Supervisors:
Prof. L.M.S. Palni, Senior Scientific Advisor, Biotechnology Government of Uttaranchal
Dr.
S.K. Nandi, GBPIHED, Kosi-Katarmal, Almora
Summary:
Forest
cover in central Himalaya is depleting rapidly due to various biotic
pressures and lack of efficient management leading to a threat to
ecological security of the region. Recent survey by forest department
(Govt. of India) revealed that forest cover in Uttaranchal is much less
than the proposed minimum limit of 60% of total area. This state of
affairs underlines the need for launching massive afforestation programmes
in this region. In view of
the importance, Quercus Linn. species (Q. glauca, Q.
leucotrichophora and Q. floribunda) was selected for the
present study in order to analyze viable approaches for
mass propagation. The study involved observations on seed storage
and viability, influencing seed germination, developing in vitro
and vegetative propagation protocols, and monitoring growth performance of
seedlings in different microclimatic conditions.
It
was observed that seeds of Q. glauca and Q. leucotrichophora
lost their germinating capacity on storage. Tetrazolium staining pattern
test indicating that seed stored at 4 oC remained viable for a
longer period (12 months) than when stored at 20oC. Efforts to
improve seed germination through physical, chemical and plant growth
regulators treatments were carried out. Seeds collected from Ranman (Q.
glauca), Daulagad (Q. leucotrichophora), and Kilbury (Q.
floribunda) Kumaun Himalaya, were subjected to different physical
treatments. Among the various physical treatment, maximum germination (82
to 94%) was observed as a result of scarification along with minimum mean
time of germination. This was found to be simple, economical and practical
method, which can easily be practiced by the rural populace. Seeds treated
by chemicals showed maximum germination of 73 to 81% when treated with
1.0% KNO3. Among
the PGRs examined, maximum germination of 73 to 77% was observed when
treated by 250 µM GA3 and 70 to 80% of germination when
treated with 25 µM and 250 µM BA.
Seed
size (based on weight) and use of different soil ratio (degraded/ non
forest and rhizosphere/ forest soil) also influenced germination rate.
The effect on germination and subsequent survival of seedlings was
examined in relation to seed weight. A wide variation in seed size (based
on weight) exists and germination was found to be correlated with seed
weight. The larger and heavier seeds required short emergence time,
exhibited better germination (65.6%) and seedling performance (survival
70%) in comparison to medium and smaller size seeds.
Highest
germination was observed in soil ratio mixture of degraded and rhizosphere
(1:1) soil, and it showed a maximum germination of 55%.
Maximum mycorhizal infection (70%) was observed in seedlings, which
were planted in rhizosphere soil.
Multiple shoots of Q. glauca Thunb, Quercus leucotrichophora L. A. Camus and Q. floribunda Lindl. ex A. Camus were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and (MS; Murashige and Skoog, 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA3)/ indole-3-butyric acid (IBA). BA (22.19 mM) was effective for induction of multiple shoots and addition of GA3 to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 mM). Nearly 67.0% and 78.0% rooting was obtained in Q. glauca and Q. leucotrichophora microshoots (3 - 4 cm height), respectively on 1/2 strength WP medium supplemented with IBA (14.76 mM). However, this was associated with basal callus formation. Subsequently a two-step method was adopted where treatment with IBA (25-100 mM) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 mM for 24 h was most effective (100% in Q. glauca, 90% in Q. leucotrichophora and 83.3% in Q. floribunda).
In vitro rooted plants were hardened and established in garden soil. The percent survival of plantlets decreased with an increase in the period of hardening. After 12 months, 80% plantlets derived from the first crop shoot and 70% from the second crop shoot of Q. glauca species survived. In case of Q. leucotrichophora the percent survival of plantlets from first and second crop shoot was 80% each. Even after 9 months, 90% of the plantlets were found growing in mist and nursery conditions in case of Q. floribunda.
Growth performance of 6 months old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light intensities (0, 600, 900, 1200, 1500 and 2000 mmol m-2 s-1) and temperature (20, 25, 30, 35 and 40 oC). Light optimum for photosynthesis was around 2000 mmol m-2 s-1 in Q. leucotrichophora and around 1500 mmol m-2 s-1 in Q. glauca whereas optimum temperature for photosynthesis was 25 oC in Q. leucotrichophora and 30 oC in Q. glauca. The rate of transpiration at different temperatures (20-40 oC), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 mmol m-2 s-1. Temperatures beyond 35 oC adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species.
In general higher
light and lower temperature optima for photosynthesis was recorded in Q.
leucotrichophora when
compared to that of Q. glauca and it indicated that the former
should perform better when planted at exposed sites and higher altitudes.
Although the ex vitro plants of Q. glauca and Q.
leucotrichophora exhibited slightly higher rates of transpiration and
net photosynthesis as compared to in vitro raised plants, these
differences were statistically not significant. Thus in vitro
propagated plants would appear to be quite normal in respect of the
physiological functions examined in this study.
Conventional
vegetative propagation trials were conducted on Q.
glauca, Q. leucotrichophora and
Q. floribunda viz. stem
cuttings, root stock, and air layering. Results revealed unsuitability of
stem cuttings of Q. glauca and Q. floribunda for propagation as they did not produce effective
rooting even through auxin treatments. But treatments with IBA + Coumarin
(0.5 + 5.0 mM) significantly influenced rooting
(50%) with sufficient number of roots in Q. leucotrichophora. Branch
air layering in Q. glauca showed
maximum rooting (75%) when branch (shoots) were treated with 100 ppm IBA.
This method can be used to produce true to type plants of Q. glauca.
A
protocol for somatic embryogenesis and subsequent conversion into
plantlets was developed for Q. floribunda Lindl. ex A. Camus using
mature seed (cotyledons) as a source of explant. Germination of somatic
embryos and conversion into plantlets were best achieved on a combined
Schenk and Hildebrandt (1972; SH) + Murashige and Skoog (1962; MS) medium
supplemented with BA (0.44 -2.20 µM). The formation of somatic embryos
was found to increase when casein hydrolysate (0.02%) was added to the
medium. While maximum germination of somatic embryos was achieved in SH+MS
medium supplemented with 0.88 µM BA, such plants were found to have poor
survival under ex vitro conditions. Therefore, the tap root system
was excised from somatic embryo derived plants and the microshoots so
obtained were allowed to root. Nearly 90% rooting of microshoots (2.0 –
3.0 cm height) was obtained on ½ strength Woody Plant (WP; Lloyd and
McCown, 1980) medium after treatment with 100 µM IBA for 24 h and
transfer to plant growth regulator free medium. The plants were hardened
in a mist chamber and transferred to polybags with 70% survival under ex
vitro conditions.
Availability of quality planting material in the Himalayan region is one of the major constraints limiting the progress of afforestation programmes and rehabilitation of degraded/wastelands. The poor economy, difficult terrain and inaccessibility prohibits the use of high – tech controlled condition glasshouses for raising tree saplings. Therefore, studies on performance of seedlings under different conditions i.e., polypit, polyhouse, chappar, green house and open were undertaken. Seedling growth was found best in polypit and polyhouse condition. Seedlings of Q. leucotrichophora showed best growth (71.0 cm, shoot height; 7.7 mm, shoot diameter and 30.9, number of leaves) in polyhouse when compared to open (32.8 cm, shoot height; 6.2, mm shoot diameter and 18.8 number of leaves) and seedlings Q. floribunda showed best growth (35.7 cm, shoot height; 4.5 mm shoot diameter and 25.1, number of leaves) in polypit when compared to open (21.0 cm, shoot height; 4.1 mm shoot diameter and 11.5, number of leaves) after 12 months.
Propagation package developed for Quercus spp. has potential applicability in the afforestation sector at global levels. Further the study provides options for economic betterment of the people.
Vijay Kant Purohit
Subject : Botany
Ph. D - Botany H.N.B.Garhwal University, Srinagar (Garhwal)
Correspondence Address:
V.K. Purohit, GBPIHED, Kosi-Katarmal, Almora,Uttaranchal-263 643
Work Title
Mass propagation of banj, tilonje and
phaniyat oaks