Journal of Agriculture and Food Research 4 (2021) 100126

Contents lists available at ScienceDirect

Journal of Agriculture and Food Research

journal homepage: www.journals.elsevier.com/journal-of-agriculture-and-food-research/

Development of an efficient protocol for production of healthy sugarcane

seed cane through Meristem culture
Shubhangi Salokhe
Symbiosis Foundation, Pune, India

A R T I C L E I N F O A B S T R A C T

Keywords: Healthy seeds are a prerequisite for improving the productivity of sugarcane crop. The best way to ensure the
Biotechnology production of disease-free sugarcane seeds is via a technique called ‘Meristem Culture’ (Tissue Culture). An
Protocol efficient protocol for meristem culture of sugarcane has been developed in this study. The potting mix for the
Sugarcane
survival of meristem cultured plantlets in a greenhouse was standardized, and agronomy for growing tissue
Seed
Tissue culture
culture plantlets in the field was developed. Multiple shoots were observed on Murashige and Skoog's (MS)
medium supplemented with BAP 0.2 mg/l and Kinetin 0.1 mg/l. Best rooting was observed on MS media sup-
plemented with IAA 1 mg/l and NAA 1 mg/l. Soil mixture containing [Soil þ Vermiculite þ Sand in 4: 1: 1
proportion (by volume)] þ Vermicompost @ 25% by volume of medium þ NPK is best suited for hardening tissue
cultured plants in a greenhouse. Two feet distance between 2 plants and 3 feet distance between 2 rows was found
as an appropriate method for planting tissue cultured plantlets in the field. Setts obtained from tissue cultured
plants planted at 15 cm spacing gave higher yield than the other spacing. Setts obtained from micro-propagated
plants (M) gave the highest yield as compared to setts from conventional sugarcane raising. The protocol
developed in this study, shows potential for development of sugarcane meristem cultured plantlets and use of
these plants for planting in field to obtain healthy sugarcane seed.

1. Introduction contributes to higher yields; therefore, the supply of healthy seed cane is
the main prerequisite for improving the productivity of the sugarcane
Sugarcane (Saccharum officinarum L.) is a major commercial crop of crop [13]. Availability of disease-free and genuine to type planting ma-
India, cultivated on 51.14 lakh hectares of land (Cooperative Sugar terial is essential for achieving a high yield of sugarcane cultivation.
2020). It is an industrial crop and one of the main crops of earning foreign Unfortunately, since the system of scientific seed production, i.e. breeder
exchange in India [31]. Brazil, India, Pakistan, China and Indonesia are seed (stage 1), foundation seed (stage2) and certified seed production
the largest producers of sugarcane. The biggest producer of sugarcane is (stage 3) is not being followed, the sugarcane farmers are facing a
Brazil, and as it grows over 40% of the worlds crop [28]. Because of the problem of a lack of good quality seed material for planting [2,24]. As
high production potential of sugarcane and its ability to be cultivated in sugarcane is propagated vegetatively, it favours the accumulation of
countries with low economic and social development, it could become a pathogens (Vishwanathan, 2016). Hence along with seed canes,
key source of income and improve quality of life in many countries [9]. disease-causing pathogens are also being introduced into the new areas.
The cane is supplied to sugar industries, where various products are The slow accumulation of different pathogens over a period of time
prepared from its juice (sugar, jaggery, alcohol, etc.). Sugarcane is the makes minor diseases into major ones [26]. Several epidemics due to red
best example of renewable natural agricultural resource since it provides rot, smut, wilt, grassy shoot, ratoon stunting, yellow leaf and leaf scald
sugar, besides biofuel, fibre, fertilizer and various by-products [7]. It is occurred in the past indicated that disease infected seed can play a sig-
recognized as an important energy crop because of the possibility to nificant role in their creation and further spread [17]. Affected planting
produce ethanol from molasses and directly from cellulose. It is also one material is a major problem in propagation, exchange of germplasm and
of the most efficient crops in the world for converting solar energy into distribution of superior genotypes of sugarcane [12]. Methods used for
chemical energy. the elimination of viruses and the development of good quality seeds in
For commercial cultivation, sugarcane is propagated vegetatively and sugarcane are thermotherapy (heat therapy) and tissue culture technol-
requires a considerable amount of seeds. Good planting material (seed) ogy [21]. Heat therapy can be used to eliminate many viruses from a

E-mail addresses: [email protected], [email protected], [email protected], [email protected].

https://doi.org/10.1016/j.jafr.2021.100126
Received 24 January 2020; Received in revised form 27 January 2021; Accepted 24 February 2021
2666-1543/© 2021 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

variety of sugarcane and provides a proven approach to remove patho- for the development of a protocol for meristem culture of sugarcane. MS
gens from seed canes. There are, however, some disadvantages of this media was enriched with 3% sucrose and 10% coconut water. This me-
therapy as sugarcane seed is treated with heat; it gives very low germi- dium was supplemented with different combinations of cytokinins and
nation percentage. The media compositions were equally important for auxin; pH of the medium was maintained at 5.8. The culture tubes/-
in-vitro regeneration of sugarcane [16]. bottles were incubated on illuminated racks at 16 h light and 8 h dark
Thermotherapy or meristem culture (tissue culture), or frequently a period in 24 h at the room temperature of 25  1  C in a growth chamber
combination of both techniques has been successfully used to eradicate in the tissue culture laboratory.
viruses from infected sugarcane plants [30,32]. If explants are taken from
disease-free plant material or if explants are heat-treated to eliminate 2.4. In vitro shoot multiplication
diseases, then the resultant plants are healthy and disease-free [15].
Meristem culture demonstrated to be an efficient tool for eliminating Different combinations of cytokinins and auxins were tested to select
virus from plants, giving the possibility to produce disease free propa- the best media for shoot formation and root formation in meristem cul-
gation material [22]. In order to achieve the demand for sugar by 2020 in ture of sugarcane. MS [19] media of HiMedia supplemented with
India, sugarcane production has to be increased. For increasing sugar- different concentration (0, 0.1, 0.25, 0.5, 1, 1.5 and 2 mg/l) of “6- Benzyl
cane production, availability of good quality seed material of high aminopurine (BAP)” and “Kinetin (KIN)” from MERCK was tested on
yielding varieties is essential. Tissue culture technique can be applied in shoot multiplication on 10 explants. Each experiment was repeated 3
breeding, propagation, disease elimination, rejuvenation of older vari- times to confirm the results. Subculture was carried out every 2–3 weeks.
eties, and development of clones suitable for abiotic and biotic stresses After subculture, the cultures were transferred into new glass bottles and
[18]. [5] suggested the in-vitro protocol for rapid micro propagation of kept for shaking on 120 rpm in the illuminated shakers (Steelmet
sugarcane which can be adopted for producing genetically-homogenous Novatech) in the laboratory for about 20 days.
and WLD-free planting material in establishing sugarcane nurseries. Four After 5 weeks of shoot growth actively growing shoots were trans-
sugarcane varieties and three explants derived seedlings were utilized to ferred to fresh medium (MS þ 0.25 mg/l BAP þ 0.1 mg/l Kin) in glass
assess the yield with respect to single bud setts. In this experiment in vitro bottles and kept in the illuminated shakers for shaking (120 rpm) and for
grown plants showed better response for certain characters [20]. Sug- further growth. Multiple shoots were developed on this media after 20
arcane micro propagation enables the massive multiplication of sugar- days. Then these culture bottles were transferred to illuminated racks in a
cane plants to obtain certified vitro plants and increase the sugarcane laboratory at 25  C with 16 h light and an 8 h dark period in 24 h. First
productivity per unit area (Bello-Bello et al., 2017). sub culturing was done after 30 days of growth. The culture was sub-
Therefore, in this research, we aimed at development of an efficient divided in bunches containing 4–5 shoots/bunch, and then these
protocol for production of healthy and disease free sugarcane tissue bunches were transferred into a fresh medium in glass bottles and again
cultured plants (micro propagated plants) on a large scale in the labo- kept on shakers for 15 days, finally kept on illuminated racks. In this way
ratory and for hardening of tissue cultured plantlets in the green-house. sub culturing was done for 5 to 6 times by transferring into fresh media
Development of agronomy for growing tissue culture plants in the field, bottles to obtain multiple shoots.
as well as for growing seeds obtained from tissue cultured plantlets has
also been performed. Development of protocol for tissue culture of sug- 2.5. In vitro rooting of plantlets
arcane and development of agronomy for tissue cultured plants was
essential to make use of tissue culture plantlets in three-tier system of Shoots obtained from the above culture bottles were transferred to
sugarcane seed production. rooting medium for inducing rooting to the shoots. Rooting medium was
prepared by supplementing MS basal medium with IAA or NAA. Various
2. Materials and methods concentrations (0.1, 0.25, 0.5, 1, 1.5 and 2 mg/l) of IAA and NAA were
tested. Each experiment was repeated 3 times. 10 plants per treatment
2.1. Plant material were tested for each repetition.

The present study was conducted at Vasantdada Sugar Institute (VSI), 2.6. Acclimatization of the plantlets in the greenhouse
Pune, India. Laboratory experiments were conducted to standardize a
protocol for large scale and low-cost production of meristem cultured Experiments were conducted to standardize the potting mix for the
(tissue culture) plants of sugarcane. During this study, explants (meri- survival of meristem cultured plantlets. Well rooted plantlets about
stems) from sugarcane variety ‘Co 86032’ were used from 6 months old 6–8 cm height were taken from the culture bottles and washed with tap
sugarcane seed nursery of VSI. Field experiments were conducted on the water to remove the traces of medium. These plantlets were taken to the
farms of Vasantdada Sugar Institute. greenhouse to transfer them to a suitable potting mix. The plantlets were
transplanted into poly bags of 15  10 cm size containing a mixture of
2.2. Explants source and surface sterilization soil, sand, vermiculite and Vermicompost in different proportions as
indicated below:
Meristems are the centres of plant growth located in the tips of the
new shoots and leaves. Tops of Sugarcane variety ‘Co 86032’ of 6 months 1) C (Control) - Soil: Vermiculite: Sand in 4: 1: 1 proportion (by volume).
old from field were brought to the laboratory. The upper leaves were 2) F1 – C þ Vermicompost @ 25% by volume of medium and NPK (55
removed, and sugarcane meristems along with outer cover were washed mg urea, 360 mg single super phosphate and 95 mg muriates of
with distilled water, then surface sterilized with 0.1% mercuric chloride potash/kg of the medium).
for 5 min and then washed 3 times with sterile distilled water to remove 3) F2 – C þ Vermicompost @ 50% by volume of and NPK @ double dose
the traces of chemicals. Leaf-sheaths surrounding the meristem were of fertilizer as in F1.
removed aseptically with the help of surgical blades, and then meristem 4) FU1– F1 þ 0.5% solution of urea spray at 15 days after transplanting
explants were obtained. The explants were then inoculated by proper @ 200 ml solution on 160 plantlets.
dissecting and sizing the meristem (0.5–1.0 cm). 5) FU2 – F1 þ 0.5% solution of urea spray two times i.e. at 15 days and
30 days after transplanting @ 200 ml solution each time on 160
2.3. Culture media and conditions plantlets.
6) V1– C þ Vermicompost @ 25% by volume of medium.
[19] medium was used as a basal medium in laboratory experiments 7) V2 – C þ Vermicompost @ 50% by volume of medium.

2
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

8) M  C þ Multinutrients sprays. Table 2

Sub treatments (Spacing between two setts of sugarcane when planted in the
(The multi-nutrient was made up of N, P, K, Fe, Mn and Zn in field).
9:9:5:2:2:2 proportions. Multinutrient spray was sprayed 3 times on Sub The spacing between two setts (cm)
plantlets. The first spraying was done on the 2nd day after planting treatment
@30 ml/L of water. The second spray was done at 15 days after planting TC15 Two eye bud setts (obtained from micro-propagated plantlets)
@ 50 ml/L of water, and the third spray was done 45 days after planting planted at 15 cm spacing.
@ 75 ml/L of water.) TC30 Two eye bud setts (obtained from micro-propagated plantlets)
planted at 30 cm spacing.
In the greenhouse study, the meristem cultured plantlets were
TC45 Two eye bud setts (obtained from micro-propagated plantlets)
transplanted on poly bags in the greenhouse. Each treatment consisted of planted at 45 cm spacing.
20 plantlets. All poly bags were labelled and were placed in a completely C15 Two eye bud setts (obtained from conventional sugarcane) planted at
randomized design in the greenhouse. Observations were recorded at 2 15 cm spacing
months age before transplanting in the field. C30 Two eye bud setts (obtained from conventional sugarcane) planted at
30 Cm spacing.
C45 Two eye bud setts (obtained from conventional sugarcane) planted at
2.7. Breeder's seed production from sugarcane meristem cultured 45 cm spacing
sugarcane plants

Breeder's seed nursery is the first step of a three-tier system of seed Such a result is indicated by placing -ns- on the computed -F- value in the
production. Well-hardened meristem cultured plantlets from greenhouse analysis of variance, significance level (P, 0.05).
were used for planting sugarcane nursery for breeders seeds. A procedure
was developed for planting meristem cultured plantlets in the field and 3. Results and discussions
spacing required (row to row and plant to plant).
3.1. Development of appropriate media for sugarcane meristem culture
2.8. Foundation seed production from seed obtained from meristem
Suitable nutrient media, its chemical composition and physical form,
cultured sugarcane plants
are essential for the success of in vitro culture of plants. An efficient and
low-cost protocol for meristem culture of sugarcane on a large scale using
The present field study of meristem cultured plantlet's seeds was
meristem explants has been developed. Nutrient media based on [19]
taken with a view to develop agronomy so that this technology can be
was used as a basal medium in all experiments. Multiple shoots and roots
used more efficiently. Seeds obtained from meristem cultured sugarcane
were developed by using various combinations and concentrations of
plantlets at the age of 10 months were used for the production of foun-
growth regulators. The effect of the different combinations of BAP and
dation seeds of sugarcane. Performance evaluation was done for the
Kinetin on shoot multiplication and the effect of auxins (IAA or NAA) on
seeds obtained from meristem cultured plantlets with appropriate cul-
rooting was tested.
tural practices specific to meristem cultured plantlets.
This experiment was conducted in the field in a split-plot design with
3 replications. The spacing between 2 rows (RS) served as the main plot 3.2. In vitro shoot multiplication
and spacing between 2 meristem cultured (tissue cultured) setts (), and
conventional sugarcane setts (C) was the sub-plot (Table 1) (see Table 2). In the present investigation, sugarcane apical meristem of 5 mm size
Setts obtained from 10 months old crop grown by using meristem was used as explants. Liquid media was used for this study. Initially,
cultured plants (TC), and conventional sugarcane (C) from sugarcane Co meristems were inoculated on filter paper support in test tubes. 10 ex-
86032 variety were used. The agronomical practices recommended by plants in 10 test tubes were inoculated for each treatment. These test
Vasantdada Sugars Institute for sugarcane cultivation were followed. The tubes were incubated on illuminated racks at 16 h light and 8 h dark
seed crop was harvested at 10 months age. The data related to the period in 24 h at the room temperature of 25  1  C in a growth chamber
number of canes/clumps, number of canes/ha, number of three eye bud in the tissue culture laboratory. Polyphenols were observed around
setts/ha., millable height/cane, the girth of cane, number of internodes meristem within 10 days, and then these meristems were transferred to
per cane and length of internodes was recorded. fresh medium in new test tubes. Observations were recorded after 30
days from the first inoculation for growth of meristem ex. number ex-
2.9. Statistical analysis plants showing multiple shoots, number of shoots per explants etc. These
observations on growth of explants are presented in Table 3.
The results of experiments carried in the laboratory were analysed The growth regulators (BAP and Kinetin) had a marked effect on the
statistically using Completely Randomized Design (CRD) with 10 repli- frequency of shoot initiation and establishment of shoot cultures. The
cations and significance level (P, 0.05) and the results of experiments results showed that the number of explants which showed multiple
carried in the greenhouse were analysed statistically using Completely shoots were higher in T3 medium (MS medium supplemented with BAP
Randomized Design (CRD) with 20 replications and significance level (P, 0.25 mg/l and Kinetin 0.1 mg/l); higher number of multiple shoots (3.7)
0.05). Split Plot Design with three replications was used in the field study was observed on this media (Table 3). Healthy shoots were developed on
of meristem cultured plantlets. The result is indicated by placing an this medium. Increasing the concentration of BAP and Kinetin reduced
asterisk on the –F- value in the analysis of variance. If the computed -F- the number of explants with multiple shoots, as well as the number of
value is smaller than or equal to the tabular -F- value at 5% level of shoots per explant. This may be related to the fact that higher level of
significance, the variation due to treatments is said to be non-significant. cytokinins inhibits cell division.
Result of this experiment showed that balance between cytokinins
and auxin is essential for the production of shoots and its further multi-
Table 1
plication. Many workers have reported the use of BAP with Kinetin for
Main treatments (Spacing between two rows).
shoot formation in sugarcane [10,27]. The current result obtained was
Main treatment The spacing between 2 rows
not in agreement with the earlier results reported by Ref. [2], they ob-
RS 90 Row Spacing 90 cm tained maximum shoot multiplication on MS medium with 1.0 mg/l BAP,
RS 120 Row Spacing 120 cm and 0.25 mg/l Kin. This might be related to the fact that higher levels of
RS 150 Row Spacing 150 cm
Cytokinin inhibit cell division and hence organogenesis [33]. Best

3
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

Table 3 illuminated racks for incubation.

In vitro shoot multiplication from meristem of sugarcane.
Treatment Media Number of No. of Number of 3.4. In vitro rooting of plantlets
Combination explants explants shoots per
inoculated showed explant The study was conducted for in vitro rooting of plantlets. For this
multiple
study, micro shoots developed after 4–5 subcultures were used for
shoots
inoculation on different media. For rooting studies, the regenerated
T1 MS (Control) 10 0 0.0  0.00 micro shoots were used.
T2 MS þ 0.1 mg/l 10 3 1.6  0.09
BAP þ 0.1 mg/l
The results showed that an increase in the concentration of NAA and
Kin IAA from 0.25 mg/l to 1 mg/l increased the number of planlets showing
T3 MS þ 0.2 mg/l 10 8 3.7 þ 0.56 roots and number of roots per plant. Best rooting was observed on T4 (MS
BAP þ 0.1 mg/l media supplemented with IAA 1 mg/l and NAA 1 mg/l). At this con-
Kin
centration, 100% shoots formed roots and number of roots per plants was
T4 MS þ 0.1 mg/l 10 4 1.8  0.06
BAP þ 0.25 mg/ higher (14.70) in this treatment. The current result obtained was in
Kin agreement with the earlier results reported by Ref. [3]. Further increase
T5 MS þ0.2 mg/l 10 5 2.8  0.45 in the concentration of NAA or IAA reduced the number of roots
BAP þ 0.25 mg/l noticeably [18]. also reported that NAA is the ideal growth regulator for
Kin
T6 MS þ 0.5 mg/l 10 4 1.7  0.47
induction of rooting in the sugarcane. Many researchers reported that
BAP þ 0.25 mg/l 5 mg/l of NAA was good for rooting, but increasing NAA beyond 5 mg/l
Kin inhibits rooting [25]. used 1.0 mg/l IBA as the best root initiating growth
T7 MS þ 0.2 mg/l 10 4 1.5  0.42 hormone. Conversely, further increasing the concentration of NAA and
BAP þ 0.5 mg/l
reduced the shoot length and number of roots noticeably. Best treatment
Kin
T8 MS þ 0.5 mg/l 10 3 1.6  0.06 for inducing roots to in vitro grown meristem culture plants of sugarcane
BAP þ 0.5 mg/l is MS þ NAA 1 mg/l.
Kin
T9 MS þ 1 mg/l 10 2 1.5  0.09
3.5. Hardening of meristem cultured plantlets in a greenhouse
BAP þ 1 mg/l
Kin
Transfer of in-vitro sugarcane Plantlets from their sterile culture ves-
sels to the soil requires a careful and stepwise procedure and suitable
potting mixes; as this is one of the important criteria for the survival of
Table 4
meristem cultured plants. The in vitro raised plantlets are tender and
In vitro rooting of meristem cultured plantlets of sugarcane.
fragile, and therefore their hardening requires special attention. The
Treatment Media Number of Number of Number of
present study was undertaken using meristem cultured plantlets of sug-
Combinations plants used Plants roots per plant
for rooting showed arcane variety ‘Co 860320 to develop a procedure for hardening of mer-
rooting istem cultured sugarcane plantlets. In vitro rooted plantlets were taken
T1 MS 10 0 0.00  0.00
out of glass bottles and then washed under tap water to remove traces of
T2 MS þ IAA 0.25 10 2 5.8  0.17 media then excess leaves are trimmed out. Then these plants are used for
mg/l transplanting in greenhouse.
T3 MS þ IAA 0.5 10 3 6.3  0.24 The survival and growth rates of meristem cultured plantlets of sug-
mg/l
arcane are given in Table 5 (see Table 4). The survival of plantlets which
T4 MS þ IAA 1 10 10 14.70  0.28
mg/l were grown in soil mixtures with chemical fertilizers and/or Vermi-
T5 MS þ IAA 1.5 10 7 11.40  1.22 compost (F1, FU1, FU2, V1 and V2) was higher (from 85 to 95%),
mg/l compared to the survival of plantlets (70%) in control conditions
T6 MS þ IAA 2 10 8 11.10  0.48 (Table 5). The survival of plantlets was only 45% in soil mixture in which
mg/l
multi nutrients were sprayed on plants.
T7 MS þ NAA 10 2 4.6  0.57
0.25 mg/l An effect of soil mixtures on tillering to sugarcane plantlets has been
T8 MS þ NAA 0.5 10 3 8.6  0.10 observed (Table 5). The use of fertilizers along with urea spraying two
mg/l times, i.e. at 15 days and 30 (FU2) gave tillering in 80% plants. Whereas
T9 MS þ NAA 1 10 9 15.00  0.36
FU1 gave tillering in 50% plants. In all other treatments, only 20–30%
mg/l
T10 MS þ NAA 1.5 10 7 11.90  0.75 plants showed tillering at 2 months age. Sugarcane plants height was
mg/l measured at 60 days of plants age. Plant height was significantly higher
T11 MS þ NAA 2 10 8 11.70  0.64 in other treatments as compared to the control. It was observed between
mg/l 8.73 in control to 12.31 cm in F1 treatment, followed by 11.15 cm in FU2
treatment.
The number of leaves per shoot ranged between 5.28 and 7.15. It is
higher in FU 2 treatment (7.15) where urea is sprayed twice followed by
treatment for in vitro shoot multiplication of sugarcane was MS þ 0.2
V1 treatment. The leaf width was in between 0.64 and 0.81 cm. It is
mg/l BAP þ0.1 mg/l Kin. In this treatment 8 out of 10 explants showed
higher in FU 2 followed by FU 1. However, there was no significant
multiple shoots and number of shoots per explant was higher (3.7).
difference in number and width of leaves due to different soil mixtures. In
case of the length of the 4th leaf, there was no significant difference. The
3.3. Subculture shoot weight per plant was between 1.48 g and 3.07 g (Table 5). Shoot
weight differed significantly due to the difference in soil mixture (F1, F2,
Shoot multiplication was done on the same medium (MS þ 0.2 mg/l FU1, FU2, V1, V2 and M). Use of fertilizers (F1) and Vermicompost at
BAP þ 0.1 mg/l Kin) by sub culturing on fresh medium in glass bottles higher dose, i.e. 50 % by volume (V2) gave higher shoot weight of
and kept for shaking on 120 rpm in the illuminated shakers in the lab- 2.16 gm to 3.07 gm as compared to that of 1.80 g in control. A lower dose
oratory for about 15 days. After shaking bottles were transferred to of Vermicompost i.e. 25 % by volume of soil mixture had no effect on

4
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

Table 5
The survival and growth rates of meristem cultured plantlets of sugarcane on different soil mixtures.
Treatments (Different Survival % Plants The Height of Number of leaves Length of the The Width of The Weight of The Weight of
Soil Mixtures) (%) Showing Tillers Plant (cm) per shoot 4th leaf (cm) 4th leaf (cm) Shoot (gm) Roots (gm)

C 70 20 8.73 5.28 39.11 0.64 1.80 0.36

F1 95 25 12.31 5.31 42.55 0.70 3.07 1.42
F2 85 25 10.81 6.13 44.90 0.81 2.67 1.10
FU1 90 50 10.91 5.64 35.86 0.73 2.40 0.97
FU2 95 80 11.15 7.15 41.10 0.74 2.82 1.41
V1 85 30 10.14 6.70 31.41 0.65 1.48 0.63
V2 85 30 10.19 6.33 36.35 0.67 2.16 0.90
M 45 5 10.38 5.44 39.44 0.68 1.52 0.47
CD (P ¼ 0.05) – – 0.46a NS. NS. NS. 0.12a 0.070a

NS - Non-significant.
a
Significant.

shoot weight.  The pits of the size of the plastic bags in the center of the furrow
No additional benefits on shoot growth when fertilizers were used at should be kept at a distance of 2 feet.
double the rate of spraying of urea along with fertilizers have been  The plantlet should be planted along with whole soil balls in the pits
observed. Similarly, there was no additional benefit on shoot weight and subsequently covered it with sufficient amount of soil. Immedi-
when fertilizers were used at double the rate or spraying along with ately after transplanting, irrigation needs to be given.
fertilizers. The weight of shoots in multi nutrient spray was 1.52 gm, and  All recommended intercultural operations (weeding, harrowing,
this value was lower than 1.80 gm in control (Table 5). The trend irrigation, pest control, etc.) should be performed within time.
observed for the weight of roots was similar for the weight of shoots. Root  Canes obtained from meristem cultured plantlets should be harvested
weight was observed significantly higher in soil mixture F1 and FU2 within 10 months.
(1.42 g and 1.41 g), which was higher as compared to that of control  One eye buds obtained from meristem cultured sugarcane crop should
0.36 g (Table 5). In case of root weight also, there was no additional be used to grow foundation seed.
benefit of increased fertilizer dose or urea spraying. There is a positive
relation between shoot weight and root weight. Root weight increased The agronomy for growing meristem cultured plants was also
with increase in shoot weight, except in the treatment of multi nutrients experimented with. 2 feet distance between 2 meristem culture plantlets
spray. and 3 feet distance between 2 rows of sugarcane proved to be suitable for
It has been observed that survival rate of plants was higher, as well as planting of meristem cultured plantlets in the field for the development
shoot weight and root weight of meristem cultured sugarcane plantlets of breeder seed. The current result obtained was in agreement with the
were also higher in a soil mixture C þ Vermicompost @25% by volume of earlier report of [23]. Best quality seed can be obtained at the age of 10
medium and NPK as 55 mg urea, 360 mg single super phosphate and 95 months of the meristem cultured sugarcane grown in the field.
mg muriate of potash/kg of medium. This is the best medium for hard- Performance of seeds obtained from meristem cultured sugarcane was
ening of meristem cultured plantlets in a greenhouse. There was no better as compared to conventional seeds, as the yield obtained from
additional benefit of chemical fertilizer when it was used at double the meristem cultured cane was 26% higher than conventional sugarcane.
rate. An increase in number of plants showing tillers was observed in Seeds obtained from meristem cultured cane were healthy and free from
treatment of urea spraying along with fertilizer application. There was a diseases (Fig. 1).
positive relation between root growth and shoot growth as indicated by
their weight (Table 5). The result obtained in the present study was not in
agreement with the earlier finding of [33]. 3.7. Foundation seed production from meristem cultured sugarcane seeds

Performance of seeds obtained from meristem cultured plantlets was

3.6. Sugarcane breeder's seed production from sugarcane meristem compared with seeds obtained from conventional sugarcane. This
cultured plants experiment was conducted in a split-plot design with 3 replications. Setts
obtained from 10 months old crop grown by using meristem cultured
Experiments were carried out to develop the procedure for planting plantlets (TC) and conventional sugarcane (C) from sugarcane ‘Co
meristem cultured plantlets in the field. Special attention was given for 86032” variety was used. The recommended agronomical practices
watering of plants, withholding watering, planting procedure, row to row (recommended by Vasantdada Sugarcane Institute) were followed, and
distance, plant to plant distance, intercultural operations etc. As a result the seed crop was harvested at ten months age of the crop.
of conducted experiments the procedure of planting meristem cultured Statistical tests were done to analyse the influence of row and set
plantlets in the field has been standardized: spacing and interaction of both on sugarcane seeds (for meristem
cultured plants as well as from conventional canes) for some agronomic
 The watering of plantlets should be withheld the day before planting characteristics (Milliable height, girth of cane, number of internodes/
in the field so that the soil in the plastic bag becomes hard and cane, number of canes per clump, number of canes/ha and yield of three
compact. This is necessary to avoid damage to the root system of the eye budded setts/ha).
plantlets. Influence of row spacing on agronomic traits of meristem cultured
 The plot should be ready with the application of green and organic sugarcane seed.
manures for improving soil fertility. Analysis of variance (ANOVA) revealed that the number of canes/
 Furrows should be opened at a distance of 3 feet and recommended clump and number of internodes/cane were significantly affected by row
basal doses of chemical fertilizers should be applied and mixed in the spacing (Table 6). Results showed that an increase in row spacing from
ground. Because meristem cultured plants also require nutrients like 90 cm to 150 cm resulted in an increased number of internodes/cane and
conventional sugarcane. number of canes per clump. The reason for the increased cane popula-
 The plantlets should be kept in the field on the ridges of furrows along tion, may be less competition between plants because of higher spacing
with poly bags. [23]. also reported a higher population of stalks in TC plots. Increase in

5
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

Fig. 1. Different stages of sugarcane meristem culture Following are the photographs of the stages of sugarcane meristem culture from experiments in laboratory and
greenhouses.

row spacing from 90 cm to 150 cm did not make any significant change in rate or through maintaining population to an optimum level. A number of
the number of canes/ha and the number of three eye bud setts/ha. canes/clump, number of canes/ha, number of internodes/ha and yield of
However, row spacing of 90 and 120 cm found equally effective in the 3 eye bud setts, were all highly influenced by setts spacing (Table 7).
production of the eye budded setts, and it is superior over 150 cm spacing Results showed that an increase in row spacing from 90 cm to 150 cm
(Table 6). There are references for research on the spacing of meristem resulted in an increased number of internodes/cane and number of canes
cultured plantlets, but no one has done this type of research on spacing per clump. Setts obtained from meristem culture plants (M) gave the
for seed obtained from meristem cultured plantlets. highest yield 116 thousand canes per hectare as compared to setts from
Increase in the number of canes per meristem culture raised cane, and conventional sugarcane (c) 95 thousands canes per hectare. Setts pro-
increased vigour is a phenomenon reported by many scientists. The duced from meristem cultured sugarcane plantlets at 15 cm spacing gave
current result is in contrast with the findings of [4] who reported higher yield (116 thousands canes per hectare) than any other spacing. A
increased stalk population and reduced cane and sugar yield in meristem significant decrease in the number of canes/clump, number of canes/ha,
culture seed over conventional seed sources. number of 3 eye buds setts/ha and number of internodes/ha was
Influence of spacing between two setts on some agronomic traits of observed, where setts spacing was increased from 15 to 45 cm in both
sugarcane. meristem cultured as well as conventional sugarcane setts. There are
The effect of different spacing between setts at the time of planting on references for research on the spacing of meristem cultured plants, but no
some agronomic traits (Milliable height, girth of cane, number of in- one has done this type of research on spacing for seed obtained from
ternodes/canes, number of canes per clump, number of canes/ha and meristem cultured plants.
yield of three eye budded setts/ha) is presented in Table 7. Number of canes per clump, no. of canes per hectare and yield of 3 eye
The higher cane yield could be achieved either through higher seed bud setts per hectare are the three important factors on which sugarcane

6
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

Table 6 multiplication of sugarcane and hardening of meristem cultured plantlets

Influence of row spacing on agronomic traits of meristem cultured sugarcane in a greenhouse was developed. The agronomy for using meristem
seed. cultured sugarcane plants in the field as breeder's seeds and agronomy for
SN. Agronomic Row Row Row SE CD using seeds obtained from meristem cultured plants, as the foundation for
characters and yield Spacing Spacing Spacing þ- at seeds was established.
90 cm 120 cm 150 cm 5% Murashige and Skoog medium supplemented with BAP 0. 2 mg/l and
1 Milliable height 224.32 229.65 230.18 3.84 NS. Kinetin 0.1 mg/l was the best medium for shoot multiplication in vitro.
(cm)/cane Murashige and Skoog medium supplemented with IAA or NAA 1 mg/l
2 Girth of cane (cm). 11.14 11.24 11.63 0.05 NS.
was the ideal medium for root formation to the shoots. The survival
3 No. of internodes/ 16.08 16.39 16.99 0.06 0.18a
cane. percentage, shoot and root weight of meristem cultured sugarcane
4 Length of 15.26 16.32 15.33 0.33 NS. plantlets were higher in a soil mixture C þ Vermicompost @ 25% y
internodes. volume of medium and NPK as 55 mg urea, 360 mg single super phos-
5 No. of canes/clump. 12.05 12.67 14.78 0.78 2.24a phate and 95 mg muriates of potash/kg of the medium. This was the best
6 No. of canes/ha 103 103 98 0.80 NS.
(thousand).
soil mix for increasing survival of meristem cultured plants in the
7 Yield of three eye 72.8 72.7 69.2 5.9 NS. greenhouse.
bud setts/ha Two feet distance between 2 meristem culture plants and 3 feet dis-
(thousand). tance between 2 rows of sugarcane was found suitable for planting of
NS - Not significant. meristem cultured plantlets in the field for the development of breeder
a
- Significant. seed. In foundation seed production an increase in row spacing from
90 cm to 150 cm resulted in an increased number of internodes/cane and
yields are judged. All these three are significantly higher in meristem number of canes per clump. Spacing between two setts highly influence
cultured sugarcane seed cane as compared to conventional sugarcane the number of canes/clumps, number of canes/ha, number of in-
seed cane. 116 thousands canes per hectare were observed in meristem ternodes/ha and number of 3 eyes budded setts per hectare. Setts ob-
cultured sugarcane which were higher than conventional sugarcane (96 tained from meristem cultured plants (M) gave the highest yield as
thousands canes per hectare). Number of canes per clump was also compared to sets from conventional sugarcane (C). Setts produced from
observed higher in TC (15.43) as compared to conventional (13.10). In meristem cultured sugarcane plants at 15 cm spacing gave a higher yield
TC sugarcane, yield of 3 eye bud setts per hectare was higher (81.9 than the other spacing. A significant decrease in the number of canes/
thousands) as compared to conventional sugarcane, (67.1 thousands). It clump, number of canes/ha, number of 3 eye bud setts/h and number of
shows that performance of meristem cultured sugarcane seed is excellent internodes/ha was observed where setts spacing was increased from 15
as compared to conventional sugarcane seed. to 45 cm. Number of canes per clump, no. of canes per hectare and yield
Analysis of variance showed that there was no significant interaction of 3 eye bud setts per hectare were significantly higher in meristem
between row spacing and spacing between sets (Table 8). However, in- cultured sugarcane seed cane as compared to conventional sugarcane
dependent effects were exerted on number of internode/cane, number of seed cane. Performance of seeds obtained from meristem culture was
canes/clump, yield of canes/ha and number of 3 eye bud sets/ha. The excellent as compared to conventional sugarcane seeds.
analysis of variance showed that there was no significant variation in Thus, the protocol developed in this study shows potential for the
agronomic characters when compared with row spacing except for the development of sugarcane meristem cultured plants and the use of these
number of canes/clump and number of internodes per cane (Table 8).
There are references for research on the spacing of meristem cultured
Table 8
plantlets, but no one has done this type of research on spacing for seed
Calculated F at 5% level of significance for agronomic characteristics as influ-
obtained from meristem cultured plantlets. enced by row and sett spacing.
All the above experiments helped to develop agronomy for seed
SN. Source of variation Row Setts Interaction
production from meristem cultured sugarcane plants. We have taken
spacing spacing (AaB)
demonstrations of meristem cultured plantlets on farms of sugar factories (A) (B)
in Maharashtra for showing performance of meristem cultured plantlets
1 Milliable height/cane 1.52N.S. 1.56N.S. 0.84N.S.
to farmers and sugar factories employee (MD, AO and CDO). Now 2 Girth of cane 1.78N.S. 0.56N.S. 1.21N.S.
farmers and sugar factories have realized the benefit of meristem culture 3 No. of internodes/cane 2.94a 3.48a 1.05N.S.
technology for improving sugarcane yield. 4 Length of internode 2.78N.S. 0.57N.S. 0.62N.S.
5 No. of canes/clump 6.65a 4.01a 0.15N.S.
6 Yield of canes/ha 1.02N.S. 6.40a 1.35N.S.
4. Conclusions 7 Yield. of three eye bud 0.51N.S. 3.22a 0.68N.S.
setts/ha
Meristem culture (tissue culture) of sugarcane is the most satisfactory
NS. Not significant.
and viable method for the production of healthy and pathogen-free seed a
Significant.
material of sugarcane. An efficient protocol for in vitro rapid

Table 7
Influence of spacing between 2 setts on agronomic traits of sugarcane.
S⋅N Agronomic traits TC15 TC30 TC45 C15 C30 C45 SE þ - CD at 5%

1 Milliable height (cm)/cane 222.98 233.44 231.83 221.13 224.23 223.71 5.22 NS.
2 Girth of cane (cm) 11.26 11.46 11.62 10.93 11.10 11.25 0.28 NS.
3 No. of internodes/cane 16.99 17.06 17.21 16.14 16.06 15.66 0.48 1.36a
4 Length of internodes 15.83 15.77 14.97 16.00 15.85 15.39 0.77 NS.
5 No. of canes/clump 15.43 14.17 13.73 13.10 12.43 11.95 0.88 2.53a
6 No. of canes/ha (thousand) 116 109 105 95 95 89 0.5 1.4a
7 Yield of 3 eye bud setts/ha (thousand) 81.9 76.9 74.1 67.1 66.8 62.6 4.0 11.4a

NS – Not significant.
a
- Significant.

7
S. Salokhe Journal of Agriculture and Food Research 4 (2021) 100126

plants for quality sugarcane seed production. This will help to supply [13] M. Ibrahim, J. Aman, T. Negi, Evaluation of tissue culture raised sugarcane
planting materials against their donor conventional seed sources as initial source
healthy seed cane to the sugarcane farmers, which is the main prereq-
of seed cane at tendaho sugar development project, North-Eastern Ethiopia,
uisite for improving the productivity of the sugarcane crop. We can also Journal of Horticulture 3 (1) (2016) 1–5.
make use of this technology for rapid multiplication of newly released [15] N.C. Jalaja, Micropropagation of sugarcane varieties for quality seed production, in:
varieties and for supplying seeds of the newly released varieties to the Manual on Sugarcane Production Technology, Sugarcane Breeding Institute,
Coimbatore, 2001, pp. 48–52.
sugarcane farmers within a short period of time. [16] S. Jamil, R. Shahzad, G.M. Talha, G. Sakhawat, S.U. Rahman, R. Sultana, M.Z. Iqbal,
Optimization of protocols for in vitro regeneration of sugarcane (Saccharum
Declaration of competing interest officinarum), International Journal of Agronomy (2017) 1–8.
[17] J. Kumar, S.C. Saxena, Seed Health Management for Better Productivity.
Proceedings of the 20th Training, Centre for advanced training in plant pathology,
No Conflict of Interest. ICAR, New Delhi, 2008.
[18] N. Lal, N.H. Singh, Rapid clonal multiplication of sugarcane through tissue culture,
Plant Tissue Cult. 4 (1) (1994) 1–7.
Acknowledgements [19] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with
tobacco tissue cultures, Physiol. Plantarum 15 (1962) 473–479.
The researcher is grateful to the Director-General of Vasandada Sugar [20] S.R. Naik, M.H. Kumar, N.V. Naidu, P. Latha, Yield comparisons: tissue culture
seedlings versus single bud setts, Int. J. Curr. Microbiol. App. Sci. 7 (11) (2018)
Institute (VSI), Pune for providing facilities to conduct the laboratory 2310–2315.
research in tissue culture laboratory of VSI, as well as field research trials [21] A. Panattoni, A. Luvisi, E. Triolo, Review: elimination of virus in plant: twenty years
on the farms of VSI. of progress, Spanish J. Agric. Res. 11 (1) (2013) 173–188 (S).
[22] K.A. Quiroz, M. Berríos, B. Carrasco, J.B. Retamales, P.D.S. Caligari, R.G. Gonzales,
Meristem culture and subsequent micropropagation of Chilean strawberry (Fragaria
Appendix A. Supplementary data chiloensis (L.) Duch.), Biol. Res. 50 (20) (2017) 1–11.
[23] S. Raghu, S. Jayram, P. Prabhakaran, V. Vekatesalu, Influence of spacing on growth
Supplementary data to this article can be found online at https://do and yield of sugarcane raised through in vitro micropropagation, Sugar Tech 8 (1)
(2006) 82–84.
i.org/10.1016/j.jafr.2021.100126. [24] S. Rithesh, Comparative Economics of Sugarcane Processed for Sugar Jaggery.
Thesis Submitted to the University of Agricultural Sciences, Dharwad in partial
References fulfilment of the requirements for the Degree of Master of Science (Agriculture),
2013.
[25] A.K. Sabaz, H. Rashid, C.M. Fayyaz, Z. Chaudhry, A. Afroz, Rapid micropropagation
[2] A. Ali, S. Naz, F. Siddiqui, J. Iqbal, An efficient protocol for large scale production of of three elite Sugarcane (Saccharum officinarum L.) varieties by shoot tip culture,
sugarcane through micropropagation, Pakistan J. Bot. 40 (1) (2008) 139–149. Afr. J. Biotechnol. 7 (13) (2008) 2174–2180.
[3] A. Ali, S. Naz, F.A. Siddiqui, J. Iqbql, An efficient protocol for large- scale [26] R. Sharma, S. Tamta, A review on red rot: the “cancer “of sugarcane, J. Plant Pathol.
production of sugarcane through micropropagation, Pakistan J. Bot. 40 (1) (2008) Microbiol. S1 (2015) 1–8, 003.
139–149. [27] R. Shukla, A.Q. Khan, G.K. Gang, In Vitro Clonal Multiplication of Sugarcane;
[4] R.A. Baily, G.R. Bechet, A Comparison of seed cane derived from tissue culture with Optimization of Media and Hardening of Plants, 1994, pp. 89–94. India.
conventional seed cane, Proceedings of the South African sugar technologists' [28] S. Solomon, L. Yang-Rui, The sugar industry of Asian region, Sugar Tech 16 (6)
association (1989) 125–129. (2016) 557–558.
[5] D.N. Balagalla, A. Wijesuriya, N.P. Ranathunge, A.M.M.S. Perera, A Protocol for In- [30] T.V. Srinivasan, N.C. Jalaja, Utilization of tissue culture technique in sugarcane
vitro direct plant regeneration from leaf tissues for micropropagation of sugarcane, improvement. C. Meristem culture. Annual Report, Sugarcane Breeding Institute,
Tropical Agricultural Research 29 (1) (2017) 55–64. Coimbatore, 1981, p. 68.
[7] M. Chakravarthi, Sugarcane Biotechnology: Challenges and Prospects, Springer, [31] D. Venkatesh, M. Venkateswarlu, An overview of Indian sugar industry, BIMS
Brazil, 2017, p. 2. International Journal of Social Science Research 2 (1) (2017) 11–16.
[9] S. Dabbert, I. Lewandowski, J. Weiss, A. Pyka, Knowledge-Driven Developments in [32] D.G.A. Walkey, Production of virus-free plants by tissue culture, in: J.P. Helgeson
the Bioeconomy: Technological and Economic Perspectives, Springer International (Ed.), Tissue Culture Methods for Plant Pathologists Ingram. D.A, Blackwell Oxford,
Publishing, Switzerland, 2017. 1980, pp. 109–117.
[10] S. Geeta, D. Padmanabhan, Effect of hormones on direct somatic embryogenesis in [33] P.S. Warakagoda, S. Subasinghe, D.L.C. Kumari, T.S. Neththikumara, Micro
sugarcane, Sugar Tech 3 (3) (2001) 120–121. propagation of sugarcane (Saccharum officinarum L.) through auxiliary buds,
[12] T.A. Hamza, A.L. Alebjo, Sugarcane (saccharum officinarum L) tissue culture in Proceeding of the forth academic session, 2007.
Ethiopia: opportunities for Ethiopia's sugar industries, International Journal of
Scientific and Technology Research 6 (8) (2017) 398–406.