A MICROPROPAGATION SYSTEM FOR CLONING OF HEMP

(CANNABIS SATIVA L.) BY SHOOT TIP CULTURE

REN WANG1*, LI-SI HE1, BING XIA1, JIN-FENG TONG1, NING LI2 AND FENG PENG1
1Institute of Botany, Jiangsu Province & Chinese Academy of Sciences, (Mem. Sun Yat-Sen);
Jiangsu province key laboratory for plant Ex-situ conservation, Nanjing, 210014, P.R. China
2Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay,
Hong Kong SAR, P.R. China
Abstract
This study describes the standardization of an efficient in vitro propagation and hardening
procedure for obtaining plantlets from shoot tips of Hemp (Cannabis sativa L.). Hemp seedlings
were germinated on half-strength 1/2 MS medium supplemented with 10 g·L-1sucrose, 5.5 g·L-1agar
at a pH of 6.8 under light for 16 h per day. MS medium containing 0.2 mg·L-1TDZ, 0.1 mg·L-1NAA
supported the maximal auxiliary bud multiplication rate of 3.22 per shoot tip. The proliferated buds
were successfully rooted on MS medium supplemented with 0.1 mg·L-1IBA and 0.05 mg·L-1NAA
resulting in 85% of the plantlets rooting. The procedure requires a 54 days cycle for the In vitro
clonal propagation (14 days for shoot multiplication and 40 days for root induction) which includes
35-42 days for acclimatized plantlet production.
Introduction
Hemp (Cannabis sativa L.) belongs to the Cannabidaceae family. It is an annual herb
that has been cultivated for the value of its fiber, and more recently for paper
manufacturing, oil extraction and medicinal or drug preparation (Ranalli et al., 1999). In
the 19th century, other fibre-producing plants such as cotton, jute and sisal became more
competitive and caused a decline in hemp production (Rode et al., 2005). However,
recently the interest in hemp has been renewed due to its adaptability to a wide variety of
agro-ecological conditions, its high yields compared with many other crops and the
production of medicinal components such as tetrahydrocannabinol (THC) (Struik et al.,
2000).
There are few research reports about the tissue culture of hemp. Most of these studies
were aimed at developing a cell culture system to obtain secondary metabolites,
particularly the THC class of cannabinoids that are distinctive to the genus of Cannabis
(Turner et al., 1980). Callus induction and a protocol for Agrobacterium mediated
transformation of hemp had been established (Hartsel et al., 1983; Braemer & Paris,
1987; Feeney & Punja, 2003), but no micro-propagation system had been reported until
now. In order to provide a basis for advancing the fundamental biology of hemp and
abundant raw materials for industrial production, the objective of this study was to
identify the condition for auxiliary bud induction and rooting of hemp to promote
regeneration of plantlets from shoot tips.
*Corresponding author Email: [email protected]
Phone: +86 25 84347111, Fax: +86 25 84347111

Plant material: Hemp (Cannbis satival L) varieties Changtu from Liaoning province,
China, were selected for this study. Hemp seeds were first washed for 20 min with 0.1%
antiseptic liquid detergent APSA80 (Amway Corporation, Ada, Michigan, USA). The
seeds were made free from the detergent foam by thoroughly washing under running tap
water for 60min., in beaker. The washed seeds were surface sterilized by briefly dipping
in 75% (v/v) ethanol for 30 s followed by four washes with sterile distilled water and
subsequent sterilization for 10-15 min., with 0.1% Mercuric chloride. The treated seeds
were finally rinsed 8 times with sterile distilled water to remove traces of sterilizing
agents. The sterilized seeds were germinated on 1/2 MS medium with 10 g·L-1sucrose,
5.5 g·L-1agar, pH 6.8, under light for 16 h per day.
Auxiliary bud and root induction: Two sets of experiments were conducted for
auxiliary bud induction. In both case, shoot tips were harvested from 20-day-old sterile
plantlets. The shoot tips were grown In vitro on MS medium (Murashige & Skoog, 1962)
supplemented with BA (1.0, 2.0, 5.0 mg·L-1), KT(1.0, 2.0, 5.0 mg·L-1) or TDZ(0.1, 0.2,
0.5 mg·L-1) respectively in Experiment I and 0.05, 0.1, 0.5 mg·L-1NAA in Experiment II.
Sterile plantlets produced from the shoot tips were then rooted introduction to free-of
basic nutrition medium supplemented with 0.1, 0.5 mg·L-1IBA respectively or in
combination with 0.05, 0.25 mg·L-1 of NAA or IAA (Table 1). In order to choose the
optimal medium for root development, the plantlets were introduced to 1/2MS, MS, B5
(Gamborg et al., 1968) or NN (Nitsch & Nitsch, 1969) with the optimal level of plant
growth regulators (Table 1).
Culture conditions: All the media for auxiliary bud induction and plantlet root
production contained 30 g·L-1 sucrose as carbon source, 6.8 g·L-1agar. The pH of the
medium was adjusted to 5.8 before sterilizing in an autoclave at a temperature of 121oC
and at the pressure of 151 bs psi for 15 min. The cultures were kept at 25±1oC with 16 h
light cycle of a light intensity of 2500 lux in every 24 h. Each treatment comprising of 20
explants pieces was repeated twice.
Hardening and transplantation: The In vitro raised plantlets were deflasked, when the
length of the roots reached 0.5-1.0 cm. One day after deflasking, 10 ml of sterile water was
poured into the bottle with cultured plantlets of hemp. The plantlets were cultured for a
further week in the bottle after which the hemp plantlets were thoroughly washed in
running tap water so as to remove any adhering medium from the surface of the plantlets
followed by washings with 0.2% (w/v) Bavistin1 and tap water. The washed plantlets were
transferred to root trainers consisting of 20 cells, each of 200 cm3, filled with perlite and 10
ml water and cultured for an extra 2-3 weeks. Finally, the plantlets were transferred to
plastic pot, filled with vermiculite and plant ash and grown in shade-house. After an
acclimatization period of 2 weeks, the plantlets were able to be transplanted in field.
Statistical analysis: The successful production of viable plantlets was evaluated at 40 days,
and the data was analyzed by SPSS software, version 10.0 (Supplier, location) by analysis
of variance (ANOVA). The mean differences were tested using ‘F’ test at p<0.05 level.

Result and Discussion

Auxiliary bud induction: Aseptic shoot tips were introduced to MS medium
supplemented with different types of cytokinin for auxiliary bud induction (Fig. 1A).
Table 2 shows the effect of different concentrations of BA, KT and TDZ on shooting
response in the shoot tips of hemp. Cytokinin stimulated shoot formation and stem
enlargement for each explant in these nine treatments. Among the three cytokinins tested,
TDZ (0.2mg·L-1) was found to provide the best bud induction, inducing an average of
3.22 buds with the thickest stem (Fig. 1B). Furthermore, the type of cytokinin in the
medium also affected plantlet morphology, with the plantlets grown in TDZ-containing
medium being more compact and vigorous. The suitability of TDZ for In vitro auxiliary
shoot propagation has been well established in many woody plants tissue culture (Carl &
John, 1993), and also determinated in many herbage plants (Donna & John, 2004; Chitra
& Padmaja, 2005; Peddaboina et al., 2006).
Table 3 shows the effect of different NAA concentrations on the growth of auxiliary
buds, where the supply of 0.1 mg·L-1 NAA concentration was optimal for auxiliary bud
induction.

Root establishment: When the regenerated auxiliary buds were separated and transferred
to the rooting medium with different hormones, rhizogenesis occurred followed by
enlargement of the stem during 40 days culture. The effects of environmental and medium
factors on rooting were investigated by evaluating the rooting percentage, root length, and
number of roots per plantlet. The combination and concentration of auxin forms
significantly influences the number of roots formed (Table 1). The best rooting and
elongation occurred with treatment 2 that contained 0.1 mg·L-1IBA and 0.05 mg·L-1NAA
(Fig. 1C). The combination of these two auxins for increasing rooting has previously been
observed (Amin et al., 1987; Kornova, 1995). ANOVA evaluation (Table 4), showed that
the F value was higher than the 0.05 mg·L-1 level of these auxins, demonstrating that
different treatments supplemented with different combinations and concentrations of
hormones have distinct effects on plantlet rooting performance (Table 4).
Four intermediate formulations, 1/2MS, MS, B5 and NN media, were tested for
growth media for rooting culture. Both the basic media of 1/2MS and MS were found to
have a superior root proliferation rate of 75% and 85%, while the B5 and NN basic media
did not differ in root length and root number performance (Table 5). The induction of
root numbers was maximal in those plantlets cultured on MS (2.45 roots/plantlet),
followed by those on 1/2MS (2.05 roots/plantlet), B5 (0.85 roots/plantlet) and NN
medium (0.60 roots/plantlet) (Fig. 1D).
Hardening and transplantation: The well grown In vitro plantlets, after treatment with
0.2% (w/v) Bavistin1, were hardened by the following two-step procedure: (i) controlled
conditions of culture room for 3-4 weeks and (ii) semi controlled conditions in the shade
house for 2 weeks allowed 95% of plantlets to acclimatize, after which 99% of these
plantlets were surviving for 3 months after transfer to the field.
Table 3. The effect of different NAA concentrations on the growth of auxiliary buds.

This work was supported by the National Natural Science Foundation of China (grant
no. 30700057), and the opening fund of Jiangsu Province key laboratory for plant Ex-situ
conservation (KF07001). We wish to thank Dr. Ewan from Tasmanian Institute of
Agricultural Research at University of Tasmania and Prof. Wenbiao Shen from the College
Life Sciences at Nanjing Agricultural University for critical reading and linguistic help in
the preparation of this manuscript. We also thank the members of the Laboratory of Plant
Molecular Miology, Institute of Botany, Jiangsu province & Chinese academy of sciences.

Auxiliar yema y la inducción de raíz: Dos conjuntos de experimentos se llevaron a cabo

para la inducción de yemas auxiliares. En ambos casos, los ápices se recogerán a partir
de plántulas de estériles de 20 días de edad. Los ápices se cultivaron in vitro en un
medio MS (Murashige y Skoog, 1962) suplementado con BA (1.0, 2.0, 5.0 mg • L-1),
KT (1.0, 2.0, 5.0 mg • L-1) o TDZ (0,1 , 0.2, 0.5 mg • L-1), respectivamente, en el
experimento I y 0,05, 0,1, 0,5 mg • L-1NAA en el Experimento II. Estéril plántulas
producidas a partir de los ápices fueron arraigados a continuación, introducción a la
libre medio de la nutrición básica complementada con 0,1, 0,5 mg • L-1IBA,
respectivamente, o en combinación con 0,05, 0,25 mg • L-1 de ANA o AIA (Tabla 1).
Con el fin de elegir el medio óptimo para el desarrollo de las raíces, las plántulas se
presentó a 1/2MS, MS, B5 (Gamborg et al., 1968) o NN (Nitsch y Nitsch, 1969) con el
nivel óptimo de reguladores de crecimiento vegetal (Tabla 1).

Auxiliar brote de inducción: consejos aséptica disparar fueron introducidos en un medio

MS suplementado con diferentes tipos de citoquininas para la inducción de yemas
auxiliares (fig. 1A). La Tabla 2 muestra el efecto de diferentes concentraciones de BA,
KT y TDZ en una respuesta de disparo en los ápices de cáñamo. Citoquinina stimulated
formación de brotes y tallos la ampliación para cada explante en estos nueve
tratamientos. Entre los tres citocininas probado, TDZ (0,2 mg • L-1) fue encontrado
para proporcionar la mejor inducción yemas, induciendo a un promedio de 3,22 brotes
con la parte más gruesa del tallo (Fig. 1B). Por otra parte, el tipo de citoquinina en el
medio también se ve afectada la morfología de plántulas, con las plántulas cultivadas en
TDZ que contienen medio de ser más compacto y vigoroso. La idoneidad de TDZ para
la propagación in vitro disparar auxiliares ha sido bien establecida en muchas plantas de
cultivo de tejidos leñosos (Carlos y Juan, 1993), y también se determinó en las plantas
de forraje muchos (Donna & John, 2004; Chitra y Padmaja, 2005; Peddaboina et al.,
2006). La Tabla 3 muestra el efecto de diferentes concentraciones de ANA en el
crecimiento de yemas axilares, donde la oferta de 0,1 mg • L-1 la concentración de
NAA fue óptima para la inducción de yemas auxiliares.

Establecimiento de raíz: Cuando los brotes regenerados auxiliares fueron separados y

trasladados al medio de enraizamiento con hormonas diferentes, rizogénesis ocurrido
seguido por la ampliación de la madre durante 40 días de cultivo. Los efectos de
factores ambientales y medio de enraizamiento fueron investigados por la evaluación
del porcentaje de enraizamiento, longitud de raíz, y el número de raíces por plántula. La
combinación y concentración de las formas auxina influye significativamente en el
número de raíces formadas (Tabla 1). La mejor enraizamiento y el alargamiento
producido con el tratamiento 2, que contenía 0,1 mg • L-1IBA y 0,05 mg • L-1NAA
(Fig. 1C). La combinación de estos dos auxinas para aumentar el enraizamiento ha
observado previamente (Amin et al, 1987;. Kornova, 1995). Evaluación de análisis de
varianza (Cuadro 4), mostró que el valor de F fue mayor que el 0,05 mg • L-1 de estas
auxinas, lo que demuestra que los diferentes tratamientos suplementados con diferentes
combinaciones y concentraciones de hormonas tienen efectos distintos sobre el
enraizamiento de plántulas de rendimiento (Cuadro 4) .
Cuatro fórmulas intermedias, 1/2MS, MS, B5 y los medios de comunicación NN,
fueron probados para medios de cultivo para el enraizamiento cultura. Tanto los medios
básicos de 1/2MS y los Estados miembros tenían una tasa de proliferación de la raíz
superior del 75% y 85%, mientras que la B5 y los medios de comunicación NN de base
no se diferencian en la longitud de raíz y el desarrollo de raíces número (Tabla 5). La
inducción de los números de la raíz fue máxima en las plántulas cultivadas en MS (2,45
raíces / plántula), seguidos de los de 1/2MS (2.05 raíces / plántula), B5 (0,85 raíces /
plántula) y medianas NN (0,60 raíces / plántula) (Fig. 1D).

Endurecimiento y el trasplante: El crecido bien en plántulas in vitro, después del

tratamiento con 0,2% (w / v) Bavistin1, se endurecieron por el siguiente procedimiento
de dos pasos: (i) condiciones controladas de la sala de la cultura durante 3-4 semanas, y
(ii) semi condiciones controladas en la casa de sombra durante 2 semanas permitido del
95% de las plántulas de aclimatación, tras los cuales el 99% de estas plántulas
sobrevivientes fueron durante 3 meses después de la transferencia al campo.