Introduction
Garlic (Allium
sativum. L) is a vegetatively propagated crop, as a result sexual
reproduction is absent, thus cloves are used for propagation. Hence,
vegetatively propagated plants are systemically infected with diseases, the
pathogens pass from one generation to the next through vegetative propagules.
Thereby entire variety which is propagated by clonally may be infected with the
pathogens, degenerates the planting material, in turn cause losses to bulb
production and productivity. It is infected with a number of viral diseases and
reported to cause 70% bulb yield loss (Nagakubo et al. 1991; Ayabe and
Sumi 2001). The important viruses influencing yield and quality are garlic
common latent carlavirus (GCLV), garlic dwarf reovirus, leek yellow stripe
potyvirus (LYSV), mite-borne filamentous viruses (MbFV), onion yellow dwarf
potyvirus (OYDV), shallot latent carlavirus (SLV) and serologically related
carlaviruses, shallot yellow stripe potyvirus (SYSV), tomato spotted wilt virus
(TSWV), carnation latent virus (CLV), leek yellow stripe virus (LYSV), garlic
mosaic virus (GMV), garlic virus X (GVX), shallot virus X, narcissus latent
virus, and iris yellow spot virus (Manjunathagowda et al. 2017; 2021). Symptoms of latent viruses are difficult to
detect, many viruses may not even show visible symptoms, and pathogen attack
does not always lead to death of plants, however the presence of viruses reduce
the crop yield and quality of produce (Wang and Hu 1980). The viruses causing
yield loss due to poty virus and carlaviruses, which ranged between 30–40%
(Sing 2005). Elimination of viruses from planting materials is highly desirable
to optimize the yield, and also to facilitate the hassle-free movement of
living plant materials across the international boundaries (Manjunathagowda et al. 2017; 2021). It is difficulties
to induce flowering in this species, thus possess problem for the breeder to
breed promising genotypes, there is need to develop disease tolerant and
resistant varieties against viral diseases, which is difficult task in garlic,
due to breeding is limited to clonal selection. However, disease free planting
material can be produced through meristem tip culture (Barandiaran et al. 1998).
In infected plants, apical meristems either free or carry a very low load of
viruses (Quak 1977; Wang and Hu 1980). Thus pathogen-free propagules are
further multiplied vegetatively for commercial production. However, isolation
of shoot meristem, their production has been focused mainly during long day
type of garlic cultivars, there is need to standardize technique for meristem
tip culture protocol for shoot and root multiplication in short day garlic
varieties, further virus free material can be used for multiplication in large
quantity in virus free areas. Hence, the present study was undertaken to
standardize a protocol for the micro propagation of short-day garlic through
meristem tip culture.
Materials and Methods
Study materials
Three different
genotypes of garlic namely G-41, G-282 and G-323 released by National
Horticultural Research and Development Foundation, Nasik, India
were used in the study.
Preparation
of explants and culture media
Preparation
of mother plant to provide quality explant for better establishment of aseptic
cultures. Cloves were used for raising in vitro meristem cultures; the
experiment was sorted in two sets. In first set, the explant was taken directly
from the fresh clove. In second set, explant are 15–20 old days sprouted
cloves. Meristem tip culture of garlic was initiated using meristematic region
from sprouted cloves, and fresh cloves (stored) which was excised and
inoculated on treatments of culture media.
The MS medium powder procured
from HiMedia, India, used for media preparation. Initially, container of 1 L
volume was rinsed with the distilled water, washed thoroughly. Then, 800 mL of
distilled water was taken in the container, along with MS medium powder, with
suitable treatment combinations of plant growth hormones, with TDZ and NAA in different combinations (the
concentration of stock solution was 1 mg/mL) along with control as MS medium.
The media combinations presented in Table 1, followed by stirring the water till the medium was dissolved completely
to a clear solution and then final volume was made to 1 L. Then, pH was
adjusted to 5.77 to 5.82 by using 1 N NaOH and HCl. Gelling agent agar was
added 8 g of (0.8%) per liter of media and it was boiled till the agar dissolve
completely to obtain a clear solution. Dispensed the medium in suitable
culture vessels (test tubes). These tubes containing medium were autoclaved at
15 lb/inch2 pressure and 121oC temperature for 15 min.
The medium was allowed to cool and used for the inoculation.
Sterilization,
inoculation of explants and initiation of culture
The surface
sterilization of explant (the outer dry layers removed of cloves) was done by
rinsing the cloves for three times under running tap water, followed by rinsing
with 70% alcohol for two min. Then cloves were treated with Bavistin (0.1%)
along two drops of Tween-20 for 15 min. Then washed the cloves with water for
one hour, to remove traces of Bavistin. Soon after, cloves were treated with
70% alcohol for two minutes in the laminar airflow and washed for thrice with
sterile distilled water. Then the cloves were treated with sterilant NaClO (2%
chlorine), Qualigens, India for 15 min, followed by washing with sterile distil
water for three to four times, then finally the surface sterilized cloves were
used for inoculated in the media.
The meristem tip was inoculated on 16 different media compositions of
MS media (Murashige and Skoog 1962) with the addition
of TDZ and NAA in 5 replications for initiation of the cultures. The
meristematic tip was dissected from the cloves and inoculated on treatment
media. The explants were inoculated on the medium under aseptic condition, the
cultures were incubated at 25±2oC under cool light
of 3000 lux and 16:8 h light/dark regime for 15–20 days.
Isolation and inoculation
meristems, incubation meristems culture and their
multiplication
Meristem tip were dissected from 15–20 days old, sprouted shoot tip, as well as from fresh clove
under ascetic condition, with help of sterilized micro-scalpels in laminar air
flow, using the forceps were used for isolation of meristem the microscope.
Short hypodermic needles were used to remove leaves and their primordia. This
technique was used to avoid transfer of the microorganisms to the sterile apex.
A thin layer of sterile distilled water used to create contact in between
explant and medium surface.
The
dissected meristem was inoculated on 16 different culture media composition
(Table 1) under aseptic condition and culture were incubated at 25±2oC
under cool light of 3000 lux and 16:8 h light/dark condition. Observations were
made after 15 days and 1 month of inoculation in both sets of experiment. Plant
height and number of shoots were recorded for five replications and data was
analysed in completely randomized design.
The multiple shoot formation led to the maximum number of propagules;
however, in culture, a meristem may develop either into single shoot or
multiple shoot masses or even into rooted plantlets.
Preparation
of media, inoculation, incubation and establishment of multiple shoots
The relatively high cytokinin concentrations in cultures enhance the
axillary branching. The MS medium added with varied concentration of the BA
(0.5, 1.0, 1.5, 2.0) and 2-ip (0.5, 1.0, 1.5, 2.0) was used, the plant growth
regulators and their concentration presented in the Table 2. The apical portion
of the single shoot was sliced by fine blade and inoculated on the media
compositions under aseptic condition and culture were incubated at
25±2oC under cool light of 3000 Table 1: Media composition and amount of plant growth regulators added for
initiation of cultures in garlic
|
No. |
Treatments [PGR (mg/L)] |
TDZ (µ/L) |
NAA (µ/L) |
|
1 |
MS basal medium |
- |
- |
|
2 |
MS basal medium + TDZ (0.1 mg/L) |
6 |
- |
|
3 |
MS basal medium + TDZ (0.5 mg/L) |
30 |
- |
|
4 |
MS basal medium + TDZ (1.0
mg/L) |
60 |
- |
|
5 |
MS basal medium + NAA (0.05
mg/L) |
- |
3 |
|
6 |
MS basal medium + NAA (0.1
mg/L) |
- |
6 |
|
7 |
MS basal medium + NAA (0.15
mg/L) |
- |
9 |
|
8 |
MS basal medium + TDZ (0.1
mg/L) + NAA (0.05 mg/L) |
6 |
3 |
|
9 |
MS basal medium + TDZ (0.1
mg/L) + NAA (0.1 mg/L) |
6 |
6 |
|
10 |
MS basal medium + TDZ (0.1
mg/L) + NAA (0.15 mg/L) |
6 |
9 |
|
11 |
MS basal medium + TDZ (0.5
mg/L) + NAA (0.05 mg/L) |
30 |
3 |
|
12 |
MS basal medium + TDZ (0.5
mg/L) + NAA (0.1 mg/L) |
30 |
6 |
|
13 |
MS basal medium + TDZ (0.5
mg/L) + NAA (0.15 mg/L) |
30 |
9 |
|
14 |
MS basal medium + TDZ (1.0
mg/L) + NAA (0.05 mg/L) |
60 |
3 |
|
15 |
MS basal medium + TDZ (1.0
mg/L) + NAA (0.1 mg/L) |
60 |
6 |
|
16 |
MS basal medium + TDZ (1.0
mg/L) + NAA (0.15 mg/L) |
60 |
9 |
Table 2: Media
composition used for the multiplication of cultures
|
No. |
Treatments (mg/L) |
Volume of PGR (µL) |
|
1 |
MS basal medium |
1 |
|
2 |
MS basal medium + BA (0.5 mg/L) |
50 |
|
3 |
MS basal medium + BA (1.0 mg/L) |
100 |
|
4 |
MS basal medium + BA (1.5 mg/L) |
150 |
|
5 |
MS basal medium + BA (2.0 mg/L) |
200 |
|
6 |
MS basal medium + 2-ip (0.5 mg/L) |
50 |
|
7 |
MS basal medium + 2-ip (1.0 mg/L) |
100 |
|
8 |
MS basal medium + 2-ip (1.5 mg/L) |
150 |
|
9 |
MS basal medium + 2-ip (2.0 mg/L) |
200 |
Table 3: Media
composition for the establishment of cultures and amount of PGR to be added
|
No |
Treatments (mg/L) |
PGR |
|
|
TDZ (µL) |
NAA (µL) |
||
|
1. |
MS basal medium. |
- |
- |
|
2. |
MS basal medium +
TDZ (0.1 mg/L) |
300 |
- |
|
3 |
MS basal medium +
TDZ (0.5 mg/L) |
500 |
- |
|
4. |
MS basal medium +
TDZ (0.1 mg/L) + NAA (0.05 mg/L). |
300 |
15 |
|
5. |
MS basal medium +
TDZ (0.1 mg/L) + NAA (0.1 mg/L). |
300 |
30 |
|
6. |
MS basal medium +
TDZ (0.5 mg/L) + NAA (0.05 mg/L). |
1500 |
15 |
lux and 16:8
h light/dark regime for 1 month. The nutrient medium containing the less plant
regulator concentration for vigorous growth was used. The media preparation was
done with varied plant growth regulators and their concentration for
establishment of cultures for the growth of small shoots and to increase the
number of plants (Table 3). The multiple shoots were inoculated on the media
compositions as shown in Table 3 and culture were incubated at 25±2oC
under cool light of 3000 lux and 16:8 h light/dark regime.
Results
Murashige and Skoog (1962) medium was employed for the initiation of
cultures supplemented with TDZ and NAA in 16 different media combinations in
three garlic varieties. In garlic, initiation of meristem cultures taken from
sprouted cloves started within 8–10 days, while it took 10 to 12 days from the
meristems of fresh cloves. All the varieties showed similar response in all the
combinations. The sprouted cloves are metabolically active; thus, it may show
the early response in initiation of cultures and induced the early
multiplication.
Shoot initiation, regeneration and multiplication was recorded in all
the media combinations in the established meristem cultures. Auxins and
cytokinins were induced the shoot growth individually, or in combinations, the shoot initiation from
meristem was delayed by 2–3 days from fresh cloves as compared to meristem
isolated from sprouted cloves. Average percentage of regeneration of
meristem was 91.6% from sprouted cloves and 95% from fresh cloves. In case of
meristem taken from sprouted cloves the average percentage of regeneration of
meristem was 82.5, 96.8 and 90.6% in G-41, G-282 and G-323, respectively (Table
4). There was difference in height of shoot in different varieties. The few
combinations showed 100% regeneration, elongation and multiplication of shoots
among the varieties (Table 4; Fig. 1A–C). In G-41, TDZ at 1.0 mg/L aided for
100% regeneration, and all the combinations of TDZ and NAA showed good response
and vigorous growth of garlic varieties.
Average percentage of regeneration of meristem was 95% in all the
varieties, MS medium in addition of TDZ and NAA individually or in combination
in all 16 media combinations was noted 80–100% regeneration rate. The height of
meristem was about 5 to 10 mm within 15 days of inoculation in different
treatment combinations among all varieties. The maximum shoot height of plants
derived from sprouted cloves meristems was 9.5 mm recorded in variety G-41. MS
medium supplemented with NAA gave 6.75 mm an average height but does not have
vigorous growth and the leaves become pale yellow in color, cultures become
poor and showed pale yellow coloration.
After one month, in the variety G-41, the shoot height was maximum
(31.0 mm), which was statistically at par other treatments, in the meristem
taken from sprouted cloves (Table 4). In variety G-282, the maximum shoot
height was 24.75 mm and in the variety G-323 the shoot height of 27.5 mm was
recorded and were statistically at par to other, treatment combinations (Table
4). In G-41, shoot height was increased with increase of individual
concentration of TDZ. Combinations gives significantly highest shoot height in
variety G-323 and showed vigorous growth was presented in Table 4. Shoot height
at one month of inoculation of meristem taken from fresh clove was
significantly highest in variety G-41 (33.75 mm), which was statistically at
par to other combinations. In G-41 increasing the individual concentration of
TDZ increases the height of shoots, but in combination with increasing
concentration of NAA, decreased the growth of shoots.
Maximum number of shoots was recorded at one month of
inoculation, through increasing concentration of TDZ and NAA, as well as the
number of multiple shoots was increased. The average number of shoots per
meristem from Table 4:
The performance of meristem cultures of garlic varieties in different treatment
combinations
|
Treatment |
G -41 |
G-282 |
G-323 |
|||||||||
|
|
Regenerate meristem (%) |
Shoot height (15 days) |
Shoot height (1 month) |
Shoots per meristem |
Regenerate meristem (%) |
Shoot height (15 days) |
Shoot height (1 month) |
Shoots per meristem |
Regenerate meristem (%) |
Shoot height (15 days) |
Shoot height (1 month) |
Shoots per meristem |
|
1 |
60 |
2.75 |
9.30 |
0.6 |
100 |
3.5 |
9.0 |
1.0 |
100 |
3.5 |
6.0 |
1.0 |
|
2 |
60 |
5.6 |
21.0 |
0.6 |
100 |
4.25 |
16.25 |
1.7 |
100 |
6.0 |
9.0 |
1.5 |
|
3 |
80 |
9.5 |
21.3 |
0.8 |
100 |
3.0 |
17.5 |
2.0 |
100 |
8.0 |
14.0 |
4.0 |
|
4 |
100 |
5.1 |
25.0 |
1.0 |
100 |
4.5 |
12.5 |
1.0 |
100 |
8.0 |
17.5 |
1.0 |
|
5 |
80 |
4.4 |
22.0 |
1.0 |
100 |
5.5 |
13.75 |
1.0 |
100 |
8.5 |
24.5 |
1.0 |
|
6 |
60 |
4.0 |
16.4 |
0.6 |
100 |
5.5 |
13.0 |
1.0 |
100 |
8.0 |
23.5 |
1.0 |
|
7 |
60 |
4.25 |
9.8 |
0.6 |
75 |
3.0 |
14.5 |
1.0 |
100 |
11.0 |
16.0 |
1.0 |
|
8 |
100 |
5.8 |
21.2 |
2.4 |
100 |
4.0 |
15.0 |
1.0 |
100 |
10.0 |
22.5 |
1.0 |
|
9 |
100 |
6.3 |
29.0 |
1.4 |
100 |
5.0 |
13.0 |
2.5 |
100 |
7.5 |
27.5 |
2.0 |
|
10 |
60 |
6.6 |
17.2 |
0.6 |
75 |
3.5 |
17.6 |
1.25 |
50 |
1.5 |
12.5 |
0.5 |
|
11 |
80 |
7.75 |
22.0 |
1.4 |
100 |
4.75 |
13.75 |
3.2 |
100 |
3.5 |
6.5 |
1.0 |
|
12 |
100 |
5.0 |
13.2 |
1.2 |
100 |
3.5 |
16.25 |
2.0 |
50 |
2.0 |
7.5 |
0.5 |
|
13 |
100 |
3.0 |
13.4 |
1.2 |
100 |
5.0 |
15.0 |
2.2 |
50 |
2.0 |
6.0 |
0.5 |
|
14 |
100 |
3.0 |
8.1 |
2.8 |
100 |
5.5 |
18.2 |
2.7 |
100 |
4.5 |
24.0 |
5.5 |
|
15 |
80 |
2.6 |
9.0 |
1.0 |
100 |
2.25 |
9.5 |
1.7 |
100 |
6.0 |
6.5 |
1.5 |
|
16 |
100 |
1.8 |
10.8 |
3.8 |
100 |
5.25 |
10.5 |
4.0 |
100 |
6.5 |
23.5 |
1.5 |
%20399-404_files/image002.png)
Fig.
1: A) The regeneration of meristems, B) Elongation of
shoots from the meristems, and C) multiplication of plants derived from
meristem culture
sprouted clove ranged from 1.16 in G-323 to 1.34 in G-282. In the variety G-41, maximum number of shoots and was 4.25 in the
variety G-323, number of shoots was 1.75. Overall, it was recorded that, as the
number of multiple shoots per meristem increased, it resulted in decreased
height of shoots, with reduced growth of shoot. Initiation of number of shoots
in meristem isolated from the fresh clove gave delayed response as compared
with the meristem from the sprouted cloves. Variety G-282 gave better response
with 1.23 average number of shoots as compared with G-41 (1.02) and G-323
(1.11). Average number
of shoot initiation in variety G-41 was non-significant in all the
combinations, whereas in variety G-282 the average number of shoots were
maximum.
In meristem from sprouted clove good number of multiple and healthy
shoots were obtained within one month, whereas it took about a month to
initiate the multiple shoot formation in case of meristem taken from fresh
cloves, which reduced multiplication in meristem taken from sprouted
cloves. Where the number of shoots are more at later stage, mainly after one
month, the growth of multiple shoots is affected. Hence, it is necessary to
subculture the shooted plants to establishment medium for vigorous growth.
Establishment media contain the low concentration of plant growth regulators, which support for the growth of small
shoots, while nutrients available in the media lead to the vigorous growth of
the shoots. After transferring it into the establishment media, within 15–20
days the plant grows vigorously.
Discussion
All the
combinations of TDZ and NAA showed good response and vigorous growth. But in
less concentration of TDZ (0.1 mg/L) and high concentration of NAA (0.15 mg/L)
the percentage of regeneration decreased to 60%. It was noticed that high
concentration of auxin suppressed the percentage of shoot regeneration (G-41).
But in the variety G-282 and G-323, low concentration of TDZ also induced the
shoot regeneration. The auxin induced the shoot regeneration, when applied
individually; gradually leaves become pale yellow in color, while the vigorous
growth of plants in the combinations having auxins and cytokinin may be due to
accumulation of the chlorophyll which promoted the conversion of etioplast into
chloroplast. The delayed shoot initiation of meristems may be due to the
dormant stage of meristem in fresh cloves and took 2 to 3 days to initiate the
cell division. In G-41, increasing the individual concentration of TDZ
increases the height of shoots, but in combination, with increasing
concentration of NAA, decreased the growth of shoots. The high concentration of
NAA reduces the growth of shoots and leaves become pale yellow in color which
was also supported by the findings given by (Haque et al. 1997). Average
number of meristems was comparatively more from sprouted cloves than the
meristem taken from fresh cloves. The meristem in fresh clove may be in dormant
stage as compared with that of sprouted cloves, which might have resulted in
greater number of shoots. Meristem culture in garlic gave successful results
for the establishment of plants (Messiaen et al. 1993), where plantlets
were successfully obtained. With successive shoot formation from explant
meristem size ranged from 0.4 to 0.6 mm with the 87% of plants were virus free
(Havranek and Novak 1973), virus free garlic plants were produced through shoot
tip culture (Peña-Iglesias and Ayuso 1982). For initiation of plant growth. The
best medium containing BA, IBA, and GA through meristem tip culture (Ghosh et
al. 1997). Thermotherapy followed by meristem tip culture produced virus
free garlic plants (Bruna et al. 1997; Manjunathagowda et al. 2021). The phenyl derivatives TDZ
and N-phenyl-N’-(-2-chloro-4-pyridyl) urea (4PU-30), were added to the B5 and
MS medium for subcultures to increase for the micro-propagation of garlic viz.
proliferation rate of garlic cultivar (Rossi et al. 1995).
The multiple shoot production is most important to produce the maximum
number of propagules. A meristem may develop either into single shoot or
multiple shoots and plantlets (Bajaj and Dhanju 1981). A high cytokinin
concentration is utilized to overcome the apical dominance of shoot and enhance
the branching of lateral buds from leaf axils, BA was the most effective
cytokinin for stimulating auxiliary shoot proliferation, followed by kinetin
and 2-ip (Kitto and Young 1981; Papachatzi et al. 1981). Endogenous
auxins do not promote auxiliary proliferation; however, their presence may
improve culture growth (Wang and Hu 1980). One of the possible roles of auxin
in stage II medium is to nullify the suppressive effect of high cytokinin
concentration on auxiliary shoot elongation and restore normal shoot growth.
The highest TDZ concentration increases the number of shoot number but
decreases in shoot size, although rooting was normal (Bertaccini et al.
2004), thus it aids in the regeneration and proliferation of meristems into
shoots.
Conclusion
The study inferred that, the survival percentage, initiation and multiplication
of meristem tip in MS medium supplemented with TDZ (1.0 mg/L) with NAA (0.15
mg/L), TDZ (1.0 mg/L) with NAA (0.05 mg/L) and MS medium supplemented with TDZ
(0.5 mg/L) with NAA (0.05 mg/L) gave better plant growth with a greater number
of multiple shoots from all three varieties (G-41, G-282, G-323). Hence, these
protocols can be utilized for meristem tip culture of garlic for the production
of virus free planting material.
Director, ICAR-Directorate of Onion and Garlic Research,
Rajgurunagar, Pune for mobilizing the funds extending the research facilities.
Author Contribution
Contribution to this paper
by each author be given here.
Conflicts of Interest
The authors declare no conflict of interest.
Data Availability
The text and Supplementary Materials provide the data that was used to
accomplish the study goal. On request from the relevant author, raw data used
in statistical analyses is accessible for future usage.
Ethics Approval
Not applicable in this paper
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