was reported in the alpine seeds at 30° C. under light after 100 days of sowing. The germination tests were performed on filter paper in Petri dishes. The drawbacks are (i) seed germination took a long time i.e. up to 100 days (ii) the meth
Ahuja Paramvir Singh
Bhattacharya Amita
Kharkwal Amit Chandra
Nagar Pramod Kumar
Prakash Om
Burns Doane Swecker & Mathis L.L.P.
Council of Scientific and Industrial Research
Gellner Jeffrey L.
Jordan Charles T.
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